**Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity**

Ariane Zamoner and Regina Pessoa-Pureur

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66926

#### **Abstract**

In this chapter, we deal with the current knowledge and important results on the cyto‐ skeletal proteins and their differential regulation by kinases/phosphatases and Ca2+‐ mediated mechanisms in developmental rat brain. We focus on the misregulation of the phosphorylating system associated with intermediate filament proteins of neural cells and its relevance to cell and tissue dysfunction. Taking into account our findings, we propose that intermediate‐filament proteins are dynamic structures whose regulation is crucial for proper neural cell function. Given their relevance, they must be regulated in response to extracellular and intracellular signals. The complexity and connection between signaling pathways regulating intermediate‐filament dynamics remain obscure. In this chapter, we get light into some kinase/phosphatase cascades downstream of membrane receptors disrupting the dynamics of intermediate filaments and its associa‐ tion with neural dysfunction. However, intermediate filaments do not act individually into the neural cells. Our results evidence the importance of misregulated cytoskeletal crosstalk in disrupting cytoskeletal dynamics and cell morphology underlying neural dysfunction in experimental conditions mimicking metabolic diseases and nongenomic actions of thyroid hormones and as an end point in the neurotoxicity of organic tellurium.

**Keywords:** intermediate filament, cytoskeleton, cell signaling, calcium, neurotoxicity

### **1. Introduction**

All the cell functions accomplished by the living cell are dependent on a sophisticated net‐ work of protein filaments with different compositions, distributions and roles into the cell, forming an integrated meshwork known as the cytoskeleton. However, the most striking fea‐ ture of the cytoskeleton concerns its ability to respond to signals and conditions to which cells are submitted, taking part of adaptive cell response to different stimuli. The cytoskeleton is

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an end point of signaling pathways adapting cells to immediate or long‐lasting behaviors in healthy and sick organisms.

Cytoskeleton of most animal cells is constituted by three interconnected filament subsystems: microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). Compelling evidence from the last decades has brought convincing understanding for the highly regu‐ lated and interconnected interactions between the cytoskeletal elements giving support to sculpting and maintaining cell shape and sustaining all kinds of morphological alterations or internal organization, as well as their implications for the behavior of animal cells. **Figure 1** demonstrates the organization of the cytoskeleton in neurons.

A cohort of accessory proteins and signaling machinery regulates the dynamic turnover of the cytoskeleton. Although each type of filament has specific cell distribution, molecular constitu‐ ents and equilibrium, the coordinated intertwining among the different networks provides the force for a number of coherent processes in response to all kinds of intra‐ and extracellular stimuli leading responses so decisive as cell survival or death [1].

This chapter initiates with a brief introduction about the structure and function of IFs, empha‐ sizing those from neural cells. However, the main purposes of the chapter are the experimen‐ tal evidence of our laboratory that the roles of IFs are beyond protection from mechanical and nonmechanical stress. They might be the end point of misregulated‐signaling mechanisms in neurotoxic conditions adapting their dynamics, in concert with the other cytoskeletal fibers, to cell survival or death.

**Figure 1.** Distribution of cytoskeletal constituents into neurons. Neuronal cytoskeleton is composed by microfilaments, microtubules, and intermediate filaments. The microtubules are nucleated at the centrosome, then released and delivered to either the dendrites or the axon. Neurofilaments are abundant in axons and the spacing of neurofilaments is sensitive to the level of phosphorylation. The microfilaments are dispersed within the cells and they are most abundant near the plasma membrane.

### **2. Intermediate filaments**

an end point of signaling pathways adapting cells to immediate or long‐lasting behaviors in

Cytoskeleton of most animal cells is constituted by three interconnected filament subsystems: microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). Compelling evidence from the last decades has brought convincing understanding for the highly regu‐ lated and interconnected interactions between the cytoskeletal elements giving support to sculpting and maintaining cell shape and sustaining all kinds of morphological alterations or internal organization, as well as their implications for the behavior of animal cells. **Figure 1**

A cohort of accessory proteins and signaling machinery regulates the dynamic turnover of the cytoskeleton. Although each type of filament has specific cell distribution, molecular constitu‐ ents and equilibrium, the coordinated intertwining among the different networks provides the force for a number of coherent processes in response to all kinds of intra‐ and extracellular

This chapter initiates with a brief introduction about the structure and function of IFs, empha‐ sizing those from neural cells. However, the main purposes of the chapter are the experimen‐ tal evidence of our laboratory that the roles of IFs are beyond protection from mechanical and nonmechanical stress. They might be the end point of misregulated‐signaling mechanisms in neurotoxic conditions adapting their dynamics, in concert with the other cytoskeletal fibers,

**Figure 1.** Distribution of cytoskeletal constituents into neurons. Neuronal cytoskeleton is composed by microfilaments, microtubules, and intermediate filaments. The microtubules are nucleated at the centrosome, then released and delivered to either the dendrites or the axon. Neurofilaments are abundant in axons and the spacing of neurofilaments is sensitive to the level of phosphorylation. The microfilaments are dispersed within the cells and they are most abundant near the

demonstrates the organization of the cytoskeleton in neurons.

stimuli leading responses so decisive as cell survival or death [1].

healthy and sick organisms.

234 Cytoskeleton - Structure, Dynamics, Function and Disease

to cell survival or death.

plasma membrane.

### **2.1. Molecular architecture of intermediate filaments**

IFs are flexible, rod‐shaped fibers averaging 10 nm in diameter, a size that is intermediate between MFs and MTs. They are ubiquitous constituents of the structural scaffold of the eukaryotic cells and considered mechanical integrators of cytomatrix [2]. These cytoskeletal filaments are widespread expressed in practically all animal cell types and are the most diverse cytoskeletal protein family, encoded by an estimated 70 IF genes in the humans. IFs have been grouped into six sequence homology classes (SHC) according to the degree of sequence identity: acidic keratins (SHC group I); basic keratins (SHC group II); desmin, vimentin and other mesenchymal IF proteins, such as glial fibrillary acidic protein (GFAP) (SHC group III); neurofilament proteins (SHC group IV); and lamins (SHC group V).

IF building blocks are fibrous proteins stabilized by multistranded left‐handed coiled coils giving rise to a rope‐like structure. Their structures are constituted by a long central α‐helical region, also designed rod domain, with a distinct number of equally sized coiled coils forming segments flanked by non‐α‐helical N‐terminal (the head domain) and C‐terminal domains (the tail domain). Both head and tail domains are highly varying in size and sequence, thus, the functional and molecular heterogeneity of IF proteins are a consequence of the highly variable non‐α‐helical end domains of subunits.

The central rod domain of IF subunits is α‐helical rod highly charged, with a role in the first phase of IF assembly. By contrast, the head domain enriched in basic amino acids is essential for the formation of tetramers (the polymerization units) and complete IF assembly.

The non‐α‐helical tail domain can vary drastically between different IF proteins. This domain is not essential for the assembly of cytoplasmic IFs but plays a significant role in filament width control. The functional role of the tail domain is particularly important in the neurofila‐ ments, the neuronal‐specific IFs, as discussed below.

Overall, the assembly of subunits giving rise to functional IFs is a complex and multistep process with individual specificities among the different representatives of this molecularly heterogeneous family. Taking into account the *in vitro* polymerization of vimentin, filament assembly starts with the formation of parallel, in‐register dimers. These dimers spontane‐ ously associate laterally into antiparallel, half‐staggered tetramers. Tetramers aggregate into higher‐order oligomers to form unit length filaments (ULFs) that undergo reorganization and elongation by longitudinal annealing to form immature IFs. The final step is radial compac‐ tion of the filaments from approximately16 nm to a diameter of 10–12 nm [3].

Different from the other IFs, NFs comprise three subunits with different molecular masses and distributions into the filament. They are formed by light, medium and heavy molecular mass NF triplet proteins (NF‐L, NF‐M and NF‐H), respectively. NF‐L can self‐assemble form‐ ing the core of the filament. NF‐M and NF‐H are peripherally disposed on the filament, with their long and flexible tails rich in highly charged domains and multiple phosphorylation sites, radially projecting out from the filament backbone when NF‐M and NF‐H co‐assemble with the short‐tail NF protein NF‐L. Interestingly, NF‐H and NF‐M by their own are not able to assemble into filaments, but by contrast, self‐assembled NF‐L yields normal looking 10‐nm filaments. These side arms of NF‐M and NF‐H contain multiple phosphorylation sites regulat‐ ing the interactions of NFs with each other and with other cytoskeletal structures [4].

#### **2.2. Roles of intermediate filaments in neural cell function**

Neurons are highly specialized in the transmission and processing of electrical and chemical signals. A functional nervous system is dependent of a proper axonal array, which in turn is critically dependent upon the organization of the axonal cytoskeleton. Five main subunit proteins form the neuronal specific NFs: the group IV NF‐L, NF‐M and NF‐H triplet pro‐ teins, α‐internexin and the group III peripherin. Mature filaments are composed of several combinations of these five subunits. In most differentiated neurons, α‐internexin expression precedes that of the NF triplet and declines somewhat postnatally, while the expression of the NF triplet sharply rises. Neurofilaments found in perikarya, dendrites and axons differ considerably in their organization and function. Perikarial NFs form a meshwork around the nucleus. In the axons of mature neurons, a large number of longitudinally oriented and phosphorylated NFs play a fundamental role increasing the diameter of myelinated axons and consequently nerve conductivity. Neurofilaments present in dendrites are less abundant and less phosphorylated than those of axons.

Neurofilaments are transported from the cell body, where they are synthesized, to be deliv‐ ered along the axon by a mechanism called axonal transport. The motors implicated in the anterograde transport are kinesins, while the retrograde transport is mediated in association with dynein, the same motor proteins involved in the fast axonal transport along MTs [4].

The multiple roles of cytoskeletal proteins in the neural cells imply that there is an underlying cytoskeletal pathology associated with several neurodegenerative processes. The major neu‐ rodegenerative diseases are characterized by the presence of inclusion bodies in implicated neurons. These inclusion bodies all contain elements of the cytoskeleton. In addition, muta‐ tions and/or accumulations of NFs are frequently observed in several human neurodegenera‐ tive disorders including amyotrophic lateral sclerosis, Alzheimer disease, Parkinson disease, Charcot‐Marie‐Tooth, giant axonal neuropathy, neuronal intermediate‐filament inclusion disease and diabetic neuropathy [5]. Multiple factors can potentially induce the accumulation of NF, including deregulation of NF gene expression, NF mutations, defective axonal trans‐ port, abnormal posttranslational modifications and proteolysis [4]**.** Beyond their association with neural damage in inherited or age‐dependent neurodegenerative diseases, studies from our laboratory indicated that the disruption of NF homeostasis is a response to toxic agents and abnormally accumulated metabolites in rat brain.

Astrocytes are important cytoarchitectural elements of the CNS; however, during the past few years, molecular and functional characterization of astroglial cells indicates that they have a much broader function than only support the neurons in the brain. Compelling evidence sup‐ ports that astrocytes have specialized functions in inducing and regulating the blood‐brain barrier (BBB), glutamate uptake, synaptic transmission, plasticity and metabolic homeosta‐ sis of the brain [6]. Astrocytes express 10 different isoforms of glial fibrillary acidic protein (GFAP), the specific astrocytic IF, together with vimentin, nestin and synemin. However, GFAP is the main IF protein expressed in mature astrocytes, where it helps maintaining mechanical strength, as well as cell shape. However, recent evidence has shown that GFAP plays a role in a variety of additional astrocyte functions, such as cell motility/migration, cell proliferation, glutamate homeostasis, neurite outgrowth and injury/protection [7].

Astrocytes are also involved in a wide range of CNS pathologies, including trauma, isch‐ emia and neurodegeneration. In such situations, the cells change both their morphology and their expression of many genes leading to activation of astroglia, or astrogliosis. It is accepted that the increase of IFs with accompanying cellular hypertrophy and an abnormal apparent increase in the number of astrocytes characterize astrogliosis. However, upregula‐ tion of IF proteins, in particular GFAP, but also vimentin and nestin, two IF proteins abun‐ dantly expressed in immature astrocytes, is regarded as the hallmark of astrogliosis [7]. In this regard, the most remarkable evidence of the relevance of GFAP in the physiological roles of astrocytes in maintaining normal brain function is Alexander disease, a fatal disorder in which GFAP mutations might compromise the astrocyte stress response [8].

### **3. Protein phosphorylation in signaling transduction**

with the short‐tail NF protein NF‐L. Interestingly, NF‐H and NF‐M by their own are not able to assemble into filaments, but by contrast, self‐assembled NF‐L yields normal looking 10‐nm filaments. These side arms of NF‐M and NF‐H contain multiple phosphorylation sites regulat‐

Neurons are highly specialized in the transmission and processing of electrical and chemical signals. A functional nervous system is dependent of a proper axonal array, which in turn is critically dependent upon the organization of the axonal cytoskeleton. Five main subunit proteins form the neuronal specific NFs: the group IV NF‐L, NF‐M and NF‐H triplet pro‐ teins, α‐internexin and the group III peripherin. Mature filaments are composed of several combinations of these five subunits. In most differentiated neurons, α‐internexin expression precedes that of the NF triplet and declines somewhat postnatally, while the expression of the NF triplet sharply rises. Neurofilaments found in perikarya, dendrites and axons differ considerably in their organization and function. Perikarial NFs form a meshwork around the nucleus. In the axons of mature neurons, a large number of longitudinally oriented and phosphorylated NFs play a fundamental role increasing the diameter of myelinated axons and consequently nerve conductivity. Neurofilaments present in dendrites are less abundant

Neurofilaments are transported from the cell body, where they are synthesized, to be deliv‐ ered along the axon by a mechanism called axonal transport. The motors implicated in the anterograde transport are kinesins, while the retrograde transport is mediated in association with dynein, the same motor proteins involved in the fast axonal transport along MTs [4].

The multiple roles of cytoskeletal proteins in the neural cells imply that there is an underlying cytoskeletal pathology associated with several neurodegenerative processes. The major neu‐ rodegenerative diseases are characterized by the presence of inclusion bodies in implicated neurons. These inclusion bodies all contain elements of the cytoskeleton. In addition, muta‐ tions and/or accumulations of NFs are frequently observed in several human neurodegenera‐ tive disorders including amyotrophic lateral sclerosis, Alzheimer disease, Parkinson disease, Charcot‐Marie‐Tooth, giant axonal neuropathy, neuronal intermediate‐filament inclusion disease and diabetic neuropathy [5]. Multiple factors can potentially induce the accumulation of NF, including deregulation of NF gene expression, NF mutations, defective axonal trans‐ port, abnormal posttranslational modifications and proteolysis [4]**.** Beyond their association with neural damage in inherited or age‐dependent neurodegenerative diseases, studies from our laboratory indicated that the disruption of NF homeostasis is a response to toxic agents

Astrocytes are important cytoarchitectural elements of the CNS; however, during the past few years, molecular and functional characterization of astroglial cells indicates that they have a much broader function than only support the neurons in the brain. Compelling evidence sup‐ ports that astrocytes have specialized functions in inducing and regulating the blood‐brain barrier (BBB), glutamate uptake, synaptic transmission, plasticity and metabolic homeosta‐ sis of the brain [6]. Astrocytes express 10 different isoforms of glial fibrillary acidic protein

ing the interactions of NFs with each other and with other cytoskeletal structures [4].

**2.2. Roles of intermediate filaments in neural cell function**

236 Cytoskeleton - Structure, Dynamics, Function and Disease

and less phosphorylated than those of axons.

and abnormally accumulated metabolites in rat brain.

Phosphorylation is the most widespread type of posttranslational modification of the intracel‐ lular signaling proteins. Phosphorylation of proteins occurs within seconds or minutes of a regulatory signal, typically an extracellular signal.

Phosphorylation is an enzymatic process in which the introduction of a phosphoryl group to specific amino acid residues of a protein is catalyzed by protein kinases and the removal of phosphoryl groups is catalyzed by protein phosphatases. For phosphorylation to be useful in the regulation of a protein activity, it is important to be a reversible process, in which the phosphorylated form of the protein could restore its original dephosphorylated form when signal ends, functioning therefore as a molecular switch. The addition of a phosphoryl group to the side chain of a Ser, Thr, or Tyr residue introduces a bulky, charged group into a polar region. The oxygen atoms of a phosphoryl group can hydrogen bond with one or several groups in a protein, commonly the amide groups of the peptide backbone at the α‐helix start or the charged guanidinium group of an Arg residue influencing the functionality of the protein [9].

#### **3.1. Phosphorylation of intermediate‐filament proteins**

Phosphorylation, glycosylation and transglutamination take part in the multiple mechanisms of IF regulation. However, phosphorylation/dephosphorylation is a major regulatory mecha‐ nism orchestrating IF dynamics. Phosphorylation sites of IF subunits are located on their head and tail domains and phosphorylation plays a major role in regulating the structural organi‐ zation and function of these cytoskeletal proteins in a cell‐ and tissue‐specific manner [10].

Amino‐terminal phosphorylation regulates the assembly/disassembly equilibrium of type III and IV IFs. Second messenger‐dependent protein kinases add phosphate groups on the amino‐terminal head domain on GFAP, vimentin and NF‐L. Specific phosphorylating sites for cAMP‐dependent protein kinase (PKA), Ca2+/calmodulin‐dependent protein kinase II (PKCaMII) and protein kinase C (PKC) are associated with IF disassembly; however, the action of the protein phosphatases 1, 2A and 2B (PP1, PP2A and PP2B), respectively, removes phosphate and restores the IF ability to polymerize [11].

Otherwise, the main phosphorylation sites on NF‐M and NF‐H are located in Lys‐Ser‐Pro (KSP) repeat regions of the tail domain of these subunits. The KSP repeats are phosphor‐ ylated by proline‐directed kinases such as Cdk5, the mitogen‐activated protein kinases (MAPK) such as Erk1/2, JNK, p38MAPK as well as glycogen synthase kinase 3 (GSK3). Phosphorylation of these KSP sites regulates the interactions of NFs with each other and with other cytoskeletal structures, since the tail domain of NF‐M and NF‐H protrudes later‐ ally from the filament backbone to form "side‐arms" when phosphorylated. These lateral interactions are central in the formation of a cytoskeletal lattice that supports the mature axon. Moreover, carboxyl‐terminal phosphorylation of NF‐M and NF‐H subunits has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons [12]. The axonal transport of NFs results from binding to the fast motor proteins kinesin and dynein intermitted with prolonged pauses. It is known that carboxyl‐ terminal phosphorylation of NF‐H progressively restricts the association of NFs with kinesin and stimulates its interaction with dynein. This event could represent one of the mechanisms by which aberrant carboxyl‐terminal phosphorylation would slow NF axonal transport. Both the maintenance of axonal caliber and axonal transport are dependent on the adequately phosphorylated NF subunits. Consequently, abnormally hyperphosphorylated NF subunits, commonly found in several neurodegenerative diseases, are intimately associated with neu‐ ral dysfunction and considered a hallmark of neurodegeneration. In addition, demyelinating diseases might be associated with hypophosphorylated NFs, compromised axonal transport and decreased axonal diameter, since the phosphorylation of NFs occurs in close proximity to myelin sheaths, which release signals needed to induce phosphorylation of NFs in mature axons [13].

In the next sections, we discuss the recent findings from our laboratory indicating that sig‐ naling mechanisms involved in the regulation of IF phosphorylation/dephosphorylation are important targets of neurotoxins, metabolites accumulating in neurodegenerative diseases as well as thyroid hormones, emphasizing the relevance of cytoskeletal homeostasis on the brain function/dysfunction. To assess the effects of the neurotoxicants on the phosphorylation level of IF proteins, we developed an approach to measure the *in vitro* incorporation of radioactive phosphate (<sup>32</sup>P‐orthophosphate) into these proteins [14]. In order to shed light onto the sig‐ naling cascades targeted by them, we used pharmacological and immunological approaches, specific enzyme inhibitors, channel blockers, or glutamate antagonists as well as monoclonal antibodies directed to signaling cascades or specific phosphorylation sites. We conclude that misregulated cell signal transduction interferes with the phosphorylation/dephosphorylation of IFs disrupting the homeostasis of the cytoskeleton of astrocytes and neurons and this is associated with cell dysfunction and neurodegeneration in experimental models of neurotox‐ icity. **Figure 2** corresponds to a schematic representation of the consequences of misregulated NF phosphorylation for neuronal function.

Intermediate Filaments as a Target of Signaling Mechanisms in Neurotoxicity http://dx.doi.org/10.5772/66926 239

amino‐terminal head domain on GFAP, vimentin and NF‐L. Specific phosphorylating sites for cAMP‐dependent protein kinase (PKA), Ca2+/calmodulin‐dependent protein kinase II (PKCaMII) and protein kinase C (PKC) are associated with IF disassembly; however, the action of the protein phosphatases 1, 2A and 2B (PP1, PP2A and PP2B), respectively, removes

Otherwise, the main phosphorylation sites on NF‐M and NF‐H are located in Lys‐Ser‐Pro (KSP) repeat regions of the tail domain of these subunits. The KSP repeats are phosphor‐ ylated by proline‐directed kinases such as Cdk5, the mitogen‐activated protein kinases (MAPK) such as Erk1/2, JNK, p38MAPK as well as glycogen synthase kinase 3 (GSK3). Phosphorylation of these KSP sites regulates the interactions of NFs with each other and with other cytoskeletal structures, since the tail domain of NF‐M and NF‐H protrudes later‐ ally from the filament backbone to form "side‐arms" when phosphorylated. These lateral interactions are central in the formation of a cytoskeletal lattice that supports the mature axon. Moreover, carboxyl‐terminal phosphorylation of NF‐M and NF‐H subunits has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons [12]. The axonal transport of NFs results from binding to the fast motor proteins kinesin and dynein intermitted with prolonged pauses. It is known that carboxyl‐ terminal phosphorylation of NF‐H progressively restricts the association of NFs with kinesin and stimulates its interaction with dynein. This event could represent one of the mechanisms by which aberrant carboxyl‐terminal phosphorylation would slow NF axonal transport. Both the maintenance of axonal caliber and axonal transport are dependent on the adequately phosphorylated NF subunits. Consequently, abnormally hyperphosphorylated NF subunits, commonly found in several neurodegenerative diseases, are intimately associated with neu‐ ral dysfunction and considered a hallmark of neurodegeneration. In addition, demyelinating diseases might be associated with hypophosphorylated NFs, compromised axonal transport and decreased axonal diameter, since the phosphorylation of NFs occurs in close proximity to myelin sheaths, which release signals needed to induce phosphorylation of NFs in mature

In the next sections, we discuss the recent findings from our laboratory indicating that sig‐ naling mechanisms involved in the regulation of IF phosphorylation/dephosphorylation are important targets of neurotoxins, metabolites accumulating in neurodegenerative diseases as well as thyroid hormones, emphasizing the relevance of cytoskeletal homeostasis on the brain function/dysfunction. To assess the effects of the neurotoxicants on the phosphorylation level of IF proteins, we developed an approach to measure the *in vitro* incorporation of radioactive phosphate (<sup>32</sup>P‐orthophosphate) into these proteins [14]. In order to shed light onto the sig‐ naling cascades targeted by them, we used pharmacological and immunological approaches, specific enzyme inhibitors, channel blockers, or glutamate antagonists as well as monoclonal antibodies directed to signaling cascades or specific phosphorylation sites. We conclude that misregulated cell signal transduction interferes with the phosphorylation/dephosphorylation of IFs disrupting the homeostasis of the cytoskeleton of astrocytes and neurons and this is associated with cell dysfunction and neurodegeneration in experimental models of neurotox‐ icity. **Figure 2** corresponds to a schematic representation of the consequences of misregulated

phosphate and restores the IF ability to polymerize [11].

238 Cytoskeleton - Structure, Dynamics, Function and Disease

axons [13].

NF phosphorylation for neuronal function.

**Figure 2.** Schematic representation of disrupted neurofilament phosphorylation. The hyperphosphorylation of neurofil‐ aments can change the cytoskeleton architecture and lead to neurofilament aggregation in perikarya and in axon accounting for cell damage.

### **3.2. Central roles of Ca2+ and glutamate receptors on the regulation of cytoskeletal dynamics in neural cells**

Changes in the cytoplasmic free Ca2+ concentration constitute one of the main pathways by which information is transferred from extracellular signals received by animal cells to intra‐ cellular sites. However, an augmented Ca2+ influx through the NMDA receptor or voltage‐ dependent calcium channels (VDCCs) can be responsible for the activation of lethal metabolic pathways in neural cells. Overactivation of glutamate receptors produces neuronal membrane depolarization. This causes the influx of Ca2+ into the cytoplasm and subsequently triggers cas‐ cade events leading to excitotoxic neuronal death. Excitotoxicity is recognized as a major patho‐ logical process of neuronal death in neurodegenerative diseases involving the CNS. In this regard, compelling findings point to the cytoskeleton as an end point of excitotoxic mechanisms.

Different toxins and stress conditions are implicated in the misregulation of intracellular Ca2+‐dependent processes in cells and different cell types exhibit a diverse range of transient responses to their stimuli. Exposure of tissue slices to neurotoxicants or metabolites in toxic concentrations triggers the activation of ionotropic and metabotropic glutamate receptors as well as L‐VDCC and the endoplasmic reticulum (ER) Ca2+ channels. These receptors and channels activate several intracellular‐signaling complexes altering cell behavior in a spatio‐ temporally regulated manner. Metabolism of cyclic nucleotides, membrane phospholipids as well as endogenous enzymatic regulators are the key biochemical steps coordinating cell response to an extracellular stimulus [15].

Calcium is a critical regulator of cytoskeletal dynamics. Dysregulation of Ca2+ homeosta‐ sis is an important event in driving the disruption of assembly/disassembly equilibrium as well as the interaction of cytoskeletal proteins with regulatory proteins or cell organelles. In particular, IF proteins are directly regulated by Ca2+ levels, which crosslink signaling cas‐ cades and connect physiological or pathological extracellular signals with the IF cytoskeleton influencing multiple aspects of cell behavior. Consequently, abnormally elicited Ca2+ signals provoking misregulation of key phosphorylation cascades are able to disrupt cytoskeletal homeostasis and this is commonly associated with the cell damage.

### **4. Toxicity of diphenyl ditelluride on the cytoskeleton of neural cells**

Many processes in the organic synthesis, vulcanization of rubber and in metal‐oxidizing solu‐ tions to tarnish metals, such as silver, extensively use tellurium. Diphenyl ditelluride (PhTe)<sup>2</sup> is the simplest of the aromatic, diorganoyl ditelluride compounds used in organic synthesis. Indeed, developmental exposure to (PhTe)<sup>2</sup> is teratogenic and is associated with long‐term behavioral and neurochemical changes in rats. Until recently, the general toxicity of (PhTe)<sup>2</sup> was considered to be exclusively related to the oxidation of thiol‐containing proteins (for review, see [16]). However, compelling evidence from our laboratory points to an important role played by signaling mechanisms involved in regulating IF phosphorylation/dephosphor‐ ylation as target of (PhTe)<sup>2</sup> neurotoxicity. In addition, we evidence a remarkable role of Ca2+ mediating these actions secondary to glutamate receptors and L‐VDCC activation.

The neurotoxicity of (PhTe)<sup>2</sup> is spatiotemporally regulated, consistent with the window of sus‐ ceptibility of signaling cascades as well as the structural and functional heterogeneity of neu‐ rons in different brain regions. In this regard, exposure of cortical slices from 18‐ and 21‐day‐old rats to (PhTe)<sup>2</sup> shows unaltered phosphorylation of IF proteins, while IFs of acute cortical slices from younger pups (9 and 15 days old) are hypophosphorylated. Activated ionotropic gluta‐ mate receptors, L‐VDCC and ryanodine channels result in PP1‐mediated hypophosphoryla‐ tion of GFAP and NF subunits pointing to the cortical cytoskeleton as a preferential target of the action of phosphatases in this window of vulnerability. Activation of PP1 is modulated by dopamine and cyclic AMP‐regulated neuronal phosphoprotein 32 (DARPP‐32), an important endogenous Ca2+‐mediated inhibitor of PP1 activity. Depending on the site of phosphorylation, DARPP‐32 is able to produce opposing biochemical effects, that is, inhibition of PP1 activity or inhibition of protein kinase A (PKA) activity [17]. Decreased cAMP and PKA catalytic subunits support that (PhTe)<sup>2</sup> disrupts the cytoskeletal‐associated phosphorylating/dephosphorylating system of neurons and astrocytes through PKA‐mediated inactivation of DARPP‐32, promot‐ ing PP1 release and hypophosphorylation of IF proteins of those neural cells [18]. Regarding neurons, hypophosphorylation of IF proteins could be associated with cell dysfunction since decreased phosphorylation of KSP repeats in the carboxyl‐terminal domains of NF‐M and NF‐H correlates with impaired axonal transport and increased NF‐packing density.

In contrast with younger rats, hippocampal slices of 21‐day‐old rats acutely exposed to (PhTe)<sup>2</sup> result in hyperphosphorylated IFs. Hippocampal IF hyperphosphorylation is par‐ tially dependent on L‐VDCC, NMDA and ER Ca2+ channels. The signal evoked by (PhTe)<sup>2</sup> is also transduced through metabotropic glutamate receptors on the plasma membrane, leading to the activation of phospholipase C (PLC) that produces the intracellular messengers inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to specific receptors on the ER changing the conformation of IP3 receptors and opening the channel. Released Ca2+ and DAG directly activate PKCaMII and PCK, resulting in the hyperphosphorylation of some of the critical amino acid residues in the carboxyl‐terminal tail domain of NF‐L known to interfere with filament assembly. In addition, the activation of Erk1/2 and p38MAPK results in hyper‐ phosphorylation of KSP repeats of NFM. Interestingly, PKCaMII and PKC are upstream of MAPK activation implying in a significant cross‐talk among signaling pathways elicited by (PhTe)<sup>2</sup> that connect the glutamate metabotropic cascade with the activation of Ca2+ channels. The final molecular result is the extensive phosphorylation of amino‐ and carboxyl‐terminal sites on IF proteins and deregulated cytoskeletal homeostasis [19].

well as the interaction of cytoskeletal proteins with regulatory proteins or cell organelles. In particular, IF proteins are directly regulated by Ca2+ levels, which crosslink signaling cas‐ cades and connect physiological or pathological extracellular signals with the IF cytoskeleton influencing multiple aspects of cell behavior. Consequently, abnormally elicited Ca2+ signals provoking misregulation of key phosphorylation cascades are able to disrupt cytoskeletal

**4. Toxicity of diphenyl ditelluride on the cytoskeleton of neural cells**

Many processes in the organic synthesis, vulcanization of rubber and in metal‐oxidizing solu‐ tions to tarnish metals, such as silver, extensively use tellurium. Diphenyl ditelluride (PhTe)<sup>2</sup> is the simplest of the aromatic, diorganoyl ditelluride compounds used in organic synthesis.

behavioral and neurochemical changes in rats. Until recently, the general toxicity of (PhTe)<sup>2</sup> was considered to be exclusively related to the oxidation of thiol‐containing proteins (for review, see [16]). However, compelling evidence from our laboratory points to an important role played by signaling mechanisms involved in regulating IF phosphorylation/dephosphor‐

ceptibility of signaling cascades as well as the structural and functional heterogeneity of neu‐ rons in different brain regions. In this regard, exposure of cortical slices from 18‐ and 21‐day‐old

from younger pups (9 and 15 days old) are hypophosphorylated. Activated ionotropic gluta‐ mate receptors, L‐VDCC and ryanodine channels result in PP1‐mediated hypophosphoryla‐ tion of GFAP and NF subunits pointing to the cortical cytoskeleton as a preferential target of the action of phosphatases in this window of vulnerability. Activation of PP1 is modulated by dopamine and cyclic AMP‐regulated neuronal phosphoprotein 32 (DARPP‐32), an important endogenous Ca2+‐mediated inhibitor of PP1 activity. Depending on the site of phosphorylation, DARPP‐32 is able to produce opposing biochemical effects, that is, inhibition of PP1 activity or inhibition of protein kinase A (PKA) activity [17]. Decreased cAMP and PKA catalytic subunits

system of neurons and astrocytes through PKA‐mediated inactivation of DARPP‐32, promot‐ ing PP1 release and hypophosphorylation of IF proteins of those neural cells [18]. Regarding neurons, hypophosphorylation of IF proteins could be associated with cell dysfunction since decreased phosphorylation of KSP repeats in the carboxyl‐terminal domains of NF‐M and

In contrast with younger rats, hippocampal slices of 21‐day‐old rats acutely exposed to

also transduced through metabotropic glutamate receptors on the plasma membrane, leading to the activation of phospholipase C (PLC) that produces the intracellular messengers inositol

tially dependent on L‐VDCC, NMDA and ER Ca2+ channels. The signal evoked by (PhTe)<sup>2</sup>

result in hyperphosphorylated IFs. Hippocampal IF hyperphosphorylation is par‐

NF‐H correlates with impaired axonal transport and increased NF‐packing density.

shows unaltered phosphorylation of IF proteins, while IFs of acute cortical slices

disrupts the cytoskeletal‐associated phosphorylating/dephosphorylating

is

mediating these actions secondary to glutamate receptors and L‐VDCC activation.

is teratogenic and is associated with long‐term

neurotoxicity. In addition, we evidence a remarkable role of Ca2+

is spatiotemporally regulated, consistent with the window of sus‐

homeostasis and this is commonly associated with the cell damage.

Indeed, developmental exposure to (PhTe)<sup>2</sup>

240 Cytoskeleton - Structure, Dynamics, Function and Disease

ylation as target of (PhTe)<sup>2</sup>

The neurotoxicity of (PhTe)<sup>2</sup>

rats to (PhTe)<sup>2</sup>

support that (PhTe)<sup>2</sup>

(PhTe)<sup>2</sup>

#### **4.1. Diphenyl ditelluride disrupts the cytoskeleton and provokes neurodegeneration in acutely injected young rats**

The *in vivo* exposure to (PhTe)<sup>2</sup> , in which the neurotoxicant reaches the brain via systemic circulation, also results in different susceptibilities of the IF proteins from neural cells. This can be evidenced in cerebral cortex and hippocampus of 15‐day‐old rats acutely injected with a toxic dose of (PhTe)<sup>2</sup> (0.3 µmol/kg body weight) [20]. Cortical hyperphosphorylation of neu‐ ronal and glial IF proteins is an early and persistent event up to 6 days after injection, accom‐ panied by increased levels of GFAP and NF‐L. Upregulated gene expression as well as GFAP and vimentin hyperphosphorylation could be a response to injury and take part in the pro‐ gram of reactive astrogliosis, as further demonstrated in striatum [21] and cerebellum [22] of (PhTe)<sup>2</sup> ‐injected rats. In addition, hippocampal IFs are not responsive to the insult until wean‐ ing. A strong evidence supports an important role of astrocytes in a more severe cortical than hippocampal damage following the *in vivo* (PhTe)<sup>2</sup> insult. This supports a direct action of the neurotoxicant on intracellular signaling pathways and highlights the relevance of the inter‐ play between glial and neuronal cells to adapt the cellular metabolic response to the insult even when the brain connections are only partially preserved, as shown in acute brain slices.

Of importance, neurodegeneration is part of the deleterious *in vivo* effects of (PhTe)<sup>2</sup> tox‐ icity, as demonstrated in the striatum [23] and cerebellum [22] of (PhTe)<sup>2</sup> ‐injected rats. Neurodegeneration is associated with alterations in Ca2+ homeostasis and glutamatergic neurotransmission, upstream of inhibited Akt and activated caspase 3. We therefore propose that excitotoxicity is a main mechanism of neurodegeneration caused by this compound in the developing rat brain. On the other hand, most of the actions of (PhTe)<sup>2</sup> disrupting the homeostasis of the cytoskeleton in neural cells are mediated by high Ca2+ levels. Moreover, a link among disrupted IF homeostasis, activated astrocytes and neuronal apoptosis in (PhTe)<sup>2</sup> ‐injected rats has been demonstrated by immunohistochemical approaches. In addi‐ tion, MAPK pathway might be a link between altered IF equilibrium and neural cell damage, since MAPK is implicated in IF hyperphosphorylation and neurodegeneration as well in the brain structures attained by (PhTe)<sup>2</sup> toxicity. Further supporting the cytoskeleton as an end point of neurotoxicity, hyperphosphorylated NFs can inhibit their proteolytic breakdown by calpain, a Ca2+‐activated protease. In addition, abnormally phosphorylated NFs accumulate in the perikarya and the phospho‐NF aggregates can thus become cytotoxic by the enduring impairment of axonal transport of NFs (see **Figure 2**). The increased time the NF spent in the cell body is thought to result in further aberrant phosphorylation and may prevent them from entering the axon, resulting in a deleterious feedback loop [24].

In summary, we propose that complex and integrated actions mediate the (PhTe)<sup>2</sup> toxicity directed to the cytoskeleton of neural cells. These molecular mechanisms induce spatiotem‐ poral responses of the cells because of the different windows of susceptibility of the develop‐ mental brain. Nonetheless, the Ca2+ ‐initiated events highlight a role for this neurotoxicant as a disruptor of the cytoskeleton.

### **5. Cytoskeleton as a target of amino acids and their metabolites**

Misregulated cytoskeletal homeostasis is among the molecular mechanisms underlying the neural cell dysfunction in brain tissue exposed to high levels of amino acids and/or their metabolites. In humans, several neurological impairments are associated with enzymatic deficiencies or defects in proteins involved in cellular metabolism of neural cells, causing accumulation of metabolic intermediates associated with neuronal damage. We discuss some aspects of the molecular mechanisms underlying the disruption of cytoskeletal homeosta‐ sis in response to branched‐chain keto acids (BCKAs) derived from leucine, isoleucine and valine. We also addressed the effects of homocysteine and quinolinic acid (QUIN), a metabo‐ lite of tryptophan metabolism, directed to the cytoskeleton.

#### **5.1. Branched chain α‐keto acids and the cytoskeleton of neural cells**

The branched‐chain ketoacids, α‐ketoisocaproic acid (KIC), α‐keto‐β‐methylvaleric acid (KMV) and α‐keto‐isovaleric acid (KIV) are produced from the respective branched‐chain amino acids (BCAAs) leucine, isoleucine and valine, in the reaction catalyzed by the branched‐ chain α‐keto acid dehydrogenase (BCKAD) complex. A deficiency of the BCKAD complex is an inherited metabolic disease known as maple syrup urine disease (MSUD) which lead to the accumulation of BCAAS and BCKAs in tissues and body fluids resulting in dramatic cerebral symptoms [25].

Curiously, cortical slices of young rats exposed to high levels of the BCAAs individually pre‐ serve the homeostasis of the cytoskeleton. On the other hand, their respective keto acids pro‐ vide an interesting example of the fine‐tune regulation of the cytoskeleton, since KIC [26] and KMV [27] were differently deleterious to the homeostasis of the cytoskeleton. KIC and KMV alter the dynamics of IF proteins of astrocytes and neurons through different transduction mechanisms dependent on excessive intracellular Ca2+ influx, while KIV appears not to be involved in the disruption of the IF cytoskeleton [28].

The effect of KIC is outlined by hypophosphorylation of GFAP, NF‐M and NF‐L in very young rats (up to 12 days of age) changing to hyperphosphorylation of the same proteins later in development (17 days of age). Nonetheless, both responses of the cytoskeletal‐associated phosphorylating system are regulated by Ca2+ currents through the NMDA and L‐VDCC, as well as by the intracellular Ca2+storage release from the ER, leading to a differential activation of protein phosphatases or kinases [28]. These paradoxical findings provide an interesting insight into the differential susceptibility of cortical IF cytoskeleton to the exposure to pathological levels of this metabolite. The different vulnerabilities of the cytoskeleton of cortical cells during development might be ascribed to the temporal maturation mediated by a multitude of developmental processes and signaling pathways. It is conceivable that they are associated with the pathological role of the developmentally regulated glutamate receptors in neural cells since the expression patterns of glutamate receptor subunit genes change during the ontogeny of the brain. Distinct regional and temporal patterns of the expression of types and subtypes of the glutamate ionotropic receptors during ontogeny may possibly explain the different signaling pathways targeting the cytoskeleton of corti‐ cal neural cells during development.

Interestingly, KMV disturbs the IF‐associated cytoskeletal phosphorylation only in 12‐day‐old rats without changing the phosphorylation level of these proteins in younger or older animals, showing a specific window of vulnerability of cytoskeleton to KMV insult in the cerebral cor‐ tex of developing brain. Strikingly, this effect was dependent on intracellular Ca2+ concentra‐ tions; however, in this case ɣ‐amino butyric acid A and B (GABAA and GABAB, respectively) rather than glutamate receptors were involved in this action. This is in agreement with GABA<sup>A</sup> and GABAB receptors mediating the induction and maintenance of Ca2+ levels [27].

Overall, we propose that BCKAs in supra‐physiological concentrations disrupt the cytoskeleton of rat brain through misregulation of the phosphorylating system associated with the IF cyto‐ skeleton. We evidenced developmentally regulated mechanisms in which Ca2+‐mediated excito‐ toxicity plays a critical role in destabilizing the cytoskeleton that may ultimately disrupt normal cell function and viability. Although evidence from animal models should be taken with cau‐ tion, we can propose that the disrupted cytoskeleton is part of the physiopathology of MSUD.

### **5.2. Hyperhomocysteinemia and the cytoskeleton of neural cells**

cell body is thought to result in further aberrant phosphorylation and may prevent them from

directed to the cytoskeleton of neural cells. These molecular mechanisms induce spatiotem‐ poral responses of the cells because of the different windows of susceptibility of the develop‐ mental brain. Nonetheless, the Ca2+ ‐initiated events highlight a role for this neurotoxicant as

Misregulated cytoskeletal homeostasis is among the molecular mechanisms underlying the neural cell dysfunction in brain tissue exposed to high levels of amino acids and/or their metabolites. In humans, several neurological impairments are associated with enzymatic deficiencies or defects in proteins involved in cellular metabolism of neural cells, causing accumulation of metabolic intermediates associated with neuronal damage. We discuss some aspects of the molecular mechanisms underlying the disruption of cytoskeletal homeosta‐ sis in response to branched‐chain keto acids (BCKAs) derived from leucine, isoleucine and valine. We also addressed the effects of homocysteine and quinolinic acid (QUIN), a metabo‐

The branched‐chain ketoacids, α‐ketoisocaproic acid (KIC), α‐keto‐β‐methylvaleric acid (KMV) and α‐keto‐isovaleric acid (KIV) are produced from the respective branched‐chain amino acids (BCAAs) leucine, isoleucine and valine, in the reaction catalyzed by the branched‐ chain α‐keto acid dehydrogenase (BCKAD) complex. A deficiency of the BCKAD complex is an inherited metabolic disease known as maple syrup urine disease (MSUD) which lead to the accumulation of BCAAS and BCKAs in tissues and body fluids resulting in dramatic cerebral

Curiously, cortical slices of young rats exposed to high levels of the BCAAs individually pre‐ serve the homeostasis of the cytoskeleton. On the other hand, their respective keto acids pro‐ vide an interesting example of the fine‐tune regulation of the cytoskeleton, since KIC [26] and KMV [27] were differently deleterious to the homeostasis of the cytoskeleton. KIC and KMV alter the dynamics of IF proteins of astrocytes and neurons through different transduction mechanisms dependent on excessive intracellular Ca2+ influx, while KIV appears not to be

The effect of KIC is outlined by hypophosphorylation of GFAP, NF‐M and NF‐L in very young rats (up to 12 days of age) changing to hyperphosphorylation of the same proteins later in development (17 days of age). Nonetheless, both responses of the cytoskeletal‐associated phosphorylating system are regulated by Ca2+ currents through the NMDA and L‐VDCC, as well as by the intracellular Ca2+storage release from the ER, leading to a differential activation of protein phosphatases or kinases [28]. These paradoxical findings provide an interesting

toxicity

In summary, we propose that complex and integrated actions mediate the (PhTe)<sup>2</sup>

**5. Cytoskeleton as a target of amino acids and their metabolites**

entering the axon, resulting in a deleterious feedback loop [24].

lite of tryptophan metabolism, directed to the cytoskeleton.

involved in the disruption of the IF cytoskeleton [28].

**5.1. Branched chain α‐keto acids and the cytoskeleton of neural cells**

a disruptor of the cytoskeleton.

242 Cytoskeleton - Structure, Dynamics, Function and Disease

symptoms [25].

Homocysteine (Hcy) is a sulfur‐containing amino acid generated during methionine metabolism. Genetic mutations impairing Hcy metabolism cause accumulation of this amino acid attaining high levels in blood, leading to severe hyperhomocysteinemia and brain damage. Otherwise, along with genetic factors, mild‐moderate hyperhomocysteinemia is associated with nutritional imbalance and hormonal factors. Mild hyperhomocysteinemia, which markedly enhance the vulnerability of neuronal cells to excitotoxicity and oxidative imbalance, is also common in older people, constituting an independent risk factor for stroke and cognitive impairment [29].

Various existing experimental evidences from our group link hyperhomocysteinemia and cyto‐ skeletal misregulation, supporting that disrupted cytoskeleton could be an end point of neural dysfunction in this neurometabolic disorder. Experiments with brain slices acutely exposed to mild Hcy levels (100 µM) showed greater vulnerability of hippocampal cytoskeleton as com‐ pared with cortical one. Moreover, a window of vulnerability of the cytoskeleton of hippocam‐ pal cells is evidenced, since misregulated phosphorylation is detected only at postnatal day 17 [30], reflecting an altered activity of the endogenous phosphorylating system associated with the IFs in this brain structure. As expected, NMDA receptors, L‐VDCC and extracellular Ca2+influx result in PKC and PKCaMII activation. The prevention of Hcy action through the inhibition of PKC and MEK, a step that is upstream of MAPK cascade (Raf‐1/MEK/MAPK), is consistent with an effect at the level of the monomeric GTPase Raf‐1, supporting a role for PKC phosphorylating and activating Raf‐1 in the Hcy‐induced modulation of the cytoskeleton.

In contrast with hypophosphorylation found in hippocampal slices, the chemically induced chronic hyperhomocysteinemia differently alters the signaling mechanisms directed to the cytoskeleton, producing PP1‐, PP2A‐ and PP2B‐mediated hypophosphorylation of NF sub‐ units and GFAP in hippocampal slices of 17‐day‐old rats without affecting the cerebral cortex [31] through glutamate and Ca2+‐mediated mechanisms. Further evidence that homocysteine targets the cytoskeleton came from cytoskeletal reorganization in primary astrocytes and neu‐ rons exposed to homocysteine [32]. Dramatically altered actin cytoskeleton in primary astro‐ cytes exposed to 100 µM Hcy is consistent with the role of actin as a main determinant of cell morphology. Concomitant disrupted GFAP meshwork underlies the remodeled actin cyto‐ skeleton and altered cell morphology. These findings provide further evidence of the cross‐talk among the different cytoskeletal subsystems and the roles played by the toxic levels of Hcy.

Therefore, taking into account our experimental evidence it is conceivable that disturbed cell signaling is an important determinant of the disrupted homeostasis of the cytoskeleton as a whole, with widespread consequences on cell function that could be associated with human hyperhomocysteinemia.

### **6. Cytoskeleton is a target of quinolinic acid neurotoxicity**

Quinolinic acid is a neuroactive metabolite of the kynurenine pathway normally found in nanomolar concentrations in human brain and cerebrospinal fluid (CSF). QUIN is antagonist of NMDA receptor and it has a high *in vivo* potency as an excitotoxin supporting involvement in the pathogenesis of a variety of human neurological diseases. The neurotoxicity of QUIN results from complex mechanisms including presynaptic receptors, energetic dysfunction, oxidative stress, transcription factors and behavior [33]. We experimentally demonstrate that the disruption of the cytoskeleton, in particular, misregulation of the phosphorylation system associated with the IFs, is a target of QUIN toxicity in injected rat striatum, tissue slices and primary astrocytes and neurons in culture.

#### **6.1. Effects of intrastriatally injected quinolinic acid on the cytoskeleton of neural cells**

Acute intrastriatal injection of QUIN (150 nmol/0.5 µL) in adolescent rats (30 days old) pro‐ vokes NF‐L and GFAP hyperphosphorylation 30 min after infusion, evidencing the suscepti‐ bility of the cytoskeleton of both neurons and astrocytes in the early events of QUIN toxicity. Hyperphosphorylated NF‐LSer55 destabilizes the NF structure and this might represent an early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum. Experimental insights to get light on the molecular mechanisms underlying this effect point to NMDA‐mediated Ca2+ events and oxidative stress upstream of activated second messenger‐ dependent protein kinases PKA, PKC and PKCaMII, but not MAPKs after QUIN infusion [34].

A link between misregulation of cell‐signaling mechanisms, disruption of IF phosphory‐ lation and cell damage as part of QUIN toxicity becomes more evident analyzing the long‐lasting effect of the acute intrastriatal injection of QUIN in adolescent rats on the dynam‐ ics of the phosphorylating system until 21 days after injection [35]. The acutely injected QUIN alters the homeostasis of IF phosphorylation in a selective manner, progressing from stria‐ tum to cerebral cortex and hippocampus. Twenty‐four hours after QUIN injection, the IFs are hyperphosphorylated in the striatum. This effect progresses to cerebral cortex causing hypo‐ phosphorylation at day 14 and appears in the hippocampus as hyperphosphorylation at day 21 after QUIN infusion, PKA and PKCaMII mediating this effect. However, MAPKs (Erk1/2, JNK and p38MAPK) are hyperphosphorylated/activated only in the hippocampus, suggest‐ ing different signaling mechanisms in these two brain structures during the first weeks after QUIN infusion. Also, PP1 and PP2B‐mediated hypophosphorylation of the IF proteins in the cerebral cortex 14 days after QUIN injection reinforces the selective signaling mechanisms in different brain structures. Increased GFAP immunocontent in the striatum and cerebral cortex 24 h and 14 days after QUIN injection, respectively, suggests reactive astrocytes in these brain regions. Yet, we observe biochemical and histopathological alterations in the striatum, cortex and hippocampus, as well as altered behavioral tests in response to the long‐lasting exposure to QUIN through glutamate and Ca2+‐mediated mechanisms. Thus, it is tempting to propose that the long‐lasting deleterious effect of intrastriatal QUIN injection could be due to the fact that QUIN interferes with the highly regulated signaling mechanisms targeting the cytoskel‐ eton in the immature brain [36].

is consistent with an effect at the level of the monomeric GTPase Raf‐1, supporting a role for PKC phosphorylating and activating Raf‐1 in the Hcy‐induced modulation of the cytoskeleton. In contrast with hypophosphorylation found in hippocampal slices, the chemically induced chronic hyperhomocysteinemia differently alters the signaling mechanisms directed to the cytoskeleton, producing PP1‐, PP2A‐ and PP2B‐mediated hypophosphorylation of NF sub‐ units and GFAP in hippocampal slices of 17‐day‐old rats without affecting the cerebral cortex [31] through glutamate and Ca2+‐mediated mechanisms. Further evidence that homocysteine targets the cytoskeleton came from cytoskeletal reorganization in primary astrocytes and neu‐ rons exposed to homocysteine [32]. Dramatically altered actin cytoskeleton in primary astro‐ cytes exposed to 100 µM Hcy is consistent with the role of actin as a main determinant of cell morphology. Concomitant disrupted GFAP meshwork underlies the remodeled actin cyto‐ skeleton and altered cell morphology. These findings provide further evidence of the cross‐talk among the different cytoskeletal subsystems and the roles played by the toxic levels of Hcy.

Therefore, taking into account our experimental evidence it is conceivable that disturbed cell signaling is an important determinant of the disrupted homeostasis of the cytoskeleton as a whole, with widespread consequences on cell function that could be associated with human

Quinolinic acid is a neuroactive metabolite of the kynurenine pathway normally found in nanomolar concentrations in human brain and cerebrospinal fluid (CSF). QUIN is antagonist of NMDA receptor and it has a high *in vivo* potency as an excitotoxin supporting involvement in the pathogenesis of a variety of human neurological diseases. The neurotoxicity of QUIN results from complex mechanisms including presynaptic receptors, energetic dysfunction, oxidative stress, transcription factors and behavior [33]. We experimentally demonstrate that the disruption of the cytoskeleton, in particular, misregulation of the phosphorylation system associated with the IFs, is a target of QUIN toxicity in injected rat striatum, tissue slices and

**6.1. Effects of intrastriatally injected quinolinic acid on the cytoskeleton of neural cells**

Acute intrastriatal injection of QUIN (150 nmol/0.5 µL) in adolescent rats (30 days old) pro‐ vokes NF‐L and GFAP hyperphosphorylation 30 min after infusion, evidencing the suscepti‐ bility of the cytoskeleton of both neurons and astrocytes in the early events of QUIN toxicity. Hyperphosphorylated NF‐LSer55 destabilizes the NF structure and this might represent an early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum. Experimental insights to get light on the molecular mechanisms underlying this effect point to NMDA‐mediated Ca2+ events and oxidative stress upstream of activated second messenger‐ dependent protein kinases PKA, PKC and PKCaMII, but not MAPKs after QUIN infusion [34]. A link between misregulation of cell‐signaling mechanisms, disruption of IF phosphory‐ lation and cell damage as part of QUIN toxicity becomes more evident analyzing the

**6. Cytoskeleton is a target of quinolinic acid neurotoxicity**

hyperhomocysteinemia.

244 Cytoskeleton - Structure, Dynamics, Function and Disease

primary astrocytes and neurons in culture.

#### **6.2. Insight into the molecular basis of quinolinic acid action toward the cytoskeleton**

Studies in acute brain slices further support the role of glutamatergic signaling and Ca2+ over‐ load disturbing the cytoskeletal equilibrium downstream of QUIN exposure. Moreover, this experimental approach brings light on the cell‐specific mechanisms targeting the cytoskeleton in astrocytes and neurons when the cell connections are partially preserved. In astrocytes, the QUIN action is mainly due to increased Ca2+ influx through NMDA and L‐VDCC. In neuronal cells, QUIN acts through the activation of metabotropic glutamate receptors and influx of Ca2+ through NMDA receptors and L‐VDCC, as well as Ca2+ release from intracellu‐ lar stores. These mechanisms then set off a cascade of events including the activation of PKA, PKCaMII and PKC, which phosphorylate head domain sites on GFAP and NFL. Moreover, Cdk5 is activated downstream of mGluR5, phosphorylating the KSP repeats on NFM and NFH. Metabotropic glutamate receptors type 1 (mGluR1) is upstream of PLC, which, in turn, produce DAG and IP3 promoting hyperphosphorylation of KSP repeats on the tail domain of NFM and NFH [37].

### **6.3. The cytoskeleton of astrocytes and neurons responds differently to quinolinic acid toxicity**

The susceptibility of the cytoskeleton to toxic levels of QUIN is also detectable in isolated astro‐ cytes and neurons growth in primary cultures [38]. In astrocytes, Ca2+‐mediated glutamate mech‐ anisms target the endogenous phosphorylating system, since metabotropic glutamate receptors and Ca2+ influx through NMDA receptors are upstream of PKA, PKCaMII and PKC activa‐ tion, provoking GFAP hyperphosphorylation. Interestingly, the misregulated phosphorylation system leads to a reversible and dramatically altered actin cytoskeleton with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the GFAP meshwork [39] supporting the dynamic behavior of the cytoskeleton.

Interestingly, neurons show greater vulnerability to QUIN than astrocytes (10×). Neurons exposed to QUIN presented PKA‐ and PKC‐mediated hyperphosphorylation of NF subunits. These effects are also downstream of ionotropic and metabotropic glutamate signaling and Ca2+ influx through NMDA receptors and L‐VDCC. The misregulated signaling pathways dis‐ rupt the neuronal cytoskeleton, evaluated by altered neurite/neuron ratios and neurite out‐ growth. It is important to consider that microtubules play a central role in cell polarity [40]. In particular, microtubules are the main determinants of neuronal polarity and regulation of microtubule dynamics includes tubulin posttranslational modifications [40] and phosphoryla‐ tion of microtubule‐associated proteins (MAPs), whose binding to microtubules is essential for neurite formation [41]. As an example, activated GSK‐3β leads to increased phosphoryla‐ tion of some MAPs, destabilizing microtubules with consequence for neurite stabilization [42]. Therefore, the neurite destabilization could derive from both NFs and microtubules disruption.

Interestingly, we found a protective role of astrocyte‐conditioned medium on the disrupted neuronal cytoskeleton and morphometric alterations, suggesting that QUIN‐induced trophic factors secreted by astrocytes are able to modulate signaling mechanisms targeting the neu‐ ronal cytoskeleton. More interestingly, co‐cultured astrocytes and neurons preserve their cytoskeletal organization and cell morphology together with unaltered activity of the phos‐ phorylating system associated with the cytoskeleton. In other words, co‐cultured astrocytes and neurons tightly and actively interact with one another reciprocally protecting themselves against QUIN injury [38]. This evidence raise the question about the role played by the acti‐ vated microglia eliciting signals essential to destabilize the astrocytic and neuronal cytoskel‐ eton but this hypothesis remains to be clarified.

All together, we conclude that among the multiple mechanisms through which accumulated QUIN is able to induce cell damage, our experimental evidence points to Ca2+‐mediated mechanisms directed to the cytoskeletal disruption as an end point of QUIN toxicity. Both *in vivo* and *ex vivo* approaches clearly demonstrate a wide spectrum of misregulated signaling mechanisms downstream of QUIN action directly affecting the cytoskeleton and disrupting cell homeostasis. We also provide evidence that impaired physiological equilibrium of the signaling cascades directed to the cytoskeleton underlies QUIN cytotoxicity and is associated with neurodegeneration. The *in vitro* results showing disorganized cytoskeleton and altered cell morphology further support the cytoskeleton as a hallmark of stress condition that could be implicated in the human brain disorders associated with high QUIN levels.

### **7. Cytoskeleton of neural cells is a target of thyroid hormones**

Thyroid hormones are essential for the development and function of central nervous sys‐ tem. In brain, these hormones are essential for myelination [43, 44], neuritogenesis [45], synaptic plasticity [46–48], IF phosphorylation [49–54], cell differentiation and maturation [55]. Considering the role of these hormones on brain development, thyroid diseases might account for brain injury as well as alteration in mood and cognition [56].

The classical mechanism of thyroid hormone action involves the modulation of nuclear receptors by 3,5,3′‐triiodo‐l‐thyronine (T<sup>3</sup> ). The nuclear receptors are ligand‐dependent tran‐ scription factors, which are involved in the genomic‐dependent effects of thyroid hormones. However, there are numerous physiological effects of these hormones that cannot be medi‐ ated by the genome‐like mechanism, due the short time frame in which the response occurs. Nongenomic actions of thyroid hormones are defined as events that (i) do not primarily involve the cell nucleus, (ii) are rapid in onset (minutes or a few hours) relative to transcrip‐ tion and translation and (iii) do not require gene transcription and protein synthesis [57]). These events are triggered by rapid/nongenomic responses that are frequently associated with secondary messenger‐signaling pathways.

of morphology to fusiform and/or flattened cells with retracted cytoplasm and disruption of the

Interestingly, neurons show greater vulnerability to QUIN than astrocytes (10×). Neurons exposed to QUIN presented PKA‐ and PKC‐mediated hyperphosphorylation of NF subunits. These effects are also downstream of ionotropic and metabotropic glutamate signaling and Ca2+ influx through NMDA receptors and L‐VDCC. The misregulated signaling pathways dis‐ rupt the neuronal cytoskeleton, evaluated by altered neurite/neuron ratios and neurite out‐ growth. It is important to consider that microtubules play a central role in cell polarity [40]. In particular, microtubules are the main determinants of neuronal polarity and regulation of microtubule dynamics includes tubulin posttranslational modifications [40] and phosphoryla‐ tion of microtubule‐associated proteins (MAPs), whose binding to microtubules is essential for neurite formation [41]. As an example, activated GSK‐3β leads to increased phosphoryla‐ tion of some MAPs, destabilizing microtubules with consequence for neurite stabilization [42]. Therefore, the neurite destabilization could derive from both NFs and microtubules disruption. Interestingly, we found a protective role of astrocyte‐conditioned medium on the disrupted neuronal cytoskeleton and morphometric alterations, suggesting that QUIN‐induced trophic factors secreted by astrocytes are able to modulate signaling mechanisms targeting the neu‐ ronal cytoskeleton. More interestingly, co‐cultured astrocytes and neurons preserve their cytoskeletal organization and cell morphology together with unaltered activity of the phos‐ phorylating system associated with the cytoskeleton. In other words, co‐cultured astrocytes and neurons tightly and actively interact with one another reciprocally protecting themselves against QUIN injury [38]. This evidence raise the question about the role played by the acti‐ vated microglia eliciting signals essential to destabilize the astrocytic and neuronal cytoskel‐

All together, we conclude that among the multiple mechanisms through which accumulated QUIN is able to induce cell damage, our experimental evidence points to Ca2+‐mediated mechanisms directed to the cytoskeletal disruption as an end point of QUIN toxicity. Both *in vivo* and *ex vivo* approaches clearly demonstrate a wide spectrum of misregulated signaling mechanisms downstream of QUIN action directly affecting the cytoskeleton and disrupting cell homeostasis. We also provide evidence that impaired physiological equilibrium of the signaling cascades directed to the cytoskeleton underlies QUIN cytotoxicity and is associated with neurodegeneration. The *in vitro* results showing disorganized cytoskeleton and altered cell morphology further support the cytoskeleton as a hallmark of stress condition that could

be implicated in the human brain disorders associated with high QUIN levels.

**7. Cytoskeleton of neural cells is a target of thyroid hormones**

account for brain injury as well as alteration in mood and cognition [56].

Thyroid hormones are essential for the development and function of central nervous sys‐ tem. In brain, these hormones are essential for myelination [43, 44], neuritogenesis [45], synaptic plasticity [46–48], IF phosphorylation [49–54], cell differentiation and maturation [55]. Considering the role of these hormones on brain development, thyroid diseases might

GFAP meshwork [39] supporting the dynamic behavior of the cytoskeleton.

246 Cytoskeleton - Structure, Dynamics, Function and Disease

eton but this hypothesis remains to be clarified.

### **7.1. Insight into the molecular basis of genomic and nongenomic action of thyroid hormones toward the cytoskeleton of neural cells**

The first evidence of nongenomic actions of thyroid hormones targeting the cytoskeleton demonstrated the thyroxine‐dependent modulation of actin polymerization in cultured astro‐ cytes. Thyroxine (T<sup>4</sup> ) was involved in the conversion of soluble actin to a fibrous form through nongenomic mechanism [58].

While many of the T<sup>3</sup> actions are mediated by genomic‐dependent mechanisms, T<sup>4</sup> and reverse 3,3′,5′‐triiodothyronine (reverse T<sup>3</sup> , rT<sup>3</sup> ) exert direct, The nongenomic effects in neural cells. Both T<sup>4</sup> and rT<sup>3</sup> hormones control actin polymerization in cultured astrocytes without affect‐ ing gene expression. The authors suggested that these events might contribute to thyroid hormone's influence on brain development. Subsequently, the same research group showed that both T<sup>4</sup> and rT<sup>3</sup> , but not T<sup>3</sup> , directly regulate the F‐actin content of elongating neurites of cerebellar neurons. These results provide a molecular mechanism for the influence of thyroid hormones on brain development that is independent of regulated gene expression [59].

Trentin and Moura Neto [60] demonstrated that T<sup>3</sup> altered the organization of GFAP in cer‐ ebellar astrocytes in culture. GFAP filaments that normally spread in the cytoplasm of astro‐ cytes became organized around the cell nucleus. In addition, Zamoner and coworkers [51] showed that both T<sup>3</sup> and T<sup>4</sup> induced GFAP phosphorylation and reorganization in glioma C6 cells through the inhibition of RhoA GTPase. The modulation of GFAP was accompanied by increased proliferation of glioma cells. Taking together, these results suggest that thyroid hormones may be important regulators of astrocyte growth and differentiation.

Despite the evidence that nongenomic actions of thyroid hormones initiated at the plasma membrane via integrin αVβ3 [57, 61], the complexity of the processes underlying the differen‐ tial mechanisms of action to thyroid hormones suggests the existence of multiple binding sites for these hormones. In this context, it has been previously demonstrated that both T<sup>3</sup> and T<sup>4</sup> may modulate the GABAergic system and induce PKA‐ and PKCaMII‐mediated hyperphos‐ phorylation of vimentin, GFAP, NF‐M and NF‐L in cerebral cortex from very young rats (up to 10 days of age) [50]. However, only T<sup>4</sup> caused hyperphosphorylation of the same proteins later in development (15 days of age) through GABA‐independent mechanisms [49]. These paradoxical findings provide an interesting insight into the differential susceptibility of corti‐ cal IF cytoskeleton to thyroid hormone exposure.

Calcium‐dependent mechanisms play a central role on the thyroid hormone‐induced modula‐ tion of the phosphorylating system associated with IFs. Zamoner and colleagues [49] demon‐ strated that the nongenomic mechanisms underlying the effects T<sup>4</sup> targeting the IF‐associated phosphorylating system in cerebral cortex from 15‐day‐old rats are dependent on extracel‐ lular Ca2+ influx through VDCC, as well as Ca2+ release from ER stores.

Taking into account that in rat the myelination peak is coincident with postnatal day 15 and that this is a period of intense synaptogenesis, the NF hyperphosphorylation induced by T<sup>4</sup> in cerebral cortex from 15‐day‐old rats appears to be correlated to synaptogenesis and myelination (for review, see [53]).

In summary, we could suggest that nongenomic actions of T<sup>4</sup> targeting the cytoskeleton of glial cells and neurons might account for neuronal cell migration, myelination, synaptogen‐ esis and synaptic plasticity. Moreover, the modulation of NF phosphorylation by thyroid hor‐ mone may control axonal caliber.

### **7.2. Hypothyroidism and the cytoskeleton of neural cell**

The effects of thyroid hormones in central nervous system during development include the modulation of the cytoskeleton dynamics. Hypothyroidism in the developing rat brain is asso‐ ciated with oxidative stress and aberrant intraneuronal accumulation of NFs in the perikaryon of Purkinje neurons (see **Figure 2**). The authors suggested that the neuron alterations observed in the developing hypothyroid brain are comparable to those seen in neurodegenerative dis‐ eases [62]. Corroborating these findings, it has been shown that the effects of hypothyroidism on neuronal cytoskeleton involve the developmental modulation of specific isoforms of protein expression, which induce stoichiometric imbalance between the NF triplet [52]. In addition, thyroid hormone deficiency induces a delay and a partial arrest of astrocyte differentiation, supported by the decreased expression of GFAP both in cortical [52] and in hippocampal astro‐ cytes [54], which was accompanied by downregulation of the astrocyte glutamate transport‐ ers. These findings are associated with the extracellular signal‐regulated kinase (ERK)1/2 and c‐jun terminal kinase (JNK) activation. NF hyperphosphorylation might account for the aber‐ rant intraneuronal accumulation of these cytoskeletal structures previously described [62].

Our research group demonstrated the hyperphosphorylation of tail KSP repeats on NF‐H in hypothyroid cortical and hippocampal neurons [52, 54]. The carboxyl‐terminal phosphoryla‐ tion of NF‐H progressively restricts association of NFs with kinesin, the axonal anterograde motor protein and stimulates its interaction with dynein, the axonal retrograde motor protein [63]. This event could represent one of the mechanisms by which carboxyl‐terminal phos‐ phorylation would slow NF axonal transport.

Taking into account our experimental evidence, we propose that the consequences of congeni‐ tal hypothyroidism to neural cells involve IF hyperphosphorylation, misregulation of gluta‐ mate‐glutamine cycle, oxidative stress and glutamate excitotoxicity. These events suggest a compromised astroglial defense system that is probably playing a role in the physiopathology of the neurological dysfunction of hypothyroidism (**Figure 3**).

Calcium‐dependent mechanisms play a central role on the thyroid hormone‐induced modula‐ tion of the phosphorylating system associated with IFs. Zamoner and colleagues [49] demon‐

phosphorylating system in cerebral cortex from 15‐day‐old rats are dependent on extracel‐

Taking into account that in rat the myelination peak is coincident with postnatal day 15 and that this is a period of intense synaptogenesis, the NF hyperphosphorylation induced by T<sup>4</sup>

cerebral cortex from 15‐day‐old rats appears to be correlated to synaptogenesis and myelination

glial cells and neurons might account for neuronal cell migration, myelination, synaptogen‐ esis and synaptic plasticity. Moreover, the modulation of NF phosphorylation by thyroid hor‐

The effects of thyroid hormones in central nervous system during development include the modulation of the cytoskeleton dynamics. Hypothyroidism in the developing rat brain is asso‐ ciated with oxidative stress and aberrant intraneuronal accumulation of NFs in the perikaryon of Purkinje neurons (see **Figure 2**). The authors suggested that the neuron alterations observed in the developing hypothyroid brain are comparable to those seen in neurodegenerative dis‐ eases [62]. Corroborating these findings, it has been shown that the effects of hypothyroidism on neuronal cytoskeleton involve the developmental modulation of specific isoforms of protein expression, which induce stoichiometric imbalance between the NF triplet [52]. In addition, thyroid hormone deficiency induces a delay and a partial arrest of astrocyte differentiation, supported by the decreased expression of GFAP both in cortical [52] and in hippocampal astro‐ cytes [54], which was accompanied by downregulation of the astrocyte glutamate transport‐ ers. These findings are associated with the extracellular signal‐regulated kinase (ERK)1/2 and c‐jun terminal kinase (JNK) activation. NF hyperphosphorylation might account for the aber‐ rant intraneuronal accumulation of these cytoskeletal structures previously described [62].

Our research group demonstrated the hyperphosphorylation of tail KSP repeats on NF‐H in hypothyroid cortical and hippocampal neurons [52, 54]. The carboxyl‐terminal phosphoryla‐ tion of NF‐H progressively restricts association of NFs with kinesin, the axonal anterograde motor protein and stimulates its interaction with dynein, the axonal retrograde motor protein [63]. This event could represent one of the mechanisms by which carboxyl‐terminal phos‐

Taking into account our experimental evidence, we propose that the consequences of congeni‐ tal hypothyroidism to neural cells involve IF hyperphosphorylation, misregulation of gluta‐ mate‐glutamine cycle, oxidative stress and glutamate excitotoxicity. These events suggest a compromised astroglial defense system that is probably playing a role in the physiopathology

targeting the IF‐associated

targeting the cytoskeleton of

in

strated that the nongenomic mechanisms underlying the effects T<sup>4</sup>

In summary, we could suggest that nongenomic actions of T<sup>4</sup>

**7.2. Hypothyroidism and the cytoskeleton of neural cell**

phorylation would slow NF axonal transport.

of the neurological dysfunction of hypothyroidism (**Figure 3**).

(for review, see [53]).

mone may control axonal caliber.

248 Cytoskeleton - Structure, Dynamics, Function and Disease

lular Ca2+ influx through VDCC, as well as Ca2+ release from ER stores.

**Figure 3.** Role of glutamate excitotoxicity on intermediate‐filament dynamics and cell damage. Congenital hypothyroid‐ ism leads to glutamate excitotoxicity, calcium overload, and oxidative stress. These events are related to intermediate‐ filament (GFAP and NF) hyperphosphorylation and neural cell damage.

### **8. General conclusion**

Studies of our group on the endogenous phosphorylating system associated with the IF pro‐ teins of neural cells point to a critical role of disrupted cytoskeleton in response to a variety of signals both in physiological and in pathological conditions. Our findings highlight the IFs as a preferential target of the signal transduction pathways. Importantly, a large body of evidence shows a link among misregulation of cell‐signaling mechanisms, disruption of IF phosphorylation and cell damage in response to different stress signals. While the exact sig‐ naling pathways regulating NF phosphorylation remains elusive, there is increasing evidence that known signal transduction cascades are involved. These actions can be initiated by the activation of NMDA‐, L‐VDCC, or G protein‐coupled receptors and the signal is transduced downstream of Ca2+ mobilization or monomeric GTPase activation through different kinase/ phosphatase pathways, regulating the dynamics of the cytoskeleton. **Figure 4** summarizes

**Figure 4.** Summary of calcium‐associated mechanisms triggered by thyroid hormones, quinolinic acid, diphenyl ditelluride, branched‐chain keto acids, and homocysteine targeting intermediate‐filament phosphorylation in neural cells. Calcium influx through the NMDA receptor or voltage‐dependent calcium channels (VDCC) can be responsible for the activation of lethal metabolic pathways in neural cells. Augmented intracellular Ca2+ levels might be associated with the modulation of diverse cell‐signaling pathways and exhibit a diverse range of responses to their stimuli.

the calcium‐associated mechanisms triggered by thyroid hormones, quinolinic acid, (PhTe)<sup>2</sup> , BCKAs and homocysteine targeting IF phosphorylation in neural cells.

Despite the focus on the misregulation of IF dynamics in response to signaling mechanisms downstream of metabolites and neurotoxicants, we should consider that cytoskeleton is a complex meshwork of interconnecting filaments [1]. In this regard, the morphological altera‐ tions demonstrated in primary cells in culture mainly reflect the reorganization of the mesh‐ work of filaments. Taking into account our findings, we propose that misregulation of kinase/ phosphatase cascades downstream of stressors could disrupt the cytoskeleton as a whole and this might be an important determinant of neural dysfunction associated with the action of neurotoxicants and in neurometabolic conditions.

### **Author details**

**8. General conclusion**

250 Cytoskeleton - Structure, Dynamics, Function and Disease

Studies of our group on the endogenous phosphorylating system associated with the IF pro‐ teins of neural cells point to a critical role of disrupted cytoskeleton in response to a variety of signals both in physiological and in pathological conditions. Our findings highlight the IFs as a preferential target of the signal transduction pathways. Importantly, a large body of evidence shows a link among misregulation of cell‐signaling mechanisms, disruption of IF phosphorylation and cell damage in response to different stress signals. While the exact sig‐ naling pathways regulating NF phosphorylation remains elusive, there is increasing evidence that known signal transduction cascades are involved. These actions can be initiated by the activation of NMDA‐, L‐VDCC, or G protein‐coupled receptors and the signal is transduced downstream of Ca2+ mobilization or monomeric GTPase activation through different kinase/ phosphatase pathways, regulating the dynamics of the cytoskeleton. **Figure 4** summarizes

**Figure 4.** Summary of calcium‐associated mechanisms triggered by thyroid hormones, quinolinic acid, diphenyl ditelluride, branched‐chain keto acids, and homocysteine targeting intermediate‐filament phosphorylation in neural cells. Calcium influx through the NMDA receptor or voltage‐dependent calcium channels (VDCC) can be responsible for the activation of lethal metabolic pathways in neural cells. Augmented intracellular Ca2+ levels might be associated with

the modulation of diverse cell‐signaling pathways and exhibit a diverse range of responses to their stimuli.

Ariane Zamoner<sup>1</sup> and Regina Pessoa‐Pureur<sup>2</sup> \*

\*Address all correspondence to: rpureur@ufrgs.br

1 Department of Biochemistry, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Brazil

2 Department of Biochemistry, Institute of Basic Sciences of Health, Federal University of Rio Grande do Sul, Porto Alegre, Brazil

### **References**


cytoskeletal proteins in cerebral cortex and hippocampus of rats. Neurotoxicology. 2008;29(1):40–7.

[21] Heimfarth L, Reis KP, Loureiro SO, de Lima BO, da Rocha JB, Pessoa‐Pureur R. Exposure of young rats to diphenyl ditelluride during lactation affects the homeosta‐ sis of the cytoskeleton in neural cells from striatum and cerebellum. Neurotoxicology. 2012;33(5):1106–16.

[8] Der Perng M, Su M, Wen SF, Li R, Gibbon T, Prescott AR, et al. The Alexander disease‐ causing glial fibrillary acidic protein mutant, R416W, accumulates into Rosenthal fibers by a pathway that involves filament aggregation and the association of alpha B‐crystal‐

[9] Ubersax JA, Ferrell JE, Jr. Mechanisms of specificity in protein phosphorylation. Nat Rev

[10] Omary MB, Ku NO, Tao GZ, Toivola DM, Liao J. "Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights. Trends Biochem Sci.

[11] Sihag RK, Inagaki M, Yamaguchi T, Shea TB, Pant HC. Role of phosphorylation on the structural dynamics and function of types III and IV intermediate filaments. Exp Cell

[12] Holmgren A, Bouhy D, Timmerman V. Neurofilament phosphorylation and their pro‐ line‐directed kinases in health and disease. J Peripher Nerv Syst. 2012;17(4):365–76. [13] McLean NA, Popescu BF, Gordon T, Zochodne DW, Verge VM. Delayed nerve stimula‐ tion promotes axon‐protective neurofilament phosphorylation, accelerates immune cell clearance and enhances remyelination in vivo in focally demyelinated nerves. PLoS One.

[14] Funchal C, de Almeida LM, Oliveira Loureiro S, Vivian L, de Lima Pelaez P, Dall Bello Pessutto F, et al. In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of

[15] Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol. 2013;698(1–3):6–18.

[16] Pessoa‐Pureur R, Heimfarth L, Rocha JB. Signaling mechanisms and disrupted cyto‐ skeleton in the diphenyl ditelluride neurotoxicity. Oxid Med Cell Longev. 2014;2014:

[17] Hakansson K, Lindskog M, Pozzi L, Usiello A, Fisone G. DARPP‐32 and modulation of cAMP signaling: involvement in motor control and levodopa‐induced dyskinesia.

[18] Heimfarth L, Loureiro SO, Reis KP, de Lima BO, Zamboni F, Lacerda S, et al. Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modula‐ tion of DARPP‐32‐dependent pathways in cerebral cortex of young rats. Arch Toxicol.

[19] Heimfarth L, Loureiro SO, Reis KP, de Lima BO, Zamboni F, Gandolfi T, et al. Cross‐talk among intracellular signaling pathways mediates the diphenyl ditelluride actions on the

hippocampal cytoskeleton of young rats. Chem Res Toxicol. 2011;24(10):1754–64. [20] Heimfarth L, Loureiro SO, Zamoner A, Pelaez PDL, Nogueira CW, Da Rocha JBT, et al. Effects of in vivo treatment with diphenyl ditelluride on the phosphorylation of

lin and HSP27. Am J Hum Genet. 2006;79(2):197–213.

rats. Brain Res Brain Res Protoc. 2003;11(2):111–8.

Parkinsonism Relat Disord. 2004;10(5):281–6.

Mol Cell Biol. 2007;8(7):530–41.

252 Cytoskeleton - Structure, Dynamics, Function and Disease

Res. 2007;313(10):2098–109.

2006;31(7):383–94.

2014;9(10):e110174.

458601.

2012;86(2):217–30.


filament and induces glutamate‐ and calcium‐mediated mechanisms in rat brain during development. Int J Dev Neurosci. 2010;28(1):21–30.


rat brain: implications for remyelination‐enhancing therapies. Int J Dev Neurosci. 2009;27(8):769–78.

[45] Martinez R, Gomes FC. Neuritogenesis induced by thyroid hormone‐treated astrocytes is mediated by epidermal growth factor/mitogen‐activated protein kinase‐phosphati‐ dylinositol 3‐kinase pathways and involves modulation of extracellular matrix proteins. J Biol Chem. 2002;277(51):49311–8.

filament and induces glutamate‐ and calcium‐mediated mechanisms in rat brain during

[32] Loureiro SO, Romao L, Alves T, Fonseca A, Heimfarth L, Moura Neto V, et al. Homocysteine induces cytoskeletal remodeling and production of reactive oxygen spe‐

[33] Lugo‐Huitron R, Ugalde Muniz P, Pineda B, Pedraza‐Chaverri J, Rios C, Perez‐de la Cruz V. Quinolinic acid: an endogenous neurotoxin with multiple targets. Oxid Med

[34] Pierozan P, Zamoner A, Soska AK, Silvestrin RB, Loureiro SO, Heimfarth L, et al. Acute intrastriatal administration of quinolinic acid provokes hyperphosphorylation of cyto‐ skeletal intermediate filament proteins in astrocytes and neurons of rats. Exp Neurol.

[35] Pierozan P, Goncalves Fernandes C, Ferreira F, Pessoa‐Pureur R. Acute intrastriatal injection of quinolinic acid provokes long‐lasting misregulation of the cytoskeleton in the striatum, cerebral cortex and hippocampus of young rats. Brain Res. 2014;1577:1–10.

[36] Pierozan P, Fernandes CG, Dutra MF, Pandolfo P, Ferreira F, de Lima BO, et al. Biochemical, histopathological and behavioral alterations caused by intrastriatal admin‐

[37] Pierozan P, Zamoner A, Soska AK, de Lima BO, Reis KP, Zamboni F, et al. Signaling mechanisms downstream of quinolinic acid targeting the cytoskeleton of rat striatal neu‐

[38] Pierozan P, Ferreira F, de Lima BO, Pessoa‐Pureur R. Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte‐neuron interac‐

[39] Pierozan P, Ferreira F, Ortiz de Lima B, Goncalves Fernandes C, Totarelli Monteforte P, de Castro Medaglia N, et al. The phosphorylation status and cytoskeletal remodeling of

striatal astrocytes treated with quinolinic acid. Exp Cell Res. 2014;322(2):313–23.

[40] Etienne‐Manneville S. From signaling pathways to microtubule dynamics: the key play‐

[41] Caceres A, Kosik KS. Inhibition of neurite polarity by tau antisense oligonucleotides in

[42] Witte H, Bradke F. The role of the cytoskeleton during neuronal polarization. Curr Opin

[43] Younes‐Rapozo V, Berendonk J, Savignon T, Manhaes AC, Barradas PC. Thyroid hor‐ mone deficiency changes the distribution of oligodendrocyte/myelin markers during

[44] Fernandez M, Paradisi M, Del Vecchio G, Giardino L, Calza L. Thyroid hormone induces glial lineage of primary neurospheres derived from non‐pathological and pathological

oligodendroglial differentiation in vitro. Int J Dev Neurosci. 2006;24(7):445–53.

istration of quinolic acid to young rats. FEBS J. 2014;281(8):2061–73.

rons and astrocytes. Exp Neurol. 2012;233(1):391–9.

tion. J Neurosci Res. 2015;93(2):268–84.

ers. Curr Opin Cell Biol. 2010;22(1):104–11.

Neurobiol. 2008;18(5):479–87.

primary cerebellar neurons. Nature. 1990;343(6257):461–3.

development. Int J Dev Neurosci. 2010;28(1):21–30.

Cell Longev. 2013;2013:104024.

254 Cytoskeleton - Structure, Dynamics, Function and Disease

2010;224(1):188–96.

cies in cultured cortical astrocytes. Brain Res. 2010;1355:151–64.


## **Acting on Actin During Bacterial Infection**

### Elsa Anes

[57] Davis PJ, Goglia F, Leonard JL. Nongenomic actions of thyroid hormone. Nat Rev

[58] Siegrist‐Kaiser CA, Juge‐Aubry C, Tranter MP, Ekenbarger DM, Leonard JL. Thyroxine‐ dependent modulation of actin polymerization in cultured astrocytes. A novel, extra‐

[59] Farwell AP, Dubord‐Tomasetti SA, Pietrzykowski AZ, Stachelek SJ, Leonard JL. Regulation of cerebellar neuronal migration and neurite outgrowth by thyroxine and

[60] Trentin AG, Moura Neto V. T3 affects cerebellar astrocyte proliferation, GFAP and fibro‐

[61] Bergh JJ, Lin HY, Lansing L, Mohamed SN, Davis FB, Mousa S, et al. Integrin alphaV‐ beta3 contains a cell surface receptor site for thyroid hormone that is linked to activa‐ tion of mitogen‐activated protein kinase and induction of angiogenesis. Endocrinology.

[62] Rahaman SO, Ghosh S, Mohanakumar KP, Das S, Sarkar PK. Hypothyroidism in the developing rat brain is associated with marked oxidative stress and aberrant intraneu‐

[63] Motil J, Chan WK, Dubey M, Chaudhury P, Pimenta A, Chylinski TM, et al. Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events. Cell

nuclear action of thyroid hormone. J Biol Chem. 1990;265(9):5296–302.

3,3',5'‐triiodothyronine. Brain Res Dev Brain Res. 2005;154(1):121–35.

ronal accumulation of neurofilaments. Neurosci Res. 2001;40(3):273–9.

nectin organization. Neuroreport. 1995;6(2):293–6.

Motil Cytoskeleton. 2006;63(5):266–86.

Endocrinol. 2016;12(2):111–21.

256 Cytoskeleton - Structure, Dynamics, Function and Disease

2005;146(7):2864–71.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66861

#### **Abstract**

Bacterial resistance to antibiotics is becoming a major threat to public health. It is imperative to find new therapeutic interventions to fight pathogens. Thus, deciphering host-pathogen interactions may allow defining targets for new strategies for effective treatments of infectious diseases. This chapter focuses on the bacterial manipulation of the host cell actin cytoskeleton. We discuss three infectious processes. The first is pathogen establishment of infection/invasion, explaining cellular uptake pathways that rely on actin, such as phagocytosis and macropinocytosis. The second process focus on the establishment of a replication niche, a process that subverts cytoskeletal functions associated with membrane trafficking namely phagosome maturation and cellular innate immune responses. Finally, pathogen dissemination is an emerging field that microfilaments have shown to participate: pathogen motility through the cytoplasm and from cell-to-cell or on the outer surface of the plasma membrane mimicking a receptor tyrosine kinase signaling pathway that helps the projection of pathogens to neighboring cells. It also establishes a connection with the innate immunity related with induction of cell signaling to inflammation, inflammasome activation, and programmed cell death. These studies revealed several potential targets related to actin cytoskeleton manipulation to design new therapeutic strategies for bacterial infections.

**Keywords:** actin, Rho GTPases, bacterial pathogens, phagocytosis, macropinocytosis, virulence mechanisms, innate immunity

### **1. Introduction**

The cell cytoskeleton is composed of three distinct protein families each of which is assembled from monomers to form polymer networks namely from actin, tubulin, or intermediate-filament proteins. Host and pathogens have developed intrinsic interactions with the cytoskeletal system, playing a central role in several stages of their life cycles. Deciphering the complexity of these interactions is revealing new insights about the mechanisms of bacterial pathogenicity but also on defining new host targets for alternative therapies to available antibiotics.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Indeed, clarifying these bacterial mechanisms of host subversion has led to many discoveries about host cell biology, including the identification of new cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Microorganisms exploit actin, microtubules, and intermediate filaments in diverse ways, however, it is mainly the actin cytoskeleton that appears to play a critical role in infection and is the topic of this chapter.

In host cells, actin is involved in the polymerization of stable filaments to assure the cell architecture; at the cell surface originates dynamic movements mediated via assembly and disassembly of microfilaments contributing to contour changes as well cellular locomotion,

**Figure 1.** Schematic diagram of host cell actin rearrangements during bacterial infection. In red: actin filaments and actin polymerization promoting Rho GTPases. In brown: cell responses to bacterial infection. In blue: bacteria hijacking mechanisms of the host actin cytoskeleton.

cell-to-cell adhesion, and signaling. In the cytoplasm, the actin skeleton provides tracks and tails to direct vesicle trafficking. Thus, the importance of the actin cytoskeleton for eukaryotic host physiology from cell movement, cell-to-cell adherence, endocytosis, vesicle trafficking, and cell signaling, among others, has provided pathogenic bacteria with a plethora of opportunistic chances to be exploited.

Indeed, clarifying these bacterial mechanisms of host subversion has led to many discoveries about host cell biology, including the identification of new cytoskeletal proteins, regulatory pathways, and mechanisms of cytoskeletal function. Microorganisms exploit actin, microtubules, and intermediate filaments in diverse ways, however, it is mainly the actin cytoskeleton

In host cells, actin is involved in the polymerization of stable filaments to assure the cell architecture; at the cell surface originates dynamic movements mediated via assembly and disassembly of microfilaments contributing to contour changes as well cellular locomotion,

**Figure 1.** Schematic diagram of host cell actin rearrangements during bacterial infection. In red: actin filaments and actin polymerization promoting Rho GTPases. In brown: cell responses to bacterial infection. In blue: bacteria hijacking

mechanisms of the host actin cytoskeleton.

that appears to play a critical role in infection and is the topic of this chapter.

258 Cytoskeleton - Structure, Dynamics, Function and Disease

The roles of the actin cytoskeleton in host-pathogen interactions can be summarized according to groups of pathogens and how they interact with this system. Some promote attachment to the plasma membrane, forming specialized actin structures (pedestals), allowing strong adherence to host epithelial surfaces. Others induce actin polymerization to enter into nonprofessional phagocytic cells; while others prevent polymerization to avoid uptake by professional phagocytic cells. A few pathogens use the actin cytoskeleton to allow other specialized internalization processes to occur in phagocytic cells as an alternative or in addition to phagocytosis. Intracellular pathogens manipulate the cytoskeleton to prevent membrane trafficking or fusion events leading to the establishment of a niche inside a vacuole often avoiding delivery into the degradative environment of the lysosome. Finally, some pathogens escape from the phagosome vacuole to the cytosol and use the actin machinery to move within cells and to spread directly from the cytoplasm of one cell into the cytoplasm of an adjacent cell. Recently, actin dynamics during infection was related to innate immune responses that rely on activation of cytosolic pattern recognition receptors (cytosolic PRRs) for inflammasome or autophagy assembly and programmed cell death.

This chapter provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack the host actin cytoskeleton (**Figure 1**).

### **2. Acting on actin during pathogen establishment of infection/invasion**

Pathogens often have to overcome epithelial barriers to gain entry into the host cells. The first of which is the epithelial mucosae and a few pathogens, along their evolution, have developed strategies to overcome these barriers by means of active invasion mechanisms. Therefore some intracellular pathogens have evolved strategies to induce or modulate their uptake into these nonprofessional phagocytic cells. Alternatively, as a barrier circumventing mechanism, they may use the cells of the immune system (professional phagocytic cells such as macrophages, neutrophils, and dendritic cells) that patrols those epithelia. Here pathogens may or not play an active role in host cell internalization. Usually professional phagocytes recognize pattern signatures of pathogens (e.g., lipopolysaccharides: LPS), or opsonized bacteria (e.g., complement C3 or IgGs), by means of surface receptors. Likewise phagocytes play an active role in bacteria internalization. As part of the immune system these cells are equipped with a series of insult mechanisms designed to clear pathogens (as the proteolysis at low pH in the phagolysosome). Likewise, extracellular pathogens modulate the host cell plasma membrane for attachment and inhibition of phagocytosis in order to survive. In contrast, intracellular pathogens developed strategies to circumvent the bactericidal mechanisms of immune cells via establishing a protective vacuolar niche.

Several actin dependent mechanisms exist for allowing the establishment of infection: (1) Conventional phagocytosis meaning the entry into professional phagocytes by bilateral membrane pseudopodia formation that tightly encloses the bacteria. Phagocytosis always involves close contact between particle and plasma membrane by multivalence receptor-ligand interactions following morphological changes assembling a zipper mechanism. The host plays a central role for the internalization event while no action is required from the pathogen; (2) induced phagocytosis, a process of active induction of internalization into nonprofessional phagocytes such as epithelial cells, by pathogen manipulation of the host cell contractile system; both the host and the pathogen have active roles in the event. Mechanistically the process occurs by strong interactions between bacterial ligands with cell receptors as in conventional phagocytosis; (3) macropinocytosis: here there may be no direct contact between ligand-pathogen and cell-receptors. Literally, macropinocytosis means—cell drinking—and always involves extensive signaling (e.g., via EGF receptor, a type of tyrosine kinase receptor) that induces pseudopodia unilateral formation surrounding large amount of extracellular volume. So particles including bacteria go in passively along with extracellular fluid. Conventional macropinocytosis may occurs in several types of cells including professional and nonprofessional phagocytes leading to the formation of a large vacuole, the macropinosome; (4) induced macropinocytosis involves pathogen manipulation of the host cell cytoskeleton through growth factor induced signaling or directly using secretion systems that injects virulence factors into the cytosol. While referred classically as trigger phagocytosis, according to the type of morphological changes (with multiple ruffles at the cell surface), there is no direct connection between pathogen and plasma membrane. Finally, (5) an unconventional form of phagocytosis may be used for the establishment of infection via actin cytoskeleton. This is termed as coiling phagocytosis and involves single folds of the phagocyte plasma membrane wrapping around microbes in multiple turns **(Figure 1)**.

#### **2.1. Phagocytosis of bacteria and inhibition of phagocytosis by pathogens**

Phagocytosis is a universal phenomenon involving the recognition and binding of a particle (over 0.5 μm in diameter), in a multivalence receptor-dependent manner, to its internalization and degradation within the phagocytic cell [1]. Mechanistically the process of particle internalization from the plasma membrane is clathrin independent and requires actin polymerization [2]. Phagocytosis of one particle does not signal or permit the indiscriminate phagocytosis of other particles bound to the cell surface. In fact particle ingestion is not automatically triggered by initial particle binding, but requires the sequential recruitment of cell surface receptors into interactions with the remainder of the particle surface. The forming phagosome conforms to the shape of the particle as a close-fitting sleeve of plasma membrane, held in place by interactions between surface receptors and the particle surface, much as teeth hold a zipper together [3]. Phagocytosis can be broadly categorized into three steps: particle binding (along with receptor-cell signaling), internalization (i.e., phagosome formation and invagination) and phagosome maturation (i.e., biogenesis of the degradative compartment: the phagolysosome).

The phases prior to the establishment of interactions between bacterial ligands and phagocytic receptors may involve pathogen fishing by cell structures—this process is also dependent of filamentous actin (F-actin), filopodia extensions **(Figure 1)**. Filopodia serves differently in pathogens and immune cells: pathogens will use it to approach cell membranes for invasion while macrophages will take advantage of these structures for fishing surrounding molecules in order to patrol the environment for possible invaders [4].

Several actin dependent mechanisms exist for allowing the establishment of infection: (1) Conventional phagocytosis meaning the entry into professional phagocytes by bilateral membrane pseudopodia formation that tightly encloses the bacteria. Phagocytosis always involves close contact between particle and plasma membrane by multivalence receptor-ligand interactions following morphological changes assembling a zipper mechanism. The host plays a central role for the internalization event while no action is required from the pathogen; (2) induced phagocytosis, a process of active induction of internalization into nonprofessional phagocytes such as epithelial cells, by pathogen manipulation of the host cell contractile system; both the host and the pathogen have active roles in the event. Mechanistically the process occurs by strong interactions between bacterial ligands with cell receptors as in conventional phagocytosis; (3) macropinocytosis: here there may be no direct contact between ligand-pathogen and cell-receptors. Literally, macropinocytosis means—cell drinking—and always involves extensive signaling (e.g., via EGF receptor, a type of tyrosine kinase receptor) that induces pseudopodia unilateral formation surrounding large amount of extracellular volume. So particles including bacteria go in passively along with extracellular fluid. Conventional macropinocytosis may occurs in several types of cells including professional and nonprofessional phagocytes leading to the formation of a large vacuole, the macropinosome; (4) induced macropinocytosis involves pathogen manipulation of the host cell cytoskeleton through growth factor induced signaling or directly using secretion systems that injects virulence factors into the cytosol. While referred classically as trigger phagocytosis, according to the type of morphological changes (with multiple ruffles at the cell surface), there is no direct connection between pathogen and plasma membrane. Finally, (5) an unconventional form of phagocytosis may be used for the establishment of infection via actin cytoskeleton. This is termed as coiling phagocytosis and involves single folds of the phagocyte plasma

260 Cytoskeleton - Structure, Dynamics, Function and Disease

membrane wrapping around microbes in multiple turns **(Figure 1)**.

**2.1. Phagocytosis of bacteria and inhibition of phagocytosis by pathogens**

Phagocytosis is a universal phenomenon involving the recognition and binding of a particle (over 0.5 μm in diameter), in a multivalence receptor-dependent manner, to its internalization and degradation within the phagocytic cell [1]. Mechanistically the process of particle internalization from the plasma membrane is clathrin independent and requires actin polymerization [2]. Phagocytosis of one particle does not signal or permit the indiscriminate phagocytosis of other particles bound to the cell surface. In fact particle ingestion is not automatically triggered by initial particle binding, but requires the sequential recruitment of cell surface receptors into interactions with the remainder of the particle surface. The forming phagosome conforms to the shape of the particle as a close-fitting sleeve of plasma membrane, held in place by interactions between surface receptors and the particle surface, much as teeth hold a zipper together [3]. Phagocytosis can be broadly categorized into three steps: particle binding (along with receptor-cell signaling), internalization (i.e., phagosome formation and invagination) and phagosome maturation (i.e., biogenesis of the degradative compartment: the phagolysosome). The phases prior to the establishment of interactions between bacterial ligands and phagocytic receptors may involve pathogen fishing by cell structures—this process is also dependent of filamentous actin (F-actin), filopodia extensions **(Figure 1)**. Filopodia serves differently in Phagocytosis was first discovered in the lower eukaryote amoebae that use it for feeding. In higher organisms, phagocytosis is fundamental for host defence against invading pathogens and contributes to the immune and inflammatory responses [5] including turnover and remodeling of tissues and disposal of dead cells. All cells may to some extent perform phagocytosis [6]. However in mammals, phagocytosis is the hallmark of specialized cells including macrophages, dendritic cells, and polymorphonuclear neutrophils—these cells are collectively referred to as professional phagocytes [6]. In certain circumstances, other cell types, such as fibroblasts engulfing apoptotic cells and bladder epithelial cells consuming erythrocytes, are able to perform conventional phagocytosis as efficiently as professional phagocytes [6].

Professional phagocytes express a series of cell surface receptors which recognize a variety of microbial ligands. Receptors on the surface of the phagocytic cell orchestrate a set of signaling events that are required for particle internalization. However, most pathogens possess many different ligands on their surface. Their phagocytic uptake occurs via multiligand interactions, which induce the engagement of many receptors at the same time.

Two major categories of receptors involved in pathogen recognition are opsonic receptors and nonopsonic receptors (pattern-recognition receptors: PRRs) [1]. Receptors for opsonins such as IgG antibodies and the complement fragment C3bi engage Fc*γ*Rs and complement receptors (CR), respectively. PRRs include toll-like receptors (TLRs) and other receptor families as C-type lectins receptors that recognize sugar residues as mannose or fucose and lipopolysaccharides (LPS). TLRs often function as coreceptors in phagocytosis by their discrimination of a broad range of microbial products, including LPS and peptidoglycan. The role of TLRs in accelerating and modulating phagosome maturation is still a matter of debate [7].

Bacteria opsonized by complement C3b, by IgG or having lipoarabinomannans at the cell wall surface will be recognized by complement receptors such as CR1 and CR3/4, Fc receptors or Man-6P receptors respectively, each triggering phagocytosis without stimulating a strong superoxide burst. The entry via these phagocytic receptors leads to the maturation of the forming phagosome into a very degradative lysosomal compartment that will destroy microbes [8]. All these receptors will be downregulated during phagocyte activation either through bacterial proinflammatory components as in the case of LPS or cytokines as IFNγ [8].

Activated macrophages will in turn reprogram their expression profile in order to increase the ability to kill pathogens via oxidative bursts and decrease protein digestion extension from amino-acids to small peptides, for antigen presentation [9].

Phagocytosis uses the actin cytoskeleton to construct a cup and close the cup by contractile activities [10]. Latter along phagosome maturation the actin cytoskeleton is also utilized for vesicle trafficking and fusion along the endocytic pathway [11]. The induced polymerization of filamentous actin (F-actin) from globular actin (G-actin) beneath the site of attachment of the particle is the driving force behind ingestion and proceeds from signal transduction downstream of the phagocytic receptors [1]. The precise signaling cascades linking activated receptors to actin polymerization are not fully understood yet it is well known that Rho GTPase family plays critical roles in controlling these cytoskeletal rearrangements [1]. These, RhoA, Rac1, and cell division cycle 42 (Cdc42) act as molecular switches in controlling actin dynamics by regulating the actin-related protein 2/3 (Arp2/3) complex [12]. Arp2/3 requires activation by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome protein (WASP) family. Nucleation-promoting factors exist in an autoinhibited conformation until activated by Cdc42 and Rac1, as well as by phosphoinositide (PI) signaling (discussed latter in this chapter). Effectors such as Cdc42 and the phosphoinositide 4,5-bisphosphate PI(4,5)P2 (PIP2) synergize to activate WASP homolog N-WASP which triggers actin polymerization via Arp2/3 [13]. As the newly formed actin branch grows, the plasma membrane is forced out, extending the membrane as pseudopodia **(Figure 1)**.

Various extracellular and intracellular cues including those from pathogens stimulate Rho GTPases, leading to actin-mediated membrane manipulation. RhoA, Rac1, and Cdc42 have all been shown to accumulate at the nascent phagosome cup. These proteins are preferred targets for bacterial toxins that in turn modulate the organization of the actin skeleton allowing invasion into nonprofessional phagocytic cells and preventing phagocytosis into professional phagocytes. These toxins modify the activity of Rho GTPases through covalent modification or regulation of the nucleotide state. Toxins such as *Clostridium difficile* toxin A and B modify Rho leading to inactivation of its function. This bacterium and the toxin it produces are a global health problem especially affecting the elderly who need to be prescribed prolonged doses of antibiotics. In fact extracellular bacteria, such as *Clostridium* spp., release toxins that glycosylate Rho GTPases in order to disorganize actin to reduce immune cell migration and phagocytosis and also to break down epithelial cell barriers [14].

Another group of toxins regulates the nucleotide state and thus the function of various Rho GTPases by acting as GTPase-activating proteins (GAPs). *Yersinia* spp. an enteropathogenic group of bacteria have secretion systems that inject a type of these Rho GAP toxins, Yop virulence factors leading to actin filamentation blocking and consequently to inhibition of phagocytosis in all host cells to where a contact is established with either professional or nonprofessional phagocytic cells [15].

Pseudomonas has the capacity to inactivate all Rho GTPases [16]. *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen that causes life-threatening infections in cystic fibrosis patients, individuals with burn wounds, and the immuno-compromised. *P. aeruginosa* pathogenicity involves cell-associated and secreted virulence factors as ExoS one of four type III cytotoxins injected into the cytosol. *In vivo* the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibiting of Rho [16]. The consequences are the prevention of phagocytosis. Moreover, the perturbation of F-to G-actin content together with cytosolic stress is sensed by the PRR pyrin triggering caspase 1 and inflammasome assembly leading to inflammation and cell death by pyroptosis.

Many intracellular bacterial pathogens have evolved to survive and even proliferate within immune phagocytic cells. Depending on the route of entry, the fate of intracellular bacteria varies significantly. Some opsonized bacteria as *Brucella*, the agent of brucellosis, for example, are destroyed efficiently within macrophages while the nonopsonised survive [17]. An essential feature of the pathogenicity of *Salmonella* is its capacity to cross a number of barriers requiring invasion of a large variety of phagocytic and nonphagocytic cells (reviewed in Ref. [18]). Virulent *Salmonella enterica* serovar Thyphimurium infection of macrophages triggers cell lysis while opsonized noninvasive mutants do not thus reinforce the idea that distinct overcomes depend on the internalization route [19]. The cytotoxicity of serovar Typhimurium is related to the capacity of this organism to invade cells. Mutants lacking invasion proteins encoded by the salmonella pathogenicity island 1 genome region (SPI-1) failed to induce cell lysis in murine macrophages [20]. This is an important step of salmonella infection allowing the pathogen escaping to macrophages to reach the basolateral membrane of the gut cells for invasion.

receptors to actin polymerization are not fully understood yet it is well known that Rho GTPase family plays critical roles in controlling these cytoskeletal rearrangements [1]. These, RhoA, Rac1, and cell division cycle 42 (Cdc42) act as molecular switches in controlling actin dynamics by regulating the actin-related protein 2/3 (Arp2/3) complex [12]. Arp2/3 requires activation by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome protein (WASP) family. Nucleation-promoting factors exist in an autoinhibited conformation until activated by Cdc42 and Rac1, as well as by phosphoinositide (PI) signaling (discussed latter in this chapter). Effectors such as Cdc42 and the phosphoinositide 4,5-bisphosphate PI(4,5)P2 (PIP2) synergize to activate WASP homolog N-WASP which triggers actin polymerization via Arp2/3 [13]. As the newly formed actin branch grows, the plasma membrane is forced out,

Various extracellular and intracellular cues including those from pathogens stimulate Rho GTPases, leading to actin-mediated membrane manipulation. RhoA, Rac1, and Cdc42 have all been shown to accumulate at the nascent phagosome cup. These proteins are preferred targets for bacterial toxins that in turn modulate the organization of the actin skeleton allowing invasion into nonprofessional phagocytic cells and preventing phagocytosis into professional phagocytes. These toxins modify the activity of Rho GTPases through covalent modification or regulation of the nucleotide state. Toxins such as *Clostridium difficile* toxin A and B modify Rho leading to inactivation of its function. This bacterium and the toxin it produces are a global health problem especially affecting the elderly who need to be prescribed prolonged doses of antibiotics. In fact extracellular bacteria, such as *Clostridium* spp., release toxins that glycosylate Rho GTPases in order to disorganize actin to reduce immune cell migration and

Another group of toxins regulates the nucleotide state and thus the function of various Rho GTPases by acting as GTPase-activating proteins (GAPs). *Yersinia* spp. an enteropathogenic group of bacteria have secretion systems that inject a type of these Rho GAP toxins, Yop virulence factors leading to actin filamentation blocking and consequently to inhibition of phagocytosis in all host cells to where a contact is established with either professional or non-

Pseudomonas has the capacity to inactivate all Rho GTPases [16]. *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen that causes life-threatening infections in cystic fibrosis patients, individuals with burn wounds, and the immuno-compromised. *P. aeruginosa* pathogenicity involves cell-associated and secreted virulence factors as ExoS one of four type III cytotoxins injected into the cytosol. *In vivo* the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibiting of Rho [16]. The consequences are the prevention of phagocytosis. Moreover, the perturbation of F-to G-actin content together with cytosolic stress is sensed by the PRR pyrin triggering caspase 1 and inflammasome assembly leading to inflammation

Many intracellular bacterial pathogens have evolved to survive and even proliferate within immune phagocytic cells. Depending on the route of entry, the fate of intracellular bacteria varies significantly. Some opsonized bacteria as *Brucella*, the agent of brucellosis, for example, are destroyed efficiently within macrophages while the nonopsonised survive [17]. An essential

extending the membrane as pseudopodia **(Figure 1)**.

262 Cytoskeleton - Structure, Dynamics, Function and Disease

phagocytosis and also to break down epithelial cell barriers [14].

professional phagocytic cells [15].

and cell death by pyroptosis.

The uptake of *Mycobacterium* spp. by phagocytes has been intensively studied since these cell types, especially macrophages, are the preferred targets of this successful pathogen. An important class of *Mycobacterium* pathogens includes tuberculosis bacilli. This intracellular facultative pathogen controls the bacterial load during macrophage internalization by interfering with actin polymerization at the phagocytic cup [21]. This is a necessary step in virulence for preventing apoptosis and therefore to prevent pathogen intracellular killing [22]. For this, during early phases of *Mycobacterium* infection, the microRNA 142-3p is overexpressed in response to phagocytosis and interferes with the expression of N-WASP and consequently with the Arp2/3 complex required for actin nucleation at the cell membrane [21]. Therefore, a low bacterial load is accomplished intracellularly, preventing the apoptosis of the infected cells. In addition, recently, miR-142-3p was shown to directly regulate protein kinase Cα (PKCα), a key gene involved in phagocytosis [23].

The heterodimeric host surface receptor complement-receptor 3 (CR-3), mediates uptake of opsonized and nonopsonized mycobacteria. Interestingly, CR-3 is targeted by other intracellular pathogens, such as *Coxiella burnetii*, the Q-fever agent, in order to avoid phagocytosis. This strategy is based on ensuring a spatial location of CR-3 outside the pseudopod extensions [24].

Lipid modification by receptor signaling creates the potential for radiating signals that can affect large areas of the plasma membrane. Phospholipid kinases, lipid phosphatases, and hydrolases are activated during phagocytosis. Classes of phospholipids typically found on the inner face of biomembranes include phosphatidylinositol (PI). The generation of phosphoinositides derived from PI via phosphorylation events will generate classes of important lipids enrolled in cell signaling and phagocytosis as example of phosphatidylinositol (4)-phosphate (PI(4)P=PIP), PI(5)P, PI(4,5)P2 (PIP2), PI (3,4)P2, and PI(3,4,5)P3 (PIP3). As mentioned previously in this chapter, these phosphoinositides, especially PIP2 and PIP3, are capable of binding and increasing the activity of proteins that modify membrane chemistry and the actin cytoskeleton. As an example, PIP2 increases the activity of WASP, a protein that stimulates actin polymerization via Arp2/3.

This class of PIs in addition to their relevance in particle internalization is important during the phase of phagosome maturation into a degradative compartment, the phagolysosome. In phagosomal membranes PIP2 activates the actin nucleators of the Ezrin, Moesin, and Radixin family inducing polymerization of F-actin and therefore phagosome maturation [11]. This will be addressed later in this chapter in the context of the manipulation of the actin cytoskeleton by pathogens in order to establish an intracellular niche.

#### **2.2. Induced phagocytosis by invasive pathogens**

Classically, the manipulation of the actin cytoskeleton by invasive pathogens was classified into two general mechanisms according to the type of morphological changes that occur in the host cell—the zipper and trigger phagocytosis [3]. Entry of uropathogenic *Escherichia coli*, *Yersinia*, *Helicobacter*, *Listeria*, and *Neisseria* into epithelial cells is reminiscent of the classical model of zipper phagocytosis. The trigger model will be addressed as macropinocytosis in the next section of this chapter as it is not in fact a phagocytosis event. Moreover, the zipper mechanism may also be triggered actively by pathogens.

Adherence to nonprofessional phagocytic cells, epithelium by a pathogen is necessary to avoid mechanical clearance and is the first step of colonization by for example enteropathogens. Thus bacterial pathogens exhibit a large variety of cell surface adhesins, including fimbriae (pili) and afimbrial adhesins some of which participate in the internalization step. Likewise, in this type of entry, a bacterial adhesin binds to a host cell surface receptor involved in cellto-cell adhesion and/or activates regulatory proteins that modulate cytoskeleton dynamics. Moreover, adherence and internalization into epithelial cells looks to be a strategy used by pathogens to escape destruction by immune cells as described below.

Most type I pili expressed by pathogenic *E. coli* bind to host mannose-containing glycoproteins some expressed in gut epithelial cells including M cells (microfold cells of Payer's Patches) [25]. Others such as FimH from uropathogenic *E. coli* can bind to β1 and α3 integrins and thereby promote bacterial internalization following a process that to date has only been described in urinary bladder epithelial cells. Uropathogenic *E. coli* (UPEC) cause the majority of community-onset urinary tract infections (UTI). Early in acute cystitis, UPEC gains access to an intracellular niche that protects a population of replicating bacteria from arriving phagocytes [26]. Transition bacillary forms of UPEC (1–2 μm in length) are readily engulfed, while filamentous UPEC resist phagocytosis, even when in direct contact with neutrophils and macrophages. Despite these strong host defenses, a subpopulation of UPEC is able to persist for months in a quiescent reservoir state which may serve as a seed for recurrent infections [27].

*Yersinia* spp. such as *Yersinia enterocolitica* and *Yersinia pseudotuberculosis* invades gut mucosae at the ileum terminal end and multiplies in the underlying lymphoid tissue. Invasin and YadA (Yersinia adhesion A) are crucial for yersinia adherence via β1 integrins and matrix components, respectively. β1 integrins exist on the basolateral face of enterocytes and on the apical surface of the epithelia derived M cells. The coalescence of integrins following bacteria invasin linkage will lead to yersinia internalization by a "zipper mechanism". Binding of invasin to β1 integrin activates focal adhesion tyrosine kinase and triggers a complex cascade implicating Rac1-Arp2/3 pathways but also phosphoinositide-3-kinase (PI3K) leading to the closure of the phagocytic cup. In contrast, YadA binds diverse extracellular matrix components, such as collagen, laminin, and fibronectin, thus indirectly mediating integrin binding [28]. Yersinia species also hijack host cell phosphoinositide metabolism for their uptake. Rac-1 recruits, and Arf6 activates the type I phosphatidylinositol-4-phosphate-5-kinase (PtdIns(4) P(5)Ka), which forms PIP2 at the entry site, and this lipid may regulate phagocytic cup formation by coordinating membrane traffic and controlling F-actin polymerization [29].

*Helicobacter pylori* is another example of pathogen that adheres to mucosa via β1integrins and invades nonphagocytic cells. Efficient infection of cultured epithelial cells seems to be restricted to certain *H. pylori* strains. This pathogen uses a type IV secretion system (T4SS) targeting β1 integrins to translocate the virulence factor CagA into the cytosol. The adhesin CagL present in the T4SS pilus surface bridge activates the integrin on the basolateral membrane of gastric epithelial cells. In all cases, however, invasion of *H. pylori* seems to involve a typical zipper-like entry process. Both PI3-K and PKC are required for bacterial uptake and induction of cytoskeletal rearrangements [30]. Curiously preinfection of cultured gastric cells with yersinia expressing Yop virulence factors that interfere with the same signaling events impaired phagocytosis of *H. pylori* [30]. Internalized *H*. *pylori* was shown to be located in tight phagosomes and in close association with condensed actin filaments and localized tyrosine phosphorylation signals. Similar to UPEC in bladder epithelial cells, invasion of epithelial cells by *H. pylori* may constitute one of the evasion strategies used by this pathogen to circumvent the host immune response and persist in stomach.

**2.2. Induced phagocytosis by invasive pathogens**

264 Cytoskeleton - Structure, Dynamics, Function and Disease

mechanism may also be triggered actively by pathogens.

tions [27].

pathogens to escape destruction by immune cells as described below.

Classically, the manipulation of the actin cytoskeleton by invasive pathogens was classified into two general mechanisms according to the type of morphological changes that occur in the host cell—the zipper and trigger phagocytosis [3]. Entry of uropathogenic *Escherichia coli*, *Yersinia*, *Helicobacter*, *Listeria*, and *Neisseria* into epithelial cells is reminiscent of the classical model of zipper phagocytosis. The trigger model will be addressed as macropinocytosis in the next section of this chapter as it is not in fact a phagocytosis event. Moreover, the zipper

Adherence to nonprofessional phagocytic cells, epithelium by a pathogen is necessary to avoid mechanical clearance and is the first step of colonization by for example enteropathogens. Thus bacterial pathogens exhibit a large variety of cell surface adhesins, including fimbriae (pili) and afimbrial adhesins some of which participate in the internalization step. Likewise, in this type of entry, a bacterial adhesin binds to a host cell surface receptor involved in cellto-cell adhesion and/or activates regulatory proteins that modulate cytoskeleton dynamics. Moreover, adherence and internalization into epithelial cells looks to be a strategy used by

Most type I pili expressed by pathogenic *E. coli* bind to host mannose-containing glycoproteins some expressed in gut epithelial cells including M cells (microfold cells of Payer's Patches) [25]. Others such as FimH from uropathogenic *E. coli* can bind to β1 and α3 integrins and thereby promote bacterial internalization following a process that to date has only been described in urinary bladder epithelial cells. Uropathogenic *E. coli* (UPEC) cause the majority of community-onset urinary tract infections (UTI). Early in acute cystitis, UPEC gains access to an intracellular niche that protects a population of replicating bacteria from arriving phagocytes [26]. Transition bacillary forms of UPEC (1–2 μm in length) are readily engulfed, while filamentous UPEC resist phagocytosis, even when in direct contact with neutrophils and macrophages. Despite these strong host defenses, a subpopulation of UPEC is able to persist for months in a quiescent reservoir state which may serve as a seed for recurrent infec-

*Yersinia* spp. such as *Yersinia enterocolitica* and *Yersinia pseudotuberculosis* invades gut mucosae at the ileum terminal end and multiplies in the underlying lymphoid tissue. Invasin and YadA (Yersinia adhesion A) are crucial for yersinia adherence via β1 integrins and matrix components, respectively. β1 integrins exist on the basolateral face of enterocytes and on the apical surface of the epithelia derived M cells. The coalescence of integrins following bacteria invasin linkage will lead to yersinia internalization by a "zipper mechanism". Binding of invasin to β1 integrin activates focal adhesion tyrosine kinase and triggers a complex cascade implicating Rac1-Arp2/3 pathways but also phosphoinositide-3-kinase (PI3K) leading to the closure of the phagocytic cup. In contrast, YadA binds diverse extracellular matrix components, such as collagen, laminin, and fibronectin, thus indirectly mediating integrin binding [28]. Yersinia species also hijack host cell phosphoinositide metabolism for their uptake. Rac-1 recruits, and Arf6 activates the type I phosphatidylinositol-4-phosphate-5-kinase (PtdIns(4) P(5)Ka), which forms PIP2 at the entry site, and this lipid may regulate phagocytic cup forma-

tion by coordinating membrane traffic and controlling F-actin polymerization [29].

Curiously the vaccinal strain for tuberculosis *Mycobacterium bovis* BCG has been used as the more effective treatment for bladder cancer [31]. The bacillus induces phagocytosis in tumor cells via their surface fibronectin attachment protein (FAP) to β1integrins. After phagocytosis a strong cytotoxic effect is displayed via T-helper CD8 stimulation leading to antitumor activity.

*Listeria monocytogenes* is a food-borne Gram-positive bacterium that makes use of two surface proteins, Internalin A (InlA) and B (InlB), to engage, in a species-specific manner, to host adhesion molecules E-cadherin and hepatocyte growth factor receptor Met respectively, to induce its internalization [32]. Only InlA is critical for invasion of the gut epithelial cells. The specific engagement of E-cadherin initiates activation of the adherens junction machinery inducing the recruitment of β-catenin, Rho GAP protein ARHGAP10, α-catenins to the site of the entry. Internalization is then further mediated by Rac- and Arp2/3-dependent actin polymerization. In contrast to this, InlB is essential for *Listeria* uptake by most nonphagocytic cell types, such as hepatocytes, endothelial cells, fibroblasts, and certain epithelial cell lines. Additionally, it is known that ActA, a *Listeria* protein required for actin-tail formation and intracellular cytosolic movement, can also mediate *Listeria* uptake by epithelial cells [32]. Recently a new phagocytic process was characterized that allows human endothelial cells to internalize listeria independent of all known pathogenic bacterial surface proteins. Here bacteria adhesion is mediated by Rho kinase and the control of the internalization step is coordinated by formins (as FHOD1 and FMNL3) a class of actin nucleation proteins. The overall control of the event is mediated by cytoskeletal proteins usually enrolled in cell shape and locomotion including Rho, focal adhesions, and PI kinases [33].

*Neisseria gonorrhoeae*, is an exclusive human pathogen that primarily infects the urogenital epithelia, causing the sexually transmitted disease gonorrhoea. Entry of *N. gonorrhoeae* into human epithelial cells is multifactorial. Initial attachment is mediated by pili (a T4SS), followed by tight adherence via the phase-variable colony opacity (Opa) proteins. These are a family of 11 outer membrane proteins variably expressed at the surface of the bacterium. However, only OpaA confers invasion into epithelia [34]. This entry is mediated by heparan sulfate proteoglycan (HSPG) receptors of the syndecan family expressed on the target cell surface. Pilus engagement has also been demonstrated to play a role in host cell cytoskeletal rearrangements inducing microvilli formation at the cell surface to surround the bacteria for a zipper mechanism of internalization [35].

In endothelial cells, the T4SS-pilus-mediated adhesion of *Neisseria meningitidis* induces the formation of membrane protrusions similar to microvilli leading to bacterial uptake. These protrusions result from a Rho- and Cdc42-dependent cortical actin polymerization, and from the activation of the ErbB2 tyrosine-kinase receptor and the Src kinase, leading to tyrosine phosphorylation of cortactin, an activator of Arp2/3 [36]. Adhesion of *N. meningitidis* to endothelial cells promotes the local formation of membrane protrusions reminiscent of epithelial microvilli structures that surround bacteria and provoke their internalization within intracellular vacuoles.

#### **2.3. Macropinocytosis, induced macropinocytosis, and coiling phagocytosis**

Unique molecular properties associated with the process of macropinocytosis are beginning to be elucidated. Because of their size and the fact that they may be formed without activation by ligands, the large vacuoles (macropinosomes) formed during this pinocytosis event can contain extracellular fluid and pathogens. At the mechanistic level, phagocytosis and macropinocytosis present many similarities including the involvement of phosphoinositol phosphate signaling and actin cytoskeleton reorganization. During macropinocytosis it is not observed a direct connection between bacteria/cargo and multiple receptors but it was demonstrated the relevance of tyrosine kinase receptors involved in responses to growth factors as the epidermal growth factor and platelet-derived growth factor. The consequence of intensive actin remodeling results in ruffling protrusions at the cell surface, or in unilateral large pseudopodia formation leading to the formation of large macropinosomes. Activated receptor tyrosine kinases, as well as the Src family kinases, are clearly observed on newly formed macropinosomes. Therefore in concert with the morphological definition provided by Lewis in 1931 based on ruffling formation, and elevation in response to growth factor stimulation can be used to define macropinocytosis [37].

Macropinocytosis has been observed in professional phagocytes as well in epithelial cells. Immature dendritic cells and activated macrophages display high levels of constitutive macropinocytosis [38]. The consequent internalization of large volumes of extracellular solute that accompanies macropinocytosis facilitates their capacity to continuously survey the extracellular space for foreign material. In fact, this increased levels of macropinocytosis upon encounter with the antigen/pathogen enhances both antigen capture and antigen presentation by dendritic cells as well as the complete clearance of pathogens after macrophage activation by inflammatory stimulus [38].

In epithelial cells, an induced form of macropinocytosis was observed after infection with pathogens such as *Shigella*, *Salmonella*, enterophatogenic *E.coli* (EPEC), and *Mycobacterium tuberculosis*. Therefore, individual pathogens have developed a range of strategies to modulate the host's normal macropinocytic pathways both to invade the host cells and to manipulate the lipid and protein composition of the encapsulating macropinosome to promote cell uptake and then survival. A few virulence factors secreted by pathogens are able to induce ruffling similar to the growth factors named above. The closure of ruffles back to themselves will entrap pathogens into a large vacuole (micropinosome) incorrectly named in distinct publications as "spacious phagosome".

surface. Pilus engagement has also been demonstrated to play a role in host cell cytoskeletal rearrangements inducing microvilli formation at the cell surface to surround the bacteria for

In endothelial cells, the T4SS-pilus-mediated adhesion of *Neisseria meningitidis* induces the formation of membrane protrusions similar to microvilli leading to bacterial uptake. These protrusions result from a Rho- and Cdc42-dependent cortical actin polymerization, and from the activation of the ErbB2 tyrosine-kinase receptor and the Src kinase, leading to tyrosine phosphorylation of cortactin, an activator of Arp2/3 [36]. Adhesion of *N. meningitidis* to endothelial cells promotes the local formation of membrane protrusions reminiscent of epithelial microvilli structures that surround bacteria and provoke their internalization within intracellular

Unique molecular properties associated with the process of macropinocytosis are beginning to be elucidated. Because of their size and the fact that they may be formed without activation by ligands, the large vacuoles (macropinosomes) formed during this pinocytosis event can contain extracellular fluid and pathogens. At the mechanistic level, phagocytosis and macropinocytosis present many similarities including the involvement of phosphoinositol phosphate signaling and actin cytoskeleton reorganization. During macropinocytosis it is not observed a direct connection between bacteria/cargo and multiple receptors but it was demonstrated the relevance of tyrosine kinase receptors involved in responses to growth factors as the epidermal growth factor and platelet-derived growth factor. The consequence of intensive actin remodeling results in ruffling protrusions at the cell surface, or in unilateral large pseudopodia formation leading to the formation of large macropinosomes. Activated receptor tyrosine kinases, as well as the Src family kinases, are clearly observed on newly formed macropinosomes. Therefore in concert with the morphological definition provided by Lewis in 1931 based on ruffling formation, and elevation in response to growth factor stimulation can be used to define macropino-

Macropinocytosis has been observed in professional phagocytes as well in epithelial cells. Immature dendritic cells and activated macrophages display high levels of constitutive macropinocytosis [38]. The consequent internalization of large volumes of extracellular solute that accompanies macropinocytosis facilitates their capacity to continuously survey the extracellular space for foreign material. In fact, this increased levels of macropinocytosis upon encounter with the antigen/pathogen enhances both antigen capture and antigen presentation by dendritic cells as well as the complete clearance of pathogens after macrophage activa-

In epithelial cells, an induced form of macropinocytosis was observed after infection with pathogens such as *Shigella*, *Salmonella*, enterophatogenic *E.coli* (EPEC), and *Mycobacterium tuberculosis*. Therefore, individual pathogens have developed a range of strategies to modulate the host's normal macropinocytic pathways both to invade the host cells and to manipulate the lipid and protein composition of the encapsulating macropinosome to promote cell uptake and then survival. A few virulence factors secreted by pathogens are able to induce

**2.3. Macropinocytosis, induced macropinocytosis, and coiling phagocytosis**

a zipper mechanism of internalization [35].

266 Cytoskeleton - Structure, Dynamics, Function and Disease

vacuoles.

cytosis [37].

tion by inflammatory stimulus [38].

Invasive enteropathogens, such as *Shigella flexneri* and *S. enterica* serovar Typhimurium, use the trigger mechanism of invasion in epithelial cells to induce membrane ruffles and macropinocytosis. This is a phenomenon dependent on a type III secretion system encoded by both bacteria. The T3SS effectors activate host Cdc42 and Rac1 albeit via distinct cellular relays. In *Salmonella*, SopE acts as a guanyl-nucleotide-exchange factor for Rho [39]. This induced Rho GTPase perturbation is recognized in the cytosol by PRRs (NOD1 sensor) inducing a proinflammatory response and innate immune responses. SigD/SopB is another protein secreted by the SPI-1 T3SS of *Salmonella* to invade nonphagocytic cells. The phosphatidyl-inositol phosphatase activity of SigD/SopB induces rapid disappearance of PIP2 from invaginating regions of the cytoplasmic membrane leading indirectly to Rho activation and macropinocytosis. Once inside the host cell, *Salmonella* induces the recovery of normal cytoskeleton dynamics via SptP, a SPI-1 effector with Cdc42 and Rac1 GAP activity that returns these proteins to their nonactivated state.

In comparison, the effectors IpaC, IpgB1, and VirA of *Shigella* bind to initiate a focal adhesion structure required for internalization via a process that recruit Rho isoforms [40]. Consequently, the injection of the effectors IpaC, IpgB1, and VirA by *S. flexneri* induces Rac1/ Cdc42-dependent actin polymerization. Finally, the translocated effector IpaA binds vinculin and enhances its association to actin filaments, thus mediating the localized depolymerization of actin, which is required to close the phagocytic cup [40].

*S. flexneri* invasion has been classically described as a macropinocytosis-like process, however the role of macropinosomes in intracellular bacterial survival remains elusive. There is evidence that bacterial entry and membrane ruffling are associated with different bacterial effectors and host responses during *S. flexneri* invasion. Rho isoforms are recruited differentially to either entering bacteria or membrane ruffles, and entry has been proposed to occur initially via effector mediated contact of *S. flexneri* to specific receptors suggesting entry is akin to receptor mediated phagocytosis. In fact, the host surface molecules β1-integrins and CD44 (hyaluronic acid receptor) are needed for *Shigella* entry [40].

Recently, the mechanism of *Shigella* invasion of epithelial cells was observed using advanced large volume correlative light electron microscopy (CLEM) indicating a combination of induced phagocytosis and macropinocytosis [41]. Here, the macropinocytic event instead of being the major effector for internalization was in fact shown to be required for release of the bacteria from the phagosome and cytosolic escape later in phagocytosis. Macropinocytic vesicles formed at the invasion site are functionally involved in vacuolar rupture. This unique and surprising pathogenic strategy stands in stark contrast to other invasive pathogens that induce direct lysis of their surrounding vacuole via the action of destabilizing bacterial proteins.

*S. enterica* is an invasive, T3SS-employing pathogen and shares many common host entry characteristics with *S. flexneri*. It was hypothesized that salmonella containing vacuole and macropinosomes may be distinct, as they are sorted into different intracellular routes [42]. These evidence suggest that pathogen induced enhanced uptake of extracellular fluid in *S. enterica serovar* Typhimurium-infected epithelial cells is an event related to the invasion mechanisms used by this pathogen but not the major mechanism for bacteria internalization as referred in most published data.

Surface-adherent pathogens, such as enteropathogenic or enterohaemorrhagic *E. coli* (EPEC or EHEC, respectively), use their T3SS to secrete a transmembrane receptor into the host membrane to stimulate actin polymerization and generate cellular extensions called pedestals. EPEC uses the T3SS apparatus to inject the intimin receptor (Tir). Tir acts as a cell receptor of host kinases activating N-WASP and the actin nucleator Arp2/3 resulting in actin polymerization and pedestal formation at the site of the attachment. While stabilizing bacteria connection to epithelial cells the actin pedestal formation promotes T3SS mediated injection of additional effector proteins able to subvert other host pathways. Where bacteria are attached, microvilli are lost; the epithelial cells form cup-like pedestals upon which the bacteria rest. The underlying cytoskeleton of the epithelial cell is disorganized, with a proliferation of filamentous actin. Although EPEC have traditionally been considered to be noninvasive, accumulating evidence casts doubt on this assumption. From the earliest published electron micrographs of EPEC infection, bacteria have been observed within epithelial cells at the sites of attaching [43]. The virulence factor dependent on Tir signaling EspG contributes to the ability of EPEC pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the *S. flexneri* VirA protein [28, 44].

Patients with inflammatory bowel disease exhibited an increased number of mucosae-associated *E. coli* with invasive properties. The adherent-invasive *E. coli* (AIEC) uses M cells to reach macrophages of Payer's Patches where they survive and replicate inside large macropinosomes that share features of phagolysosomes. To survive, these bacteria, inside the vacuoles, adapted to the harsh acidic environment that is the key signal to activate virulence genes. In fact infected macrophages with AIEC secrete large amounts of tumor necrosis factor alpha leading to local granuloma formation. Those macrophages will subsequently aggregate and fuse releasing bacteria that then will reach the basolateral domain of gut epithelial cells for invasion. Epithelial cell invasion is a key virulence factor only for EIEC, which may lead to a dysentery-like illness similar to that caused by *S. flexneri* [45].

Alveolar macrophages constitute the main defense against *M. tuberculosis* infection. However, tuberculosis bacilli resist phagocytic cell bactericidal mechanisms and replicate within them. Although *M. tuberculosis* survives within phagocytic cells, this bacterium may also bind and invade alveolar epithelial cells [46] and endothelial lymphatic cells [47]. Infection of epithelial cells was concomitant with large lamellipodia projections (ruffles) similar to macropinocytosis. Likewise, *Mycobacterium* can induce formation of macropinosomes however; this does not depend on a bacterial secretion system, as the culture media in the absence of pathogen was sufficient to induce this process. Since nonviable bacteria fail to induce macropinocytosis in opposition to live bacteria, the most prominent candidate to induce ruffling is pointed as being secretory products actively produced by life bacilli. There are no requirements for bacteria to attach directly to the plasma membrane. In endothelial cells, scanning electron microscopy (SEM) micrographs show that mycobacteria were internalized by characteristic phagocytosis-like and macropinocytosis events [47]. However the mycobacterial determinants leading to actin reorganization and pathogen active internalization are not clarified. It is very likely that the invasion and survival in epithelial and endothelial cells contributes to the one-third of the human population latently infected with this microorganism.

These evidence suggest that pathogen induced enhanced uptake of extracellular fluid in *S. enterica serovar* Typhimurium-infected epithelial cells is an event related to the invasion mechanisms used by this pathogen but not the major mechanism for bacteria internalization as

Surface-adherent pathogens, such as enteropathogenic or enterohaemorrhagic *E. coli* (EPEC or EHEC, respectively), use their T3SS to secrete a transmembrane receptor into the host membrane to stimulate actin polymerization and generate cellular extensions called pedestals. EPEC uses the T3SS apparatus to inject the intimin receptor (Tir). Tir acts as a cell receptor of host kinases activating N-WASP and the actin nucleator Arp2/3 resulting in actin polymerization and pedestal formation at the site of the attachment. While stabilizing bacteria connection to epithelial cells the actin pedestal formation promotes T3SS mediated injection of additional effector proteins able to subvert other host pathways. Where bacteria are attached, microvilli are lost; the epithelial cells form cup-like pedestals upon which the bacteria rest. The underlying cytoskeleton of the epithelial cell is disorganized, with a proliferation of filamentous actin. Although EPEC have traditionally been considered to be noninvasive, accumulating evidence casts doubt on this assumption. From the earliest published electron micrographs of EPEC infection, bacteria have been observed within epithelial cells at the sites of attaching [43]. The virulence factor dependent on Tir signaling EspG contributes to the ability of EPEC pathogens to establish infection through a modulation of the host cytoskeleton involving transient microtubule destruction and actin polymerization in a manner akin to the

Patients with inflammatory bowel disease exhibited an increased number of mucosae-associated *E. coli* with invasive properties. The adherent-invasive *E. coli* (AIEC) uses M cells to reach macrophages of Payer's Patches where they survive and replicate inside large macropinosomes that share features of phagolysosomes. To survive, these bacteria, inside the vacuoles, adapted to the harsh acidic environment that is the key signal to activate virulence genes. In fact infected macrophages with AIEC secrete large amounts of tumor necrosis factor alpha leading to local granuloma formation. Those macrophages will subsequently aggregate and fuse releasing bacteria that then will reach the basolateral domain of gut epithelial cells for invasion. Epithelial cell invasion is a key virulence factor only for EIEC, which may lead to a

Alveolar macrophages constitute the main defense against *M. tuberculosis* infection. However, tuberculosis bacilli resist phagocytic cell bactericidal mechanisms and replicate within them. Although *M. tuberculosis* survives within phagocytic cells, this bacterium may also bind and invade alveolar epithelial cells [46] and endothelial lymphatic cells [47]. Infection of epithelial cells was concomitant with large lamellipodia projections (ruffles) similar to macropinocytosis. Likewise, *Mycobacterium* can induce formation of macropinosomes however; this does not depend on a bacterial secretion system, as the culture media in the absence of pathogen was sufficient to induce this process. Since nonviable bacteria fail to induce macropinocytosis in opposition to live bacteria, the most prominent candidate to induce ruffling is pointed as being secretory products actively produced by life bacilli. There are no requirements for bacteria to attach directly to the plasma membrane. In endothelial cells, scanning electron

dysentery-like illness similar to that caused by *S. flexneri* [45].

referred in most published data.

268 Cytoskeleton - Structure, Dynamics, Function and Disease

*S. flexneri* VirA protein [28, 44].

Coiling phagocytosis is an actin dependent endocytic event, morphologically accompanied by a typical pseudopodia that looks like whorls or wrapps around the bacteria in several turns (**Figure 1**). A definition of the phenomena is complex as it presents similarities to macropinocytosis and conventional phagocytosis: for the first due to the large pseudopodia; for the second due to cargo specific entrapment. In coiling phagocytosis, the single pseudopodia do not trap fluid droplets but enclose microbes; however, the multiple pseudopod whorls have largely self-apposed surfaces instead of those that are microbe-apposed surfaces. *Legionella pneumophila* and *Borrelia burgdorferi* the agents of Legionellosis and Lyme disease, respectively, use this form of endocytosis for establishment of the infection within macrophages. It was demonstrated that coiling phagocytosis is an active and selective process of the phagocytes, initially triggered by heat- and aldehyde-insensitive moieties of the microbial surface [48], suggesting that coiling and conventional phagocytosis are very closely related, most likely starting from the same phagocytosis-promoting receptor(s). The lack of difference between viable and killed microbes indicates that coiling phagocytosis is actively driven by the phagocytes and not by the microbes. This distinguishes coiling phagocytosis from nonclassical uptake mechanisms such as the induced phagocytosis or macropinocytosis. In this respect, the identification of granulocyte macrophage colony-stimulating factor (GM-CSF) and phorbol esters such as PMA as coiling-promoting substances may be a clue as to the regulatory mechanisms involved in coiling phagocytosis [48]. On the side of the phagocytes, coiling phagocytosis obviously is clearly a regulated mechanism, because the monocytes used it selectively for certain spirochetes, which is inconsistent with simply an accidental trapping of pericellular microbes.

In summary, deciphering the players that induce or prevent phagocytosis in one infection context may be used as strategies to clear pathogens in other context. It is an interesting observation that preinfection of cultured gastric cells with yersinia expressing Yop virulence factors that interfere with the same signaling events, impaired phagocytosis of *H. pylori*. This may be a potential starting strategy to fight gastric cancer due to this pathogen.

Define what receptors stimulate to induce a more bactericidal response of infected cells, how to control bacterial load that is internalized to induce apoptosis, as is the case of microRNAs that control WASP in tuberculosis context; how to neutralize factors that prevent Rho family of GTPases to modify actin in order to induce phagocytosis of extracellular pathogens, these are a few targets to explore deeply. Other relevant area to act is how to neutralize bacterial adhesins, secretion systems or their access to surface receptors as integrins to prevent epithelia invasion. It is imperative to decipher what are the virulence factors that mimics or induce growth factors that leads to induced macropinocytosis. In addition, it is important to find how to neutralize secretion systems that reorganize the actin cytoskeleton for macropinosome formation and therefore for pathogen invasion of epithelial and endothelial cells, important reservoirs of latent infections.

### **3. Acting on actin for the establishment of an intracellular niche**

In addition to particle binding and internalization, phagocytosis includes the process of phagosome maturation leading to pathogen destruction in the acidic hydrolytic environment of the phagolysosome. These events are important innate immune mechanisms. Indeed a consequence of phagosome maturation is the activation of the antigen presentation machinery. Macropinocytosis culminates in the appearance of a large vacuole that, indeed follows the fate of the phagosome. Some pathogens have evolved to establish sustained infection in professional phagocytes preventing phagosome maturation as is the case of *M. tuberculosis* and *S. enterica*. Other's diverts the endocytic pathway into a distinct vacuole more similar to the secretory pathway (e.g., *Legionella pneumophila* associates with the endoplasmic reticulum). By doing this, pathogens establish an intracellular niche were they survive, escape the immune bactericidal responses and have access to nutrients. Finally, a group of pathogens are able to escape the endocytic pathway by lysing the vacuole and move to the cytosol (e.g., *Mycobacterium marinum* within macrophages; *M. tuberculosis* within endothelial cells; *Shigella*, listeria within epithelial cells) **(Figure 1)**.

The material in endosomes or phagosomes that is destined for lysosome degradation by endocytosis or phagocytosis reaches this compartment by fusing with the organelle. Critical for this is the membrane composition of the correct repertoire of lipids, membrane-bound proteins, and also proteins that shuttle on and off membranes. The manipulation of the phagosomal membrane by pathogens may block the ability of fusion with lysosomes leading to a vacuole that may be trafficked apart from the endocytic route. In alternative, the vacuole may be arrested from maturation along the endocytic pathway by pathogen membrane manipulation leading to continuous transient fusion events with upper compartments.

Phagosome maturation is known to be influenced by the lipid species present on the outer and most likely inner membrane, and published studies have focused mostly on kinases that generates PIP, and PIP2, which binds actin nucleation proteins [49]. Additionally, the ability to nucleate actin leading to F-actin polymerization from phagosomal membranes was associated to the formation and availability of actin tracks for organelles to move towards the actinnucleating source, increasing vesicle trafficking, fusion events, and phagolysosome biogenesis **(Figure 1)** [50]. Identifying key roles for PIP and PIP2 opened the door for the analysis of several other lipids that interconnected with these phosphoinositides in the actin assembly process, as well as sphingolipids and fatty acids favouring phagosome maturation [11, 51]. Examples of F-Actin stimulatory factors includes the eicosanoide omega 6 arachadonic acid, ceramide and sphingosine-1-phosphate.

Several groups have explored the role of actin cytoskeleton during *Mycobacterium* late phases of phagocytosis. Pioneering work by de Chastellier and co-workers shows that *Mycobacterium*  avium a pathogen common in AIDS patients, disrupt the macrophage actin filament network highlighting here the target for the bacterium that allows sustained intracellular survival. It was demonstrated that in contrast to nonpathogenic mycobacteria, pathogenic *M. tuberculosis* prevents actin polymerization on phagosomal membranes [11, 52]. Therefore, the enrichment of *M. tuberculosis* phagosomal membranes with classes of lipids that leads to PIP2 was shown to induce F-actin tracks from the vacuole membrane. This is concomitant with an increase of fusion events, phagolysosome biogenesis and, consequently *M. tuberculosis* intracellular killing [11]. Drug-induced manipulation of the pathogen actin nucleation-induced blockade represents interesting alternative therapies for tuberculosis.

**3. Acting on actin for the establishment of an intracellular niche**

listeria within epithelial cells) **(Figure 1)**.

270 Cytoskeleton - Structure, Dynamics, Function and Disease

sphingosine-1-phosphate.

In addition to particle binding and internalization, phagocytosis includes the process of phagosome maturation leading to pathogen destruction in the acidic hydrolytic environment of the phagolysosome. These events are important innate immune mechanisms. Indeed a consequence of phagosome maturation is the activation of the antigen presentation machinery. Macropinocytosis culminates in the appearance of a large vacuole that, indeed follows the fate of the phagosome. Some pathogens have evolved to establish sustained infection in professional phagocytes preventing phagosome maturation as is the case of *M. tuberculosis* and *S. enterica*. Other's diverts the endocytic pathway into a distinct vacuole more similar to the secretory pathway (e.g., *Legionella pneumophila* associates with the endoplasmic reticulum). By doing this, pathogens establish an intracellular niche were they survive, escape the immune bactericidal responses and have access to nutrients. Finally, a group of pathogens are able to escape the endocytic pathway by lysing the vacuole and move to the cytosol (e.g., *Mycobacterium marinum* within macrophages; *M. tuberculosis* within endothelial cells; *Shigella*,

The material in endosomes or phagosomes that is destined for lysosome degradation by endocytosis or phagocytosis reaches this compartment by fusing with the organelle. Critical for this is the membrane composition of the correct repertoire of lipids, membrane-bound proteins, and also proteins that shuttle on and off membranes. The manipulation of the phagosomal membrane by pathogens may block the ability of fusion with lysosomes leading to a vacuole that may be trafficked apart from the endocytic route. In alternative, the vacuole may be arrested from maturation along the endocytic pathway by pathogen membrane manipula-

Phagosome maturation is known to be influenced by the lipid species present on the outer and most likely inner membrane, and published studies have focused mostly on kinases that generates PIP, and PIP2, which binds actin nucleation proteins [49]. Additionally, the ability to nucleate actin leading to F-actin polymerization from phagosomal membranes was associated to the formation and availability of actin tracks for organelles to move towards the actinnucleating source, increasing vesicle trafficking, fusion events, and phagolysosome biogenesis **(Figure 1)** [50]. Identifying key roles for PIP and PIP2 opened the door for the analysis of several other lipids that interconnected with these phosphoinositides in the actin assembly process, as well as sphingolipids and fatty acids favouring phagosome maturation [11, 51]. Examples of F-Actin stimulatory factors includes the eicosanoide omega 6 arachadonic acid, ceramide and

Several groups have explored the role of actin cytoskeleton during *Mycobacterium* late phases of phagocytosis. Pioneering work by de Chastellier and co-workers shows that *Mycobacterium*  avium a pathogen common in AIDS patients, disrupt the macrophage actin filament network highlighting here the target for the bacterium that allows sustained intracellular survival. It was demonstrated that in contrast to nonpathogenic mycobacteria, pathogenic *M. tuberculosis* prevents actin polymerization on phagosomal membranes [11, 52]. Therefore, the enrichment of *M. tuberculosis* phagosomal membranes with classes of lipids that leads to PIP2 was shown to induce F-actin tracks from the vacuole membrane. This is concomitant with an increase of

tion leading to continuous transient fusion events with upper compartments.

Another pathogen that blocks phagosome maturation is *Salmonella*. Several hours after bacterial uptake into different host cell types, *Salmonella* induces the formation of an F-actin meshwork around the *Salmonella*-containing vacuole (SCV), which is a modified phagocytic compartment. SCV integrity is closely linked to a surrounding meshwork of actin that in contrast to what happens during mycobacteria infection, acts as a barrier that prevents membrane contact and, therefore vacuole fusion with other endocytic organelles [53]. This process does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion. A second T3SS, the salmonella pathogenicity island 2 (SPI2), translocate effectors from the phagosomal membrane to the cytosol. The consequence of this event is the induced polymerization of actin around the SCV that will allow salmonella intravacuolar survival. The spv virulence locus will express the SpvB protein and ADP-ribosyl transferase that will promote actin depolymerisation in latter stages of infection. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of salmonella. Furthermore, after this treatment, bacteria were released into the host cell cytosol, whereas SPI-2 mutant bacteria remained within vacuoles [53]. In conclusion, while during *M. tuberculosis* infection actin assembly is prevented or F-actin is disrupted to allow the establishment of an intracellular niche, in the case of salmonella infection the generation of an F-actin induced mesh is required to maintain and position a vacuole that sustains bacterial growth.

### **4. Acting on actin for pathogen dissemination: actin-based motility of pathogens and innate immunity**

Early after host invasion some pathogens escape lysosomal destruction and antigen presentation by escaping into the cytosol. Thereafter, actin polymerization is manipulated by several cytosolic pathogens such as *L. monocytogenes*, *S. flexneri*, *Burkholderia pseudomallei*, *Rickettsia* spp., and *M. marinum*. These generate and use actin tails to move within and between cells.

When intracellular moving bacteria reaches the plasma membrane, they push out long protrusions that are taken up by neighboring cells, facilitating the infection to spread from epithelial cell to cell in the absence of immune surveillance. At the cell-to-cell cytoplasmic membranes sites, the cytosolic actin-based moving pathogens induce the formation of surface protrusions that force the internalization from the infected cell into noninfected neighbor cells. The process of engulfment is called paracytophagy and involves internalization of a double membrane containing pathogen: the inner from the donor cell and the outer from the recipient cell (**Figure 1**) [54, 55]. At this point the pathogen may escape again to cytosol to start a new infection process.

In the case of enterophatogenic *E. coli* EPEC it was found that some actin pedestal of the attached EPECs also translocate along the cell surface, reaching speeds of 0.007 μm/s allowing bacteria to spread between attached cells [34] (**Figure 1**). While this model shares similarities with the *Listeria* or *Shigella* systems, the main difference is the presence of a membrane between the pathogen and the cell cytoskeleton (**Figure 1**: as in the case of filopodia fishing compared to paracytophagy). The actin polymerization system Arp2/3 complex has been manipulated by several pathogens differently. Some mimics the Wiskott-Aldrich syndrome protein (WASP) family [56], while other's recruit WASP directly to activate Arp2/3 [57]. Examples of the first include the actA protein of listeria and RickA of riquetsia. For the second examples exist as is the case of IcsA of *S. flexneri* and nondetermined factors of *M. marinum* but dependent on the ESAT-6 secretion system 1 [57]. *M. marinum* is a water-borne bacterium that naturally infects fish and amphibians and is an opportunistic pathogen for humans causing tuberculosis while *Rickettsia conorii* belongs to the spotted fever group of *Rickettsia* species transmitted by ticks [55].

The actin-based motility of *B. pseudomallei* the causative agent of melioidosis occurs by a mechanism distinct to that used by other intracytoplasmic pathogens. In fact, the actin tails induced by this pathogen contains Arp2/3 components but it is not clear in the enrollment of the intracellular motility of *B. pseudomallei* [58]. The overexpression of Scar1 a cellular actin nucleating promoting factor that in the context of *S. flexneri*, *L. monocytogenes* and *R. conorii*, blocks actin tail formation and motility, during *B. pseudomallei* infection as no effect on actin-based motility [58].

The predominance of a membrane surrounding vacuole during the infection of most intracellular pathogens looks to be related to immune protection from the defensive mechanisms that exist in the cytosol. The arrival of a pathogen or their PAMPs to the cytosol could "wake up" several patrol mechanisms that include cytosolic PRRs. The sensing by cytosolic innate receptors leads to an inflammatory response by secretion of proinflammatory cytokines and chemokines or a interferon type I response that overall leads to antimicrobial response; the stress in the cytosol induce inflammasome assembly [59].

Therefore, the arrival of the pathogens in the cytosol establishes a bridge to the innate immune response by contact of the pathogen-associated molecular patterns (PAMPS) with PRRs, such as NLRPs (Nod like, similar to Toll like receptors- TLRs on cell membranes). Additionally, and by causing cytosol stress, PAMPS will activate (via PRRs) the inflammasome, a complex structure of proteins similar to the apoptosome [60]. Inflammasome assembly will lead to pro-Interleukin1β (pro-IL-1β) and pro-IL-18 inflammatory cytokine activation via caspase 1 and to the programmed cell death dependent on caspase 1, as it is pyroptosis and pyronecrosis [22]. This is a natural immune response in gut and respiratory epithelial cells but not in endothelial vascular and lymphatic cells that lakes these cytosolic receptors and constitutes important host niches for intracellular pathogen survival [33, 47].

Rickettsiae possess a tropism to endothelial cells, a tissue that usually serves as barrier to intravascuolar blood from surrounding tissues. This tropism leads to the endothelial cell injury associated with complications of the disease. RickA (mentioned previously in this chapter) is a protein present in the pathogenic species *R. conorii*, but absent in *Rickettsia thyphi* [56]. This absence is responsible for an erratic actin-based motility of *R. thyphi* leading to the hypothesis of existence of multiple actin-polymerization mechanisms in pathogenic rickettsia. A consequence of this erratic movement may be the delayed spread from cell to cell and continuous replication of thyphi species leading to bacterial overload and necrotic cell lysis [56]. For *R. conorii* paracytophagy cell-to-cell-spread is the common mechanism for pathogen dissemination [55].

Macrophages, in contrast to endothelial cells, possess NLRs and other PRRs families. During *M. tuberculosis* as well as for *M. marinum* infection phagolysosomal rupture and bacteria escape to the cytosol usually leads to necrotic cell death [61, 62]. The existence of a functional RD1 region expressing ESAT-6 is relevant for the activation of the inflammasome, the necrotic cell death and the secretion of proinflammatory cytokines IL-1β [21]. In endothelial cells, however, the tubercle bacilli survives [47].

The detection of cytosolic LPS, as a consequence of disruption of replication vacuoles harboring Gram-negative bacteria was shown to trigger the activation of murine caspase-11 that leads to the assembly of a noncanonical inflammasome [63]. Caspase-11 (Casp-4 in humans) is also crucial for clearance of bacteria that escape the vacuole, such as *Burkholderia*. In addition, detection of *sdhA* mutants of *Legionella* and *sifA* mutants of *Salmonella* activate caspase-11-dependent pyroptosis [63]. Detection of cytosolic pathogens thus leads to caspase-1- or caspase-11-mediated pyroptosis and restricts bacterial growth.

Another potent host defense mechanism that restricts intracellular pathogens is autophagy. Some intracellular bacteria cause the formation of ubiquitinated aggregates around either bacterial structures or replication vacuoles, and the autophagic machinery can recognize these. The process of bacterial clearance by selective autophagy is called xenophagy. *Listeria* moves within the host cytoplasm through actin-based motility, promoted by the bacterial ActA protein, which is important for avoiding recognition by autophagy [64]. In contrast to the ActA protein, the *Shigella* IcsA protein that also promotes actin-based motility from one pole of the bacterium binds to the autophagy protein Atg5 thus targeting the bacterium to a phagophore. *Shigella* uses two different mechanisms to escape the host autophagic response: first, it secretes IcsB, a protein that competitively binds to IcsA and prevents its recognition by Atg5 thus preventing LC3 recruitment and the process of autophagy [65].

All together these findings let us to postulate that important strategies to fight pathogens will pass by control their life cycle in the cytosol. Either addressing the linkage of actin tails to Arp2/3 or WASP proteins or neutralizing the bacteria actin nucleators to prevent motility and spread to neighbor cells; either to induce death of the infected cell by apoptosis, pyroptosis, or necrotic lysis; either by exposition of pathogen signatures that leads to xenophagy; altogether these are a few potential strategies to address in the future.

### **5. Concluding remarks**

compared to paracytophagy). The actin polymerization system Arp2/3 complex has been manipulated by several pathogens differently. Some mimics the Wiskott-Aldrich syndrome protein (WASP) family [56], while other's recruit WASP directly to activate Arp2/3 [57]. Examples of the first include the actA protein of listeria and RickA of riquetsia. For the second examples exist as is the case of IcsA of *S. flexneri* and nondetermined factors of *M. marinum* but dependent on the ESAT-6 secretion system 1 [57]. *M. marinum* is a water-borne bacterium that naturally infects fish and amphibians and is an opportunistic pathogen for humans causing tuberculosis while *Rickettsia conorii* belongs to the spotted fever group of *Rickettsia* species

The actin-based motility of *B. pseudomallei* the causative agent of melioidosis occurs by a mechanism distinct to that used by other intracytoplasmic pathogens. In fact, the actin tails induced by this pathogen contains Arp2/3 components but it is not clear in the enrollment of the intracellular motility of *B. pseudomallei* [58]. The overexpression of Scar1 a cellular actin nucleating promoting factor that in the context of *S. flexneri*, *L. monocytogenes* and *R. conorii*, blocks actin tail formation and motility, during *B. pseudomallei* infection as no effect on actin-based motility [58]. The predominance of a membrane surrounding vacuole during the infection of most intracellular pathogens looks to be related to immune protection from the defensive mechanisms that exist in the cytosol. The arrival of a pathogen or their PAMPs to the cytosol could "wake up" several patrol mechanisms that include cytosolic PRRs. The sensing by cytosolic innate receptors leads to an inflammatory response by secretion of proinflammatory cytokines and chemokines or a interferon type I response that overall leads to antimicrobial response; the

Therefore, the arrival of the pathogens in the cytosol establishes a bridge to the innate immune response by contact of the pathogen-associated molecular patterns (PAMPS) with PRRs, such as NLRPs (Nod like, similar to Toll like receptors- TLRs on cell membranes). Additionally, and by causing cytosol stress, PAMPS will activate (via PRRs) the inflammasome, a complex structure of proteins similar to the apoptosome [60]. Inflammasome assembly will lead to pro-Interleukin1β (pro-IL-1β) and pro-IL-18 inflammatory cytokine activation via caspase 1 and to the programmed cell death dependent on caspase 1, as it is pyroptosis and pyronecrosis [22]. This is a natural immune response in gut and respiratory epithelial cells but not in endothelial vascular and lymphatic cells that lakes these cytosolic receptors and constitutes

Rickettsiae possess a tropism to endothelial cells, a tissue that usually serves as barrier to intravascuolar blood from surrounding tissues. This tropism leads to the endothelial cell injury associated with complications of the disease. RickA (mentioned previously in this chapter) is a protein present in the pathogenic species *R. conorii*, but absent in *Rickettsia thyphi* [56]. This absence is responsible for an erratic actin-based motility of *R. thyphi* leading to the hypothesis of existence of multiple actin-polymerization mechanisms in pathogenic rickettsia. A consequence of this erratic movement may be the delayed spread from cell to cell and continuous replication of thyphi species leading to bacterial overload and necrotic cell lysis [56]. For *R. conorii* paracytophagy cell-to-cell-spread is the common mechanism for pathogen dis-

transmitted by ticks [55].

272 Cytoskeleton - Structure, Dynamics, Function and Disease

semination [55].

stress in the cytosol induce inflammasome assembly [59].

important host niches for intracellular pathogen survival [33, 47].

During evolution, higher eukaryotic organisms have developed epithelial barriers and phagocytic immune cells to resist and fight infections. The discovery of antibiotics in the early part of the last century led to predictions that bacterial infections would be kept under tight control via natural systems and treatment with drugs. But the capacity of bacteria to evade natural protective systems and rapidly develop resistance to antibiotics had led to the current situation of bacteria posing major health problems in both the developed and underdeveloped world. There is now a major requirement to find alternative treatments to fight bacterial pathogens. Over the years, various studies have elucidated the mechanisms by which bacterial PAMPs, adhesins, and secretion systems together with their translocated effectors target and alter the host actin dynamics. Targeting the host actin machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Studying the manipulation of host actin by pathogens has vastly improved our understanding of various basic cell biological processes in host cells while giving key insights into both bacterial pathogenesis and host innate immunity. Together this opens a new and exciting field of research with the objective of discovering new classes of antibiotics that directly or indirectly interfere with this actin-modulating mechanism.

### **Author details**

Elsa Anes

Address all correspondence to: eanes@ff.ulisboa.pt

Research Institute for Medicines iMed-ULisboa, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal

### **References**


[10] Mercanti V, Charette SJ, Bennett N, Ryckewaert J-J, Letourneur F, Cosson P. Selective membrane exclusion in phagocytic and macropinocytic cups. Journal of Cell Science 2006;119:4079–87.

adhesins, and secretion systems together with their translocated effectors target and alter the host actin dynamics. Targeting the host actin machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Studying the manipulation of host actin by pathogens has vastly improved our understanding of various basic cell biological processes in host cells while giving key insights into both bacterial pathogenesis and host innate immunity. Together this opens a new and exciting field of research with the objective of discovering new classes of antibiotics that directly or indirectly interfere with this

Research Institute for Medicines iMed-ULisboa, Faculty of Pharmacy, Universidade de Lisboa,

[1] Niedergang F, Chavrier P. Regulation of phagocytosis by Rho GTPases. Current Topics

[2] Cannon GJ, Swanson JA. The macrophage capacity for phagocytosis. Journal of Cell

[3] Swanson JA, Baer SC. Phagocytosis by zippers and triggers. Trends in Cell Biology

[4] Bornschlögl T. How filopodia pull: what we know about the mechanics and dynamics of

[5] Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annual Review

[6] Rabinovitch M. Professional and non-professional phagocytes: an introduction. Trends

[7] Yates RM, Russell DG. Phagosome maturation proceeds independently of stimulation of

[8] Yates RM, Hermetter A, Taylor GA, Russell DG. Macrophage activation downregulates

[9] Russell DG, VanderVen BC, Glennie S, Mwandumba H, Heyderman RS. The macrophage marches on its phagosome: dynamic assays of phagosome function. Nature

actin-modulating mechanism.

274 Cytoskeleton - Structure, Dynamics, Function and Disease

Science 1992;101:907–13.

1995;5:89–93.

Address all correspondence to: eanes@ff.ulisboa.pt

in Microbiology and Immunology 2005;291:43–60.

toll-like receptors 2 and 4. Immunity 2005;23:409–17.

the degradative capacity of the phagosome. Traffic 2007;8:241–50.

filopodia. Cytoskeleton 2013;70:590–603.

of Immunology 1999;17:593–623.

Reviews Immunology 2009;9:594–600.

in Cell Biology 1995;5:85–7.

**Author details**

Lisbon, Portugal

**References**

Elsa Anes


[39] Guiney DG, Lesnick M. Targeting of the actin cytoskeleton during infection by Salmonella strains. Clinical Immunology 2005;114:248–55.

[25] Miller H, Zhang J, Kuolee R, Patel GB, Chen W. Intestinal M cells: the fallible sentinels?

[26] Olson PD, Hunstad DA. Subversion of host innate immunity by uropathogenic

[27] Justice SS, Hung C, Theriot JA, Fletcher DA, Anderson GG, Footer MJ, et al. Differentiation and developmental pathways of uropathogenic *Escherichia coli* in urinary tract pathogenesis. Proceedings of the National Academy of Sciences of the United States of America

[28] Reis RSD, Horn F. Enteropathogenic *Escherichia coli*, *Samonella*, *Shigella* and *Yersinia*: cellular aspects of host-bacteria interactions in enteric diseases. Gut Pathogens 2010;2:8.

[29] Wong K-W, Isberg RR. Arf6 and phosphoinositol-4-phosphate-5-kinase activities permit bypass of the Rac1 requirement for beta1 integrin-mediated bacterial uptake. The

[30] Kwok T, Backert S, Schwarz H, Berger J, Meyer TF. Specific entry of *Helicobacter pylori* into cultured gastric epithelial cells via a zipper-like mechanism. Infection and Immunity

[31] Sinn HW, Elzey BD, Jensen RJ, Zhao X, Zhao W, Ratliff TL. The fibronectin attachment protein of bacillus Calmette-Guerin (BCG) mediates antitumor activity. Cancer

[32] Cossart P, Pizarro-Cerda J, Lecuit M. Invasion of mammalian cells by Listeria monocytogenes: functional mimicry to subvert cellular functions. Trends in Cell Biology

[33] Rengarajan M, Hayer A, Theriot JA. Endothelial cells use a formin-dependent phagocytosis-like process to internalize the bacterium listeria monocytogenes. PLoS Pathogens

[34] Dramsi S, Cossart P. Intracellular pathogens and the actin cytoskeleton. Annual Review

[35] Griffiss JM, Lammel CJ, Wang J, Dekker NP, Brooks GF. *Neisseria gonorrhoeae* coordinately uses pili and opa to activate hec-1-b cell microvilli, which causes engulfment of

[36] Lambotin M, Hoffmann I, Laran-Chich M-P, Nassif X, Couraud PO, Bourdoulous S. Invasion of endothelial cells by Neisseria meningitidis requires cortactin recruitment by a phosphoinositide-3-kinase/Rac1 signalling pathway triggered by the lipo-oligosaccha-

[38] BoseDasgupta S, Pieters J. Inflammatory stimuli reprogram macrophage phagocytosis to macropinocytosis for the rapid elimination of pathogens. PLoS Pathogens

[37] Kerr MC, Teasdale RD. Defining macropinocytosis. Traffic 2009;10:364–71.

World Journal of Gastroenterology 2007;13:1477–86.

Journal of Experimental Medicine 2003;198:603–14.

Immunology, Immunotherapy 2008;57:573–9.

of Cell and Developmental 1998;14:137–66.

ride. Journal of Cell Science 2005;118:3805–16.

the gonococci. Infection and Immunity 1999;67:3469–80.

*Escherichia coli*. Pathogens 2016;5(1):2.

276 Cytoskeleton - Structure, Dynamics, Function and Disease

2004;101:1333–8.

2002;70:2108–20.

2003;13:23–31.

2016;12:e1005603.

2014;10:e1003879.

