Conventional lysosomes in many cell types are uniquely capable of responding to elevations in cytosolic free Ca2+ by fusing with the plasma membrane. Our previous studies identified the Ca2+-sensing lysosomal protein synaptotagmin VII (Syt VII) as a key regulator of this process, and defined the components of Syt VII- interacting SNARE complexes mediating lysosomal exocytosis. Genetic ablation and dominant negative approaches revealed that Syt VII/Ca2+-dependent lysosomal exocytosis plays an important role in the resealing of plasma membrane wounds. We now plan to extend these studies into a more detailed understanding of the cellular mechanisms involved in plasma membrane resealing. Syt VII also mediates the delivery of lysosomal membrane to nascent phagosomes, providing us with an unique opportunity to characterize in detail the regulated transport of intracellular membrane to the cell surface.
Our specific aims are: 1. Define the role of Ca2+ and Syt Vll-containing microdomains in the delivery of intracellular membrane to nascent phagosomes;2. Assess the contributions of evolutionarily conserved regulatory proteins in the delivery of lysosomal membrane to the cell surface during phagocytosis and plasma membrane repair;3. Elucidate the Ca2+-dependent mechanism by which eukaryotic cells repair plasma membrane lesions caused by pore-forming toxins. To this end we will utilize high resolution imaging techniques to characterize the Syt Vll-containing compartment in phagocytic cells, thereby defining membrane domains according to the location of various late endosomal/lysosomal regulatory molecules. The reorganization of membrane domains during particle uptake will be followed in space and time, and the role of Syt VII as a Ca2+ sensor and a putative component of CDeS-containing tetraspanin webs will be investigated. Transcriptional silencing will be applied in parallel to Drosophila hemocytes and to mammalian cells, in order to functionally define the role of the evolutionarily conserved proteins Syt VII, VAMP7 and CD63, and to identify additional molecules involved in the Ca2+-dependent transfer of lysosomal membrane microdomains to the cell surface. We will also investigate in detail the mechanism by which eukaryotic cells repair lesions induced by pore-forming toxins. These studies will significantly increase our understanding of how Ca2+-regulated transport of intracellular membrane to the cell surface is regulated. They will also provide important new insights into the mechanism by which eukaryotic cells survive membrane-damaging lesions produced by pathogenic bacteria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Ainsztein, Alexandra M
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University of Maryland College Park
Anatomy/Cell Biology
Schools of Earth Sciences/Natur
College Park
United States
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Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina et al. (2016) Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes. PLoS One 11:e0152583
Corrotte, M; Castro-Gomes, T; Koushik, A B et al. (2015) Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses. Methods Cell Biol 126:139-58
Miller, Heather; Castro-Gomes, Thiago; Corrotte, Matthias et al. (2015) Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes. J Cell Biol 211:1193-205
Fernandes, Maria Cecilia; Corrotte, Matthias; Miguel, Danilo C et al. (2015) The exocyst is required for trypanosome invasion and the repair of mechanical plasma membrane wounds. J Cell Sci 128:27-32
Andrews, N W; Corrotte, M; Castro-Gomes, T (2015) Above the fray: Surface remodeling by secreted lysosomal enzymes leads to endocytosis-mediated plasma membrane repair. Semin Cell Dev Biol 45:10-7
Andrews, N W; Perez, F (2015) The plasma membrane repair shop: Fixing the damage. Semin Cell Dev Biol 45:1
Castro-Gomes, T; Koushik, A B; Andrews, N W (2014) ESCRT: nipping the wound in the bud? Trends Biochem Sci 39:307-9
Andrews, Norma W; Almeida, Patricia E; Corrotte, Matthias (2014) Damage control: cellular mechanisms of plasma membrane repair. Trends Cell Biol 24:734-42
Hissa, Barbara; Pontes, Bruno; Roma, Paula Magda S et al. (2013) Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes. PLoS One 8:e82988
Flannery, Andrew R; Renberg, Rebecca L; Andrews, Norma W (2013) Pathways of iron acquisition and utilization in Leishmania. Curr Opin Microbiol 16:716-21

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