Septins are a family of GTP-binding, membrane-interacting cytoskeletal proteins, highly conserved and essential in all eukaryotes (with the exception of plants). Septins play important roles in a number of cellular events that involve membrane remodeling and compartmentalization. One such event is cytokinesis, the last stage of cell division. While cytokinesis is ultimately achieved via the mechanical contraction of an actomyosin ring at the septum, determination of the location where cytokinesis will take place, and recruitment of factors involved in signaling events leading to septation requires the activity of septins. We are working towards dissecting the properties of septins from the budding yeast Saccharomyces cerevisiae, where they were first discovered as cell cycle mutants. In our studies we have employed several complementary electron microscopy techniques to describe the organization and structure of septins both in vitro and in situ.

Septins are essential for the completion of cytokinesis. In budding yeast, Saccharomyces cerevisiae, septins are located at the bud neck during mitosis and are closely connected to the inner plasma membrane. In vitro, yeast septins have been shown to self-assemble into a variety of filamentous structures, including rods, paired filaments, bundles, and rings (Bertin et al. Proc Natl Acad Sci U S A, 105(24):8274-8279, 2008; Garcia et al. J Cell Biol, 195(6):993-1004, 2011; Bertin et al. J Mol Biol, 404(4):711-731, 2010). Using electron tomography of freeze-substituted sections and cryo-electron tomography of frozen sections, we determined the three-dimensional organization of the septin cytoskeleton in dividing budding yeast with molecular resolution (Bertin et al. Mol Biol Cell, 23(3):423-432, 2012; Bertin and Nogales. Commun Integr Biol 5(5):503-505, 2012). Here, we describe the detailed procedures used for our characterization of the septin cellular ultrastructure.

Septins are conserved guanosine triphosphate-binding cytoskeletal proteins involved in membrane remodeling. In budding yeast, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1), which are essential for cytokinesis, transition during bud growth from a patch to a collar, which splits into two rings in cytokinesis and is disassembled before the next cell cycle. Cdc3, Cdc10, Cdc11, and Cdc12 form an apolar octameric rod with Cdc11 at each tip, which polymerizes into straight paired filaments. We show that Shs1 substitutes for Cdc11, resulting in octameric rods that do not polymerize into filaments but associate laterally, forming curved bundles that close into rings. In vivo, half of shs1Δ mutant cells exhibit incomplete collars and disrupted neck filaments. Importantly, different phosphomimetic mutations in Shs1 can either prevent ring formation or promote formation of a gauzelike meshwork. These results show that a single alternative terminal subunit is sufficient to confer a distinctive higher-order septin ultrastructure that can be further regulated by phosphorylation.

Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys-less septins--or where one of the Cys-less septins contained a single Cys introduced at a new site--displayed assembly properties in vitro indistinguishable from wild-type.

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.