Damer Cynthia K, Bayeva Marina, Hahn Emily S, Rivera Javier, Socec Catherine I
Biology Department, Vassar College, Poughkeepsie, NY 12604, USA.
BMC Cell Biol. 2005 Dec 12;6:46. doi: 10.1186/1471-2121-6-46.
Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways.
We have identified six copines genes in the Dictyostelium discoideum genome, cpnA-cpnF, and have focused our studies on cpnA. CpnA is expressed throughout development and was shown to be capable of binding to membranes in a calcium-dependent manner in vitro. A GFP-tagged CpnA was also capable of binding to membranes in a calcium-dependent manner in vitro. In live wildtype Dictyostelium cells expressing GFP-CpnA, GFP-CpnA was typically found in the cytoplasm without any specific localization to membranes. However, in a small subset of starved cells, GFP-CpnA was observed to bind transiently (typically approximately 1-10 s) to the plasma membrane and intracellular vacuoles. In some cells, the transient membrane localization of GFP-CpnA was observed to occur multiple times in an oscillatory manner over several minutes. In plasma membrane disrupted cells, GFP-CpnA was observed to associate with the plasma membrane and intracellular vacuoles in a calcium-dependent manner. In fixed cells, GFP-CpnA labeled the plasma membrane and intracellular vacuoles, including contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes.
Our results show that Dictyostelium has multiple copine homologs and provides an excellent system in which to study copine function. The localization of a GFP-tagged CpnA to the plasma membrane, contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes suggests that CpnA may have a role in the function of these organelles or the trafficking to and from them. In addition, we hypothesize that the observed transient oscillatory membrane localization of GFP-CpnA in a small subset of starved cells is caused by fast calcium waves and speculate that CpnA may have a role in development, particularly in the differentiation of stalk cells.
柯平蛋白是在多种生物体中发现的可溶性、钙依赖性膜结合蛋白。柯平蛋白的特征是在N端区域有两个C2结构域，随后在C端区域有一个“A结构域”。“A结构域”在序列上与整合素中发现的血管性血友病因子A（VWA）结构域相似。C2结构域的存在表明柯平蛋白可能参与细胞信号传导和/或膜运输途径。
我们在盘基网柄菌基因组中鉴定出六个柯平蛋白基因，cpnA - cpnF，并将研究重点放在cpnA上。CpnA在整个发育过程中都有表达，并且在体外显示出能够以钙依赖性方式与膜结合。带有绿色荧光蛋白（GFP）标签的CpnA在体外也能够以钙依赖性方式与膜结合。在表达GFP - CpnA的活野生型盘基网柄菌细胞中，GFP - CpnA通常存在于细胞质中，没有任何特定的膜定位。然而，在一小部分饥饿细胞中，观察到GFP - CpnA会短暂地（通常约1 - 10秒）与质膜和细胞内液泡结合。在一些细胞中，观察到GFP - CpnA的短暂膜定位会在几分钟内以振荡方式多次发生。在质膜破裂的细胞中，观察到GFP - CpnA以钙依赖性方式与质膜和细胞内液泡结合。在固定细胞中，GFP - CpnA标记了质膜和细胞内液泡，包括收缩液泡、内溶酶体途径的细胞器和吞噬体。
我们的结果表明盘基网柄菌有多个柯平蛋白同源物，并提供了一个研究柯平蛋白功能的优秀系统。带有GFP标签的CpnA定位于质膜、收缩液泡、内溶酶体途径的细胞器和吞噬体，这表明CpnA可能在这些细胞器的功能或与之相关的运输过程中发挥作用。此外，我们推测在一小部分饥饿细胞中观察到的GFP - CpnA的短暂振荡膜定位是由快速钙波引起的，并推测CpnA可能在发育中发挥作用，特别是在柄细胞的分化过程中。
