Cell and molecular biology of septins.

CHAPTER SEVEN

Cell and Molecular Biology of Septins Karen Y.Y. Fung*,†,1, Lu Dai*,‡,1, William S. Trimble*,†,‡,2 *Cell Biology Program, Hospital for Sick Children, Toronto, Canada † Department of Biochemistry, University of Toronto, Toronto, Canada ‡ Department of Physiology, University of Toronto, Toronto, Canada 1 Equal contribution 2 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Septin Discovery and General Properties 2.1 Septin domain structure 2.2 Polymerization 3. Functions of Septins 3.1 Diffusion barrier 3.2 Septins and microtubule stability 3.3 Septins as macromolecular scaffold 3.4 Septins in vesicle trafficking 3.5 Septins as regulator for cortical rigidity 4. Septins in Complex Biological Processes 4.1 Cell division in budding yeast 4.2 Mammalian cytokinesis 4.3 Septins and pathogen invasion 4.4 Cell polarity 4.5 Septins and primary cilia 4.6 Cell migration 5. Septin-Associated Diseases 5.1 Hereditary neuralgic amyotrophy 5.2 Male sterility 5.3 Cancer 6. Concluding Remarks References

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Abstract Septins are a family of GTP-binding proteins that assemble into cytoskeletal filaments. Unlike other cytoskeletal components, septins form ordered arrays of defined stoichiometry that can polymerize into long filaments and bundle laterally. Septins associate directly with membranes and have been implicated in providing membrane stability

International Review of Cell and Molecular Biology, Volume 310 ISSN 1937-6448 http://dx.doi.org/10.1016/B978-0-12-800180-6.00007-4

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and serving as diffusion barriers for membrane proteins. In addition, septins bind other proteins and have been shown to function as multimolecular scaffolds by recruiting components of signaling pathways. Remarkably, septins participate in a spectrum of cellular processes including cytokinesis, ciliogenesis, cell migration, polarity, and cell– pathogen interactions. Given their breadth of functions, it is not surprising that septin abnormalities have also been linked to human diseases. In this review, we discuss the current knowledge of septin structure, assembly and function, and discuss these in the context of human disease.

1. INTRODUCTION Septins were first discovered more than 40 years ago in screens for cell division defects in yeast, but research into their properties and functions has become increasingly intense in recent years due to a convergence of scientists from different fields. As the intensity of their study has increased, septins have been recognized as an important component of the cytoskeleton in a wide range of eukaryotic species. These filament-forming GTPases are unique from other components of the cytoskeleton in that they assemble into precisely organized heterooligomeric complexes that in turn are thought to polymerize into filaments. Unfortunately, little is currently known about what controls their assembly and disassembly. Septins also interact with both the microtubule and microfilament skeletons and can influence their dynamics. In addition, they bind to membranes and have been suggested to define membrane compartments by limiting the lateral diffusion of membrane proteins. Not surprisingly, septins are now appreciated to participate in wide range of cellular and organismal functions. Given this diversity of function, they have also been implicated in many diseases. In this review, we will provide an overview of the current understanding of how septins assemble into filaments, the types of general properties associated with these filaments, and how they regulate complex biological processes with an emphasis on mammalian systems. In addition, we will present examples where septins have been implicated in disease and discuss emerging concepts and future directions within the field.

2. SEPTIN DISCOVERY AND GENERAL PROPERTIES The septin family of filament-forming GTPases was first discovered in Saccharomyces cerevisiae in temperature-sensitive screens aimed at identifying

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genes involved in cell division (Hartwell, 1971). Four mutants, Cdc3, Cdc10, Cdc11, and Cdc12, were identified that all gave rise to a similar failure in yeast cytokinesis. Electron and immunofluorescence microscopy performed in budding yeast further revealed the localization of septins as a filamentous collar encircling the bud neck (Byers and Goetsch, 1976a,b; Haarer and Pringle, 1987) where the septum, or the cell wall separating the mother and daughter cell, is found, leading them to be named “septins” (Pringle, 2008). Phylogenetic analysis of septins shows that they are found in fungi, animals, protists but not in plants (Nishihama et al., 2011; Pan et al., 2007). Additionally, the number of septins present varies between organisms (Kinoshita, 2003; Nguyen et al., 2000). For example, Caenorhabditis elegans have 2 septin members (Unc-59 and Unc-61), Saccharomyces cerevisiae have 7 members (Cdc10, Cdc3, Cdc11, Cdc12, Shs1, Spr3, and Spr28), Drosophila melanogaster have 5 members (Pnut, Sep1, Sep2, Sep4, and Sep5), while humans have 13 members (septins 1–12 and 14, hereafter designated SEPT1–SEPT14). In S. cerevisiae, Cdc3, Cdc10, Cdc11, Cdc12, and Shs1 are expressed during vegetative growth, while Spr3 and Spr28 are sporulation specific. In mammals, many of the septins are expressed in tissue specific patterns. The number of mammalian septins is further expanded by the expression of isoforms that result from alternative promoters and transcript splicing of the septin genes. An extreme case of highly complex alternative splicing occurs in SEPT9 where there are potentially 15 different isoforms that could result from shuffling of exons located at the N- and C-termini (McIlhatton et al., 2001). While the function of the different isoforms is not known, work performed by Estey et al. suggests that not all SEPT9 isoforms serve the same function (Estey et al., 2010). Irregardless, they undoubtedly increase the diversity of septin filaments and may alter their functions in different tissues and cell types.

2.1. Septin domain structure Septins are P-loop containing GTP-binding proteins whose general structural features are shown in Fig. 7.1. Both the N-terminus and C-terminus are variable between septin family members. In many species, septins have an N-terminal polybasic region that has been shown to bind phosphoinositides (Casamayor and Snyder, 2003; Zhang et al., 1999). Downstream of this is the GTP-binding domain, which ends with the Septin Unique Element that

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Septin unique element

Variable N-terminus

N

Poly basic NC–NC interface

GTP-binding domain

G–G interface

Variable C-terminus

Coiled coil

C

NC–NC interface

Figure 7.1 Basic features of septins. The different domains are illustrated, while the NC– NC and G–G interface that are involved in septin–septin interaction are outlined in red.

distinguishes septins from other members of the P-loop containing GTPase family (Pan et al., 2007; Steels et al., 2007; Versele et al., 2004). The variable C-terminus following the GTPase domain may contain zero, one, two, or three coiled-coil domains. It has been suggested that these may either be necessary for interactions with other septins or other substrates (Casamayor and Snyder, 2003; Versele and Thorner, 2005). Mammalian septin members can be categorized into four subgroups based on sequence similarity and the number of coiled-coil domains present. The four subgroups are not orthologously related to the septins expressed in vegetatively growing S. cerevisiae (Pan et al., 2007) but they do share structural similarity (Versele and Thorner, 2005). The mammalian subgroups and their yeast equivalents are shown in Table 7.1. The GTP-binding domain can bind to and hydrolyze GTP (Field et al., 1996; Mendoza et al., 2002; Sheffield et al., 2003; Versele and Thorner, 2004), although both GTP hydrolysis and exchange occur very slowly and these rates differ among different septins (Farkasovsky et al., 2005; Field et al., 1996; Huang et al., 2006; Mendoza et al., 2002; Sheffield et al., 2003; Sirajuddin et al., 2009; Vrabioiu and Mitchison, 2006). The purpose of GTP hydrolysis remains unclear although septin mutants that are not able to bind GTP showed altered formation, appearance, subcellular localization and/or function (Casamayor and Snyder, 2003; Hanai et al., 2004; Kinoshita et al., 1997; Nagaraj et al., 2008; Robertson et al., 2004; Steels et al., 2007; Vega and Hsu, 2003). However, SEPT6 family members lack a conserved threonine residue implicated in GTP hydrolysis (Sirajuddin et al., 2009). It has recently been suggested that GTP hydrolysis contributes to the assembly of septin complexes, which will be discussed further below.

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Table 7.1 Categorization of mammalian septin family members along with their structural feature and their yeast equivalents Septin group Members Features Yeast equivalent

Two coiled-coil domains

Cdc11p

Septin 3 Septin 9 Septin 12

No coiled-coil domain

Cdc10p

Septin 6

Septin Septin Septin Septin Septin

6 8 10 11 14

One coiled-coil domain

Cdc3p

Septin 7

Septin 7 Septin 13a

One coiled-coil domain

Cdc12p

Septin 2

Septin Septin Septin Septin

Septin 3

a

1 2 4 5

A pseudogene.

2.2. Polymerization 2.2.1 Septin–septin interactions: The septin complex From early in their discovery, septins were thought to function as complexes rather than single protein entities. The first biochemical characterization of septins from Drosophila revealed that they existed in a hexameric complex composed of two copies each of Pnut, Sep1, and Sep2 (Field et al., 1996; Oegema et al., 1998). Similarly, the yeast septin complex appears to be an octamer containing stoichiometric ratios of Cdc3, Cdc10, and Cdc12, with substoichiometric levels of Cdc11 and Shs1 (Frazier et al., 1998; Mortensen et al., 2002), which are thought to compete for the terminal positions in the octamer (Bertin et al., 2008; Garcia et al., 2011). Similar compositions were seen with septin complexes isolated from Candida albicans (Kaneko et al., 2004). Since different organisms express different numbers of septins, it is not surprising that they produce septin heteromers of different sizes. In the case of C. elegans, which has only two septins, a heterotetramer with two copies of each septin was observed ( John et al., 2007). While there is ample evidence that septins function within complexes, there is currently no evidence that they can or do function outside of a complex. As mammals have 13 genes encoding septins, there exists the possibility of very large or complicated complex structures. The first septin preparations

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isolated from rat brain contained at least five septins and their stoichiometry was not clear (Hsu et al., 1998). Mass spectrometry analysis of immunoisolated septins from the brain identified as many as eight different septins present at varying levels (Kinoshita et al., 2002). In contrast, biochemical isolation of brain septins led to the purification of a complex of just three septins, which was predicted to contain stoichiometric amounts of SEPT3, SEPT5, and SEPT7 (Lukoyanova et al., 2008). Human septin complexes isolated from HeLa cells are able to associate in an octameric complex (Kim et al., 2011; Sellin et al., 2011b) although they are also found as hexamers. Since mammalian septins can be grouped into four related subgroups or families, it is tempting to speculate that septin complexes are comprised of a combination of members of each family. However, it is not known if all septin complexes require members of each family or if all complexes have defined stoichiometry. Septins isolated from tissues with many cell types, such as the brain, may exist in distinct complexes in each cell type, resulting in stoichiometries that represent the averages of the mixture of complexes isolated. Extensive immunoblotting studies have revealed that while at least 10 different septins can be detected in brain tissue, pure cultures of primary hippocampal neurons express only five, SEPT3, SEPT5, SEPT6, SEPT7, and SEPT11 (Tsang et al., 2011), all of which were found to coimmunoprecipitate. Clearly the issue of complex size and stoichiometry remains a complicated one that requires further study. A major advance in our understanding of the septin complex was achieved when it was determined that septin assembly is highly ordered. The best characterized endogenous septin complex is that of S. cerevisiae. Electron microscopy (EM) studies revealed that this complex exists as a rod-like structure consisting of septins Cdc3, Cdc10, Cdc11, and Cdc12 (Frazier et al., 1998). By tagging individual septins with bulky domains or antibodies in the EM preparations, it was determined that this rod consists of two tetramers positioned in mirror symmetry in the following order: Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 (Bertin et al., 2008). The molecular structure of the septin complex was solved through X-ray crystallography of a recombinant complex of mammalian SEPT2, SEPT6, and SEPT7 which form a hexamer when expressed in E. coli (Sirajuddin et al., 2007). Like the yeast complex, the crystal structure revealed that the mammalian septins also exhibited mirror symmetry resulting in a nonpolar rod with the order: SEPT7–SEPT6–SEPT2–SEPT2–SEPT6–SEPT7 (Sirajuddin et al., 2007). As illustrated in Fig. 7.2A, the crystal structure also revealed the contact points between the septins in the complex. The septin

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A

Septin 6 + 7 coiled coil

Septin 6 + 7 coiled coil SEPT7

SEPT6

SEPT2

G–G NC–NC

SEPT2

SEPT6

G–G

SEPT7

NC–NC

NC–NC

B G–G interphase

NC–NC interphase

9 7

6 2

2 6

7 9

Figure 7.2 Structure of the septin complex. (A) Surface representation of the mammalian septin heterohexamer as solved through X-ray crystallography. Indicated above this representation is the type of interface between each septin subunit (G–G stands for G–G interface, while NC–NC represents NC–NC interface. (B) Cartoon illustration of the order of the septin heterooctamer where each jellybean represents a septin. Image (A) is the crystal structure obtained from the Protein Data Bank - structure 2QAG (Sirajuddin et al., 2007) visualized through the program Chimera (Pettersen et al., 2004). Image (B) is the cartoon illustration of the septin hetero-octamer where each jelly bean represents a septin.

complex consisted of alternating face-to-face interactions between the GTP-binding domain (referred to as the G-G interface) and back-to-back interactions at a surface comprised by the N and C portions (termed the NC-NC interface) of the protein. The GTP binding domain of SEPT2SEPT6 and SEPT7-SEPT9 and the N- and C- termini of SEPT2-SEPT2 and SEPT6-SEPT7 are associated (Sirajuddin et al., 2007). The N- and C-termini were not resolved in the crystal but the C-termini projected orthogonally to the hexamer axis (Sirajuddin et al., 2007). Since the C-terminal coiled-coils of SEPT6 and SEPT7 have been shown to directly interact with each other (Low and Macara, 2006; Shinoda et al., 2010), they are predicted to aid in septin–septin interactions.

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Mammalian septins were also found to have an octameric organization as determined by biochemical means (Kim et al., 2011; Sellin et al., 2011b). The septin complex consisted of SEPT2, SEPT6, SEPT7, SEPT9, and SEPT11, as determined through immunoprecipitation experiments in HeLa cells (Estey et al., 2010; Sellin et al., 2011b; Surka et al., 2002). Sedimentation analysis revealed that the average septin complex was consistent with an octamer, and depletion of SEPT9 resulted in oligomers with a lower average mass (Sellin et al., 2011b). Since this mass is comparable to that of the SEPT2–SEPT6–SEPT7 hexamer, it suggests that SEPT9 also participates in the septin complex that is larger than a hexamer (Sellin et al., 2011b). The order of the subunits within the complex was determined by coexpressing wild type or mutant forms of SEPT2, SEPT6, SEPT7, and SEPT9 where the NC-NC and G-G interfaces were mutated to prevent their dimerization. Additionally, one septin was tagged with His6 and a second with maltose-binding protein. By using tandem affinity purification, the sequential order of the octamer was determined to be SEPT9–SEPT7– SEPT6–SEPT2–SEPT2–SEPT6–SEPT7–SEPT9 as shown in Fig. 7.2B (Kim et al., 2011). In the studies performed by Sellin et al., it was also found that SEPT6 and SEPT11 were interchangeable in the septin complex. Examination of lysates from K562 cells transfected with a C-terminally Flag tagged SEPT6 (SEPT6–FLAG) revealed a selective degradation of endogenous SEPT6 and SEPT11 only (Sellin et al., 2011b). Sedimentation analysis showed that the size of the SEPT6–FLAG containing septin complex was indistinguishable from the endogenous septin complex (Sellin et al., 2011b). Altogether, these results suggest that the overexpressed SEPT6–FLAG competed with endogenous SEPT6 and 11 for a specific position in the septin heteromer. It was later found that septin subgroup members are not interchangeable within preassembled septin complexes (Sellin et al., 2011a), which appear to be stable. The mechanism of octamer assembly was investigated in a recent study by Kim et al. that suggests a role for GTP hydrolysis. Briefly, an N-terminally truncated SEPT2 (SEPT2D15) was found to homooligomerize and form homomeric filaments. This mutant was then used as a model to study septin–septin interaction at the G–G and NC–NC interfaces. SEPT2D15 mutants were made that disrupted the G–G interface (G mutant) or NC– NC interface (NC mutant). Immunofluorescence and immunoprecipitation experiments showed that the NC mutants were able to disrupt SEPT2D15 filament formation through interaction with nonmutated SEPT2D15 while

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G mutants were not able to do so. Coimmunoprecipitation of two differently tagged SEPT2D15 NC mutants was possible, but two differently tagged G mutants were not, indicating that the interaction at the G-G interface is the dominant one. Indeed, when any pair of mammalian septins was coexpressed in HeLa cells, all combinations were found to coimmunoprecipitate by a G-G interface interaction except for the SEPT6–SEPT6 pair. This finding was unexpected as the solved crystal structure showed that some of these combinations normally interact via their NC-NC interface. However, those pairs that normally formed G-G interface interactions bound to each other with the highest affinity (Kim et al., 2011). Altogether, these data suggest that the affinity of the G–G domain interaction is much stronger than the NC–NC interaction which leads to the hypothesis that there is a sequential order of interaction as follows: first, interactions would be between the pairs of septins that interact canonically at the G-G interface (as predicted by the crystal structure). This interaction would then trigger GTP hydrolysis leading to a change in the G-G and NC-NC interfaces (Sirajuddin et al., 2009) that would facilitate the interaction of dimers with each other and ultimately result in the formation of the octamer. Intriguingly, the mammalian septin family containing SEPT6 does not hydrolyze GTP and lacks the threonine in the switch I region (Thr78), the equivalent threonine that is also missing in Cdc3 and Cdc11 in yeast (Sirajuddin et al., 2009). GTP hydrolysis was found to be nonessential in these genes (Sirajuddin et al., 2009). This had led to the suggestion that the presence of a GTP moiety might stabilize the dimer interface at the core of the septin complex (Sirajuddin et al., 2009). Although much work has been done to uncover how septins behave with each other in vivo, much remains to be determined. For example, the range of different septin complexes that can be formed and how easily different members of the same subgroup can replace each other is not known, nor is the specific contribution of alternatively spliced isoforms. To date, it has not been possible to develop a purely in vitro octamer assembly system but such methods would help to address these issues. In any case, although septin complexes from different organisms vary in composition and size, a common theme is the symmetric, nonpolar organization of septin complexes. 2.2.2 High-order structures The septin heterooligomeric complexes described above are thought to be the building blocks of more complex septin ultrastructures. Given the rodshaped nature of the septin oligomer, filaments are likely formed by the

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joining of each oligomer end-on-end, while bundles can also be formed from a lateral interaction between the oligomers; both structures are illustrated in Fig. 7.3. Bundled filaments can then form even higher order structures such as rings and gauzes. These are illustrated in Fig. 7.3 and described in more detail below. 2.2.2.1 Filaments

Evidence that septins can form filaments in vivo stems largely from EM studies. Initial EM studies on yeast showed the presence of a series continuous filaments at the mother–bud neck (Byers and Goetsch, 1976a) that were lost in Cdc3, Cdc10, Cdc11, and Cdc12 mutants but not in other Cdc mutants following a shift to the nonpermissive temperature (Byers and Goetsch, 1976b; Pringle, 2008). Subsequently, yeast spheroplasts, where the cell wall is removed through enzymatic digestion, were shown to have septin filaments in ring and gauze-like structures (Rodal et al., 2005). Additionally, septin filaments were seen by EM in A. gossypii after treatment with forchlorfenuron, a plant growth regulator thought to stabilize septin filaments (DeMay et al., 2011). These septins were also seen to bundle together laterally to form thicker filaments with similar widths and periodicity as the filaments that associated with the membrane (DeMay et al., 2011). Most recently, the appearance of a threedimensional network of septin filaments was observed at the mother–bud junction by electron tomography, revealing that septin filaments ran both perpendicular and parallel with the mother–bud axis (Bertin et al., 2012). Septins can also be induced to polymerize into filaments in vitro. Immunoaffinity isolated septin complexes purified from yeast (Frazier ...

...

... ... ...

Gauze

Ring

... ... ...

Filament

Figure 7.3 High-order structures of septins. Illustration of how septins can form filaments through end-on-end connections of multiple septin oligomers. This can then be bundled to form thicker filaments or alternately overlaid to form gauzes. Septin rings have also been observed yet the orientation of septin oligomers in the ring (parallel or perpendicular to the ring axis) remains to be determined.

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et al., 1998), and Drosophila (Field et al., 1996) were shown to polymerize into paired filaments under low salt conditions as determined by EM. Additionally, recombinant yeast septin octamers under low salt conditions formed long paired filaments with periodic densities connecting the two filaments (similar to railroad tracks) (Bertin et al., 2008). These periodic densities were proposed to result from the interaction of the coiled-coil projections orthogonal to the septin complex. Prolonged incubation of septin complexes under low salt conditions led to lateral bundling of the paired filaments (Bertin et al., 2008). Moreover, polymerization was significantly enhanced by the presence of lipid monolayers containing phosphatidylinositol-4,5-bisphosphate (PIP2) and occurred under conditions where filaments did not form in solution, such as in the presence of high salt or the absence of Cdc11. The presence of phosphatidylinositol-3,4,5-trisphosphate in the monolayer also supported lateral filament bundling (Bertin et al., 2010). In the case of mammalian cells, septins colocalize with actin or tubulin polymers (Kinoshita et al., 1997; Nagata et al., 2003; Surka et al., 2002), making it more difficult to determine if they are filaments themselves or merely complexes associated with a filamentous structure. In fibroblasts, the filamentous appearance of endogenous septins was lost when actin organization was disrupted by the treatment with cytochalasin D or latrunculin B (Kinoshita et al., 2002; Xie et al., 1999). However, this treatment induces the formation of rings that lack actin (Kinoshita et al., 2002; Xie et al., 1999), suggesting that the actin-associated septins may have been filaments held straight by their association with actin, but that collapsed into rings in its absence. Direct binding of septins with myosin IIa ( Joo et al., 2007) may explain how septin filaments could be associated with actin in linear structures. Recombinant mammalian SEPT2, SEPT6, and SEPT7 coexpressed in bacteria can be induced to polymerize into long filaments in vitro (Kinoshita et al., 2002; Low and Macara, 2006) as can SEPT2 alone (Huang et al., 2006; Mendoza et al., 2002) and these long filaments can also bundle into thick structures. 2.2.2.2 Regulation of filament formation

The mechanism by which basic septin filaments bundle to form these large fibrous structures is unknown but certain regulators have been implicated. One class of regulator is the family of proteins called Borgs, which are able to directly interact with septins through their BD3 domains ( Joberty et al., 2001). Specifically, it has been found through in vitro pulldown assays that Borg3 binds to the SEPT6–SEPT7 heterodimer at the interface of their

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coiled-coil domains ( Joberty et al., 2001; Sheffield et al., 2003). Overexpression of GFP-tagged Borg3 resulted in septin fibers that were longer and thicker than those in GFP-transfected cells ( Joberty et al., 2001). Borgs were first identified as effectors of the Rho family GTPase Cdc42 and the Borg–septin interaction was found to be inhibited by constitutively active Cdc42 ( Joberty et al., 2001). Moreover, overexpression of constitutively active Cdc42 led to a loss of septin filaments and septins were mislocalization to the perinuclear region ( Joberty et al., 2001). These results have been interpreted to mean that Borgs control septin organization, and that Cdc42–GTP inhibits this function of Borgs. In addition to being regulated by another party, septin polymerization may also be a self-regulated. This is suggested by the discovery that a single amino acid (Q4) in SEPT2 prevents this protein from self-assembling into filaments. Mutation of this residue to alanine, or deletion of the first 15 amino acids results in robust SEPT2 filaments in several cell lines (Kim et al., 2012). This homotypic polymerization was dependent on both the NC–NC and G–G interfaces as well as the coiled-coil domain since several mutations (F20A, V27D (NC-NC interface), W260A, H270D (G-G interface), and C-terminal truncation) prevented the formation of SEPT2 filaments (Kim et al., 2012). Previous studies have shown that a single septin could polymerize into long filaments (Huang et al., 2006; Mendoza et al., 2002; Nagata et al., 2003; Schmidt and Nichols, 2004) and one study demonstrated that this could be controlled by GTP binding (Mendoza et al., 2002). Additional studies are required to fully understand how septin polymerization is controlled by intrinsic and extrinsic factors. 2.2.2.3 Rings

Septins were long known to form a ring-like structure at the mother–bud neck in yeast, but it was not known if this ring-like pattern was a result of associating with the curved membrane, or if septins themselves had some intrinsic curvature resulting in ring-like structures. In vitro studies using recombinant yeast have raised the possibility that curvature may be imparted by the specific septins found in the complex. These studies showed that the yeast septin Shs1, which is frequently found in substoichiometric ratios in purified septin complexes and is thought to substitute for Cdc11 at the ends of septin complexes, can alter septin ultrastructure in vitro. Shs1 was considered to be a nonessential gene as Shs1 deletions are viable. Interestingly, however, when septin complexes containing Cdc11 were incubated in

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low salt, long linear filaments formed, but when Shs1 was substituted at the ends of the complexes, the ocatmeric units assembled laterally into a curved structure that resolved into rings (Garcia et al., 2011). Further work studying the fungus Ashbya gossypii Shs1 showed that the removal of the C-terminal coiled-coil domain led to septin rings of greater diameter, suggesting a role of the C-terminus in limiting the size of Shs1 containing rings (Meseroll et al., 2012). The ability of septins to assemble into ring-like structures was first noted in mammalian cells following actin filament disruption by cytochalasin D (Kinoshita et al., 2002). Under these circumstances septins reorganize into uniform ring structures with outer diameters of 0.66 0.08 mm (Kinoshita et al., 2002). Time lapse microscopy of GFP–SEPT6 in the Chinese hamster ovary (CHO) cell line with cytochalasin D treatment showed relocation of SEPT6 containing rings to the cytoplasm, free from other cytoskeletal components (Kinoshita et al., 2002) although rings are often also associated with the membrane. Myosin IIA, but not actin localizes with these rings ( Joo et al., 2007). Additionally, ring formation was found to be reversible as washing out the cytochalasin D treatment restored septin localization to the reformed actin stress fibers (Kinoshita et al., 2002). Spontaneous ring formation is also observed in many cells and cell types. Altogether, these experiments suggest that the linear septins dissociated from actin fibers can roll up to form ring structures and that assembly into ring structures is independent of actin. (Kinoshita et al., 2002). In addition, recombinant septins polymerizing in vitro have also been observed to form spiral ribbons and rings (Kinoshita et al., 2002). Septin rings were also observed in nonadherent K562 cells expressing a C-terminally GFP-tagged SEPT7 by live cell imaging. In these experiments, there was an average of 141 29 septin disks per interphase cell with an average diameter of 0.8 mm. The stability of the septin rings was dependent on intact microtubules as microtubule depolymerization following treatment with nocodazole or cooling led to disintegration of the septin disks. In the presence of the microtubule stabilization drug, taxol, the septin complex was preserved following cell permeabilization. These findings were also seen in immortalized T-lymphocytes Jurkat cells. Actin was not needed for septin ring stabilization as treatment with cytochalasin D did not change the septin ring organization. Additionally, the septin rings were found mutually exclusive to actin localization in the uropods in Jurkat cells and in filopodia of K562 cells (Sellin et al., 2011a).

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2.2.2.4 Gauzes

In addition to filaments and rings, septins can also form structures referred to as gauzes, which consist of a meshwork of septin filaments. Gauzes were initially seen by quick freeze EM of “unroofed”yeast spheroplasts by Rodal and colleagues. Inspection of the fibers that make up the gauzes revealed structural similarities to metal shadowed septin filaments. Immunoelectron microscopy revealed that Cdc3 was localized predominantly to the gauze structures. In addition to its localization at septin filaments, Cdc10–GFP also localized to patches in intact yeast which may represent the previously observed gauze structures. Temperature-sensitive alleles of Cdc12, a septin essential for yeast septin complexes, lack gauze structures when grown at the nonpermissive temperature. Altogether these results indicate that the gauzes are composed of septins. The cross-linking filaments of the gauzes were typically 5–8 nm in diameter and 0.3–0.4 mm long while the gauzes varied in length (Rodal et al., 2005). Yeast septin complexes containing Cdc11 were also observed in vitro to form a mesh-like arrangement on PIP2-containing lipid monolayers (Bertin et al., 2010). The length of the shortest cross-bridging filament was approximately the same as that of a septin octamer (36.7 7 nm), suggesting that the formation of the septin gauzes required the interaction of the terminal septin, Cdc11. Further mutational analysis identified the C-terminus of Cdc11 as the region needed for a mesh-like septin organization (Bertin et al., 2010). When Shs1 replaced Cdc11 at the ends of octamers, septins typically assembled into rings. However, when phosphomimicking mutations were introduced into sites on the NC interface of Shs1, this caused a switch in morphology from rings to gauzes (Garcia et al., 2011). These residues are known to be phosphorylated by Cdk1. Intriguingly, septin filaments at the mother–bud neck were observed to exist both parallel and perpendicular to the mother–bud axis by EM, suggesting that gauzes exist at this location. Whether the structure of the gauze is regulated by Cdk1 phosphorylation remains to be determined.

3. FUNCTIONS OF SEPTINS 3.1. Diffusion barrier Compartmentalization allows cells to develop specialized structures that are dedicated to specific tasks. This can occur by separating parts of the cell using additional membrane, which give rise to various organelles such as the nucleus, or the Golgi apparatus. In other cases, large multiprotein complexes

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can form diffusion barriers that restrict the diffusional movement of proteins. Such compartmentalization is crucial during the development of budding yeast, where cell growth needs to be restricted to the bud (Barral et al., 2000; Gladfelter, Moskow, et al., 2001a). When yeast cells enter asexual reproduction, growth is concentrated at a selected area, which becomes the site of bud emergence. After the bud has been established, the cell enters isotropic growth, during which growth over the entire bud is observed. This polarized growth is made possible by a ring of septins at the bud neck separating the bud from the mother. Proteins required for isotropic growth, such as the polarisome and exocyst complexes, are localized specifically to the bud (Barral et al., 2000). In a temperature-sensitive Cdc12–6 mutant strain of yeast, the septin ring disassembled when the cells were moved to the restrictive temperature, and as a result, markers for the polarisome and exocyst complexes were no longer excluded from the mother (Finger and Novick, 1997; TerBush et al., 1996). Barral and colleagues demonstrated that this redistribution of proteins still took place when de novo protein synthesis was prevented in the dividing cell, suggesting the pool of redistributed protein came from the bud rather than from de novo protein synthesis in the mother (Barral et al., 2000). Hence, the septin ring plays a still undefined role in compartmentalizing the mother and bud cells in yeast. It was initially believed that septins could also restrict the movement of integral plasma membrane proteins at the bud neck. Using GFP-tagged Ist2p as a membrane marker, Takizawa and colleagues demonstrated that the protein localized to the cortex of the bud in Cdc12 temperature-sensitive mutants at the permissive temperature. Fluorescence Recovery After Photobleaching (FRAP) analysis revealed that the bud restricted GFP–Ist2p was not fixed to the bud, but actually freely mobile within the bud, suggesting that the protein freely diffused in the plane of the cortex but diffusion into the mother was restricted at the bud neck. However, as soon as the cells were moved to the restrictive temperature to disrupt the septin rings between the mother and daughter, Ist2p redistributed into the mother (Takizawa et al., 2000). At the time, it seemed reasonable to conclude that the phenotype was caused by a loss of a septin-based plasma membrane diffusion barrier at the bud neck. However, it was later shown that Ist2p permanently resides in the cortical endoplasmic reticulum (ER) and not in the plasma membrane (Manford et al., 2012; Wolf et al., 2012). Hence, a septin-based diffusion barrier could be acting in the ER. Indeed, more recent studies from the Barral lab supported the notion of a septin-based diffusion barrier in the ER that blocks the movement of ER membrane proteins from traversing

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the mother–bud neck (Luedeke et al., 2005) raising the possibility that Ist2p diffusion was limited by this barrier. In addition to an ER diffusion barrier, the Barral lab has demonstrated that a septin barrier or filter is responsible for limiting the movement of old nuclear pore complexes into the bud during cell division, ensuring that buds receive only new nuclear pore complexes (Shcheprova et al., 2008). This process appears to be important to ensure a full lifespan for the bud cell, although it remains unclear whether this filtration process is identical to the ER diffusion barrier. Interestingly, Shs1p is required for the ER diffusion barrier, raising the possibility that some aspect of septin bundling or gauze formation may be involved. The recent three-dimensional septin network described at the mother–bud neck revealed that the septin ring appeared to associate much closer to the ER membrane than the plasma membrane (Bertin et al., 2012). Much further work is needed to characterize the nature of these diffusion barriers and to determine if one exists for yeast plasma membrane proteins. In mammals, septin-mediated diffusion barriers have been proposed for several subcellular compartments. Initially, a membrane protein diffusion barrier demonstrated to exist at the midbody between two dividing cells was speculated to be septin based (Schmidt and Nichols, 2004), but no studies have directly tested this possibility. Septins were also shown to accumulate at the neck of dendritic spines (Tada et al., 2007; Xie et al., 2007), a location where diffusion barriers had also been predicted to compartmentalize transmembrane and membrane-associated molecules (Ashby et al., 2006), but to date no direct evidence has been reported to show that a diffusion barrier exists at that location. Septins were also implicated as forming a ring-like diffusion barrier at the base of the primary cilium in mammalian cells (Hu and Nelson, 2011; Hu et al., 2010) but more recent studies have implicated components of the transition zone as forming the diffusion barrier (Chih et al., 2012) and septins are not always seen at the base of cilia in mammalian cells (Ghossoub et al., 2013). Perhaps the best evidence for a mammalian septin-based plasma membrane diffusion barrier comes from studies of spermatozoa from SEPT4 null mice. The mammalian sperm cell consists of three parts: the head, the midpiece, and the tail/flagella. The latter two are connected by a septin-enriched ring structure called the annulus. SEPT4 null spermatozoa lack a clear annulus and have a sharp bend at the site of the annulus resulting in the loss of motility. While the structural phenotype suggests that septins provide membrane rigidity at the annulus, Kwitny et al. showed that they are also critical for forming a cortical barrier

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between the midpiece and the tail. In wild-type sperm cells, a protein essential for spermatogenesis, basigin, is freely diffusing but confined to the region distal to the annulus at the early stage, and later redistributes to the midpiece as the sperm matures. This typical membrane restriction of basigin was lost in SEPT4 null sperm, and basigin became localized throughout the whole tail at all stages (Kwitny et al., 2010). While these data are consistent with the loss of a septin-based diffusion barrier, they do not rule out the possibility of indirect effects on basigin localization through changes in basigin transport, or alterations in sperm maturation due to the lack of SEPT4. Clearly, much more work is needed to confirm the role of septins as diffusion barriers in the compartmentalization of the plasma membrane.

3.2. Septins and microtubule stability The interaction between septins and microtubules was first revealed when Drosophila septins Pnut1, Sep1, and Sep2 were found to copurify with microtubules in vitro (Sisson et al., 2000). Subsequently, septins in other model organisms and various mammalian cell types were found to associate with microtubules. Many septins have been reported to colocalize with microtubules in various cell types at different cell cycle stages: in HMEC and HeLa cells, SEPT9 (Nagata et al., 2003; Surka et al., 2002) and SEPT11 (Hanai et al., 2004) localized with microtubules during interphase. SEPT2 also localized with a subset of stable microtubules during interphase in MDCK cells (Spiliotis et al., 2008). In addition, SEPT2 and SEPT6 have been found to localize to mitotic spindles in MDCK and HeLa cells (Spiliotis, 2005); a similar distribution along spindles was also found for SEPT9 in HMEC (Nagata et al., 2003) and HeLa cells (Surka et al., 2002). SEPT1 also localized to the spindle pole throughout mitosis and to the midbody in telophase (Qi et al., 2005). FRAP analysis revealed that septin dynamics were approximately threefold slower than that of microtubules. Interestingly, the localization of these seemingly more stable septins could be displaced upon microtubule depolymerization in some cells, while septin depletion and overexpression often had effects on microtubule dynamics and posttranslational modifications (Kremer, 2005; Neufeld and Rubin, 1994; Sisson et al., 2000; Spiliotis et al., 2008). For example, SEPT7-depleted HeLa cells increased resistance of microtubules to depolymerization by nocodazole (Kremer, 2005). SEPT9_i3 depletion led to a decrease in microtubule depolymerization without affecting overall tubulin levels (Nagata et al., 2003). In contrast, overexpression of SEPT9_i4 conferred resistance to microtubule stabilization by paclitaxel treatment and caused cells

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to repolymerize microtubules more slowly after cold treatment (Chacko et al., 2012). Similarly, tumors expressing higher levels of SEPT9_i1 were also resistant to paclitaxel (Amir and Mabjeesh, 2007) suggesting that septins affect tubulin dynamics. While for most of the septins, it is still not clear whether association with microtubules occurs via direct interaction or by means of adaptor proteins, SEPT9 has been reported to directly associate with microtubule via a central region that contains the GTP-binding domain (Nagata et al., 2003), and its localization along microtubule was sensitive to nocodazole-induced microtubule depolymerization (Surka et al., 2002). The N-terminal extension of SEPT9 also appeared to be required for microtubule-association as demonstrated by Sellin et al. using a nonadherent human myeloid cell line. In K562 cells, endogenous septins can be induced to precipitate with microtubules by treating the cells with paclitaxel. However, association with nonbundled microtubules in the absence of paclitaxel occurred when endogenous SEPT9 was replaced with a single isoform SEPT9_i1 but not when replacement was done with short N-terminal isoforms (SEPT9_i4 and SEPT9_i5), suggesting the N-terminal extension is required for this property (Sellin et al., 2012). Further investigation is required to fully reveal the molecular mechanism of SEPT9–microtubule interaction. The Macara group provided evidence of how septins may regulate microtubule dynamics. They demonstrated that SEPT2 affected microtubule stability by directly binding to microtubule-associated protein 4 (MAP4), a promoter of microtubule stability, and inhibiting its binding to microtubules. Immunoprecipitation using MAP4 as the bait pulled down SEPT2/6/7 trimers and vice versa. The region that binds SEPT2 is a proline-rich region of MAP4 that also binds microtubules. As a result, association with septin filaments sequesters MAP4 away from microtubules, leading to microtubule depolymerization. Consistent with this model, depletion of septins in HeLa cells resulted in accumulation of acetylated microtubules (a marker of microtubule stability) that were resistant to nocodazole treatment, indicating increased microtubule stability (Kremer, 2005). Spiliotis et al. proposed an alternative model for the interaction between septins, MAP4, and microtubules where SEPT2 competes with MAP4 for binding to microtubules. They demonstrated through a competitive blot overlay assay that when tubulin-containing nitrocellulose blots were first overlaid with increasing concentrations of purified SEPT2/6/7 and then overlaid with MAP4, binding of MAP4 decreased in a concentration-dependent manner. This suggests that septin complexes occupy the binding sites of MAP4 and directly

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interfere with MAP4–tubulin binding (Spiliotis et al., 2008). In addition, SEPT2 was shown to associate specifically with the polyglutamylatedtubulin tracks in MDCK cells and SEPT2 depletion led to the loss of this subset of stable microtubules, suggesting a role of septins in the maintenance of polyGlu-microtubules. The extent to which SEPT2 participates in the regulation of microtubules seems to be cell-type dependent, since SEPT2 and tubulin showed distinct distributions in NRK cells, and SEPT2 depletion did not affect their microtubule organization (Schmidt and Nichols, 2004).

3.3. Septins as macromolecular scaffold Their ability to form stable filamentous complexes and to interact with various proteins allows septins to function as macromolecular scaffolds and facilitate protein–protein interactions in the cell. This is best characterized in budding yeast, where septins act as a scaffold during bud-site selection, chitin deposition, and cytokinesis. When haploid yeast cells undergo budding, septins form a collar around the bud site prior to bud emergence (Kim et al., 1991), and this septin ring functions as a scaffold that marks the position of the bud neck and recruits downstream molecules (DeMarini et al., 1997), including other bud site landmarks and regulators of actin cytoskeleton organization (Chant and Herskowitz, 1991; Chant and Pringle, 1995; Drees et al., 2001; Fujita et al., 1994; Halme et al., 1996; Roemer et al., 1996; Sanders and Herskowitz, 1996). The sole type II myosin in S. cerevisiae, Myo1, is an essential component of the actomyosin ring. It colocalizes with the septin ring from bud emergence to cytokinesis (Bi et al., 1998; Lippincott and Li, 1998), and its neck localization prior to cytokinesis is dependent on septins (Balasubramanian et al., 2004). The primary septum is a chitinous cell wall structure required for maintaining neck integrity during bud growth (Schmidt et al., 2003). The ring of chitin is formed just before bud emergence, and it remains at the base of the bud throughout cell division (Wloka and Bi, 2012). The chitin ring is largely formed by chitin synthase III, which localizes to the mother side of the bud neck and deposits chitin in the cell wall (DeMarini et al., 1997). The catalytic subunit in the complex, Chs3, and its activator, Chs4, require septins for their neck localization (DeMarini et al., 1997). In addition to functioning as a scaffold to concentrate these proteins to the division site, septins were found to be required for the localization of nearly 100 proteins to the bud neck (Gladfelter, Pringle,

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et al., 2001b; McMurray and Thorner, 2009). However, only a few of these proteins were reported to directly associate with individual septins; perhaps septin higher order structures are required for interaction with these proteins or their recruitment may be indirect via other recruited proteins. Septins have also been found to act as a scaffold during cytokinesis in mammalian cells. Joo and colleagues demonstrated direct binding between nonmuscle myosin II and SEPT2 in CHO cells; when this interaction was disrupted, it resulted in reduced phosphorylation of myosin II regulatory light chain and led to instability of the ingressed cleavage furrow. During interphase, myosin II activation is required for the formation of stress fibers. As expected, cells with diminished septin–myosin II interaction also had a loss of stress fibers. These studies suggest that septins provide a molecular platform for myosin II activation by various kinases ( Joo et al., 2007).

3.4. Septins in vesicle trafficking Amongst proteins that interact with septins, some are essential for synaptic vesicle trafficking and membrane fusion. In the mammalian nervous system, exocytic vesicles are generated at the Golgi apparatus and guided toward the plasma membrane by cytoskeletal tracks and motor proteins (Schmoranzer and Simon, 2003). Once the vesicles are in the vicinity of their target sites, the exocyst, an evolutionarily conserved octameric protein complex, is responsible for tethering the vesicles to their target membranes (TerBush et al., 1996). Eventually, vesicle fusion is facilitated by interactions of the v-SNARE VAMP/synaptobrevin with the t-SNARE syntaxin and SNAP-25 (Rizo and Sudhof, 2012). The first indication of septins interacting with components of the exocytic machinery was provided by Hsu et al. Using antibodies against Sec8, a component of the exocyst complex, they coimmunoprecipitated four mammalian septins from total rat brain proteins: SEPT2, SEPT4, SEPT6, and SEPT7; partial colocalization between the exocyst and septin complex was also observed in cultured hippocampal neurons (Hsu et al., 1998). Shortly thereafter, SEPT2 and SEPT5 were found to directly interact with syntaxin in a complex. The C-terminal region of syntaxin that binds to septins is the same region that interacts with SNAP-25 and VAMP (Beites et al., 1999), raising the possibility that septins are potential regulators of SNARE protein interactions. To explore this, Beites and colleagues transiently transfected cells with human growth hormone (hGH), measured as a secretion reporter in HIT-T15 cells. Intriguingly, transfection of wild-type

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SEPT5 attenuated evoked secretion, whereas the dominant negative form of SEPT5, which was defective in GTP binding, potentiated it. When a GTPase mutant of SEPT5 was cotransfected with tetanus toxin that cleaves VAMP, exocytosis was abolished, indicating that the potentiation mediated by mutant SEPT5 was upstream of SNARE interaction (Beites et al., 1999). Taken together, these data support the idea that septins are acting as a physical barrier that regulates exocytosis and prevents unwanted fusion events. Yang and colleagues provided further evidence for this hypothesis using the Calyx of Held synapses from SEPT5-null mice (Yang et al., 2010). By staining the wild-type synapse with antibodies against SEPT5, it was found that SEPT5 occupies the active zone (AZ) in immature neurons, but is excluded from the AZ following development. This developmental reorganization of SEPT5 coincides with an increase in the number of synaptic vesicles docked at the AZ, as well as an increase in vesicular release. Functional inhibition of SEPT5 in wild type, immature mice led to a rapid increase in release, consistent with the notion that SEPT5 acts as a spatial barrier separating synaptic vesicles from docking to AZs in immature synapses. Overall, septins may have two roles in exocytosis. By interacting with components of the exocyst complex, they may regulate the transport of vesicles to sites of membrane fusion at the plasma membrane; and as an interacting partner of syntaxin, septins may also play a role governing membrane fusion events.

3.5. Septins as regulator for cortical rigidity One indication that septins regulate cortical rigidity of cells is found in spermatozoa, where septins constitute the ring-like annulus structure. As previously described, in mice lacking SEPT4, the annulus is absent and the tail bends sharply or breaks at that location as they develop and become motile (Ihara et al., 2005). Septins are also thought to provide cortical rigidity in amoeboid T cells. Immunofluorescence microscopy revealed a fibrous septin array through the mid-zone of the T cells, lying perpendicular to the axis of cell migration. SEPT7 depletion resulted in more protrusive cell bodies and extensive blebbing, indicating increased flexibility of the membrane. The SEPT7 depleted cells were also more efficient at transmigrating through very small pores than control cells. Similar effects could be achieved by treating cells with nocodazole to relax their cell cortex, and could be suppressed by rigidifying the cortex with paclitaxel (Tooley et al, 2009).

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These data all support a role of septins in regulating cortical rigidity either directly or through microtubules. Septins may regulate membrane flexibility by directly binding to phospholipids. Purified septins have been shown to have affinities for negatively charged lipids such as PIP, PIP2, and PIP3. In particular, a short polybasic sequence on the N-terminal side of the GTPase domain of SEPT4 was shown to be necessary for PIP2 binding (Zhang et al., 1999). This basic-rich sequence is found in most septins at the same position, and likely contributes to septin–lipid interaction. By associating with lipids, septins can dramatically alter the shape of the membrane in vitro. When stable giant unilamellar liposomes containing PC, PI, PIP, and PIP2 were exposed to septincontaining porcine brain extract or recombinant septin complexes, tubules immediately protruded from the liposome until the entire surface was converted into tubules. EM revealed that tubules were trussed with septin filaments (Tanaka-Takiguchi et al., 2009). Septin self-assembly in vitro is very slow, but the tubule formation was rapid, suggesting that the membranous platform facilitates in septin interaction, and the septin filaments in turn generate a curved, rigid surface that draws tubules from the liposomes. In fact, binding to membranes to rigidify them might be how septins regulate cortical stability in amoeboid T cells. In crawling T cells, septins are mostly absent from protrusions, but a transient enrichment of septins at these protrusions can be observed during retraction. To determine if septins play an active role in retracting membrane, Gilden and colleagues induced mimicked blebbing in T cells by exposing them to hypotonic medium. The cells rapidly swelled up as a result of the increased internal hydrostatic pressure, this was followed by a slower regulatory volume decrease phase, during which the cells adjust the tonicity of the cytosol and cell volume shrinks. SEPT7-depleted cells were indistinguishable from control cells during the initial swelling phase but exhibited significantly slower recovery, suggesting that septins are required to effectively retract the cortex (Gilden et al., 2012). While the cortical distribution of septins in blebbing T cells suggests that phosphoinositides might play a role in recruiting septins to the plasma membrane, the study by Gilden et al. also pointed out that the actomyosin cytoskeleton is involved in the process. Treatment with a MyoIIA inhibitor or an actin polymerization inhibitor (blebbistatin and Latrunculin B respectively) both caused the cells to recover at a slower rate during the swelling assay, similar to that seen in SEPT7-depleted cells. Shrinkage of SEPT7-depleted cells treated with latrunculin B did not lead to additional impairment, indicating septins and the actomyosin cytoskeleton may be collaborating to

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promote cortical retraction (Gilden et al., 2012). In fact, septins are found to associate with actin through myosin II in HeLa and NIH3T3 cells, and their filamentous appearance in these cells depend on stress fibers, which are actomyosin bundles found in many cultured nonmuscle cells ( Joo et al., 2007; Kinoshita et al., 1997, 2002). Although anillin has also been reported to connect septins to actin, it is exclusively found in the nucleus during interphase, thus is not likely to be acting as an adaptor protein between actin and septins in nonmitotic cells. Disruption of polymerized actin with latrunculin B or cytochalasin D eliminates stress fibers and causes septin arrays to eventually transform into septin rings (Xie et al., 1999). Conversely, displacing septins from actin by expressing dominant negative anillin or Borg3 causes cells to lose stress fibers (Kinoshita et al., 1997, 2002). As mentioned previously, septins can directly interact with myosin II and regulate its activation by kinases such as ROCK and CRIK ( Joo et al., 2007). As a result, septins could control cortex rigidity by regulating stress fiber formation and actin-based protrusions. Consistent with this model, septin-depleted HeLa cells have disrupted stress fibers and decreased membrane tethering and rigidity (Mostowy et al., 2011).

4. SEPTINS IN COMPLEX BIOLOGICAL PROCESSES We have mentioned in previous sections that septins are implicated in cell division and provided specific examples where septins are required as a diffusion barrier or as a molecular scaffold during division. In this section, we will provide a more integrated overview on the involvement of septins in cell division in S. ceresiviae and mammalian cells.

4.1. Cell division in budding yeast Budding yeast undergo asymmetric division, resulting in two cells with distinct sizes. The daughter cell is approximately two-thirds of its mother in size. Upon entering a new cell cycle, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) are recruited to the presumptive bud site in a Cdc42-dependent manner (Iwase et al., 2006). The septins are first seen as unorganized clouds or patches, but soon arranged into a dynamic cortical septin ring (Longtine and Bi, 2003). Septins remain at the bud neck throughout division and are implicated in several events, which collaboratively ensure proper separation of mother and daughter cells: (1) Bud-site specification, (2) bud growth, (3) nuclear positioning, and (4) cell cycle checkpoint regulation.

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4.1.1 Bud-site specification S. cerevisiae adopt one of two budding patterns depending on their mating type. Diploid cells undergo bipolar budding where daughter cells bud on the opposite end of the previous bud site, and mother cell buds at either end. On the other hand, haploid cells undergo axial budding where the new bud is formed adjacent to the previous bud site. The axial budding pattern requires the recruitment to the bud neck of several axial budding landmark proteins, such as Bud3, Bud4, Axl1, and Axl2/Bud10 (Adames and Cooper, 2000; Chant and Herskowitz, 1991; Chant et al., 1995; Fujita et al., 1994; Halme et al., 1996; Roemer et al., 1996; Sanders and Herskowitz, 1996). Mutations in these proteins do not affect budding of diploid cells, but cause haploid cells to adopt the bipolar budding pattern. Of these landmark proteins, the localization of Bud3 and Bud4 has been found to be septin dependent. However, the mechanisms by which the septins determine the localization of Bud3 and Bud4 remain unknown. Interestingly, the septin ring is also particularly required for bud-site selection during axial budding, as haploid cells with a nonfunctional septin ring bud in a bipolar fashion (Chant and Pringle, 1995; Flescher et al., 1993). Recent studies have shown that the septin ring forms from a patch due to localized inhibition of Cdc42 and focal exocytosis at the center of the patch, driving the septins to the periphery of the patch where they assemble into a ring (Okada et al., 2013). 4.1.2 Polarized bud growth As the bud emerges, the septin ring is transformed into a stable hourglass structure at the bud neck, and the bud enters isotropic growth. Bud growth requires the insertion of new plasma membrane and the synthesis of new cell wall. The material and remodeling enzymes involved in these processes need to be delivered to the bud via the secretory pathway. In other words, isotropic growth relies heavily on exocytosis at the bud, which is controlled by vesicle delivery and docking/fusion (Finger and Novick, 1997). Vesicle delivery towards the bud cortex requires polarized actin cables that extend along these actin tracks. The polarisome is a protein complex that plays a crucial role in the polarization of actin tracks. In the absence of the polarisomes, buds grow as spheres rather than ellipsoids, indicating that growth is no longer focused at the bud tip (Chenevert et al., 1994; Evangelista et al., 1997). After transport along actin cables, the fusion of the vesicle with the plasma membrane requires the exocyst complex. Using a temperature-sensitive septin mutant, Barral et al. demonstrated that

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components of the polarisome and exocyst localize to the bud cortex at the permissive temperature; but upon shifting the cells to the restrictive temperature and disrupting their septin rings, both complexes cross the bud neck boundary and diffuse into the mother cell (Barral et al., 2000). Thus, by restricting the movement of polarisome and the exocyst complex, the septin ring helps maintain polarity during bud growth. Consistent with this, septin defects prevent the cell from entering isotropic growth (Barral et al., 1999). 4.1.3 Nuclear positioning Budding yeast undergo a closed mitosis without nuclear envelope breakdown. The microtubule-organizing centers (spindle pole bodies or SPBs) embedded in the nuclear envelope are responsible for emanating cytoplasmic microtubules (cMT) that extend to the cell cortex. Microtubule motors tether cMTs to cortical receptors at the bud neck and the bud cortex, and generate movement by depolymerizing cMT at the plus-end (Adames and Cooper, 2000; Carminati and Stearns, 1997). Another pathway that contributes to nuclear positioning involves dynein, which promotes the lateral sliding of the cMT plus-end along the bud cortex. The proper localization of both Bud6 and dynein is septin dependent, suggesting an indirect involvement of septins in nuclear positioning. Indeed, the septin ring is required for cortical interaction of cMTs with the bud neck and therefore for nuclear positioning. 4.1.4 Cell Cycle Checkpoint We have mentioned in the previous section that the septin ring at the bud neck acts as a scaffold to anchor Myo1 and components of CSIII complex, which are involved in actomyosin ring assembly and primary septum formation respectively (Balasubramanian et al., 2004; DeMarini et al., 1997). While interactions with Myo1 and CSIII components are important under normal conditions, septins also play a crucial role when bud formation is impaired. Normal cell cycle progression requires Swe1p to be phosphorylated and degraded in G2/M phase (Barral et al., 1999; Lew, 2003; Longtine et al., 2000; McMillan et al., 1998). Swe1p is localized to the nucleus and to the daughter side of the bud neck. Its localization and degradation requires Hsl1p and its binding partner Hsl7p, both of which localize to the daughter side of the bud neck in a septin-dependent manner. If bud morphogenesis proceeds normally, Swe1p would become phosphorylated and degraded. In case of abnormal morphogenesis, or in septin mutant cells, Hsl1p and Hsl7p are released from the bud neck; this leads to Swe1p stabilization and cell cycle arrest in G2 (Barral et al., 1999).

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4.2. Mammalian cytokinesis Although septins were first discovered as critical components in yeast cytokinesis, they also play a role in mammalian cytokinesis. Briefly, mammalian cytokinesis begins after the onset of anaphase when a contractile ring made of actin and myosin is constructed at the cell cortex between the segregated chromosomes. This ring constricts, drawing in the membrane at the middle of the cell to create an indentation referred to as the cleavage furrow. The ring continues to contract until only a thin bridge (termed intercellular bridge) made up of stabilized acetylated tubulin remains at late telophase. The midbody is the protein dense structure located at the middle of the intercellular bridge where proteins involved in the severing of the intercellular bridge (a process known as abscission) are recruited. The midbody also dictates the site of abscission as the intercellular bridge breakage typically occurs on one side of the midbody. The mechanism of abscission is still under debate but three nonexclusive models have been proposed. In the first model, abscission is mediated by soluble N-ethylmalemide-sensitive factor attachment protein receptor (SNARE)dependent vesicle fusion at sites where the exocyst complex has tethered vesicles near the midbody. This model is supported by the fact that approximately one third of the midbody proteome is composed of proteins involved in vesicle tethering and fusion (Skop et al., 2004) and abscission was found to take place about 10 min after vesicle fusion at the midbody (Gromley et al., 2005; Guizetti et al., 2011). The exocyst and SNAREs are recruited to the midbody by centriolin, and depletion of any of these components results in an abscission defect (Gromley et al., 2005). Two types of vesicles have been shown to accumulate to the midbody: recycling endosomes and Golgi-derived vesicles. Endosomes targeted to the midbody carry GTPases Rab11 or Rab35 and the depletion of either Rab protein led to protracted cytokinesis or binucleation (Chesneau et al., 2012; Kouranti et al., 2006; Wilson et al., 2005), while prevention of post-Golgi secretion also led to a failure in abscission (Skop et al., 2001). While vesicle delivery to the midbody was found to be needed for abscission, it is not clear whether vesicles provide membrane or deliver cargo proteins needed for abscission. A second model involves the ingression of membrane at the site of abscission mediated by the Endosomal Sorting Complex Required for Transport (ESCRT). The ESCRT machinery is well known to be involved in membrane scission to form multivesicular bodies and has been implicated in viral budding (Henne et al., 2011; Wollert et al., 2009), and

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this processes is topologically comparable to membrane fission during abscission. Recently, it has been found through high resolution live and fixed imaging that part of the ESCRT machinery is localized at both sides of the intercellular bridge as helical filaments (Elia et al., 2011; Guizetti et al., 2011). Depletion of the ESCRT protein Tsg101 and ESCRT associated protein Alix led to impaired abscission (Agromayor et al., 2009; Bajorek et al., 2009; Carlton and Martin-Serrano, 2007; Dukes et al., 2008; Morita et al., 2007). In addition, it was found that the microtubule severing protein spastin is required for abscission and is recruited by ESCRT protein CHMP1B (Connell et al., 2009). The third model is a hybrid of the previously described two, where newly added membrane from vesicle fusion, and the action of spastin, lead to an even narrower midbody bridge that is then the target of ESCRTmediated membrane breakage (Schiel et al., 2013). Septins have been long implicated in cytokinesis where their depletion has led to cytokinetic failure as well as chromosome congression and alignment impairment (Kremer et al., 2005; Spiliotis et al., 2005). Most recently, Estey et al., discovered that different septins act during different stages of mammalian cytokinesis. In HeLa cells, it was found that the expression levels of the different septins were consistent throughout cytokinesis, yet specific septins acted at distinct stages of cytokinesis (Estey et al., 2010). The roles that septins play in mammalian cytokinesis will be discussed in four parts: (1) chromosome segregation, (2) cleavage furrow function, (3) abscission, and (4) mitotic checkpoint.

4.2.1 Chromosome segregation Proper chromosome segregation is mediated by kinetochores, which allow for the attachment of the microtubule based mitotic spindle to the aligned chromosomes at the cell equator. Centromere protein E (CENP E) is a microtubule based kinetochore motor protein that stabilizes and positions the chromosome, ensuring correct attachment by the mitotic spindle. CENP E also serves as part of the mitotic checkpoint machinery which delays the next step of the cell cycle when replication errors are detected, allowing for the correction of the error. The cell cycle machinery and progression are heavily regulated by phosphorylation of key proteins and a critical regulator is Aurora B. SEPT1 (Qi et al., 2005), SEPT2, SEPT6 (Spiliotis et al., 2005), and SEPT9 (Nagata et al., 2003) were found to be localized to

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the mitotic spindles. Furthermore, Septins have been found to be involved in the localization of CENP E to kinetochores (Zhu et al., 2008). Depletion of septins led to delays in chromosome congression and degradation, consistent with a role in scaffolding CENP E to kinetochores (Spiliotis et al., 2005). 4.2.2 Cleavage furrow function Septin localization was analyzed through immunofluorescence experiments which indicated that during anaphase, SEPT2, SEPT6, SEPT7, SEPT9, and SEPT11 are found along the cleavage furrow (Estey et al., 2010; Joo et al., 2007). This was also seen in Drosophila embryos as well as dividing cells where Peanut was found at the furrow canal and the cleavage furrow respectively (Field et al., 2005; Neufeld and Rubin, 1994; Oegema et al., 2000). Cells depleted of each individual septin by siRNA were followed through cytokinesis by time lapse microscopy; this led to the conclusion that at least SEPT2 and SEPT11 are involved in cleavage furrow ingression as their absence led to abnormal cleavage furrow constriction where ingression occurred at one side of both segregated nuclei (Estey et al., 2010). The role of SEPT2 may be linked to MyosinII as inhibiting this interaction led to unstable cleavage furrows and binucleated daughter cells ( Joo et al., 2007). Interestingly, this phenotype is similar to that resulting from loss of anillin and anillin is required for septin recruitment to the furrow (Piekny and Glotzer, 2008), suggesting that their functions are related. 4.2.3 Abscission Through immunofluorescence, it was found that SEPT2, SEPT6, SEPT7, SEPT9, and SEPT11 localized to either side of the intercellular bridge as well as at the midbody (Estey et al., 2010; Joo et al., 2007). SEPT1 was also found at the midbody colocalizing with the kinase Aurora B, suggesting that the function of SEPT1 may be regulated by phosphorylation (Qi et al., 2005). As stated in the previous section, in contrast to other septins, the knockdown of SEPT9 did not lead to a cleavage furrow defect, but instead resulted in a defect in abscission. Abscission defects were quantified as an increase in the number of cells with persistent midbodies and in SEPT9 depleted cells there was a significant increase in cells observed with midbodies compared to control siRNA treated cells. Using time lapse microscopy, it was seen that 100% of the control knockdown cells completed abscission within 3.9 h

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after metaphase entry, compared to SEPT9 depleted cells where only 70% of the cells completed abscission within 9 h. Of the 30% that failed to abscise, 20% of the cells had midbodies that persisted into the next round of mitosis or did not break even after 40 h postcytokinesis initiation. The remainder either experienced a midbody regression to become binucleated or went through apoptosis (Estey et al., 2010). The role that SEPT9 plays in abscission remains to be determined, but the absence of SEPT9 led to a mislocalization of the exocyst component Sec8 from the intercellular bridge (Estey et al., 2010), suggesting that a major role may to be to ensure the proper tethering of vesicles at the midbody. Much additional work will be required to further define this cellular function. 4.2.4 Mitotic checkpoint The mitotic checkpoint is interconnected with the cell cycle machinery to ensure proper segregation of DNA and cytoplasmic content by delaying the progression into the next stage of mitosis. One trigger for this delay is through the detection of damaged DNA and interestingly septins have been linked to repair of damaged DNA. Specifically, it was found that the septin complex (SEPT2, SEPT6, and SEPT7) interacts with SOCS7 (Suppressor of Cytokine Signaling-7) (Kremer et al., 2007). SOCS7 contains a nuclear localization signal and acts as a transporter to move Nck (an actin-associated adaptor protein) into the nucleus. Surprisingly, septin depletion resulted in nuclear localization of SOCS7 and Nck (Kremer et al., 2007). It was also shown that the nuclear localization of both proteins is required for activating the mitotic checkpoint and cellular arrest (Kremer et al., 2007). Therefore this suggests a mechanism in which septins regulate the DNA damage response during mitosis by controlling the cellular distribution of SOCS7 and Nck.

4.3. Septins and pathogen invasion Several pathogenic bacteria can cause diseases by entering into nonphagocytic cells. A few of them, including Listeria monocytogenes and Shigella flexneri, are capable of exploiting the host actin cytoskeleton and use it for their own motility both intra- and intercellularly (Welch and Way, 2013). These invasive bacteria are seen with long actin tails in the cytosol of the host. Interestingly, septins have been found to form a cage around the actin tails and surround bacterial bodies. Treatment with cytochalasin D or latrunculin B revealed that actin polymerization is critical to both tail

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formation and septin caging. Moreover, the septin cages seem to prevent tail formation, as siRNA-mediated septin depletion increased the number of internalized bacteria with actin tails. In contrast, treatment with TNF-a, a pleiotropic cytokine that plays a role in host defense against pathogens, stimulated septin caging, and restricted actin tail formation. This indicates that recruitment of septins is a cellular defense mechanism against pathogens (Mostowy et al., 2010). In addition to preventing the actin-based motility, Mostowy et al. also demonstrated a role for the host septins in targeting intracytosolic pathogens to the autophagy pathway. Many markers of the autophagy pathway associate with Shigella in septin cages; inhibition of septin cage formation prevents accumulation of autophagy markers, and vice versa (Mostowy et al., 2010). This suggests septin assembly and autophagy are two interdependent processes, but the mechanisms underlying septin recruitment at the site of autophagosome formation require further investigation.

4.4. Cell polarity From unicellular to multicellular organisms, cell polarity is crucial for differentiation, proliferation, and morphogenesis. The asymmetric organization of cellular compartments allows cells to develop specialized structures that are essential to their survival. In yeast, septins are especially implicated in the establishment and maintenance of cortical polarity, that is, the asymmetric organization of the plasma membrane and the intracellular structures associated with it. Two forms of cortical polarity can be observed: apical polarity is when asymmetry is established around one point, resulting in a gradient; and cortical compartmentalization is when the cortex is divided into domains separated by boundaries. Apical polarity is crucial during bud emergence when cortical markers are concentrated at the presumptive bud site and cortical compartmentalization is important during bud growth when the cortex of the mother and the daughter must be separated. We have discussed in detail how septins are involved in these processes in previous sections. In short, septins form a molecular scaffold at the bud site, and contribute to apical polarity by recruiting other bud site makers; during bud growth, the septin ring at the bud neck restrict the movement of proteins across the bud neck, thereby play a key role in maintaining cortical asymmetry. In addition to cell division, polarized growth in yeast is also observed in response to pheromones when mating partners extend a projection towards each other. The Cdc12–6 mutant is defective in projection

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formation, likely caused by a mislocalization of key proteins, such as Afr1p, to the base of the mating projection. Examples of septins affecting cell polarity can also be found in the mammalian system as seen in amoeboid T cells. As discussed above, septins provide membrane rigidity and limit protrusions to the front and the back of the cell. By doing so, septins promote efficient chemotaxis by facilitating the directionality of cell movement. SEPT7-depleted T cells were found to have more protrusions extended outside the path of motility, which do not contribute to directional movements (Tooley et al., 2009). In MDCK cells, vesicles that are destined for the membrane travel along microtubule tracks that are decorated by septins. Spiliotis et al. reported a decrease in delivery from the trans Golgi network to the membrane upon SEPT2 depletion in MDCK cells. Consequently, apical and basolateral membrane markers end up accumulating intracellularly instead of reaching the membrane, and the cells fail to exhibit morphology characteristic of a polarized epithelium cell (Spiliotis et al., 2008). The implication of septins in cell polarity is closely associated with their ability to form diffusion barriers and molecular scaffolds. The relatively stable and filamentous nature of septins allows them to maintain stable positions, define distinct cellular structures, and recruit specific effectors; thus granting them a crucial role in establishing and maintaining cell polarity.

4.5. Septins and primary cilia Primary cilia are nonmotile solitary protrusions found on the surface of most eukaryotic cell types. The primary cilium stems from a centriole-derived structure called the basal body; and the length, or the axoneme, of the cilium is composed of a cylindrically organized ring of microtubules. The ciliary membrane is embedded with receptor proteins. Its unique composition allows the primary cilium to act as a multisensory antenna of the cell, capable of detecting fluid flow, pressure, light, and odor. In addition to sensing environmental inputs, cilia also plays a role in transducing intercellular signals, regulating key signaling pathways such as Hedgehog and Wnt (Kim and Dynlacht, 2013). Since the primary cilium is not separated from the rest of the cell by membrane, the entry and exit of ciliary proteins needs to be tightly regulated in order to control its content. Hu et al. suggested that septins play a part in the compartmentalization of the primary cilium, depletion of which led to mislocalization of ciliary membrane proteins and inhibited ciliogenesis

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(Hu et al., 2010). Using IMCD3 cells, they determined that SEPT2 localizes to the base of the cilium, in a region known as the transition zone, which is just distal to the basal bodies. In some optical sections, the patch of SEPT2 appears as a ring-like structure of 500 nm diameter; and in about 10% of the cells examined, SEPT2 staining was also found along the axoneme. Upon siRNA-mediated SEPT2 depletion, cells that completely lack SEPT2 failed to form a cilium, whereas cells with partial depletion of SEPT2 had a short cilium. FRAP analysis of fluorescently tagged ciliary membrane proteins showed that when the entire cilium was photobleached, cells with a partial SEPT2 depletion had more rapid recovery compared to control cells, suggesting SEPT2 is a component of the diffusion barrier at the base of the cilium and is required for ciliogenesis (Hu et al., 2010). Consistent with this finding, in Xenopus embryos, SEPT2 or SEPT7 mutations were also found to result in ciliogenesis defects (Kim et al., 2010). While the involvement of septins in ciliogenesis is undisputed, there seems to be controversies regarding the mechanism through which they contribute to this process. A recent study by Ghossoub et al. examined the role of septins in the primary cilium of RPE cells. Surprisingly, SEPT2, SEPT7, and SEPT9 were all found localized to the axoneme, with no colocalization with basal body or transition zone markers. SEPT7 appears to be required for ciliogenesis, depletion of which led to a significant decrease in ciliated cells; whereas SEPT9 depletion resulted in shorter cilia. FRAP analysis showed no recovery of GFP–SEPT2 when the entire cilium, or part of the cilium, was photobleached, suggesting septin complexes in the cilium are not dynamic, and are likely a structural component (Ghossoub et al., 2013). MAP4 was also found in the axoneme, and previous studies have suggested that septins regulated MAP4 function (Kremer et al., 2005) so they examined the association of septins and MAP4 in cilia length. They found that MAP4-depleted cells had longer cilia than control cells and MAP4 overexpression seemed to displace septins from the primary cilium. This led them to propose that MAP4 inhibits cilia elongation and that septins were competing with it to regulate cilium length (Ghossoub et al., 2013). While these studies provide further insight into the connection between septins and the primary cilium, they seem to suggest that the role of septins in ciliogenesis is cell specific. On one hand, septins form a ring at the transition zone and act as a diffusion barrier to regulate ciliary protein content in IMCD3 cells; on the other, they are a stable structural component of the axoneme in RPE cells. Are there different types of primary cilium? Could

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septins be playing different roles depending on the cell type? Hopefully, ongoing investigations will provide insights in the near future.

4.6. Cell migration It is no surprise that septins are implicated in cell migration given that they are involved in the regulation of the cytoskeleton as well as cell polarity, two factors important in cell migration. Cell migration strongly relies on cytoskeletal dynamics, protrusions at the leading edge and coordination of cell polarity for the cell to travel in one direction. When cell polarity is disrupted, as seen in septin-depleted migrating T cells, protrusions are no longer limited to the front and the back of the cell, thus the directionality of movement is lost (Tooley et al., 2009). Intriguingly, altering the expression of SEPT9_i4 isoform alone is sufficient to affect cell motility and polarity. When SEPT9_i4 was overexpressed, endogenous SEPT9 became delocalized from filamentous structures, and actin processes were formed around the cell periphery. SEPT9_i4 expressing cells also had enhanced cell motility but directional movement was perturbed and a Golgi reorientation assay revealed loss of normal polarity in these cells (Chacko et al., 2012).

5. SEPTIN-ASSOCIATED DISEASES Given the range of cellular functions attributed to septins, it is not surprising that the septins might be linked to a variety of diseases due to the loss or gain of such cellular functions. Below we discuss specific diseases where septins have been associated.

5.1. Hereditary neuralgic amyotrophy The strongest link between septins and human disease has been identified for hereditary neuralgic amyotrophy (HNA), a rare autosomal dominant recurrent peripheral neuropathy characterized by the onset of severe pain in the shoulder and/or arm as well as weakness, sensory loss, and atrophy of the arm muscles (Kuhlenbaumer et al., 2005). Affected patients usually experience full recovery, but this can take weeks to years. Genetic analysis of several HNA patients and their families identified mutations in the SEPT9 locus. To date, missense mutations R88W or S93F were found in some HNA pedigrees (numbered from the longest SEPT9 isoform) (Hannibal et al., 2009; Kuhlenbaumer et al., 2005). In addition, duplications encompassing the SEPT9 gene were also detected, where the portion SEPT9 duplicated varied

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in length and location (Collie et al., 2010). While complete duplications did not alter protein expression patterns, internal gene duplications often led to altered protein expression patterns in lymphoblastoid cells derived from the patients (Collie et al., 2010). Moreover the duplicated region in all studied cases consists of the exon containing the R88W and S93F mutation (Collie et al., 2010). Interestingly, the point mutations and gene duplication occur at the N-terminal region common to isoforms 1, 2, and 3 but not 4 and 5 of SEPT9 suggesting the importance of this region to the onset of HNA. Unfortunately it remains unclear which component of the peripheral nervous system is affected by the mutations.

5.2. Male sterility A group of patients that are sterile due to reduced sperm motility (athenospermic) showed a disorganization of the annulus and septin rings (Ihara et al., 2005; Lhuillier et al., 2009; Sugino et al., 2008). As explained in previous sections, septins provide rigidity at the annulus and act as a diffusion barrier to compartmentalize the different parts of the sperm. Specifically, SEPT4 null mice resulted in a loss of sperm motility and SEPT4 was found to be critical for generating the barrier between the midpiece and tail. Although the loss of SEPT4 has been detected in infertile human males (Lhuillier et al., 2009; Sugino et al., 2008), the cause of SEPT4 loss remains to be determined and whether the significance of SEPT4 is to provide cellular rigidity or diffusion barrier function still needs to be verified. In addition to SEPT4, SEPT12 was also found to be linked to male infertility, where again there was a reduced level of SEPT12 in athenospermic patients (Lin et al., 2009). From this study, it was found that SEPT12 expression levels were critical for human sperm development. Additional studies by Miyakawa linked SEPT12 to cases of Sertoli-cell-only syndrome where the patients do not produce sperm. Karyotyping these patients revealed eight single nucleotide polymorphisms in the SEPT12 locus yet the functional significance was not determined (Miyakawa et al., 2012). A separate group identified two missense mutations that were located in the predicted GTP-binding domain of SEPT12 (Kuo et al., 2012). Additionally, these two SEPT12 mutants blocked the ability of wild type SEPT12 to form filaments in a dose-dependent dominant negative manner suggesting that the mutants were also able to participate in filament formation (Kuo et al., 2012). The T89M mutation showed a reduced GTP hydrolysis in vitro and associated sperm had an abnormal morphology and reduced motility

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(Kuo et al., 2012). The D197N mutation interfered with GTP binding and patients had reduced sperm count and sperm motility (Kuo et al., 2012). Microscopy revealed a loss of SEPT12 from the annulus, resulting in a defective annulus and a bent tail (Kuo et al., 2012).

5.3. Cancer It is no surprise that septins are associated with cancer given their participation in a wide range of cellular processes. Below we describe some forms of cancer to which septins have been linked, and discuss possible functional links between septins and cancer progression. 5.3.1 Leukemia Septins have been associated with a variety of human leukemias that result from translocation of the mixed lineage leukemia (MLL) oncogene into a septin gene locus. MLL is a gene that encodes for a histone-lysine N-methyltransferase and is involved in positively regulating gene transcription. MLL maps to chromsome 11q23 and this locus is frequently involved in chromsomal translocations associated with leukemias. While more than 60 different translocations of the MLL gene have been identified to date, 5 of these involve members of septin family making it very unlikely that this has occurred by chance. These translocations have resulted in a variety of acute leukemias including acute lymphoblastic leukmia, acute myeloid leukemia, chronic neutrophilic leukemia, and several others. The first described septin fusion with the MLL gene located at chromosome 11 involved the SEPT9 gene on chromosome 17 resulting in a chimeric protein consisting the N-terminal region of the MLL attached to SEPT9 (Osaka et al., 1999). Other septins (SEPT2, SEPT5, SEPT6, and SEPT11) were later reported to undergo a similar translocation and created fusion proteins with MLL (Borkhardt et al., 2001; Cerveira et al., 2006; Fu et al., 2003; Kadkol et al., 2006; Kim et al., 2004; Kojima et al., 2004; Kreuziger et al., 2007; Megonigal et al., 1998; Ono et al., 2002; Slater et al., 2002; Strehl et al., 2006; Taki et al., 1999; van Binsbergen et al., 2007; Yamamoto et al., 2002). This fusion protein is thought to contribute to the progression of leukemia by the overactivation of MLL leading to unwanted transcription of certain genes including members of the HOX family. The contribution of septins to this activation is not understood, but may involve their self-interacting properties which could lead to dimerization of MLL. In addition, their association with the membrane or with other components of the cytoskeleton could also play a role.

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5.3.2 Lymphoma SEPT9 was also associated with lymphoma when it was identified as a locus commonly targeted in mice by the T-cell lymophoma inducing virus SL3-3. The frequent association of insertion into the SEPT9 locus in T-cell lymphomas led to the suggestion that the SEPT9 locus may be a protooncogene (Sorensen et al., 2000). 5.3.3 Breast and ovarian cancer In addition to leukemias and lymphomas, the SEPT9 locus has also been implicated in sporadic human breast and ovarian cancers. The human SEPT9 locus was determined to be a hot spot for allelic alterations in ovarian and breast cancer (Kalikin et al., 2000; Russell et al., 2000). In addition, amplification of the locus was observed in human and mouse breast cancer cell lines (Montagna et al., 2003) and during tumor progression (Connolly et al., 2011). Although no mutations were found in the coding sequence of the gene, changes in the overall expression level and of specific isoforms have been observed (Burrows et al., 2003; Gonzalez et al., 2007; Montagna et al., 2003; Scott et al., 2006). For example, it is frequently seen that SEPT9 is upregulated in ovarian and breast tumors (Montagna et al., 2003). Similarly, upregulation of SEPT9_i1 has also been seen (Amir and Mabjeesh, 2007; Gonzalez et al., 2007; Scott et al., 2005, 2006). While these results may appear contradictory, deregulation of the balance of SEPT9 isoforms may be the critical feature and overexpression of individual isoforms may contribute differently to cancer progression. For example, overexpression of SEPT9_i4 increases cell migration (Chacko et al., 2005) and may link to metastatic properties, while overexpression of SEPT9_i1 inhibits the action of microtubule destabilizing drugs and may support tumor survival following chemotherapy (Amir and Mabjeesh, 2007; Chacko et al., 2012). In addition, SEPT9_i1 stabilizes Jun kinase, increasing signaling through this proliferative signaling pathway (Gonzalez et al., 2009). 5.3.4 Head and squamous carcinoma Head and squamous carcinoma is a cancer of the head and neck region where the tumor originated as squamous epithelial cells. High expression of SEPT9_i1 has been associated with poor outcomes of this cancer (Stanbery et al., 2010) and the SEPT9 locus is frequently methylated in this disease (Bennett et al., 2008) suggesting that, as in breast cancer, alterations in SEPT9 isoforms may contribute to malignant phenotypes. In addition, elevated expression of SEPT1 has also been observed in squamous cancer

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Table 7.2 Circumstantial links connecting different septin members to head and squamous carcinoma Septin Link to head and neck squamous carcinoma

Septin 1

Spectral Karyotyping identified involvement in Carcinoma (Squire et al., 2002)

Septin 3

Located between the DIA1 gene and microsatellite marker D22S274, both of which were seen lost in patients with Carcinoma suggesting the lost of Septin 3 as well (Reis et al., 2002)

Septin 6

Circumstantial evidence show a link between a mutation of this gene with esophageal carcinoma (Ueno et al., 2002)

Septin 12 Mapped to a locus frequently deleted in esophageal carcinoma (Hirasaki et al., 2007) Septin 14 Located at a locus that is involved in tongue carcinoma (Tsui et al., 2009)

cell lines and in some tumors (Mizutani et al., 2013). These results are summarized in Table 7.2. 5.3.5 Colorectal cancer Colorectal cancer is the third most commonly diagnosed cancer in the world and is frequently fatal due to its typically late detection. As with breast, ovarian and squamous cancers, colorectal cancers show an altered pattern of SEPT9 isoform expression. Specifically, expression of splice variant 1 of SEPT9 was reduced, while splice variants 2, 4, 4*, and 5 were elevated in the cancerous epithelial cells of the patients (Toth et al., 2011). The decrease in SEPT9 expression is likely due to methylation at the CpG islands within the SEPT9 promoter which decreases its transcriptional frequency. Consistent with this, treatment of cells in culture with demethylating agents led to an increase in SEPT9 mRNA and protein levels (Toth et al., 2011). This methylation-based alteration in SEPT9 expression has been recently used to develop diagnostic colon cancer screens using a blood-based assay for methylation of the SEPT9 promoter (Lofton-Day et al., 2008). This promising method has the potential to allow earlier diagnoses since it is less invasive and would therefore likely to have a higher participation rate than colonoscopy. Hence, not only do these findings point to a new therapeutic approach, but have led to a novel, effective, and noninvasive diagnostic protocol for the detection of colorectal cancer.

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5.3.6 Possible mechanistic links to cancer 5.3.6.1 Aneuploidy

A link between septins and cancer might be expected, given their roles also in cell division, yet the lines of evidence supporting an association between septins and cancer is largely circumstantial. An obvious link would be that defects in chromosome segregation or cell division, functions septins have been implicated in, as described previously, could lead to aneuploidy. Aneuploidy is commonly associated with cancer progression and tetraploidy, as would arise following a failure of cell division, and has been linked to malignant transformation in a mouse model (Fujiwara et al., 2005). In the following section, we will outline the examples where septins have been associated with cancer and discuss their potential roles in the disease.

5.3.6.2 Metastasis

One way that septins could contribute to cancer progression is through tumor metastasis as septins are found to be involved in cell migration. Metastasis commonly involves a shift of the cancerous epithelial cell to a motile mesenchymal state to allow for cell migration (through a process called mesenchymal-epithelial transition). The spread of tumors requires the formation of a pseudopodial protusion and invadapodia to give the cancerous cell a more intrusive behavior as well as migration abilities to invade and travel to the new organ. This is dependent on a dynamic actin cytokskeleton as metastatic tumors showed an increase expression of actin-regulator genes. One study looked at pseudopod specific proteins in six metastatic epithelial cell lines of different tumorigenic origin, and identified SEPT9 as one of those proteins (Shankar et al., 2010). Depletion of SEPT9 in metastatic cancer cells inhibited its migration and invasion, caused the withdrawal of pseudopods as well as decreased actin dynamics and mesenchymal-epithelial transition (Shankar et al., 2010). The link between SEPT9 and cell migration is complex as specific isoforms of SEPT9 may be involved. One study looked at isoform 4 of SEPT9 and showed that overexpression of this isoform alters actin organization, increased the generation of protrusions around the cell, and increased cell motility where migration lacked directionality (Chacko et al., 2005). Alternatively, other studies have showed that isoform 1 of SEPT9 also increased the motility (Connolly et al., 2011; Gonzalez et al., 2007) as well as invasiveness of cultured cells (Gonzalez et al., 2007). Altogether, these data point to a complex association of SEPT9

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to cell migration. More work will be needed to determine the role of isoform expression in tumor metastasis. 5.3.6.3 Angiogenesis

HIF-1a is a transcription factor that senses low oxygen levels and translocates into the nucleus to activate transcription of a host of important genes including factors important for angiogenesis. SEPT9_i1 was shown to bind specifically to HIF-1a and promote its activity in vitro and in vivo (Amir et al., 2006). Enhancement of this pathway resulted from both increased HIF1a transcriptional activity and also stabilized the protein from degradation (Amir et al., 2009, 2010). One means of SEPT9-mediated activation appears to be through the promotion of HIF-1a association with a importin, which is necessary for the nuclear translocation of HIF-1a and its transcriptional activity (Golan and Mabjeesh, 2013). Hence, one role of septins in cancers may be to promote angiogenesis by activation of the HIF-1a pathway. 5.3.6.4 Failure of apoptosis

As part of a housekeeping mechanism, apoptosis is the process of programmed cell death that is activated when cellular abnormalities are detected. SEPT4_i2, also known as ARTS, was first detected as an apoptosis-related protein in the TGF-b signaling pathway (Larisch et al., 2000). It has been implicated in the apoptotic pathway through its interaction with inhibitor of apoptosis proteins (IAPs) which lead to the activation of certain caspases (Gottfried et al., 2004; Larisch et al., 2000) and ultimately regulate apoptosis. Interestingly, ARTS has been shown to be silent in human leukemia, while the loss of SEPT4 function in mice was shown to support spontaneous leukemia or lymphoma (Garcia-Fernandez et al., 2010), linking this septin family member to cancer. Altogether this characterizes SEPT4_i2 as potentially a tumor suppressor and raises the possibility that one role of septins in cancer may be to affect apoptosis.

6. CONCLUDING REMARKS Septins are a widely conserved, yet so far poorly characterized filamentous component of the cytoskeleton found in diverse organisms. Unlike other cytoskeletal components, they are composed of a mixture of different septin proteins such that different types of filaments could be formed in different tissues through unique combinations of septins. As discussed above, their interactions with membranes, actin, and microtubule structures are

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important in many different biological processes and therefore it is not surprising that they are linked to many human diseases. In particular, their association with cell division and cell migration may explain their frequently altered expression in cancers. Future studies on the biochemical and biological properties of septins will provide new insights into their functions, provide new biomarkers for disease diagnosis, and may ultimately provide new therapeutic targets for a host of diseases.

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