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

Abstract

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9. Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9 thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled the first-time isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin/microtubule cytoskeleton.SummaryHuman septins in cells form hetero-octameric complexes containing the ubiquitously expressed SEPT9. Iv et al. describe the first-time isolation of recombinant human septin octamers with distinct SEPT9 isoforms. Reconstitution studies show that octamers with either a long or a short SEPT9 isoform form higher-order filament assemblies and directly bind and cross-link actin filaments.

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

“…The discrepancy is most likely linked to the fact that in these in vitro experiments, Sept9 was used outside of its natural octameric context, often at high concentrations (up to 10-30 µM in pelleting assays) and using paclitaxel or GMPCPP stabilized microtubules in very low salt buffers, which likely favored electrostatic interactions. Using recombinant octamers harboring Sept9_i1 and Sept9_i3 12 , we observed that only those harboring Sept9_i1 associated with dynamics microtubules at low nanomolar concentrations, in vitro. Our in vitro reconstitution experiments results are the first one to recapitulate the specific binding of Sept9_i1 octamers to microtubules observed in cells here and by others 40,52 .…”

“…Recombinant septin octamers harboring Sept9_i1 interact directly and specifically with microtubules and slow down their depolymerization. In order to assess if recombinant octamers harboring (Oct_9i1) are able to bind directly to microtubules, we purified recombinant septin octamers (Sept2-Sept6-Sept7-Sept9_i1-Sept9_i1-Sept7-Sept6-Sept2 and Sept2-Sept6-Sept7-Sept9_i3-Sept9_i3-Sept7-Sept6-Sept2) 12 (Supplementary Fig. 1b) and reconstituted dynamic microtubules made from purified tubulin /-heterodimers in the presence of each type of octamer.…”

“…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.…”

“…Human septin hexameric and octameric complexes with either Sept9_i1 or Sept9_i3 were produced, purified and analyzed as detailed in Iv et al 12 . No difference in apparent MW was observed between recombinant septin octamers or hexamers and those extracted from cells after native gel separation and WB (Supplementary Fig.…”

“…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] .…”