Cytoskeleton club - Feng-Ching Tsai

Speaker:  Feng-Ching Tsai

Title: ASYMMETRIC BRANCHED ACTIN ASSEMBLY DRIVEN BY N-WASP AROUND A MEMBRANE NECK LEADS TO NECK BREAKAGE

Simli Dey¹; Ann-Sophie Mace¹; Aurelie Bertin^1,2; John Manzi¹; Patricia Bassereau¹; Christophe Le Clainche ² Julien Berro³; Julien Heuvingh⁴; Feng-Ching Tsai¹

¹Curie Institute, Physics of Cells and Cancer (PCC), PARIS, France;

 ²Université Paris-Saclay, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France; 

³Yale University, epartment of Molecular Biophysics & Biochemistry, Department of Cell Biology, New Haven, CT, USA; 

⁴Université Paris Diderot, Laboratoire PMMH, Paris, France 

Abstract: Actin assembly plays a critical role in various membrane remodelling processes, including intracellular trafficking. In this process, the membranes often form vesicular buds connected to the plasma membrane by saddle-shaped necks. However, the molecular mechanisms regulating actin assembly at these membrane necks remain elusive. Moreover, it is still unclear how this dense actin network produces force to facilitate membrane fission at the necks. To address this fundamental question, in this work, we developed a reconstituted system featuring saddle-shaped membrane necks, generated from wrapping the lipid bilayers around beads grafted onto a glass substrate. The formation of these curved membrane necks at the base of the beads was confirmed by the enrichment of the curvature sensing BAR protein SNX9. Our results show that upon introducing actin nucleation-promoting factor N-WASP, together with Arp2/3 complex and actin monomers, branched actin network assembles asymmetrically around the membrane necks. This asymmetric actin assembly exert force on the necks, leading to their displacement, as evidenced by the lateral and vertical displacement of the beads. This suggests that the neck structure was disrupted. In contrast, when using a truncated formed of N-WASP (pWA), which still activates Arp2/3, the actin network grows symmetrically around the necks while no bead displacement was observed. These findings align with recent reports showing that in mammalian cells, actin assembly at stalled clathrin-mediated endocytosis sites is asymmetric, acting like a “bottle opener” to pull off endocytic buds. Together, our findings provide new insights into how actin nucleation-promoting factors spatially regulate actin assembly at complex membrane geometries, advancing our understanding of processes such as endocytosis.