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

脊髓灰质炎在肌动蛋白细胞骨架和膜运输调节中的作用

• 批准号: 10203493
• 负责人: CYNTHIA K DAMER
• 金额: $ 42.41万
• 依托单位: CENTRAL MICHIGAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10203493
• 关键词: Actins; Adhesions; Amoeba genus; Animal Model; Autophagocytosis; Binding; Binding Proteins; Biochemical; Biological Assay; Brain; Caenorhabditis elegans; Calcium; Cell Polarity; Cell membrane; Cell model; Cell physiology; Cells; Chemotaxis; Complex; Cytokinesis; Cytoplasmic Protein; Cytoskeleton; Defect; Dendritic Spines; Dictyostelium; Dictyostelium discoideum; Drosophila genus; Event; Exocytosis; Family; Fluorescence Microscopy; Genes; Goals; Human; Knock-out; Knowledge; Lysosomes; Malignant Neoplasms; Membrane; Membrane Fusion; Microfilaments; Microscopy; Modification; Molecular; Mus; Organelles; Organism; Phagocytosis; Phospholipids; Process; Property; Proteins; Regulation; Research; Research Project Grants; Role; SNAP receptor; Surface; Synapses; System; Techniques; Testing; Time; Vacuole; Yeasts; base; cell behavior; copine; experimental study; human disease; mutant; neurotransmitter release; postsynaptic; presynaptic; response; tool; trafficking; vesicle-associated membrane protein

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, identification of copine protein binding partners, and fluorescence microscopy techniques 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. 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.

项目摘要 大多数在大多数真核生物中发现的钙依赖性膜结合蛋白的家族 包括人类，其中有九种不同的copine基因。许多人与许多人有关 癌症。但是，目前尚无关于这些难以捉摸蛋白的功能的统一主题。 该研究项目的主要目标是确定是否可以使用常见的基本机械功能 归因于所有美联体蛋白。我们一直在使用模型生物dictyostelium discoideum进行研究 这些神秘的蛋白质。 dictyostelium是研究这些蛋白质的好模型生物体，因为它们具有 六个章节基因，而其他模型生物具有几个或没有cosine基因。我们的研究有 主要专注于美联社A（CPNA）。但是，我们现在有许多研究其他五个副本的工具 （CPNB-F）。我们对dictyostelium中CPNA敲除突变体的研究表明，CPNA参与了许多 细胞功能，包括需要肌动蛋白细胞骨架的过程（即细胞因子，趋化性，细胞 极性和粘附），需要膜融合（即收缩液泡驱动和凸面 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 细丝以钙依赖性方式。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的膜的蛋白质和 内溶血系统的细胞器响应钙浓度升高。两个主要假设 为了从我们的研究中出现CPNA的功能：CPNA在钙依赖性调节中的功能1） 肌动蛋白细丝动力学和/或2）膜融合。因此，我们建议使用dictyostelium探索 从单细胞生物到人类的所有copines都参与钙依赖性的想法 调节肌动蛋白丝动力学和/或膜融合。我们计划使用几种包括 Copine敲除突变体的功能表征，鉴定Copine蛋白结合伴侣的功能表征， 以及可视化肌动蛋白丝动力学和膜融合的荧光显微镜技术 美联社敲除突变体。我们还将探讨更具体的假设，即copines调节肌动蛋白 膜表面上的细丝以调节膜融合。如果我们发现并不所有的copines在 调节肌动蛋白细丝动态和/或膜融合，统一的主题很可能很可能 当我们表征每个occine敲除突变体以识别任何常见缺陷并识别时出现 Copine蛋白的常见结合零件。然后可以使用这些新知识来了解 许多人类癌症的分子机制高度表达。