Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Abstract

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

“…This altered bleb-adjacent localization supports a requirement for inter-oligomer polymerization in the formation of curvature-independent stable septin structures, and suggests polymerized septin structures have an increased affinity for non-bleb plasma membrane. This bleb exclusion might be driven by polymerization-dependent crosslinking of the plasma membrane and actomyosin cortex by septins and associated factors, as has been previously observed 62 .…”

Septins and K63 chains form separate bacterial microdomains during autophagy of entrappedShigella

“…Note that HeLa cells produce SEPT6 and SEPT7, that together with SEPT2 and SEPT9, produce a hetero-oligomer SEPT2-SEPT6-SEPT7-SEPT9 that assembles into filaments and higher-order structures (Kim et al, 2011; Mostowy and Cossart, 2012). SEPT7 is a septin essential for heter-oligomer formation, and therefore SEPT7 staining represent the cellular distribution of the other septins (Martins et al, 2022). Cells were then washed 3 − 6X in PBS and incubated 45 min with Alexa-555-conjugated anti-rabbit or Alexa-647-conjugated anti-rabbit secondary antibody diluted 0.1% Triton X-100 (in PBS).…”

A specific mesh-like organization of human septin octameric complex drives membrane reshaping and curvature sensitivity

“…In humans, 13 septin subunits are expressed in a tissue dependent manner 15 . They can assemble into linear palindromic complexes either hexameric or octameric 16 . Hence, multiple septin subunit combinations can possibly be observed.…”

“…In budding yeast, septins were shown to be essential for cell division and localized at the constriction sites between the mother cell and the bud 8 . As opposed to yeast septins which have been already thoroughly studied using bottom-up in vitro assays, the behavior of human septins at the membrane has not been examined in controlled environments in vitro and barely examined in cellular contexts 16 . Indeed, until very recently human septin octamers including the essential Sept9 were not available for in vitro studies.…”