The role of copines in the regulation of the actin cytoskeleton and membrane trafficking

项目摘要 Copines是在大多数真核生物中发现的钙依赖性膜结合蛋白家族 包括人类，他们有九种不同的copine基因。Copines与许多人类 癌的然而，在这个时候，没有统一的主题关于这些难以捉摸的蛋白质的功能。 本研究项目的主要目标是确定一个共同的基本机械功能是否可以 所有的Copine蛋白质。我们一直在使用模式生物盘基网柄藻（Dictyosteelium discoideum）来研究 这些神秘的蛋白质网骨藻是研究这些蛋白质的良好模式生物，因为它们具有 六个copine基因，而其他模式生物要么有几个copine基因，要么没有copine基因。我们的研究 主要集中在Copine A（CpnA）。然而，我们现在有许多工具来研究其他五个copines （CpnB-F）。我们对CpnA基因敲除突变体的研究表明，CpnA基因参与了许多 细胞功能，包括需要肌动蛋白细胞骨架的过程（即胞质分裂、趋化性、细胞 极性和粘附），并需要膜融合（即收缩性空泡排出，和后溶酶体 成熟和胞吐作用）。生化研究表明CpnA与酸性磷脂和肌动蛋白结合 纤维以钙依赖的方式。定位研究表明，CpnA是一种可溶性的细胞质 转移到质膜和收缩液泡系统膜的蛋白质， 内溶酶体系统的细胞器响应钙浓度的上升。两个主要假设 对于CpnA的功能，从我们的研究中出现：CpnA在钙依赖性调节1） 肌动蛋白丝动力学和/或2）膜融合。因此，我们建议使用网骨藻来探索 所有的copines，从单细胞生物到人类，都参与了钙依赖性的 调节肌动蛋白丝动力学和/或膜融合。我们计划使用几种策略，包括 copine敲除突变体的功能表征和copine蛋白结合配偶体的鉴定。 全内反射荧光显微镜（TIRF）将用于可视化肌动蛋白丝动力学， Copine敲除突变体中的膜融合。我们还将探讨更具体的假设， copines调节膜表面的肌动蛋白丝以调节膜融合。TIRF显微镜是一种 一种特殊类型的荧光显微镜，将荧光团的激发和发射限制在非常薄的 紧邻盖玻片的标本区域，用于对细胞过程成像 发生在质膜附近。如果我们发现并非所有的辅蛋白都能调节肌动蛋白 细丝动力学和/或膜融合，copines的统一主题很可能会出现，因为我们 表征每种Copine敲除突变体，以鉴定任何共同缺陷并鉴定共同的 copine蛋白的结合伴侣。这些新的知识可以用来理解分子 许多人类癌症的潜在机制，其中copines高度表达。

PROJECT SUMMARY Copines are a family of calcium-dependent membrane binding proteins found in most eukaryotic organisms including humans, which have nine different copine genes. Copines have been implicated in numerous human cancers. However, at this time there is no unifying theme with respect to the function of these elusive proteins. The main goal of this research project is to determine whether a common basic mechanistic function can be attributed to all copine proteins. We have been using the model organism Dictyostelium discoideum to study these enigmatic proteins. Dictyostelium is a good model organism to study these proteins because they have six copines genes, while other model organisms either have a few or no copine genes. Our studies have mostly focused on Copine A (CpnA). However, we now have many of the tools to study the other five copines (CpnB-F). Our studies on cpnA knockout mutants in Dictyostelium indicate that CpnA is involved in many cellular functions including processes that require the actin cytoskeleton (i.e. cytokinesis, chemotaxis, cell polarity, and adhesion) and require membrane fusion (i.e. contractile vacuole expulsion, and postlysosome maturation and exocytosis). Biochemical studies indicate that CpnA binds to acidic phospholipids and actin filaments in a calcium-dependent manner. Localization studies indicate that CpnA is a soluble cytoplasmic protein that translocates to the plasma membrane and the membranes of the contractile vacuole system and organelles of the endolysosomal system in response to a rise in calcium concentration. Two main hypotheses for the function of CpnA emerge from our studies: CpnA functions in the calcium-dependent regulation of 1) actin filament dynamics and/or 2) membrane fusion. Therefore, we propose to use Dictyostelium to explore the idea that all copines, from single-celled organisms to humans, are involved in the calcium-dependent regulation of actin filament dynamics and/or membrane fusion. We plan to use several strategies that include the functional characterization of copine knockout mutants and identification of copine protein binding partners. Total Internal Reflection Fluorescence Microscopy (TIRF) will be used to visualize actin filament dynamics and membrane fusion in the copine knockout mutants. We will also explore the more specific hypothesis that copines regulate actin filaments on membrane surfaces to regulate membrane fusion. TIRF Microscopy is a special type of fluorescence microscopy that restricts excitation and emission of fluorophores to a very thin region of the specimen immediately adjacent to the glass coverslip and is used to image cellular processes happening close to the plasma membrane. If we find that not all copines function in the regulation of actin filament dynamics and/or membrane fusion, a unifying theme for copines will most likely emerge as we characterize each of the copine knockout mutants to identify any common defects and identify common binding-partners of copine proteins. This new knowledge can be then be used to understand the molecular mechanisms underlying many of the human cancers in which copines are highly expressed.