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.

Septins are essential for membrane compartmentalization and remodeling. Electron tomography of yeast bud necks shows filaments perpendicular and parallel to the mother-bud axis that resemble in vitro septin arrays. Filaments are still present, although disordered, in mutants lacking a single septin, underscoring the importance of septin assembly.

A structural characterization of multicomponent cellular assemblies is essential to explain the mechanisms governing biological function. Macromolecular architectures may be revealed by integrating spatial information collected from various biophysical sources. For instance, low-resolution electron cryomicroscopy (cryo-EM) reconstructions of entire assemblies can be interpreted in relation with the crystal structures of the constituent fragments. A simultaneous