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

项目总结 铜绿素是一类钙依赖的膜结合蛋白，存在于大多数真核生物中。 包括人类，他们有九种不同的Copine基因。铜绿假单胞菌与许多人类 癌症。然而，关于这些难以捉摸的蛋白质的功能，目前还没有统一的主题。 本研究项目的主要目的是确定一个共同的基本机构函数是否可以 归因于所有的Copine蛋白质。我们一直在利用模式生物盘基网柄菌进行研究 这些神秘的蛋白质。网柄苔藓是研究这些蛋白质的一个很好的模式生物，因为它们有 六个Copine基因，而其他模式生物要么只有几个Copine基因，要么没有Copine基因。我们的研究已经 主要集中在铜绿素A(CpnA)。然而，我们现在有许多工具来研究其他五个铜矿 (CpnB-F)。我们对网柄网柄菌中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、细胞 极性和粘附性)，需要膜融合(即收缩空泡排出和溶酶体后 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 花丝以一种依赖钙的方式。定位研究表明CpnA是一种可溶性细胞质 转位到质膜和收缩液泡系统的膜上的蛋白质 内溶酶体系统的细胞器对钙浓度升高的反应。两个主要假设 对于我们的研究中出现的CpnA的功能：CpnA在钙依赖的调节中发挥作用 肌动蛋白细丝动力学和/或2)膜融合。因此，我们建议利用Dictyostelius来探索 认为从单细胞生物体到人类，所有的铜绿素都参与了钙依赖 肌动蛋白细丝动力学和/或膜融合的调节。我们计划使用几种策略，包括 Copine基因敲除突变体的功能特征和Copine蛋白结合伙伴的鉴定。 全内反射荧光显微镜(TIRF)将用于可视化肌动蛋白细丝动力学和 Copine基因敲除突变体中的膜融合。我们还将探索更具体的假设，即 Copins调节膜表面的肌动蛋白细丝，从而调节膜融合。TIRF显微镜是一种 一种特殊类型的荧光显微镜，它将荧光团的激发和发射限制在非常薄的 紧靠玻璃盖片的样品区域，用于对细胞过程进行成像 发生在靠近质膜的地方。如果我们发现并不是所有的Copine都在肌动蛋白的调节中起作用 细丝动力学和/或膜融合，一个关于Copines的统一主题很可能会随着我们的出现而出现 确定每个Copine基因敲除突变体的特征，以确定任何共同的缺陷和共同的 Copine蛋白的结合伙伴。这一新知识可以用来理解分子 许多人类癌症的潜在机制，在这些癌症中，Copine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.