Munc13-4 regulates Ca2+-stimulated exosome release during EMT

 描述(申请人提供)：外体是分泌的细胞外小泡，对应于多囊泡小体(MVB)的腔内小泡(ILV)，将生物活性化合物输送到靶细胞的细胞质。Exosome通过转移癌基因、诱导肌成纤维细胞分化、促进侵袭足的形成和阻止肿瘤细胞的免疫监视，在肿瘤转移中发挥着多种作用。促转移的行为依赖于经历上皮细胞向间充质细胞转变(EMT)的细胞，在EMT中，许多钙离子通道异常表达，导致不适当的钙刺激。钙离子调节胞吐的机制在所有类型的后生动物细胞中都是保守的，其中圈套促进了脂质双层融合。Munc13蛋白家族的成员增强了SNARE相互作用，因此极大地加速了囊泡融合。MVB与质膜的融合导致外切体释放，我的初步数据首次表明，急性钙升高刺激外切体释放，并进一步暗示Munc13-4是一个主要的钙感受器。免疫印迹法诱导EMT可增加胰腺癌细胞系(PANC-1)和肺癌细胞系(A549)Munc13-4的水平。此外，通过使用转化生长因子-1获得间充质表型，可以将外切体的释放从基础状态转变为钙敏感状态。这一建议的主要假设是，钙离子刺激的外切体释放是通过与Munc13-4结合来调节的。目的1利用超分辨显微镜首次确定Munc13-4-Ca~(2+)结合是否调节一组独特的外切体，并首次确定Ca~(2+)刺激的外切体释放的分泌动力学。基础的或受钙离子刺激的外切体将被提纯，以确定刺激后是否释放了一组独特的RNA和蛋白质。AIM2将决定Munc13-4调控的融合事件，该融合事件控制外切体的释放。在shRNA下调Munc13-4或使用新的Munc13-4钙依赖抑制剂后进行EMT的PANC-1和A549细胞将使用超分辨率显微镜成像，以确定钙和Munc13-4调节的外体分泌中的内体转运步骤。了解调控钙离子刺激的外切体分泌和细胞内转运的分子机制可能为阻止肿瘤的进展和转移提供治疗靶点。这项提议还将提供该领域专家在尖端分子生物学和超分辨率显微镜方面的培训。活细胞的超分辨率成像现在是高端细胞生物学研究的一项要求。马丁实验室在揭示分泌的基本机制方面拥有丰富的经验，在该实验室的培训将使我对外体分泌的机制产生新的想法。此外，马丁实验室在培养独立研究员级别的科学家方面有着良好的记录，为申请人领导学术实验室的职业生涯做好准备。

 DESCRIPTION (provided by applicant): Exosomes are secreted extracellular vesicles corresponding to intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) that deliver bioactive compounds to the cytoplasm of target cells. Exosomes play numerous roles in cancer metastasis by transferring oncogenes, inducing myofibroblast differentiation, promoting invadopodia formation and preventing immune surveillance of tumor cells. Pro-metastatic behavior is dependent on cells undergoing epithelial to mesenchmyal transition (EMT) where a number of Ca2+ channels are aberrantly expressed resulting in inappropriate Ca2+-stimulations. The mechanisms of Ca2+-regulated exocytosis are conserved across all metazoan cell types where SNAREs promote lipid bilayer fusion. Members of the Munc13 family of proteins enhance SNARE interactions and therefore greatly accelerate vesicle fusion. Fusion of MVB with the plasma membrane causes exosome release and my preliminary data are the first to show that acute Ca2+ elevation stimulates exosome release and further implicates Munc13-4 as a major Ca2+- sensor. Induction of EMT increases Munc13-4 levels in pancreatic (Panc-1) and lung (A549) carcinoma cell lines by immunoblot. Moreover, acquisition of a mesenchymal phenotype by use of TGFß-1 shifts exosome release from exclusively basal to Ca2+-sensitive. The primary hypothesis of this proposal is that Ca2+-stimulated exosome release is regulated by Ca2+-binding to Munc13-4. AIM 1 will determine if Munc13-4-Ca2+-binding regulates a unique population of exosomes and determine the secretory dynamics of Ca2+-stimulated exosome release for the first time using super-resolution microscopy. Basal or Ca2+-stimulated exosomes will be purified to identify if a unique set of RNA and protein is released following stimulation. AIM2 will determine the fusion event regulated by Munc13-4 that controls exosome release. Panc-1 and A549 cells undergoing EMT following shRNA downregulation of Munc13-4 or use of a novel Munc13-4 Ca2+-dependent inhibitor will be imaged using super-resolution microscopy to determine the endosomal trafficking steps in exosome secretion regulated by Ca2+ and Munc13-4. Understanding the molecular mechanisms that regulate Ca2+-stimulated exosome secretion and intracellular trafficking may provide therapeutic targets to block tumor progression and metastasis. This proposal will also provide training on cutting edge molecular biology and super resolution microscopy from experts in the field. Imaging of live cells in super resolution is now a requirement for high end cell biological studies. Training in the Martin laboratory which has extensive experience in uncovering the basic mechanisms of secretion will allow me to generate novel ideas on the mechanisms of exosome secretion. In addition, the Martin laboratory has a strong track record of producing scientists at the independent investigator level preparing the applicant for a career leading an academic laboratory.