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 membrane binding 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 septins 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.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

“…Given the high density of filaments and the 2D projection character of negative-stain EM, we cannot conclude if these are truly paired filaments like budding yeast septin filament pairs (Bertin et al, 2008); if so, these would have to be more tightly paired given their narrow interfilament spacing (~5 nm). Similarly narrow interfilament spacing was observed recently for paired filaments formed by human septin hexamers in solution (Leonardo et al, 2021) and by membrane-bound Drosophila septin hexamers (Szuba et al, 2021). Septin filament bundles exhibited a high degree of interconnectivity, with a given bundle often showing splayed ends that could connect to one or more different bundles, or with septin filaments forming meshes (Fig.…”

Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

“…membranes, actin fibers and microtubules 6 . Septin octamers and hexamers co-associate to form septin filaments 7,8,[10][11][12][13] , whose properties may depend in part on the relative abundance of incorporated octamers and hexamers. In addition, the expression profile of different septin isoforms, including those of Sept9, may direct both localization and function of septin octamers.…”

“…Septins are GTP-binding protein that form heteropolymeric complexes associating with membranes, actin filaments and a subset of microtubules 6 . Like yeast and Drosophila septins, human septin hexameric and octameric protomers can polymerize into filaments [7][8][9][10][11][12][13] . Septins are thought to function as diffusion barriers for protein compartmentalization or as scaffolds for protein-protein interactions during cells division and in an increasing number of processes during interphase [14][15][16] .…”

Spatial regulation of microtubule-dependent transport by septin GTPases

“…Septins associate with microtubules in vitro, and increasing septin concentrations exert biphasic effects on microtubule dynamics, which correlate with a shift from an oligomeric to a higher-order polymeric state [32]. On membrane bilayers, septins form networks of parallel paired filaments whose repeatability and density enable them to function as protein scaffolds [51,52]. Filamentous networks of septins assemble on domains of micro-scale curvature, but septins are also seen in nanometer-scale domains and patches of membrane organelles which may consist of septin oligomers [15,35].…”