Regulated Exocytosis of Lysosomes

描述（由申请人提供）：许多细胞类型中的常规溶酶体能够通过与质膜融合对胞质游离Ca 2+升高做出独特反应。我们以前的研究确定了Ca 2+敏感的溶酶体蛋白突触结合蛋白VII（Syt VII）作为这一过程的关键调节因子，并定义了Syt VII相互作用的SNARE复合物介导溶酶体胞吐作用的组分。遗传消融和显性负性方法显示，Syt VII/Ca 2+依赖性溶酶体胞吐作用在质膜伤口的再密封中起着重要作用。我们现在计划将这些研究扩展到更详细地了解质膜重新密封所涉及的细胞机制。Syt VII还介导溶酶体膜向新生吞噬体的递送，为我们提供了一个独特的机会来详细表征细胞内膜向细胞表面的调节运输。我们的具体目标是：1.确定Ca 2+和Syt VII-含有微域在细胞内膜向新生吞噬体递送中的作用; 2.评估在吞噬和质膜修复过程中，进化保守的调节蛋白在溶酶体膜向细胞表面递送中的贡献; 3.阐明真核细胞修复由成孔毒素引起的质膜损伤的Ca 2+依赖性机制。为此，我们将利用高分辨率成像技术来表征吞噬细胞中含Syt VII的隔室，从而根据各种晚期内体/溶酶体调节分子的位置来定义膜结构域。在颗粒摄取过程中的膜结构域的重组将在空间和时间上进行，并将研究Syt VII作为Ca 2+传感器和含CDeS的四跨膜蛋白网的推定组分的作用。转录沉默将应用于果蝇血细胞和哺乳动物细胞平行，以功能上定义的进化保守的蛋白质Syt VII，VAMP 7和CD 63的作用，并确定其他分子参与的钙离子依赖性转移的溶酶体膜微结构域的细胞表面。我们还将详细研究真核细胞修复由成孔毒素诱导的损伤的机制。这些研究将显着增加我们的理解如何Ca 2+调节的细胞内膜到细胞表面的运输受到调节。它们还将为真核细胞在致病菌造成的膜损伤中存活的机制提供重要的新见解。

DESCRIPTION (provided by applicant): 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.