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.

Biochemical characterization of a human septin octamer

“…This is reflected by the concentration dependency of the hexamer curve shifts in our assay. Whether SEPT9 is a bundling factor on its own is currently under debate (Mavrakis et al 2014;Iv et al 2021;. Octamers, but not hexamers have an influence on actin monomer turnover in the elongation phase as detected by our assay.…”

“…The C-terminus of SEPT9 contributes to filament formation Human hexameric septin rods are available for in vitro experimentation and even crystallization since several years (Sheffield et al 2003;Sirajuddin et al 2007). However, purified octameric rods containing also SEPT9 were only very recently obtained (Iv et al 2021). We introduce here an additional simple, two step purification protocol for human octameric rods harboring a hexa-histidine tag at only one of its subunits.…”

“…Human septin complexes interfere with actin in the early stages of polymerization Several studies have already demonstrated the capability of SEPT9, septin hexamers and octamers to bind and bundle filamentous actin (Mavrakis et al 2014;Iv et al 2021;. All these studies monitored the effects of septins on filamentous actin.…”

“…All other structures represent septin monomers or dimers. An octameric rod for in vitro experimentation was only presented recently (Iv et al 2021) and in this work. We present here a first biochemical characterization of the nucleotide binding-and hydrolysis properties of purified human octameric septin rods as well as a kinetic assay that allows to measure the influence of septin complexes on actin filament assembly in real time.…”

DataSheet1.pdf

“…For many years, the subunit order of the human hexamer (and therefore that implied for the octamer) was erroneously considered to be SEPT7-6-2-2-6-7. New studies have shown the correct order to be SEPT2-6-7-7-6-2 (Mendonça et al, 2019;Mendonça et al, 2021) or SEPT2-6-7-3-3-7-6-2 in the case of octamers (Figure 1B; DeRose et al, 2020;Soroor et al, 2021;Iv et al, 2021).…”