Abstract:

The spectrin-actin membrane skeleton is a ubiquitous membrane-associated 2D cytoskeleton underneath the lipid membrane of metazoan cells. It promotes the clustering and spatial organization of membrane proteins, thereby regulating the functions of the plasma membrane and its associated proteins. Here we performed structural analysis of the membrane skeleton in its intact networked form (isolated from erythrocytic plasma membrane) using cryo-EM. We determined the high-resolution structures of the spectrin-actin junctional complex, a highly decorated short F-actin that serves as the central organizational unit of membrane skeleton. These structures reveal general principles underlying the organization and assembly of the skeleton and elucidate specific molecular roles of the junctional complex components. Intriguingly, our structural analysis revealed a novel skeleton factor, SH3BGRL2, which caps the pointed end of short F-actin. This finding highlights the power of  ex vivo structural analysis of endogenous macromolecular assemblies in dissecting their organizations and mechanisms. Subsequent  in vivo pull-down assays confirmed that SH3BGRL2 serves as a universal F-actin capping factor, underscoring its broader biological significance. In this talk, I will also show my efforts over the past decade to advance and integrate cryo-EM methods in overcoming the challenges of structural studies on membrane-associated macromolecular assemblies and the regulation of DNA replication.