**4. Cytoskeleton as the main responsible for displacement of** *Nostoc* **and** *Sporisorium scitamineum* **cells**

Directed cell migration is a physical process that involves dramatic modifications in cell shape and, generally, adhesion to the extracellular matrix [37]. Chemoattractive displacement is typically linked to the reorganization of actin filaments in cells, since polarization is the triggering event of cell migration [38]. A ligand on cell surface must activate a signaling pathway that leads to contraction/

divide, and this assures the recognition of new phycobiont produced after cell division by its fungal partner [22]. This should be interpreted as meaning that the polypeptide sequence of arginase (the lectin produced by the mycobiont) possesses an amino acid domain capable of stereochemically recognizing the remains of D-β-

the domain defined as site for the sugar binding and, for the same reason, the structure of the catalytic site for arginine. The ability to bind both cations together in order to develop their binding capacity to specific galactose ligands has been demonstrated for other lectins, such as that purified and crystallized from *Spatholobus*

According to this, Marx and Peveling [27] found that many cultured

lichen lectins, previously identified as a polygalactosylated urease.

phycobionts isolated from several lichen species bind to commercial lectins, including Con A and RCA. In addition, Fontaniella et al. [24] found that ConA is able to bind to the cell wall of algal cells recently isolated from *E. prunastri* and *X. parietina* thalli. This binding involves a ligand, probably a glycoprotein containing mannose, which has been isolated by affinity chromatography. Analysis by SDS-PAGE of the purified ligand revealed that it is a dimeric protein composed by two monomers of 54 and 48 kDa. This ligand shows to be different from the receptor for natural

The binding of sugarcane glycoproteins to their cell wall ligands in the bacterial

*Effect of secreted sugarcane glycoproteins on the cytoagglutination of* Xanthomonas albilineans*. (A) Bacterial*

*cells immediately after the contact with plant defense glycoproteins and (B) 3 h after the contact.*

endophyte *Gluconacetobacter diazotrophicus* [28] and in the bacterial pathogen *Xanthomonas albilineans* [29] results in cell recruitment (**Figure 4**) rather than a defense mechanism. Similar results on cytoagglutination were obtained using *Herbaspirillum rubrisubalbicans* treated with sugarcane glycoproteins of

This mode of binding a lectin to the polysaccharide moiety of its ligand by an affinity reaction equals, at the level of action mechanism, the secreted lichen arginases with other, well-known lectins from higher plants, such as concanavalin A (ConA) from *Canavalia ensiformis*, and ricin A (RCA) from *Ricinus communis*. Studies carried out by using α-methyl-mannose as a ligand suggest that the sugar forms seven hydrogen bonds with the peptide of ConA, four with –NH groups of Lys99, Tyr100, Arg228 and Lys229, and three with amino acids interacting with Ca2+, Asn 14 and Asp208 [23]. On the other hand, Fontaniella et al. [24] showed that a commercial ConA was able to develop arginase activity that increased more than 40 times in the presence of 1.7 mM Mn2+. Another similarity between ConA and fungal arginases lies in the fact that their activity as enzymes requires Mn2+**,** while their activity as lectin is dependent on Ca2+ and both cations, at the level of biological activity, are mutually excluding. The comparison between crystalline structures of ConA-containing or not Ca2+ suggests that the cation pulls from Tyr12, Asp208, and Arg228 to conform the site to bind the specific sugar [25]. It is probable that the binding of Ca2+ to the specific domain for the cation changes the tertiary structure of

galactose in β-1,3 bonds of the glycosylated, algal urease.

*Parasitology and Microbiology Research*

*parviflorus* [26].

**Figure 4.**

**8**

relaxation of the cytoskeleton. Then, cell polarizes and as a consequence, it moves to the chemoattractant source.

Moreover, many intracellular signaling molecules are involved in cell motility, such as MAPK cascades, lipid kinases, phospholipases, Ser/Thr and Tyr kinases, and scaffold proteins. Specially, GTP molecules play an essential role in both signal transduction and actin organization through Rho GTPases, which appear as the most important components of signaling cascade related to cell migration [38, 39].

Cell migration is the core to modern cell biology. However, progress has been hindered by experimental limitations and the complexity of the process. This has led to the popularity of *Dictyostelium discoideum*, with its experimentally friendly lifestyle and small, haploid genome, as a tool to dissect the pathways involved in migration. *Dictyostelium* has the potential to unlock many fundamental questions in the cell motility field [37]. Here, the involvement of the cytoskeleton in movement is analyzed in two very different systems, such as the compatible association fungus-alga in the lichen *Peltigera canina* and the plant-pathogen interaction between *Sporisorium scitamineum* and sugarcane plants.

#### **4.1 Cytoskeleton reorganization in** *Nostoc* **cells in response to the binding of a fungal lectin**

For symbiotic interaction, germinating hyphae of the mycobiont needs to meet a compatible photobiont cell, to recognize it, and to make contact [40]. When an isolated fungus and an isolated alga associate, the photobiont migrates toward its potential compatible partner, which implies that the cyanobiont would develop organelles to move toward the fungus. Displacement is particularly relevant in cyanolichens, in which the cyanobiont forms filaments inside the thallus, a segment of which can break off and migrate toward other locations [19]. The recognition process continues during thallus growth, since it is necessary that new generations of photobiont cells become involved in the association [9].

However, neither gliding nor blebbing can explain the invaginations observed by electron microscopy in one of the poles of *Nostoc* cells during the displacement [42], as can be seen in **Figure 6**. That is why the cytoskeleton has been revealed as responsible of migration of photobionts toward the fungus during a compatible

*receptor in* Nostoc *cell; , the signal transducing proteins; , organized photosynthetic apparatus; , disorganized photosynthetic apparatus; , the F-actin; , the anchorage proteins; , the*

*cyanobiont cell wall; and , the plasmatic membrane of cyanobiont.*

*On the right, recognition of fungus cyanobiont that leads to the cytoskeleton reorganization in* Nostoc *cells after the lectin produced by compatible exosymbiont binds to a specific receptor in cell wall. In compatible interactions, integrity of photosynthetic apparatus is maintained. On the left, non-compatible symbiotic interaction. In this case, there is no ligand-receptor specificity. Internalized fungal arginase increases putrescine cytoplasmic levels, which activates glucanase that breaks down the cell wall. In noncompatible interaction, a disorganization of photosynthetic apparatus occurs. Representing: , the fungal lectin; , compatible exosymbiont; , noncompatible exosymbiont; , the specific receptor in* Nostoc *cell; , unspecific*

*Role of the Cytoskeletal Actomyosin Complex in the Motility of Cyanobacteria and Fungal Spores*

*DOI: http://dx.doi.org/10.5772/intechopen.81299*

Some bacterial actin-like proteins or MreB have been already described in free-living cyanobacteria [46–47] but, contrary to that expected, chemotaxis assays of *Nostoc* displacement in presence of S-(3,4-dichlorobenzyl) isothiourea (A22), an inhibitor of MreB functionality, did not prevent the movement of cells toward the source of the lectin. Conversely, when *Nostoc* cells were incubated with the actin inhibitor phalloidin during chemoattraction assays, the drug inhibited chemotaxis by 50%. Also latrunculin A, which blocks actin polymerization, impedes *Nostoc* migration. The occurrence of F-actin fibers in *Nostoc* have also been found by immunocytochemical techniques associated with transmission

Interestingly, when phalloidin was combined with blebbistatin, an eukaryotic myosin II inhibitor, the negative effect on displacement increases (78%), suggesting

This means that, in the presence of compatible fungus, the binding of the lectin to its specific cell wall receptor would activate the signaling pathway that involves cytoskeleton reorganization. It must take place probably by means of GTPase activity, since the inhibition of chemotaxis produced by the combined action of phalloidin and blebbistatin is largely reversed by GTP and its analogs, GTP(γ)S and

that blebbistatin may target a molecular target related to chemotaxis in

interaction.

**Figure 5.**

electron microscopy.

cyanobacteria [42].

**11**

Lectins found in both prokaryotic and eukaryotic cells play an important role in cell interaction processes. Synthesis of fungal lectins with arginase activity and the occurrence of an algal receptor showing urease activity are absolutely required in the formation of lichen associations [41]. Urease on the algae cell wall acts as a ligand for fungal arginase, fixing it on the cell wall and preventing it to penetrate the cell [20]. So, lectins with arginase activity participate as recognizing proteins of compatible alga binding to a specific receptor on the cell wall. However, they penetrate and cause destruction of algae cells if the specific receptor does not exist [41]. This is the case of noncompatible interaction, as it is shown in **Figure 5**.

The search for the chemoattractant attracting photobiont cells leads to the discovery of the attractant properties of fungus lectin. In particular, chemotaxis of *Nostoc* cells from *P. canina* toward the lectin isolated from the same lichen species has been amply studied [42]. Many multicellular filamentous cyanobacteria move on solid surfaces by gliding, in absence of pili or fimbriae. It is the case of filaments and hormogonia of *Nostoc* [43]. This mechanism, which occurs in a parallel direction to the cell long axis, is associated with the production of polysaccharide slime and the attachment of the cell to a surface is needed. On the other hand, blebbing and the release of small vesicles by the cyanobacterial outer membrane have been observed in distantly related symbiotic and nonsymbiotic cyanobacteria such as *Nostoc*, the cyanobiont of *Peltigera* spp. [44]. Blebs are spherical membrane protrusions produced by contractions of the actomyosin cortex often considered to be a hallmark of apoptosis. However, blebs are also frequently observed during cytokinesis and migration in three-dimensional cultures and in vivo conditions [45].

*Role of the Cytoskeletal Actomyosin Complex in the Motility of Cyanobacteria and Fungal Spores DOI: http://dx.doi.org/10.5772/intechopen.81299*

#### **Figure 5.**

relaxation of the cytoskeleton. Then, cell polarizes and as a consequence, it moves to

Moreover, many intracellular signaling molecules are involved in cell motility, such as MAPK cascades, lipid kinases, phospholipases, Ser/Thr and Tyr kinases, and scaffold proteins. Specially, GTP molecules play an essential role in both signal transduction and actin organization through Rho GTPases, which appear as the most important components of signaling cascade related to cell migration [38, 39]. Cell migration is the core to modern cell biology. However, progress has been hindered by experimental limitations and the complexity of the process. This has led to the popularity of *Dictyostelium discoideum*, with its experimentally friendly lifestyle and small, haploid genome, as a tool to dissect the pathways involved in migration. *Dictyostelium* has the potential to unlock many fundamental questions in the cell motility field [37]. Here, the involvement of the cytoskeleton in movement is analyzed in two very different systems, such as the compatible association fungus-alga in the lichen *Peltigera canina* and the plant-pathogen interaction

the chemoattractant source.

*Parasitology and Microbiology Research*

**of a fungal lectin**

**10**

between *Sporisorium scitamineum* and sugarcane plants.

of photobiont cells become involved in the association [9].

**4.1 Cytoskeleton reorganization in** *Nostoc* **cells in response to the binding**

For symbiotic interaction, germinating hyphae of the mycobiont needs to meet a compatible photobiont cell, to recognize it, and to make contact [40]. When an isolated fungus and an isolated alga associate, the photobiont migrates toward its potential compatible partner, which implies that the cyanobiont would develop organelles to move toward the fungus. Displacement is particularly relevant in cyanolichens, in which the cyanobiont forms filaments inside the thallus, a segment of which can break off and migrate toward other locations [19]. The recognition process continues during thallus growth, since it is necessary that new generations

Lectins found in both prokaryotic and eukaryotic cells play an important role in cell interaction processes. Synthesis of fungal lectins with arginase activity and the occurrence of an algal receptor showing urease activity are absolutely required in the formation of lichen associations [41]. Urease on the algae cell wall acts as a ligand for fungal arginase, fixing it on the cell wall and preventing it to penetrate the cell [20]. So, lectins with arginase activity participate as recognizing proteins of compatible alga binding to a specific receptor on the cell wall. However, they penetrate and cause destruction of algae cells if the specific receptor does not exist [41]. This is the case of noncompatible interaction, as it is shown in **Figure 5**.

The search for the chemoattractant attracting photobiont cells leads to the discovery of the attractant properties of fungus lectin. In particular, chemotaxis of *Nostoc* cells from *P. canina* toward the lectin isolated from the same lichen species has been amply studied [42]. Many multicellular filamentous cyanobacteria move on solid surfaces by gliding, in absence of pili or fimbriae. It is the case of filaments and hormogonia of *Nostoc* [43]. This mechanism, which occurs in a parallel direction to the cell long axis, is associated with the production of polysaccharide slime and the attachment of the cell to a surface is needed. On the other hand, blebbing and the release of small vesicles by the cyanobacterial outer membrane have been observed in distantly related symbiotic and nonsymbiotic cyanobacteria such as *Nostoc*, the cyanobiont of *Peltigera* spp. [44]. Blebs are spherical membrane protrusions produced by contractions of the actomyosin cortex often considered to be a hallmark of apoptosis. However, blebs are also frequently observed during cytokinesis and migration in three-dimensional cultures and in vivo conditions [45].

*On the right, recognition of fungus cyanobiont that leads to the cytoskeleton reorganization in* Nostoc *cells after the lectin produced by compatible exosymbiont binds to a specific receptor in cell wall. In compatible interactions, integrity of photosynthetic apparatus is maintained. On the left, non-compatible symbiotic interaction. In this case, there is no ligand-receptor specificity. Internalized fungal arginase increases putrescine cytoplasmic levels, which activates glucanase that breaks down the cell wall. In noncompatible interaction, a disorganization of photosynthetic apparatus occurs. Representing: , the fungal lectin; , compatible*

*exosymbiont; , noncompatible exosymbiont; , the specific receptor in* Nostoc *cell; , unspecific*

*receptor in* Nostoc *cell; , the signal transducing proteins; , organized photosynthetic apparatus; , disorganized photosynthetic apparatus; , the F-actin; , the anchorage proteins; , the cyanobiont cell wall; and , the plasmatic membrane of cyanobiont.*

However, neither gliding nor blebbing can explain the invaginations observed by electron microscopy in one of the poles of *Nostoc* cells during the displacement [42], as can be seen in **Figure 6**. That is why the cytoskeleton has been revealed as responsible of migration of photobionts toward the fungus during a compatible interaction.

Some bacterial actin-like proteins or MreB have been already described in free-living cyanobacteria [46–47] but, contrary to that expected, chemotaxis assays of *Nostoc* displacement in presence of S-(3,4-dichlorobenzyl) isothiourea (A22), an inhibitor of MreB functionality, did not prevent the movement of cells toward the source of the lectin. Conversely, when *Nostoc* cells were incubated with the actin inhibitor phalloidin during chemoattraction assays, the drug inhibited chemotaxis by 50%. Also latrunculin A, which blocks actin polymerization, impedes *Nostoc* migration. The occurrence of F-actin fibers in *Nostoc* have also been found by immunocytochemical techniques associated with transmission electron microscopy.

Interestingly, when phalloidin was combined with blebbistatin, an eukaryotic myosin II inhibitor, the negative effect on displacement increases (78%), suggesting that blebbistatin may target a molecular target related to chemotaxis in cyanobacteria [42].

This means that, in the presence of compatible fungus, the binding of the lectin to its specific cell wall receptor would activate the signaling pathway that involves cytoskeleton reorganization. It must take place probably by means of GTPase activity, since the inhibition of chemotaxis produced by the combined action of phalloidin and blebbistatin is largely reversed by GTP and its analogs, GTP(γ)S and

**4.2 Cytoskeleton reorganization in** *S. scitamineum* **cells in response to the**

*Role of the Cytoskeletal Actomyosin Complex in the Motility of Cyanobacteria and Fungal Spores*

existence of these mechanisms and to study how they can be carried out.

Cytoskeleton reorganization in response to the binding of glycoproteins also occurs during *Sporisorium scitamineum*-sugarcane recognition. Moreover, displacement after recognition also results in cytoagglutination of smut teliospores in the same way that activation and chemotaxis of lichen photobionts induced by fungal lectins cause cell aggregation [15]. Interestingly, if glycoproteins are produced by sugarcane-resistant varieties, chemotaxis initially directed to plant invasion results

It has been proposed that at least three classes of glycoproteins exist in the mixture of sugarcane defensive glycoproteins produced by resistant cultivars: (i) a chemotactic glycoprotein, yet uncharacterized; (ii) a cytoagglutinating factor endowed with arginase activity, which also inhibits germination; and (iii) enzymatic proteins that mediate the breakdown of the teliospore cell wall. It has been demonstrated that agglutination of a lot of smut cells in a small region in contact with sugarcane glycoproteins confers resistance, since degradative activity also contained in these glycoproteins (β-1,3-, β-1,4-glucanase, and chitinase) can hydrolyze cell wall of many teliospores at the same time [15]. In this context, it must be

chemoattraction of cells. For this reason, it is very interesting to go into some depth about how the teliospores movement is stimulated by sugarcane signals. Currently, it has been found that the early chemoattractive effect is fully relevant to trigger a

Brand and Gow [53] summarize the knowledge on spore movement in plant-

submicroscopical contractions of helically arranged fibrils within the cell walls and the occurrence of motile appendages in zoospores. Other species of pathogenic fungi produce spores that are capable of gliding in the same way that it occurs for many species of cyanobacteria. Gliding is a form of cell movement that differs from crawling or swimming in which it does not rely on any obvious external organ or change in cell shape and it occurs only in the presence of a substrate [54].

Light and electron microscopy images showed the absence of motile external structures in smut teliospores. However, in the same way that it occurs for *Nostoc*, the invaginations observed during the cellular displacement suggested that cytoskeleton could be the responsible of spore displacement after the contact with sugarcane glycoproteins. Indeed, chemotactic movement of teliospores was strongly inhibited by phalloidin, latrunculin A, and blebbistatin, and the presence of actin and myosin in *S. scitamineum* teliospores has been revealed by immunohistochemi-

Teliospores do not need to develop lamellipodia in the direction of movement because they do not "crawl" on a substrate, but "swim" in solution because of the

pointed out that defensive agglutination depends necessarily on early

related to the minor capacity of these plants to defend themselves.

successful defensive response [52]. Lower levels of chemoattractant power exhibited by glycoproteins released by nonresistant cultivars have been directly

pathogen interactions. The two most frequently proposed mechanisms are

In the early stages of smut disease, spore germination occurs on the internode surface of host stalks, followed by the formation of appressoria, mainly on the inner scale of young buds and on the bases of emerging leaves [49]. Penetration into the plant meristem takes place between 6 and 36 h after fungal cells are deposited on the surface [50]. Since the pathogens normally use the opened stomata of sugarcane leaves to penetrate, it is easy to think that the teliospores deposited at random on the surface of a leaf, far from stomata, should develop a mechanism of displacement toward the way of entry [51]. For this rationale, it is important to demonstrate the

**binding of sugarcane glycoproteins**

*DOI: http://dx.doi.org/10.5772/intechopen.81299*

in a "suicide" mechanism.

cal techniques [52].

**13**

#### **Figure 6.**

*Scheme of movement of* Nostoc *cells during symbiotic interaction that explains how motility of lichen cyanobionts is due to contraction-relaxation episodes of the cytoskeleton. (1) Chemoattractant lectins released by fungus bind to specific receptors in photobiont cell walls. As a result, the transduction signal that implies cytoskeleton reorganization is activated. (2) Polar cell invaginations are produced by interaction of an ATPase with contractile ability, sensitive to blebbistatin, with F-actin cytoskeleton. (3) After this, depolymerization of F-actin is achieved at the opposite pole, repolymerization of which produces the cell advancement. Representing: , the fungal lectin; , the specific receptor in* Nostoc *cell; , the signal transducing proteins; , the actin monomeres; , the F-actin; , the contractile protein; , the anchorage proteins; , the cell wall; and , the plasmatic membrane. Ferritin-labelled F-actin can be seen in micrographs obtained by transmission electron microscopy (TEM).*

GDP(β)S, as well as by cyclic AMP [48]. On the contrary, when it is a noncompatible interaction, lectin penetrates into the cell, promoting putrescine synthesis. The diamine, which causes disorganization of photosynthetic apparatus, activates glucanase that breaks down the cell wall. Compatible and noncompatible interaction effect on cytoskeleton organization is schematized in **Figure 5**.

The absence of superficial elements (fimbriae, pili, or flagellum), related to cell movement, and the appearance of invaginated cells during or after movement, verified by scanning electron microscopy, support the hypothesis that the motility of lichen cyanobionts could be achieved by contraction-relaxation episodes of the cytoskeleton induced by fungal lectin [42]. However, other issues raised included (1) how cytoskeleton is reorganized during migration, (2) how is the mechanism of force generation of movement for cyanobacteria from *P. canina***,** and (3) how it can be related to the invaginations previously observed by electron microscopy. The answers to all of these questions have led to elaborate a proposal of migration mechanism in cyanobacteria.

**Figure 6** represents F-actin contraction/relaxing cycles in the *Nostoc* photobiont cells during migration following the lectin gradient. Firstly, binding of arginase molecules to cell wall receptors induces F-actin contraction by means of the activation of a signaling cascade where GTPases must play a main role. At the same time, contraction of filaments must be responsible for invagination appearance in one of the poles of the cell, which is followed by the actin depolarization at the opposite pole. This fact releases the tension from the actin-like cable bound to the membrane, and, finally, induces recovery of the spherical cell shape and movement of the cell [42].
