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

Carla Silva Martins, Cyntia Taveneau, Gerard Castro-Linares, Mikhail Baibakov, Nicolas Buzhinsky, Mar Eroles, Violeta Milanović, Francois Iv, Léa Bouillard, Alex Llewellyn, Maxime Gomes, Mayssa Belhabib, Mira Kuzmić, Pascal Verdier-Pinard, Stacey Lee, Ali Badache, Sanjay Kumar, Cristel Chandre, Sophie Brasselet, Felix Rico, Olivier Rossier, Gijsje H. Koenderink, Jerome Wenger, Stéphanie Cabantous, Manos Mavrakis
Preprint bioRxiv. 2022-02-24; :
DOI: 10.1101/2022.02.23.481653

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.

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