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

Abstract

Septins are cytoskeletal proteins conserved from algae and protists to mammals. 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 septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution 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 septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays 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 filaments at the plasma membrane.

The compass to follow: Focal adhesion turnover

“…In addition to, or independently from their actin filament cross-linking activity, septins could stabilize SFs and thus contribute to FA maturation by anchoring SFs at the plasma membrane. Purified mammalian septin hexamers bind lipid membranes [67][68][69], purified human septin octamers can simultaneously bind lipid membranes and actin filaments [63], thus providing actin-membrane anchoring, whereas ventral SF-associated septins are closely opposed to the plasma membrane [63], supporting altogether such a scenario. The identification of the actin-and membrane-binding domains of septins will be key for dissecting their role in FA maturation.…”

“…SEPT2 knockdown in CAFs led to a depletion of actomyosin ventral SFs, which are otherwise heavily decorated by septins in wild-type CAFs [60], whereas SEPT7 and SEPT9 knockdown in melanoma cells also affected the generation of ventral actin SFs, which are also decorated by septins in wild-type cells [61]. SEPT9 knockdown in U2OS cells, or impairing septins from polymerizing altogether, compromised both septin-SF association and actomyosin ventral SF integrity, emphasizing an essential role for septin heterooctamers, but not heterohexamers, in generating or/and maintaining SFs [63].…”

“…SEPT2 knockdown in Cancer-Associated Fibroblasts (CAFs) was further reported to lead to fewer FAs [60], whereas SEPT2 or SEPT9 knockdown in murine and human melanoma cells led to a decrease in FA size [61]. How septins affect FA maturation and thus the lifetime of FAs is not clear, in particular since septins are largely excluded from FAs, as shown by immunostainings in NRK [62], MDCK [59] and U2OS cells [63]. The role of septins in FA stabilization is most likely due to their association with FA-anchored actin SFs.…”

BORG family proteins in physiology and human disease

“…The mechanism for why BORG binding might stabilize septin filaments is currently a mystery. Membrane association plays a role in septin filament stability (Bertin et al, 2010; Bridges et al, 2014) and septins help anchor stress fibers to the membrane (Martins et al, 2023). BORGs do not generally direct septins to the membrane, suggesting they function through an alternative mechanism.…”

Septin filament assembly assist the lateral organization of membranes

“…In the case of septins, their organization into octamer-based filaments is essential for their role as actin-membrane linkers in human cells (20). Furthermore, the density and adhesion strength of the cytoskeletal linkers are crucial parameters to prevent the cell cortex slippage during mechanically active processes, such as actomyosin contraction, and allow sustained membrane deformations (22)(23)(24).…”