Measurement of physiological secretory vesicle-plasma membrane tethering forces o

描述（由申请方提供）：许多细胞类型通过胞吐作用释放包装在膜结合分泌囊泡或颗粒中的分子。该机制介导神经递质、激素以及许多其他化合物的释放，包括来自肥大细胞的组胺或来自嗜酸性粒细胞的细胞毒性蛋白。神经元和神经内分泌细胞的易于释放的囊泡以拴在质膜上的释放位点的状态等待刺激。目前还不清楚是什么分子机制形成了囊泡和其靶膜之间的物理联系。这种知识差距是由于缺乏一种技术，可以物理测量的束缚相互作用的囊泡与质膜，并定量探测这种相互作用所涉及的力量。我们建议开发一种技术，填补这一空白，允许表征囊泡质膜束缚相互作用。将具有暴露于膜片细胞质侧的栓系囊泡的PC12细胞质膜片用作制备液。将开发一种方法来绑定AFM针尖牢固地绑定到由生理拴系组件拴系到质膜的颗粒。为了精确监测垂直于质膜的囊泡运动，AFM安装在具有TIRF能力的荧光显微镜上。由此可以将系链解离事件与AFM针尖从囊泡的解离区分开，并提供关于系链相互作用的详细信息。系链解离动力学和景观将被表征为所施加的力的函数。新形成的系链的性质将与先前存在的系链的性质进行比较。这项技术的发展将为使用药理学和分子操作的研究提供基础，以阐明拴系的分子机制，并与重组系统的实验进行比较。如果成功的话，这种使能技术将大大推进我们对束缚和融合的理解，这是胞吐和细胞内运输中普遍存在的机制。 公共卫生相关性：在健康和疾病中，不同的细胞类型响应生物体的特定需要从膜结合囊泡释放各种分子。为了实现这一功能，囊泡必须在释放位点被束缚，并且当束缚被破坏时，免疫系统的激素、神经递质和介质的释放受损。该项目开发了一种能够测量将囊泡保持在细胞中正确位置的分子力的技术，以促进我们对这种复杂的生物分子束缚装置的理解。

DESCRIPTION (provided by applicant): Many cell types release molecules packaged in membrane bound secretory vesicles or granules by exocytosis. This mechanism mediates release of neurotransmitters, hormones as well as many other compounds including histamine from mast cells or cytotoxic proteins from eosinophilic granulocytes. The readily releasable vesicles of neurons and neuroendocrine cells are awaiting stimulation in a state tethered to their release site at the plasma membrane. It remains unclear what molecular mechanism forms a physical link between a vesicle and its target membrane. This knowledge gap is due to the lack of a technique that can physically measure tethering interaction of a vesicle with the plasma membrane and quantitatively probe the forces involved in this interaction. We propose to develop a technology that fills this gap allowing characterization of vesicle-plasma membrane tethering interactions. PC12 cell plasma membrane sheets with tethered vesicles exposed on the cytoplasmic side of the membrane sheet will be used as the preparation. A method will be developed to bind an AFM tip firmly bound to a granule tethered to the plasma membrane by the physiological tethering components. For precise monitoring of the vesicle movement perpendicular to the plasma membrane the AFM is mounted on a fluorescence microscope with TIRF capability. Tether dissociation events can thereby be distinguished from a dissociation of the AFM tip from the vesicle and provide detailed information on the tethering interactions. Tether dissociation kinetics and landscape will be characterized as a function of applied force. The properties of newly formed tethers will be compared with those of pre-existing tethers. This technology development will provide the foundation for studies using pharmacological and molecular manipulations to elucidate the molecular mechanism of tethering and for comparison with experiments on reconstituted systems. If successful, this enabling technology will considerably advance our understanding of tethering and fusion, which is a ubiquitous mechanism in exocytosis and intracellular trafficking. PUBLIC HEALTH RELEVANCE: In health and disease, different cell types release various molecules in response to specific needs of the organism from membrane bound vesicles. For this function, the vesicles must be tethered at the release site and release of hormones, neurotransmitters and mediators of the immune system is impaired when tethering is disrupted. This project develops a technology capable of measuring the molecular forces that hold vesicle at the right site in the cell to advance our understanding of this intricate biological molecular tethering apparatus.