Measurement of physiological secretory vesicle-plasma membrane tethering forces o

描述（由申请人提供）：许多细胞类型通过胞吐作用释放包裹在膜结合分泌囊泡或颗粒中的分子。这种机制介导神经递质、激素以及许多其他化合物的释放，包括肥大细胞的组胺或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释放囊泡在被束缚在质膜上的释放位点等待刺激。目前尚不清楚是什么分子机制形成了囊泡与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，可以物理测量囊泡与质膜的系缚相互作用，并定量探测这种相互作用所涉及的力。我们建议开发一种技术来填补这一空白，允许表征囊泡-质膜系缚相互作用。将PC12细胞质膜片与系留囊泡暴露在膜片的细胞质侧作为制备。将开发一种方法，将AFM尖端牢牢地绑定到通过生理系住成分系住在质膜上的颗粒上。为了精确监测垂直于质膜的囊泡运动，AFM安装在具有TIRF功能的荧光显微镜上。因此可以将系绳解离事件与AFM针尖与囊泡的解离区分开来，并提供系绳相互作用的详细信息。系绳解离动力学和景观将被表征为施加力的函数。将新形成的系绳与已有系绳的性质进行比较。这项技术的发展将为使用药理学和分子操作来阐明系索的分子机制以及与重组系统的实验进行比较的研究提供基础。如果成功，这项技术将极大地促进我们对栓系和融合的理解，这是胞吐和细胞内运输中普遍存在的机制。

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.