Membrane binding controls ordered self-assembly of animal septins

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.

DataSheet1.pdf

“…The wider spacing is compatible with the coiled coils of the SEPT6-and SEPT7-group members (8-11 nm) when taking into account contributions from unstructured parts of the C-domain (C N and C C ) (Leonardo et al, 2021). The former has also been proposed to arise from lateral contacts between G-domains (Szuba et al, 2021). However, in yeast, deleting the C-domain of Cdc11 or Cdc3/Cdc12 eliminated tighly-and loosely-paired filaments, respectively, supporting the involvement of specific C-domains in maintaining each type of spacing (Bertin et al, 2010).…”

“…They have also been associated with the formation of paired filaments, gauzes and stacked filament structures (Bertin et al, 2008;Bertin et al, 2010;Jiao et al, 2020;Szuba et al, 2021). Two kinds of spacing between filaments have been reported in vitro both in yeast and in mammalian septins: tight (∼5 nm) (Bertin et al, 2010;Jiao et al, 2020; Leonardo et al, 2021;Szuba et al, 2021) and loose (15-20 nm) (Frazier et al, 1998;Bertin et al, 2008Bertin et al, , 2010Leonardo et al, 2021). The former resembles the lengths of the coiled coils of the SEPT2-group members (4-5 nm).…”

“…Although apparently important for the stabilization of the NCinterface in the parallel orientation, the C-domains are not required for polymerization (Sirajuddin et al, 2007;Szuba et al, 2021), indicative of an additional role. They have also been associated with the formation of paired filaments, gauzes and stacked filament structures (Bertin et al, 2008;Bertin et al, 2010;Jiao et al, 2020;Szuba et al, 2021).…”

“…Although apparently important for the stabilization of the NCinterface in the parallel orientation, the C-domains are not required for polymerization (Sirajuddin et al, 2007;Szuba et al, 2021), indicative of an additional role. They have also been associated with the formation of paired filaments, gauzes and stacked filament structures (Bertin et al, 2008;Bertin et al, 2010;Jiao et al, 2020;Szuba et al, 2021). Two kinds of spacing between filaments have been reported in vitro both in yeast and in mammalian septins: tight (∼5 nm) (Bertin et al, 2010;Jiao et al, 2020; Leonardo et al, 2021;Szuba et al, 2021) and loose (15-20 nm) (Frazier et al, 1998;Bertin et al, 2008Bertin et al, , 2010Leonardo et al, 2021).…”

“…Nowadays, it is known that in animal cells, septins may be found at a variety of locations, depending on the type and stage of cell development and may also act to restrict the diffusion of membrane components and rigidify the cell cortex at specific sites (Barral et al, 2000;Spiliotis and Gladfelter, 2012;Palander et al, 2017;Spiliotis, 2018;Spiliotis and McMurray, 2020). It is therefore not surprising that septins are involved in many important cellular processes, such as cytokinesis, phagocytosis, ciliogenesis, cytoskeletal dynamics during bacterial entrapment, barrier formation, cellular polarization and morphogenesis, besides several others that demand membrane remodeling and/or scaffolding capabilities (Longtine et al, 2000;Hu et al, 2010;Mostowy et al, 2010;Mostowy and Cossart, 2012;Ewers et al, 2014;Beber et al, 2019;Falk et al, 2019;Robertin and Mostowy, 2020;Szuba et al, 2021).…”