**2.2 Multicilia**

*Basic and Clinical Understanding of Microcirculation*

and primary cilia.

**2.1 Ciliogenesis**

**2. Cilia type and structure**

the vasculature, but literature on the subject is scarce [1].

overview of multicilia and their motion will also be covered.

centers (MTOC) or spindle poles for mitosis [12].

signaling cascades, which are often the result of abnormal cilia-regulated signaling pathways. There is a documented connection between cilia and NO in the vasculature, as well as an overlap between signaling pathways in other pathologies. It has been postulated that there is a connection between primary cilia and NO outside of

This chapter aims to explain cilia type, structure, and function, as well as ciliogenesis, nitric oxide signaling, and finally the interplay between nitric oxide

To understand what makes primary cilia unique, it is important to understand the differences between cilia form and function. Cilia are dynamic sensory organelles present in nearly every cell in every animal, as well as most protozoa. There are two classes of cilia; motile, which possess the dynein motor complexes needed to move, and nonmotile. Motile and nonmotile cilia both contain a 25 μm diameter cytoskeletal scaffold known as the axoneme [5]. The axoneme is comprised of hundreds of proteins and houses nine peripheral microtubule doublets. These doublets are made up of A and B tubules, and they either surround a central pair of microtubules (9 + 2 pattern), or do not (9 + 0 pattern) [5]. Some motile cilia contain a 9 + 2 pattern and exist in clusters on cells called multiciliated cells (MCCs) [6]. There is also a class of motile cilia that have a 9 + 0 structure and exist as solitary monocilia on cell surfaces. The presence or absence of the central pair leads to significant functional differences in the cilia. The 9 + 2 structure commonly moves in a wave-like motion to move fluid, and an example of this are the ependymal cilia. The 9 + 2 patterned cilia also move cerebral spinal fluid, while the 9 + 0 structured most commonly moves in a rotary or corkscrew motion, as seen in flagella, which is useful for propulsion [7, 8]. There is some debate on whether sperm tail flagella should be classified as motile monocilia; regardless, they also possess a similar axonemal structure [5, 6, 9–11]. Nonmotile cilia, known as primary cilia, have a 9 + 0 structure and exist as monocilia on the surface of cells. As primary cilia can be found on vascular endothelial cells, they will be the focus of this review, but a brief

Cilia formation is known as ciliogenesis. Ciliogenesis is correlated with cell division and occurs at the G1/G0 phase of the cell cycle. Reabsorption or disassembly of the cilium starts after cell cycle re-entry. In the first step of ciliogenesis, the centrosome travels to the cell surface, whereupon a basal body is formed by the mother centriole, and it nucleates the ciliary axoneme at the G1/G0 phase of the cell cycle [12]. This first process is regulated by distal appendage proteins, such as centrosomal protein 164 [13]. During the second step, the cilium elongates; this process is regulated by nuclear distribution gene E homolog 1 (Nde1), up until the cilium is matured [14]. The third step is cilia resorption, followed by axonemal shortening during cell cycle reentry. This third process is controlled by the Aurora A-HDAC6, Nek2-Kif24, and Plk1-Kif2A pathways [15]. In the fourth step, the basal body is released from the cilia, which frees the centrioles that act as microtubule organizing

Immotile cilia formation is impacted by the coordination of the assembly and disassembly equilibrium, the IFT system, and membrane trafficking. When the axoneme nucleates from the basal body, it contains a microtubule bundle contained

**50**

In vertebrates, MCCs are present in a wide variety of different tissue types. In mammals, ependymal MCCs line brain ventricles and the airway epithelium. Multicilia are produced by specialized cells for highly specialized functions. MCCs are typically defined as having more than two cilia on their surface, although this occurrence is not well documented or understood. Recently, MCCs have been observed in unicellular eukaryotes and protists, as well as many metazoans, and even in certain plant sperms [23–25]. MCCs result in the production of motile axonemes, with the only notable exception being mammalian olfactory cilia. These olfactory MCCs lack dynein arms and are considered immotile despite having a 9 + 2

#### **Figure 1.**

*Primary cilia structure. The axonemes of primary cilia are anchored from the basal body and encapsulated within the ciliary membrane. The ciliary membrane is one continuous extension of the plasma membrane. The basal body is composed of the mother and the daughter centrioles, as well as some transition fibers that anchor the basal body to the cell membrane. The ciliary membrane houses specific membrane and protein receptors, all of which facilitate proper cilia signaling (left panel). Primary cilia that are found on vascular endothelial cells are identifiable by an immunofluorescence technique with antibody against acetylated α-tubulin (green) labeling primary cilia, and pericentrin (red) labeling the centriole or basal body. The nucleus is counterstained with DAPI (blue) to label DNA (right panel). Left panel is adopted from Ref. [1].*

structure. This occurrence is indicative of MCCs being a solution to the need for local fluid flow, possibly due to their ability for hydrodynamic coupling [6, 26, 27].

Multicilia carry out their functions by beating, and the basic machinery and organization of cilia beating seems well conserved between eukaryotes, as well as between single motile cilium and multicilia. Some parameters, such as beat frequency, are under cellular control and are varied among cell types. In addition, only motile cilia and sperm flagella contain the dynein machinery needed to power axonemal beating during ATP hydrolysis [5, 28]. The ciliary beat cycle has two phases: the effective stroke, and the recovery stroke. The effective stroke is the initially bending from its upright position, while the recovery stroke sees it return to its original, unbent position [6, 29]. Ciliary motility is controlled by outer and inner axonemal dynein arms, which slide adjacent doublets in respect to one another. The sliding is mediated by protein bridges between doublets, and by the basal anchoring of the axoneme. As a result, cilia bend [6, 29].

The phenomenon metachrony occurs when cilia are organized in such a way that each cilium, in a two-dimensional array, will beat at the same frequency, but in a phase shifted manner. As a result, a traveling wave of ciliary action moves across the array, which propels fluids in a current. Even if each cilium in an array start off in synch, hydrodynamic forces between each cilium will nudge them back towards metachrony, possibly because in a metachronal array, the work each cilium must do is reduced, and more fluid is displaced. Because of this, multiciliation is thought to be a more evolutionarily efficient way to generate fluid flow [6, 30–32].

#### **2.3 Primary cilia**

Nearly all human cells house a single nonmotile cilium on their surface, and these primary cilia serve sensory and signaling purposes. The role of primary cilia function and formation on animal health and pathophysiology has only recently been brought to researcher's attentions and is even now not fully understood. A wealth of new information about the primary cilia has been discovered within the last few decades, shedding some light on the function of the previously thought vestigial organelle [33].

Primary cilia are part of various signaling pathways in vertebrates, and usually lack a microtubule central pair (9 + 0 axoneme), as well as the structures associated with the central doublet pair, such as the inner and outer dynein arms required for ciliary movement [34, 35]. Primary cilia can be found in, but are not limited to; endothelia, epithelia, and neurons. Although found in nearly every mammalian cell, notable exceptions include the intercalated cells of the kidney collecting ducts, red blood cells, hepatocytes, and MCCs [34].

Primary cilia function mainly as sensory hubs and are host to many different groups of mechanosensory proteins, chemosensory receptors, and ion channels. These translate extracellular stimuli into an intracellular biochemical signal, which cause different cellular responses. There are two current models that attempt to explain how primary cilia function; the compartment model, and the scaffold model. The compartment model states that the ciliary structure is essential for proper signaling, while the scaffold model states that IFT molecules must play a part in either signaling itself, or the acquisition of outside transduction intermediates [36, 37]. As stated in the ciliogenesis section, primary cilia form through IFT, which transports proteins along the microtubules of the axoneme. The axoneme acts like a scaffold for certain protein complexes, including kinesins and dyneins, which facilitate back-and-forth trafficking of cargo proteins along the length of the cilium. Along with their creation, IFT is also required for maintenance of the primary cilium, and possibly even their core functions [1, 10].

**53**

**3. Nitric oxide**

**4. Cilia and nitric oxide interplay**

*Primary Cilia are Sensory Hubs for Nitric Oxide Signaling*

The ciliary membrane is an extension of the cellular membrane where a host of proteins and receptors are housed due to the ciliary transition zone, which provides docking sites for molecular transport into and out of the cilioplasm (**Figure 1**) [38–40]. While there is no confirmed mechanism by which molecules enter and exit the cilia, several mechanisms have been proposed. One such mechanism is the active transport of vesicles from the Golgi apparatus to docking sites in the transition zone [41]. The vesicles are thought to interact with exocyst complexes, where they experience soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) mediated diffusion across the cilioplasm/cytoplasm barrier [37]. The BBSome, which resides in the basal body and is an octameric protein complex, is involved in the movement of transmembrane proteins to the ciliary membrane. BBSomes are known to recognize ciliary targeting sequences and will readily interact with molecules that are upstream of Rab8 activation. BBSomes are not thought to be required for any aspect of ciliogenesis; nevertheless, if BBSomes fail to deliver certain vital proteins to the cilia, the cilia may lose functionality [42–45]. Another proposed mechanism of molecule trafficking is the action of transmembrane proteins, of which some are associated with specific protein sequences that target cilia localization, such as the N-terminal RVxP sequence on polycystin-2 (PC-2) [42, 46]. Primary cilia serve many mechanosensory functions within the body. For example, proper kidney function depends on regulated fluid flow through the nephrons and collecting ducts, which controls the glomerular filtration rate [47]. This flow is sensed by the epithelial primary cilia present in the kidney. Fluid redirection by the primary cilia causes an increase in intracellular calcium. This calcium influx is also mediated by the PC1/2 ion channel complex, and both the mechanosensitive polycystin-1 (PC-1) membrane protein and the PC-2 channel localize to the primary cilia [48–50]. In renal cells, defects in this ion channel complex, along with

complete disruption of cilia formation, is known to result in PKD [51].

Nitric oxide is a signaling molecule involved in a wide array of cellular pathways; mainly, NO contributes to the normal functions of a variety of organ systems [52]. NO is highly reactive, and readily diffuses across cellular membranes. As a result, NO is found in many paracrine signaling pathways. NO is mainly synthesized from l-arginine, oxygen, and NADPH in a redox reaction, catalyzed by nitric oxide synthase (NOS) [53]. NOS has three isoforms, but only endothelial NOS (eNOS) and neuronal NOS (nNOS) are constantly and consistently expressed in cells. Both eNOS and nNOS are calcium dependent, while the other NOS isoform, cytokine inducible NOS (iNOS), is expressed by pro-inflammatory cytokines on an as needed basis [54]. iNOS and nNOS are both soluble enzymes that exist within the cytosol. eNOS, however, is found to localize to the plasma or Golgi body membranes. Because of its unique and wide cellular and subcellular distribution, NOS has many diverse functions throughout the entirety of the body [1, 54, 55].

Although primary cilia and NO have various independent roles within the body, especially in the vasculature, their functions often intersect and cooperate with each other. Most research on the interaction between endothelial primary cilia and nitric oxide focuses on vascular homeostasis, but their interactions extend into other areas [1]. However, this chapter will focus on signaling cascades that lead

*DOI: http://dx.doi.org/10.5772/intechopen.89680*

#### *Primary Cilia are Sensory Hubs for Nitric Oxide Signaling DOI: http://dx.doi.org/10.5772/intechopen.89680*

*Basic and Clinical Understanding of Microcirculation*

of the axoneme. As a result, cilia bend [6, 29].

red blood cells, hepatocytes, and MCCs [34].

primary cilium, and possibly even their core functions [1, 10].

**2.3 Primary cilia**

vestigial organelle [33].

structure. This occurrence is indicative of MCCs being a solution to the need for local

The phenomenon metachrony occurs when cilia are organized in such a way that each cilium, in a two-dimensional array, will beat at the same frequency, but in a phase shifted manner. As a result, a traveling wave of ciliary action moves across the array, which propels fluids in a current. Even if each cilium in an array start off in synch, hydrodynamic forces between each cilium will nudge them back towards metachrony, possibly because in a metachronal array, the work each cilium must do is reduced, and more fluid is displaced. Because of this, multiciliation is thought to

be a more evolutionarily efficient way to generate fluid flow [6, 30–32].

Nearly all human cells house a single nonmotile cilium on their surface, and these primary cilia serve sensory and signaling purposes. The role of primary cilia function and formation on animal health and pathophysiology has only recently been brought to researcher's attentions and is even now not fully understood. A wealth of new information about the primary cilia has been discovered within the last few decades, shedding some light on the function of the previously thought

Primary cilia are part of various signaling pathways in vertebrates, and usually lack a microtubule central pair (9 + 0 axoneme), as well as the structures associated with the central doublet pair, such as the inner and outer dynein arms required for ciliary movement [34, 35]. Primary cilia can be found in, but are not limited to; endothelia, epithelia, and neurons. Although found in nearly every mammalian cell, notable exceptions include the intercalated cells of the kidney collecting ducts,

Primary cilia function mainly as sensory hubs and are host to many different groups of mechanosensory proteins, chemosensory receptors, and ion channels. These translate extracellular stimuli into an intracellular biochemical signal, which cause different cellular responses. There are two current models that attempt to explain how primary cilia function; the compartment model, and the scaffold model. The compartment model states that the ciliary structure is essential for proper signaling, while the scaffold model states that IFT molecules must play a part in either signaling itself, or the acquisition of outside transduction intermediates [36, 37]. As stated in the ciliogenesis section, primary cilia form through IFT, which transports proteins along the microtubules of the axoneme. The axoneme acts like a scaffold for certain protein complexes, including kinesins and dyneins, which facilitate back-and-forth trafficking of cargo proteins along the length of the cilium. Along with their creation, IFT is also required for maintenance of the

fluid flow, possibly due to their ability for hydrodynamic coupling [6, 26, 27]. Multicilia carry out their functions by beating, and the basic machinery and organization of cilia beating seems well conserved between eukaryotes, as well as between single motile cilium and multicilia. Some parameters, such as beat frequency, are under cellular control and are varied among cell types. In addition, only motile cilia and sperm flagella contain the dynein machinery needed to power axonemal beating during ATP hydrolysis [5, 28]. The ciliary beat cycle has two phases: the effective stroke, and the recovery stroke. The effective stroke is the initially bending from its upright position, while the recovery stroke sees it return to its original, unbent position [6, 29]. Ciliary motility is controlled by outer and inner axonemal dynein arms, which slide adjacent doublets in respect to one another. The sliding is mediated by protein bridges between doublets, and by the basal anchoring

**52**

The ciliary membrane is an extension of the cellular membrane where a host of proteins and receptors are housed due to the ciliary transition zone, which provides docking sites for molecular transport into and out of the cilioplasm (**Figure 1**) [38–40]. While there is no confirmed mechanism by which molecules enter and exit the cilia, several mechanisms have been proposed. One such mechanism is the active transport of vesicles from the Golgi apparatus to docking sites in the transition zone [41]. The vesicles are thought to interact with exocyst complexes, where they experience soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) mediated diffusion across the cilioplasm/cytoplasm barrier [37]. The BBSome, which resides in the basal body and is an octameric protein complex, is involved in the movement of transmembrane proteins to the ciliary membrane. BBSomes are known to recognize ciliary targeting sequences and will readily interact with molecules that are upstream of Rab8 activation. BBSomes are not thought to be required for any aspect of ciliogenesis; nevertheless, if BBSomes fail to deliver certain vital proteins to the cilia, the cilia may lose functionality [42–45]. Another proposed mechanism of molecule trafficking is the action of transmembrane proteins, of which some are associated with specific protein sequences that target cilia localization, such as the N-terminal RVxP sequence on polycystin-2 (PC-2) [42, 46].

Primary cilia serve many mechanosensory functions within the body. For example, proper kidney function depends on regulated fluid flow through the nephrons and collecting ducts, which controls the glomerular filtration rate [47]. This flow is sensed by the epithelial primary cilia present in the kidney. Fluid redirection by the primary cilia causes an increase in intracellular calcium. This calcium influx is also mediated by the PC1/2 ion channel complex, and both the mechanosensitive polycystin-1 (PC-1) membrane protein and the PC-2 channel localize to the primary cilia [48–50]. In renal cells, defects in this ion channel complex, along with complete disruption of cilia formation, is known to result in PKD [51].
