Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate.
ABSTRACT Phosphoinositides serve crucial roles in cell physiology, ranging from cell signalling to membrane traffic. Among the seven eukaryotic phosphoinositides the best studied species is phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), which is concentrated at the plasma membrane where, among other functions, it is required for the nucleation of endocytic clathrin-coated pits. No phosphatidylinositol other than PI(4,5)P2 has been implicated in clathrin-mediated endocytosis, whereas the subsequent endosomal stages of the endocytic pathway are dominated by phosphatidylinositol-3-phosphates(PI(3)P). How phosphatidylinositol conversion from PI(4,5)P2-positive endocytic intermediates to PI(3)P-containing endosomes is achieved is unclear. Here we show that formation of phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impairs the maturation of late-stage clathrin-coated pits before fission. Timed formation of PI(3,4)P2 by PI(3)K C2α is required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates. These findings provide a mechanistic framework for the role of PI(3,4)P2 in endocytosis and unravel a novel discrete function of PI(3,4)P2 in a central cell physiological process.
SourceAvailable from: Laura Santambrogio[Show abstract] [Hide abstract]
ABSTRACT: Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-deficient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 flux and dampening of macroautophagy are observed in dendritic cells from Anxa2 À / À mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.Nature Communications 02/2015; 6. DOI:10.1038/ncomms6856 · 10.74 Impact Factor
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ABSTRACT: Intracellular pathogens such as Shigella flexneri and Listeria monocytogenes achieve dissemination in the intestinal epithelium by displaying actin-based motility in the cytosol of infected cells. As they reach the cell periphery, motile bacteria form plasma membrane protrusions that resolve into vacuoles in adjacent cells, through a poorly understood mechanism. Here, we report on the role of the class II phosphatidylinositol 3-phosphate kinase PIK3C2A in S. flexneri dissemination. Time-lapse microscopy revealed that PIK3C2A was required for the resolution of protrusions into vacuoles through the formation of an intermediate membrane-bound compartment that we refer to as vacuole-like protrusion (VLP). Genetic rescue of PIK3C2A depletion with RNAi-resistant cDNA constructs demonstrated that VLP formation required the activity of PIK3C2A in primary infected cells. PIK3C2A expression was required for production of phosphatidylinositol 3-phosphate (PtdIns (3)P) at the plasma membrane surrounding protrusions. PtdIns (3)P production was not observed in the protrusions formed by L. monocytogenes, whose dissemination did not rely on PIK3C2A. PIK3C2A-mediated PtdIns (3)P production in S. flexneri protrusions was regulated by host cell tyrosine kinase signaling and relied on the integrity of the S. flexneri type 3 secretion system. We suggest a model of S. flexneri dissemination in which the formation of VLPs is mediated by the PIK3C2A-dependent production of the signaling lipid PtdIns (3)P in the protrusion membrane, which relies on the T3SS-dependent activation of tyrosine kinase signaling in protrusions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.Infection and Immunity 02/2015; DOI:10.1128/IAI.03138-14 · 4.16 Impact Factor
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ABSTRACT: The understanding of lipid membranes and their organization has undergone significant development with better techniques and therefore more resolved experiments. Many new factors and organizing principles have been discovered, and interplay between these factors is expected to result in rich functional behaviours. The major factors regulating the lateral membrane heterogeneity, apart from the well-studied phase separation, are cytoskeleton pinning, clustering of lipids and curvature. These factors are effective means to create membrane domains that provide rich biological functionality. We review the recent advances and concepts of membrane heterogeneity organization by curvature, cytoskeleton and clustering proteins.Essays in Biochemistry 02/2015; 57:109-119. DOI:10.1042/bse0570109 · 4.39 Impact Factor