Phosphatidylinositol polyphosphate binding to the mammalian septin H5 is modulated by GTP

BACKGROUND: Septins are members of a conserved family of GTPases found in organisms as diverse as budding yeast and mammals. In budding yeast, septins form hetero-oligomeric filaments that lie adjacent to the membrane at the mother-bud neck, whereas in mammals, they concentrate at the cleavage furrow of mitotic cells; in both cases, septins provide a required function for cytokinesis. What directs the location and determines the stability of septin filaments, however, remains unknown. RESULTS: Here we show that the mammalian septin H5 is associated with the plasma membrane and specifically binds the phospholipids phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P(2)) and phosphatidylinositol 3,4, 5-trisphosphate (PtdIns(3,4,5)P(3)). Deletion analysis revealed that this binding occurs at a site rich in basic residues that is conserved in most septins and is located adjacent to the GTP-binding motif. Phosphoinositide binding was inhibited by mutations within this motif and was also blocked by agents known to associate with PtdInsP(2) or by a peptide corresponding to the predicted PtdInsP(2)-binding sequence of H5. GTP binding and hydrolysis by H5 significantly reduced its PtdInsP(2)-binding capability. Treatment of cells with agents that occluded, dephosphorylated or degraded PtdInsP(2) altered the appearance and localization of H5. CONCLUSIONS: These results indicate that the interaction of septins with PtdInsP(2) might be an important cellular mechanism for the spatial and temporal control of septin accumulation.     

Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization

Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order structures. In budding yeast (Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin-membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation in vitro via atypical Cdc12-Cdc12 and Cdc3-Cdc3 interactions, respectively.     

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.     

Polymerization of Purified Yeast Septins: Evidence That Organized Filament Arrays May Not Be Required for Septin Function

The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types. In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother–bud neck, called the neck filaments. In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro. To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11. These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Δ and cdc11Δ cells did not contain detectable neck filaments. These results strengthen the hypothesis that the septins are the major structural components of the neck filaments. Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Δ cells. This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.     

Dynamics of septin ring and collar formation in Saccharomyces cerevisiae

Although the septin ring and collar in budding yeast were described over 20 years ago, there is still controversy regarding the organization of septin filaments within these structures and about the way in which the ring first forms and about how it converts into a collar at the mother-bud neck. Here we present quantitative analyses of the recruitment of fluorescently-tagged septins to the ring and collar through the cell cycle. Septin ring assembly began several minutes after polarity establishment and this interval was longer in daughter than in mother cells, suggesting asymmetric inheritance of septin regulators. Septins formed an initial faint and irregular ring, which became more regular as septins were recruited at a constant rate. This steady rate of septin recruitment continued for several minutes after the ring converted to a collar at bud emergence. We did not detect a stepwise change in septin fluorescence during the ring-to-collar transition. After collar formation, septins continued to accumulate at the bud neck, though at a reduced rate, until the onset of cytokinesis when the amount of neck-localized septins rapidly decreased. Implications for the mechanism of septin ring assembly are discussed.     

Nucleotide binding and filament assembly of recombinant yeast septin complexes

Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the gamma-phosphate.     

Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly

In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck.     

GTP binding induces filament assembly of a recombinant septin

The septins are a family of GTPases involved in cytokinesis in budding yeast, Drosophila, and vertebrates (see for review). Septins are associated with a system of 10 nm filaments at the S. cerevisiae bud neck, and heteromultimeric septin complexes have been isolated from cell extracts in a filamentous state. A number of septins have been shown to bind and hydrolyze guanine nucleotide. However, the role of GTP binding and hydrolysis in filament formation has not been elucidated. Furthermore, several lines of evidence suggest that not all the subunits of the septin complex are required for all aspects of septin function. To address these questions, we have reconstituted filament assembly in vitro by using a recombinant Xenopus septin, Xl Sept2. Filament assembly is GTP dependent; moreover, the coiled-coil domain common to most septins is not essential for filament formation. Septin polymerization is preceded by a lag phase, suggesting a cooperative assembly mechanism. The slowly hydrolyzable GTP analog, GTP-gamma-S, also induces polymerization, indicating that polymerization does not require GTP hydrolysis. If the properties of Xl Sept2 filaments reflect those of native septin complexes, these results imply that the growth or stability of septin filaments, or both, is regulated by the state of bound nucleotide.     

A purified Drosophila septin complex forms filaments and exhibits GTPase activity

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.     

Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4

Assembly at the mother-bud neck of a filamentous collar containing five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) is necessary for proper morphogenesis and cytokinesis. We show that Cdc10 and Cdc12 possess GTPase activity and appropriate mutations in conserved nucleotide-binding residues abrogate GTP binding and/or hydrolysis in vitro. In vivo, mutants unable to bind GTP prevent septin collar formation, whereas mutants that block GTP hydrolysis do not. GTP binding-defective Cdc10 and Cdc12 form soluble heteromeric complexes with other septins both in yeast and in bacteria; yet, unlike wild-type, mutant complexes do not bind GTP and do not assemble into filaments in vitro. Absence of a p21-activated protein kinase (Cla4) perturbs septin collar formation. This defect is greatly exacerbated when combined with GTP binding-defective septins; conversely, the septin collar assembly defect of such mutants is suppressed efficiently by CLA4 overexpression. Cla4 interacts directly with and phosphorylates certain septins in vitro and in vivo. Thus, septin collar formation may correspond to septin filament assembly, and requires both GTP binding and Cla4-mediated phosphorylation of septins.     

Protein-protein interactions governing septin heteropentamer assembly and septin filament organization in Saccharomyces cerevisiae

Mitotic yeast (Saccharomyces cerevisiae) cells express five related septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) that form a cortical filamentous collar at the mother-bud neck necessary for normal morphogenesis and cytokinesis. All five possess an N-terminal GTPase domain and, except for Cdc10, a C-terminal extension (CTE) containing a predicted coiled coil. Here, we show that the CTEs of Cdc3 and Cdc12 are essential for their association and for the function of both septins in vivo. Cdc10 interacts with a Cdc3-Cdc12 complex independently of the CTE of either protein. In contrast to Cdc3 and Cdc12, the Cdc11 CTE, which recruits the nonessential septin Shs1, is dispensable for its function in vivo. In addition, Cdc11 forms a stoichiometric complex with Cdc12, independent of its CTE. Reconstitution of various multiseptin complexes and electron microscopic analysis reveal that Cdc3, Cdc11, and Cdc12 are all necessary and sufficient for septin filament formation, and presence of Cdc10 causes filament pairing. These data provide novel insights about the connectivity among the five individual septins in functional septin heteropentamers and the organization of septin filaments.     

Septin Form and Function at the Cell Cortex

Septins are GTP-binding proteins that form filaments and higher-order structures on the cell cortex of eukaryotic cells and associate with actin and microtubule cytoskeletal networks. When assembled, septins coordinate cell division and contribute to cell polarity maintenance and membrane remodeling. These functions manifest themselves via scaffolding of cytosolic proteins and cytoskeletal networks to specific locations on membranes and by forming diffusional barriers that restrict lateral diffusion of proteins embedded in membranes. Notably, many neurodegenerative diseases and cancers have been characterized as having misregulated septins, suggesting that their functions are relevant to diverse diseases. Despite the importance of septins, little is known about what features of the plasma membrane influence septin recruitment and alternatively, how septins influence plasma membrane properties. Septins have been localized to the cell cortex at the base of cilia, the mother-bud neck of yeast, and branch points of filamentous fungi and dendritic spines, in cleavage furrows, and in retracting membrane protrusions in mammalian cells. These sites all possess some degree of curvature and are likely composed of distinct lipid pools. Depending on the context, septins may act alone or in concert with other cytoskeletal elements to influence and sense membrane properties. The degree to which septins react to and/or induce changes in shape and lipid composition are discussed here. As septins are an essential player in basic biology and disease, understanding the interplay between septins and the plasma membrane is critical and may yield new and unexpected functions.     

Guides to the final frontier of the cytoskeleton: septins in filamentous fungi

Recent investigations have established core principles by which septins can form non-polar filaments in vitro. How cells then assemble, regulate and use septin polymers is still only beginning to be understood. It is clear that there is plasticity and variability in septin organization across diverse species and cell types. Work in the filamentous fungi has been invaluable in discovering this variation in form and function. In particular filamentous fungi display many forms of higher order septin structures and study of septins in these systems has led to insights into septin assembly, dynamics and regulation. Importantly in many cases work in these alternative systems reveal differences to how septins may be organized, functioning or regulated in Saccharomyces cerevisiae. Here I review the novel aspects of septin biology found in filamentous fungi and raise many open questions about these enigmatic polymers that should guide future study.     

Borg/septin interactions and the assembly of mammalian septin heterodimers,trimers, and filaments

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.     

Septins recognize micron-scale membrane curvature

How cells recognize membrane curvature is not fully understood. In this issue, Bridges et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512029) discover that septins, a component of the cytoskeleton, recognize membrane curvature at the micron scale, a common morphological hallmark of eukaryotic cellular processes.Eukaryotic cells have dedicated proteins that sense membranes, depending on their curvature (Antonny, 2011). Sensors of membrane curvature are important because they organize a wide variety of cellular functions, including vesicle trafficking and organelle shaping (McMahon and Gallop, 2005). Curvature-sensing proteins, for example, the Bin-Amphiphysin-Rvs (BAR) domain–containing proteins, have been mostly described to work at the nanometer scale (Zimmerberg and Kozlov, 2006). In contrast, a clear mechanism of sensing membrane curvature at the micron scale in eukaryotic cells has not been described. In this issue, Bridges et al. discover that septins, a poorly understood component of the cytoskeleton, recognize plasma membrane curvature at the micron scale and serve as landmarks for eukaryotic cells to know their local shape.Septins are an evolutionarily conserved family of GTP-binding proteins that assemble into nonpolar filaments and rings (John et al., 2007; Sirajuddin et al., 2007; Bertin et al., 2008). Septins have been implicated in diverse membrane organization events where micron-scale curvature takes place (Saarikangas and Barral, 2011; Mostowy and Cossart, 2012), including the cytokinetic furrow, the annulus of spermatozoa, the base of cellular protrusions (e.g., cilium and dendritic spines), and the phagocytic cup surrounding invasive bacterial pathogens (Fig. 1). However, the precise role of septin–membrane interactions remains elusive. It was first suggested in 1999 that the interaction of human septins with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is important for septin localization (Zhang et al., 1999). More recently, work using recombinant septins from budding yeast Saccharomyces cerevisiae assembled on PI(4,5)P2 lipid monolayers showed that septins interact with membrane to facilitate filament assembly (Bridges et al., 2014). Membrane-facilitated septin assembly has also been observed using phospholipid liposomes, and in this case septins were also shown to induce membrane tubulation (Tanaka-Takiguchi et al., 2009). Given that (a) septins can interact with membrane, (b) septin assembly is membrane facilitated, and (c) septin assemblies are associated with a variety of membrane organization events from yeast to mammals, Bridges et al. (2016) hypothesized that septins serve as a mechanism to recognize membrane curvature.Open in a separate windowFigure 1.Morphological hallmarks of eukaryotic cells characterized by micron-scale membrane curvature and septin assembly. Septins have been implicated in membrane organization events where micron-scale curvature takes place. (A) A septin ring acts as a scaffold for cytokinesis proteins and forms a diffusion barrier at the cytokinetic furrow of a dividing cell. (B) A septin ring forms a diffusion barrier at the annulus of a mammalian spermatozoon, which separates the anterior and posterior tail. (C) A septin ring forms a diffusion barrier at the base of a cilium to separate the ciliary membrane from the plasma membrane. (D) In neurons, a septin-dependent diffusion barrier can localize at the base of dendritic spine necks. (E) During phagocytosis, a cup is formed at the plama membrane; septin rings assemble at the base of the phagocytic cup to regulate entry.In their new work, Bridges et al. (2016) provide several lines of evidence to support the hypothesis that septins recognize micron-scale curvature. First, using the filamentous fungus Ashbya gossypii, they performed in vivo localization studies and showed that the fungal septin Cdc11a concentrates in regions of positive micron-scale curvature and that the degree of concentration is proportional to the degree of curvature. Moreover, septins localize to curved membranes that also recruit septin-interacting proteins (e.g., the signaling protein Hsl7). These findings indicate that, by acting as curvature-sensing proteins, septins can localize signaling platforms in the cell. To test if septins can differentiate among micron-scale curvatures, Bridges et al. (2016) developed an elegant model system for septin assembly in vitro. They decorated silica beads with anionic phospholipid bilayers and measured the interaction affinity between purified fungal septin complexes and beads of different curvatures. Interestingly, septins were maximally recruited to “intermediate” sized beads (1.0–3.0 µm in diameter), with little to no recruitment to either very large (5.0–6.5 µm in diameter) or very small (0.3 µm in diameter) beads. These results indicate that septin filaments preferentially localize to a curvature (κ) of 0.7–2.0 µm⁻¹ in the absence of other cellular factors. To provide additional information on the mechanism of sensing, the authors purified mutant septin complexes that fail to polymerize into filaments and showed that the affinity of septins for micron-scale membrane curvature does not require filament formation per se. However, septins must polymerize into filaments for stable membrane association. Collectively, in vivo experiments using A. gossypii and in vitro experiments using silica beads highlight that septins have the intrinsic ability to recognize membrane curvature at the micron scale.Finally, to study the recognition of micron-scale membrane curvature beyond fungi, Bridges et al. (2016) turn their attention to human septins. Using tissue culture cells, they observe that the abundance of septins is associated with the degree of membrane curvature. To confirm these observations in vitro, they purified human septins and analyzed their binding affinity to silica beads with phospholipid bilayers. As seen with A. gossypii septins, human septins also showed a preference for beads ∼1.0 µm in diameter, strongly suggesting an evolutionarily conserved property of septins for sensing membrane curvature at the micron scale.Based on their findings, Bridges et al. (2016) propose that septins provide eukaryotic cells with a mechanism to recognize curvature at the micron scale. This feature differentiates septins from other sensor proteins that strictly detect curvature at the nanometer scale (e.g., BAR domain–containing proteins). However, it is likely that septins do more than recognize membrane, and the precise role of septins in membrane recognition remains unknown. The highly conserved structural and biochemical properties of septins suggest they organize, stabilize, and functionalize membrane domains (Caudron and Barral, 2009; Kusumi et al., 2012; Bridges and Gladfelter, 2015). Although we are far from knowing the full repertoire of septin function, this new work by Bridges et al. (2016) reminds us that understanding how membranes can specify septin assembly is essential to understand the role of septins in eukaryotic cells.     

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) isproposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4)P). We investigated the role these phosphatases in regulating PtdIns(4,5)P2 subcellular distribution. PtdIns(4,5)P2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Deltasac1 Deltainp54 and sac1ts Deltainp54 mutants. Furthermore, sac1ts Deltainp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P2. Under these conditions PtdIns(4,5)P2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P2. Deltasac1 Deltainp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects.     

The Caenorhabditis elegans septin complex is nonpolar

Septins are conserved GTPases that form heteromultimeric complexes and assemble into filaments that play a critical role in cell division and polarity. Results from budding and fission yeast indicate that septin complexes form around a tetrameric core. However, the molecular structure of the core and its influence on the polarity of septin complexes and filaments is poorly defined. The septin complex of the nematode Caenorhabditis elegans is formed entirely by the core septins UNC-59 and UNC-61. We show that UNC-59 and UNC-61 form a dimer of coiled-coil-mediated heterodimers. By electron microscopy, this heterotetramer appears as a linear arrangement of four densities representing the four septin subunits. Fusion of GFP to the N termini of UNC-59 and UNC-61 and subsequent electron microscopic visualization suggests that the sequence of septin subunits is UNC-59/UNC-61/UNC-61/UNC-59. Visualization of GFP extensions fused to the extremity of the C-terminal coiled coils indicates that these extend laterally from the heterotetrameric core. Together, our study establishes that the septin core complex is symmetric, and suggests that septins form nonpolar filaments.     

Localization and possible functions of Drosophila septins.

The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex.     

Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.