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.     

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.     

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.     

Deciphering the rules governing assembly order of mammalian septin complexes

Septins are conserved GTP-binding proteins that assemble into lateral diffusion barriers and molecular scaffolds. Vertebrate genomes contain 9-17 septin genes that encode both ubiquitous and tissue-specific septins. Expressed septins may assemble in various combinations through both heterotypic and homotypic G-domain interactions. However, little is known regarding assembly states of mammalian septins and mechanisms directing ordered assembly of individual septins into heteromeric units, which is the focus of this study. Our analysis of the septin system in cells lacking or overexpressing selected septins reveals interdependencies coinciding with previously described homology subgroups. Hydrodynamic and single-particle data show that individual septins exist solely in the context of stable six- to eight-subunit core heteromers, all of which contain SEPT2 and SEPT6 subgroup members and SEPT7, while heteromers comprising more than six subunits also contain SEPT9. The combined data suggest a generic model for how the temporal order of septin assembly is homology subgroup-directed, which in turn determines the subunit arrangement of native heteromers. Because mammalian cells normally express multiple members and/or isoforms of some septin subgroups, our data also suggest that only a minor fraction of native heteromers are arranged as perfect palindromes.     

Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast

Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its “G interface”) cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un­expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its “unfoldase” activity, indicating a latent “holdase” activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases.     

Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis

Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.     

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Two non-hydrolyzable analogs of GTP, guanylyl-β,γ-methylene diphosphonate and guanylyl imidodiphosphate, have been found to induce rapid and efficient microtubule assembly in vitro by binding at the exchangeable site (E-site) on tubulin. Characterization of microtubule polymerization by several criteria, including polymerization kinetics, nucleotide binding to depolymerized and polymerized microtubules, and microtubule stability, reveals strong similarities between microtubule assembly induced by GTP and non-hydrolyzable GTP analogs. Nucleoside triphosphates which bind weakly or not at all to tubulin, such as ATP, UTP and CTP, are shown to induce microtubule assembly by means of a nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6.) activity which is not intrinsic to tubulin. The NDP-kinase mediates microtubule polymerization by phosphorylating tubulin-bound GDP in situ at the E-site. Although hydrolysis of exchangeably bound GTP occurs, it is found to be uncoupled from the polymerization reaction. The non-exchangeable nucleotide binding site on tubulin (N-site) is not directly involved in microtubule assembly in vitro. The N-site is shown to contain almost exclusively GTP which is not hydrolyzed during microtubule assembly. A scheme is presented in which GTP acts as an allosteric effector at the E-site during microtubule assembly in vitro.     

Characterization of engineered actin binding proteins that control filament assembly and structure

Background

Eukaryotic cells strictly regulate the structure and assembly of their actin filament networks in response to various stimuli. The actin binding proteins that control filament assembly are therefore attractive targets for those who wish to reorganize actin filaments and reengineer the cytoskeleton. Unfortunately, the naturally occurring actin binding proteins include only a limited set of pointed-end cappers, or proteins that will block polymerization from the slow-growing end of actin filaments. Of the few that are known, most are part of large multimeric complexes that are challenging to manipulate.

Methodology/Principal Findings

We describe here the use of phage display mutagenesis to generate of a new class of binding protein that can be targeted to the pointed-end of actin. These proteins, called synthetic antigen binders (sABs), are based on an antibody-like scaffold where sequence diversity is introduced into the binding loops using a novel “reduced genetic code” phage display library. We describe effective strategies to select and screen for sABs that ensure the generated sABs bind to the pointed-end surface of actin exclusively.

Conclusions/Significance

From our set of pointed-end binders, we identify three sABs with particularly useful properties to systematically probe actin dynamics: one protein that caps the pointed end, a second that crosslinks actin filaments, and a third that severs actin filaments and promotes disassembly.     

The role of Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast

The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth.     

Regulation of septin organization and function in yeast

Septins are a conserved eukaryotic family of GTP-binding filament-forming proteins with functions in cytokinesis and other processes. In the budding yeast Saccharomyces cerevisiae, septins initially localize to the presumptive bud site and then to the cortex of the mother-bud neck as an hourglass structure. During cytokinesis, the septin hourglass splits and single septin rings partition with each of the resulting cells. Septins are thought to function in diverse processes in S. cerevisiae, mainly by acting as a scaffold to direct the neck localization of septin-associated proteins.     

Interplay of septin amphipathic helices in sensing membrane-curvature and filament bundling

The curvature of the membrane defines cell shape. Septins are GTP-binding proteins that assemble into heteromeric complexes and polymerize into filaments at areas of micron-scale membrane curvature. An amphipathic helix (AH) domain within the septin complex is necessary and sufficient for septins to preferentially assemble onto micron-scale curvature. Here we report that the nonessential fungal septin, Shs1, also has an AH domain capable of recognizing membrane curvature. In a septin mutant strain lacking a fully functional Cdc12 AH domain (cdc12-6), the C-terminal extension of Shs1, containing an AH domain, becomes essential. Additionally, we find that the Cdc12 AH domain is important for regulating septin filament bundling, suggesting septin AH domains have multiple, distinct functions and that bundling and membrane binding may be coordinately controlled.     

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.     

Septin9 is involved in septin filament formation and cellular stability

Septin9 (Sept9) is a member of the filament-forming septin family of structural proteins and is associated with a variety of cancers and with hereditary neuralgic amyotrophy. We have generated mice with constitutive and conditional Sept9 knockout alleles. Homozygous deletion of Sept9 results in embryonic lethality around day 10 of gestation whereas mice homozygous for the conditional allele develop normally. Here we report the consequences of homozygous loss of Sept9 in immortalized murine embryonic fibroblasts. Proliferation rate was not changed but cells without Sept9 had an altered morphology compared to normal cells, particularly under low serum stress. Abnormal, fragmented, and multiple nuclei were more frequent in cells without Sept9. Cell migration, as measured by gap-filling and filter-invasion assays, was impaired, but individual cells did not move less than wild-type cells. Sept9 knockout cells showed a reduced resistance to hypo-osmotic stress. Stress fiber and vinculin staining at focal adhesion points was less prominent. Long septin filaments stained for Sept7 disappeared. Instead, staining was found in short, often curved filaments and rings. Furthermore, Sept7 was no longer localized to the mitotic spindle. Together, these data reveal the importance of Sept9 for septin filament formation and general cell stability.     

Modular complexes that regulate actin assembly in budding yeast

The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts. For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell. Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles. The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.     

The cytokinesis formins from the nematode worm and fission yeast differentially mediate actin filament assembly

Formins drive actin filament assembly for diverse cellular processes including motility, establishing polarity, and cell division. To investigate the mechanism of contractile ring assembly in animal cells, we directly compared the actin assembly properties of formins required for cytokinesis in the nematode worm early embryo (CYK-1) and fission yeast (Cdc12p). Like Cdc12p and most other formins, CYK-1 nucleates actin filament assembly and remains processively associated with the elongating barbed end while facilitating the addition of profilin-actin above the theoretical diffusion-limited rate. However, specific properties differ significantly between Cdc12p and CYK-1. Cdc12p efficiently nucleates filaments that in the presence of profilin elongate at approximately the same rate as control filaments without formin (approximately 10.0 subunits/s). CYK-1 is an inefficient nucleator but allows filaments to elongate profilin-actin 6-fold faster than Cdc12p (approximately 60 subunits/s). Both Cdc12p and CYK-1 bind to pre-assembled actin filaments with low nanomolar affinity, but CYK-1 dissociates 2 orders of magnitude more quickly. However, CYK-1 rapidly re-associates with free barbed ends. Cdc12p allows barbed ends to elongate in the presence of excess capping protein, whereas capping protein inhibits CYK-1-mediated actin assembly. Therefore, these evolutionarily diverse formins can drive contractile ring assembly by a generally similar mechanism, but cells with unique dimensions and physical parameters might require proteins with carefully tuned actin assembly properties.     

Self assembly of human septin 2 into amyloid filaments

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in β-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.