Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10 -Cdc10 -Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel ''railroad tracks.'' Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.electron microscopy ͉ yeast ͉ complexes ͉ GTP

Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear hetero-oligomeric 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 hetero-octameric rod that polymerizes into filaments that are thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin-membrane interaction, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microcopy. Using this methodology we have acquired new insights concerning 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 presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions 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.

Substitution of specific terminal subunits within septin complexes and septin phosphorylation drive the formation of distinct higher-order septin assemblies in budding yeast.

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.