Ultrastructural Organization of Budding Yeast Septin Filaments Both in vitro and in situ, Analyzed by Electron Microscopy

Abstract

Septins have been discovered more than 30 years ago as temperature sensitive mutants in budding yeast Saccharomyces Cerevisiae. Septins make an hourglass shaped structure of filaments bound to the inner cell membrane. Mitotic budding yeasts express five septins: Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7. All, but Shs1, are essential for cell division.Using electron microscopy of negatively-stained samples, in vitro, we have observed that the Cdc3-Cdc10-Cdc11-Cdc12 septin complex self-assemble into octameric rods in high salt. At lower ionic strength, septins polymerize into paired filaments. The position and identity of each subunit in the rod has been determined. This analysis revealed a symmetric organization where the different subunits are arranged in the following order: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11. We have also shown that the subunit-subunit interfaces alternate between so called N-C and G interfaces.To get more insight into septin organization in situ we have studied septin-lipid interaction using a lipid monolayer model assay. We have seen that budding yeast septins interact specifically with (PI(4,5)P2). This interaction promotes filament formation and organization, even for mutants or under conditions where septins do not polymerize in solution. This interaction appears to be specifically mediated through Cdc10 and Cdc3.We have been also analyzed the organization of septin filaments in situ, using electron tomography to visualized dividing budding yeast cells. 3D reconstructions of yeast sections were obtained by electron tomography using either freeze substituted samples or cryo-sections (Cemovis). Surprisingly, an array of two sets of perpendicular filaments is present at the bud neck. Cells displaying different types of septin mutations are now being analyzed.