Membrane binding controls ordered self-assembly of animal septins.

Agata Szuba,Ralf P Richter,Manos Mavrakis,Francois Iv,Fouzia Bano,Aurélie Bertin,Gerard Castro-Linares,Gijsje H Koenderink

doi:10.7554/elife.63349

Abstract

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

Highlights

Septins are a conserved family of cytoskeletal proteins [1] capable of forming filamentous scaffolds at the cell cortex that participate in many processes such as cytokinesis, cell-cell adhesion, and phagocytosis [2,3,4,5]
We focus on septin hexamers composed of DSep1, DSep2, and Pnut from the model organism Drosophila, which have been previously characterized in vivo [39, 47, 67, 68] and in vitro [39, 56, 57], and which are highly homologous to their human septin orthologs
We investigated the influence of membrane-binding on septin hexamer assembly by reconstituting recombinant animal septins on supported lipid bilayers and imaging septin assembly with several complementary techniques

Summary

Introduction

Septins are a conserved family of cytoskeletal proteins [1] capable of forming filamentous scaffolds at the cell cortex that participate in many processes such as cytokinesis, cell-cell adhesion, and phagocytosis [2,3,4,5]. Electron microscopy of immuno-stained cells revealed localization of cortical septins with cortical actin in tissue culture cells [44, 45], but the high density of the actin cortex in animal cells has made it impossible to determine whether cortical septins directly interact with the plasma membrane. It is even unclear whether cortical septins truly form filaments. Our findings establish that membrane binding catalyzes animal septin polymerization and has a dramatic impact on septin self-assembly, with C-terminal coiled-coils playing a key role in higher-order septin filament organization

Results

Discussion

Materials and Methods