Components And Kinetics In Exocytosis

本项目主要研究钙触发胞吐作用的机制，这是一种普遍存在的真核生物过程，通过这种过程，囊泡融合到质膜上并释放其内容物。研究的重点是海胆卵分泌囊泡的皮质融合，这种融合发生在受精后。海胆卵的分离皮质被用作研究胞吐作用的最小准备，因为它既不需要ATP也不需要胞浆。分离的皮质颗粒可以重组，与卵质膜、其他皮质颗粒甚至纯脂质膜融合。因此，皮质颗粒本身有足够的蛋白质机制进行融合。在棘皮动物受精卵过程中，皮质分泌囊胞大量同步分泌。通过将生理和分子分析与多种纯化的膜分离物相结合，这些分离物含有与质膜融合或彼此融合的分泌囊泡，他们已经表征了这种钙触发的胞吐的最后步骤。虽然免疫印迹法或免疫印迹法被广泛用于特异性蛋白质的检测，但它通常被认为是一种较差的定量工具，用于测量特异性蛋白质的浓度。然而，为了在定量科学的层面上检验假设，有这样一个工具是必不可少的。为了分析和理解胞吐作用的分子机制，需要明确地识别和定量评估特定分子的表面密度。在这里，我们使用新改进的免疫印迹和分析范式，对功能性分泌囊泡的SNARE蛋白补体进行了全面的定量分析。这些发现证实了从飞摩尔到原子摩尔的已知蛋白的常规定量，并表明海胆卵的天然分泌囊泡，像其他受调节的分泌囊泡一样，经历Ca2+触发的融合，尽管内源性SNARE补体比模型囊泡系统中相对低效的融合所需的补体低约50倍。

This project is centered on the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. Research is focused on the cortical fusion of secretory vesicles of the sea urchin egg, which occurs upon fertilization. The isolated cortex of the sea urchin egg is used as a minimal preparation for the study of exocytosis, as it requires neither ATP nor cytosol. Isolated cortical granules can be reconstituted to fuse with egg plasma membrane, other cortical granules, and even purely lipid membranes. Thus cortical granules themselves have sufficient protein machinery for fusion. Upon echinoderm egg fertilization, cortical secretory vesicle exocytosis is massive and synchronous. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or each other, they have characterized the final steps of this calcium-triggered exocytosis. Although immunoblotting, or Western blotting is widely used for detection of specific proteins, it is generally thought to be poor as a quantative tool for measuring the concentration of specific proteins. However, for testing hypotheses at the level of quantitative science, it is essential to have such a tool. For analysis and understanding of the molecular mechanism of exocytosis requires one to unambiguously identify and quantitatively assess the surface density of specific molecules. Here, using newly refined immunoblotting and analysis paradigms, we provide a fully quantitative analysis of the SNARE protein complement of functional secretory vesicles. These findings demonstrate the routine quantitation of femtomole to attomole amounts of known proteins and indicate that native secretory vesicles of the sea urchin egg, like other regulated secretory vesicles, undergo Ca2+-triggered fusion despite an endogenous SNARE complement that is ~50-fold lower than required for relatively inefficient fusion in model vesicle systems. At the heart of exocytosis is membrane fusion. The energetics of a fusion pathway was considered theoretically, starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.