RUI: Investigation of Copine Function in Dictyostelium

在过去的10年里，在真核细胞中介导和调控膜泡运输的蛋白质的鉴定方面取得了长足的进步。不同的蛋白质在囊泡运输的不同步骤中发挥作用，如囊泡的萌发、运输、靶向和融合。这些蛋白质中的大多数与在从酵母到哺乳动物的有机体中发现的同源物很好地保守。然而，其中许多蛋白质的确切作用仍不清楚。此外，很可能并不是所有参与这些过程的蛋白质都被鉴定出来。在囊泡膜运输中扮演角色的一个新候选者是一类最近发现的蛋白质，称为Copines。铜绿素于1997年首次从草履虫中分离出来，因为它们能够以钙依赖的方式结合磷脂。草履虫有两个密切相关的Copine基因，对现有序列数据库的分析表明，多个Copine同源物不仅存在于纤毛虫中，还存在于黏菌、绿色植物、线虫、小鼠和人类中。不同生物体中的铜绿素高度保守，这表明它们在真核细胞中发挥着基础作用。在结构上，Copine在N-末端有两个C2结构域，在C-末端有一个类似于整合素中发现的A结构域的区域。C2结构域是最初在蛋白激酶C中发现的钙/磷脂结合基序。胞内可溶性蛋白中的A结构域是Copins的独特特征，因为该结构域通常存在于细胞外蛋白或膜蛋白的胞外部分。在A结构域之后，Copine具有一个长度可变的C-末端结构域，该结构域相对富含Pro。这个结构域可能赋予不同的Copine家族成员独特的特征，并为蛋白质-蛋白质相互作用提供一个位置。有几条证据表明，铜绿素可能在囊泡运输中发挥作用。首先，针对人类Copine产生的抗体识别一种与嗜铬颗粒结合的蛋白质，表明Copine结合分泌囊泡。此外，几种被认为与膜转运有关的蛋白质，如synaptopagmin、rabPhilin、DOC2和Munc13，都含有多个C2结构域，赋予钙/磷脂结合特性。长期的研究目标是从分子的角度确定膜泡运输的机制。在短期内，研究目标是利用模式遗传有机体盘基网眼菌来确定铜绿素在真核细胞中的一般作用。酵母中的遗传学研究一直是鉴定和表征与囊泡膜运输有关的蛋白质的基础。然而，在酵母中不存在Copine同系物。盘基菌提供了与酵母菌相同的遗传优势，初步研究表明，盘基网柄菌中存在两种铜绿素同系物。本研究的两个主要目的是：1.利用绿色荧光蛋白标记的Copins在Dictyostelials中表达，并在荧光显微镜下观察细胞，确定Copins在活细胞中的定位。这一实验方法将被用来确定不同细胞行为期间局部的瞬时变化。2.利用同源重组的方法建立了Dictyostelius的Copine基因敲除突变体，并对突变体的表型进行了分析。这一实验方法将被用来确定由于Copine基因功能丧失而导致的膜转运途径的功能丧失。一旦达到这两个目标，就有可能将Copins在细胞内的位置与Copine突变体的功能丧失表型联系起来，并对Copins在真核细胞中的功能提出更具体的假设。

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.