The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation

Yashar Sadian,Marian Farkašovský,Oliver Hofnagel,Csilla Patasi,Stefan Raunser,Roger S Goody,Christos Gatsogiannis

doi:10.7554/elife.01085

Abstract

Septins are guanine nucleotide-binding proteins that polymerize into filamentous and higher-order structures. Cdc42 and its effector Gic1 are involved in septin recruitment, ring formation and dissociation. The regulatory mechanisms behind these processes are not well understood. Here, we have used electron microscopy and cryo electron tomography to elucidate the structural basis of the Gic1-septin and Gic1-Cdc42-septin interaction. We show that Gic1 acts as a scaffolding protein for septin filaments forming long and flexible filament cables. Cdc42 in its GTP-form binds to Gic1, which ultimately leads to the dissociation of Gic1 from the filament cables. Surprisingly, Cdc42-GDP is not inactive, but in the absence of Gic1 directly interacts with septin filaments resulting in their disassembly. We suggest that this unanticipated dual function of Cdc42 is crucial for the cell cycle. Based on our results we propose a novel regulatory mechanism for septin filament formation and dissociation. DOI: http://dx.doi.org/10.7554/eLife.01085.001.

Highlights

Septins are ubiquitous guanine nucleotide-binding proteins that have been implicated in many cellular processes such as cytokinesis, spindle positioning, morphogenesis, and exocytosis, and their mutation or overexpression is associated with neoplasia, neurodegenerative diseases and male infertility (Hall and Russell, 2004)
Studying the same but not EGFPlabeled samples using electron microscopy (EM), we found that in contrast to blank septin polymers that form long, often pairwise arranged filaments (Figure 1C), Gic1-septin complexes display a regular railroad-like structure with many cross-linked filaments bundled together (Figure 1D)
Our analysis focuses on only three proteins that are important for septin recruitment, ring formation and disassembly, which are complex processes involving many proteins, we think that the new information provided by our study is coherent enough to suggest the following mechanism for the interplay between Gic1, Cdc42 and septins during budding or the cell cycle

Summary

Introduction

Septins are ubiquitous guanine nucleotide-binding proteins that have been implicated in many cellular processes such as cytokinesis, spindle positioning, morphogenesis, and exocytosis, and their mutation or overexpression is associated with neoplasia, neurodegenerative diseases and male infertility (Hall and Russell, 2004).In yeast, four essential septins (Cdc3, Cdc10, Cdc11 and Cdc12) are found at the bud neck (Haarer and Pringle, 1987; Ford and Pringle, 1991; Kim et al, 1991), where they form an ordered ring composed of membrane-adjacent filaments (Hartwell, 1971; Byers and Goetsch, 1976). To determine which septin subunit interacts with Gic1, we labeled septin-Gic1 complexes with antibodies against Cdc11 and observed that it sits exactly in the middle of a septin filament between two cross-bridges (Figure 2A,B). Gic1 formed a stable complex with Cdc42-GppNHp (non-hydrolysable GTP analogue), but did not bind to Cdc42 in the GDP-state (Figure 5).

Results

Conclusion