Regulated Exocytosis of Lysosomes

描述(申请人提供)：许多细胞类型的常规溶酶体能通过与质膜融合来对胞浆内游离钙离子的升高作出独特的反应。我们以前的研究发现，钙离子敏感的溶酶体蛋白突触素VII(SYT VII)是这一过程的关键调节因子，并确定了SYT VII相互作用的SNARE复合体的组成，介导溶酶体的胞吐作用。基因消融和显性负性方法表明，依赖于Syt VII/Ca~(2+)的溶酶体胞吐作用在质膜创面的再封闭中起着重要作用。我们现在计划将这些研究扩展到更详细地了解质膜再密封所涉及的细胞机制。SYT VII还介导溶酶体膜向新生吞噬小体的传递，为我们提供了一个独特的机会来详细描述细胞内膜到细胞表面的调节运输。我们的具体目标是：1.确定含钙和SYT Vll的微域在细胞内膜向新生吞噬体膜输送中的作用；2.评估进化上保守的调控蛋白在吞噬和质膜修复过程中将溶酶体膜输送到细胞表面的作用；3.阐明真核细胞修复由成孔毒素引起的质膜损伤的钙依赖机制。为此，我们将利用高分辨率成像技术来表征吞噬细胞中含有SYT V11的隔室，从而根据各种晚期内小体/溶酶体调节分子的位置来确定膜域。在颗粒摄取过程中膜结构域的重组将在空间和时间上进行，并将研究SYT VII作为钙离子感受器和含有CDEs的Tetrspanin网的可能成分的作用。转录沉默将同时应用于果蝇血细胞和哺乳动物细胞，以便从功能上确定进化上保守的蛋白质SYT VII、VAMP7和CD63的作用，并识别参与钙依赖的溶酶体膜微区向细胞表面转移的其他分子。我们还将详细研究真核细胞修复由成孔毒素引起的损伤的机制。这些研究将极大地增加我们对钙离子调节的胞内膜到细胞表面的运输是如何调控的理解。它们还将为真核细胞在病原菌造成的膜损伤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.