Abstract
The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP-WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.
Highlights
Actin dynamics are essential for the eukaryotic cell in providing a framework for organizing the membrane and producing force for vesicle deformation, transport of endocytic vesicles, cytokinesis, and cell protrusions, among other functions
Our previous work showed that the coincidence of PI(4,5)P2, PI(3)P, and membrane curvature stimulates actin polymerization in Xenopus egg extracts in an SNX9-dependent manner (Gallop et al, 2013)
We have discovered and fully reconstituted a SNX9-dependent signaling pathway that links its unique ability to coincidently detect PI(4,5)P2, PI(3)P, and membrane curvature with recruitment and activation of Cdc42, neural Wiskott-Aldrich syndrome protein (N-WASP), and Arp2/3 complex to trigger actin polymerization (Fig. 8 D)
Summary
Actin dynamics are essential for the eukaryotic cell in providing a framework for organizing the membrane and producing force for vesicle deformation, transport of endocytic vesicles, cytokinesis, and cell protrusions, among other functions. How the array of actin nucleators, elongators, and bundlers work together to orchestrate actin assembly when and where it is needed remains a key question (Skau and Waterman, 2015). Clathrin-mediated endocytosis (CME) in mammalian cells provides a striking example of the need for regulated actin assembly. Though it is well established that actin plays a critical role for endocytosis in yeast, where it drives inward invagination. M.R. Holst, and J.R. Gadsby contributed to this paper
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