Defining the Mammalian Intercellular Bridge Interactome

细胞间通讯对所有多细胞生物都是必不可少的。一种独特的细胞间 生殖细胞之间发生通讯; 0.5-3毫米的“通道”，称为细胞间桥，在进化上是 在基本上所有多细胞生物的生殖腺中连接生殖细胞的保守结构。 虽然已经发现了几种果蝇细胞间桥（环管）蛋白，但直到我们敲除了 睾丸表达基因14（TEX 14），没有哺乳动物或脊椎动物的蛋白质已被确定是必不可少的， 细胞间的桥我们已经表明，TEX 14是一个162.5 kD的蛋白质，具有3个N-末端锚蛋白重复序列， 定位于雄性和雌性生殖细胞间桥的激酶样结构域。TEX 14阳性 当精原细胞开始 分化并通过成熟精子的形成继续连接雄性生殖细胞。 TEX 14基因敲除破坏了雄性和雌性小鼠的细胞间桥，并导致雄性不育， 女性因此，细胞间桥对哺乳动物精子发生至关重要，但对卵子发生和TEX 14无影响。 是第一个脊椎动物蛋白质，是必不可少的细胞间的桥梁。通过使用TEX 14作为基本的 作为细胞间桥的标志物，我们能够对细胞间桥进行生物化学富集。 然后进行蛋白质组学分析。在此过程中，我们鉴定了两种中间体基质蛋白， 驱动蛋白样蛋白1（MKLP 1）和雄性生殖细胞Rac GT酶激活蛋白（MgcRacGap），即 细胞质分裂所必需的，也是细胞间桥的组成部分; TEX 14将这些中间体 蛋白质转化为稳定的细胞间桥成分。我们鉴定了另外的桥蛋白，包括 CEP 55和一种新的睾丸富集蛋白，我们称之为细胞间桥蛋白2 （IBP 2）。使用酵母双杂交分析，我们已经表明全长TEX 14与其自身MKLP 1相互作用， CEP 55和IBP 2。因此，TEX 14在细胞间桥相互作用组中起核心作用。整体 这些研究的假设是TEX 14直接将IBP 2募集到细胞间桥， TEX 14：CEP 55相互作用是形成细胞间桥和阻断完成所必需的。 胞质分裂根据我们的研究结果，这两年计划的具体目标如下：1）生产一个 敲除细胞间桥蛋白2（IBP 2）;和2）定义蛋白质：蛋白质相互作用网络， 阻止胞质分裂的完成并导致稳定的细胞间桥。我们的研究是他们的第一个 从分子上定义哺乳动物细胞间桥相互作用组，确定 体内细胞间桥的各种成分，并了解桥如何保持稳定 通过多种细胞分裂。我们提出的具体目标也将有助于我们开发更多的高 用于鉴定细胞间桥的小分子破坏物的通量筛选测定。

Intercellular communication is essential for all multicellular organisms. A unique form of intercellular communication occurs between germ cells; 0.5-3 mm “channels”, called intercellular bridges, are evolutionarily conserved structures that interconnect germ cells in the gonads of essentially all multicellular organisms. Although several fruit fly intercellular bridge (ring canal) proteins have been uncovered, until our knockout of testis expressed gene 14 (TEX14), no mammalian or vertebrate protein had been identified to be essential for the intercellular bridge. We have shown that TEX14 is a 162.5 kD protein with 3 N-terminal ankyrin repeats and a kinase-like domain that localizes to male and female germ cell intercellular bridges. TEX14-positive intercellular bridges interconnect human and mouse spermatogonia as soon as spermatogonia begin to differentiate and continue to interconnect male germ cells up through formation of mature spermatozoa. Knockout of TEX14 disrupts intercellular bridges in male and female mice and causes sterility in males but not females. Thus, intercellular bridges are critical for mammalian spermatogenesis but not oogenesis and TEX14 is the first vertebrate protein that is essential for the intercellular bridge. By using TEX14 as an essential marker for the intercellular bridge, we were able to perform a biochemical enrichment of the intercellular bridges followed by proteomic analysis. In the process, we identified two midbody matrix proteins, Mitotic Kinesin-Like Protein 1 (MKLP1) and Male germ cell Rac GTPase-activating protein (MgcRacGap), that are both necessary for cytokinesis and also components of the intercellular bridge; TEX14 converts these midbody proteins into stable intercellular bridge components. We identified additional bridge proteins including CEntrosomal Protein 55 (CEP55) and a novel testis-enriched protein that we call Intercellular Bridge Protein 2 (IBP2). Using yeast 2-hybrid analysis, we have shown that full length TEX14 interacts with itself, MKLP1, CEP55, and IBP2. Thus, TEX14 plays a central role in the intercellular bridge interactome. The overall hypotheses of these studies are that TEX14 directly recruits IBP2 to the intercellular bridge and that the TEX14:CEP55 interaction is required for both formation of an intercellular bridge and blocking the completion of cytokinesis. Based on our findings, the Specific Aims of this two year proposal are as follows: 1) Produce a knockout of Intercellular Bridge Protein 2 (IBP2); and 2) Define the protein:protein interaction network that prevents the completion of cytokinesis and leads to stable intercellular bridges. Our studies are the first of their kind to molecularly define the mammalian intercellular bridge interactome, determine the essential functions of the various components of the intercellular bridge in vivo, and understand how the bridge remains a stable entity through multiple cell divisions. Our proposed Specific Aims will also help us to develop additional high throughput screening assays for the identification of small molecule disruptors of the intercellular bridge.