RUI: Investigation of Copine Function in Dictyostelium

在过去的10年里，在真核细胞中介导和调节膜囊泡运输的蛋白质的鉴定方面取得了很大的进展。不同的蛋白质在囊泡运输的不同步骤中起作用，例如囊泡的出芽、运输、靶向和融合。这些蛋白质中的大多数与从酵母到哺乳动物的生物体中发现的同源物非常保守。然而，这些蛋白质的确切作用仍不清楚。此外，很可能并非所有参与这些过程的蛋白质都已被鉴定。一个新的候选人在囊泡膜运输的作用是一个家庭最近发现的蛋白质称为copines。1997年首次从草履虫中分离出Copines，其能够以钙依赖性方式结合磷脂。草履虫有两个密切相关的copine基因，目前的序列数据库的分析表明，多个copine同系物不仅存在于纤毛虫，而且在黏菌，绿色植物，线虫，小鼠和人类。copines在不同生物体中的高度保守性表明它们在真核细胞中起着重要作用。在结构上，Copine在N-末端具有两个C2结构域，在C-末端具有与在整联蛋白中发现的A结构域相似的区域。C2结构域是最初在蛋白激酶C中鉴定的钙/磷脂结合基序。胞内可溶性蛋白中的A结构域是copines的独特特征，因为该结构域通常存在于胞外蛋白或膜蛋白的胞外部分中。在A结构域之后，辅蛋白具有可变长度的C-末端结构域，其相对富含脯氨酸。该结构域可以赋予不同的copine家族成员独特的特征和蛋白质-蛋白质相互作用的位点。一些证据表明，copines可能在囊泡贩运的功能。首先，针对人类copine的抗体识别与嗜铬颗粒结合的蛋白质，表明copine结合分泌囊泡。此外，几种被认为参与膜运输的蛋白质，如synaptotagmin，rabphilin，DOC 2和munc 13，含有多个C2结构域，赋予钙/磷脂结合特性。长期的研究目标是在分子水平上确定膜囊泡运输的机制。在短期内，研究目标是确定copines在真核细胞中的一般作用，使用模式遗传生物，Dictyosteelium discoideum。酵母的遗传学研究是鉴定和表征参与囊泡膜运输的蛋白质的基础。然而，在酵母中不存在Copine同系物。网囊藻提供了与酵母相同的遗传优势，并且初步研究表明在网囊藻中存在两种Copine同系物。本研究的两个主要目的是：1.通过在网骨藻中表达绿色荧光蛋白标记的copines并通过荧光显微镜检查细胞来确定copines在活细胞中的定位。这种实验方法将用于确定不同细胞行为期间定位的瞬时变化。2.通过同源重组的基因置换法构建网骨藻copine基因敲除突变体，并分析突变体的表型。该实验方法将用于确定由于Copine基因功能丧失而导致的膜运输途径中的功能丧失。一旦这两个目标得到满足，它将有可能相关的copines与copine突变体的功能丧失表型的细胞内位置，并产生一个更具体的假设copines在真核细胞中的功能。

In the past 10 years, great strides have been made in the identification of proteins that mediate and regulate membrane vesicle trafficking in eukaryotic cells. Different proteins function in distinct steps of vesicular trafficking such as budding, transport, targeting, and fusion of vesicles. Most of these proteins are well conserved with homologs found in organisms from yeast to mammals. However, the exact roles of many of these proteins remain unclear. Furthermore, it is likely that not all proteins involved in these processes have been identified. One new candidate for a role in vesicle membrane trafficking is a family of recently discovered proteins called copines. Copines were first isolated from Paramecium in 1997 by their ability to bind phospholipids in a calcium-dependent manner. Paramecium have two closely related copine genes and analysis of current sequence databases indicates that multiple copine homologs exist not only in ciliates, but also in slime molds, green plants, nematodes, mice, and humans. The high degree of conservation of copines among diverse organisms suggests they play a fundamental role in eukaryotic cells. Structurally, copines have two C2 domains at the N-terminus and a region similar to the A domain found in integrins at the C-terminus. The C2 domain is a calcium-/phospholipid- binding motif originally identified in protein kinase C. The A domain in an intracellular soluble protein is a unique characteristic of copines because this domain is typically found in extracellular proteins or extracellular portions of membrane proteins. Following the A domain, copines have a variable length C-terminal domain that is relatively rich in prolines. This domain may confer unique characteristics to the different copine family members and a site for protein-protein interactions. Several lines of evidence suggest that copines may function in vesicular trafficking. First, antibodies raised against a human copine recognize a protein that binds to chromaffin granules indicating that copines bind secretory vesicles. In addition, several proteins thought to be involved in membrane trafficking, such as synaptotagmin, rabphilin, DOC2, and munc13, contain multiple C2 domains that confer calcium/phospholipid binding properties. The long-term research objective is to define in molecular terms the mechanisms underlying membrane vesicle trafficking. In the short term, the research goal is to determine the general role of copines in eukaryotic cells using the model genetic organism, Dictyostelium discoideum. Genetic studies in yeast have been fundamental to the identification and characterization of proteins involved in vesicular membrane trafficking. However, no copine homologs exist in yeast. Dictyostelium provides the same genetic advantages as yeast and preliminary research demonstrates the existence of two copine homologs in Dictyostelium. The two main objectives of the research are: 1. Determine the localization of copines in live cells by expressing green fluorescent protein tagged copines in Dictyostelium and examining the cells by fluorescence microscopy. This experimental approach will be used to determine transient changes in localization during different cellular behaviors. 2. Create copine gene knockout mutants in Dictyostelium with gene replacement by homologous recombination and analyze the mutant phenotypes. This experimental approach will be used to determine loss of function in membrane trafficking pathways due to loss of copine gene function. Once these two objectives are met, it will be possible to correlate the intracellular location of copines with loss-of-function phenotypes in copine mutants and produce a more specific hypothesis about the function of copines in eukaryotic cells.