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Defining the Mammalian Intercellular Bridge Interactome

哺乳动物细胞间桥相互作用组的定义

基本信息

批准号：
7900873
负责人：
MARTIN M. MATZUK
金额：
$ 38.38万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-27 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7900873
关键词：
Address Ankyrin Repeat Applications Grants Binding Biochemical Biological Assay Cell division Communication Contraceptive methods Cytokinesis Data Drosophila genus Drosophila melanogaster Endocrine Female Future GTPase-Activating Proteins Gap Junctions Genes Genetic Genomics Germ Cells Goals Gonadal structure Haploidy Human Hybrids Hydra Polyps In Vitro Knock-out Length Male Contraceptive Agents Male Infertility Male Sterility Mammals Marsupialia Meiosis Mitotic Mus N-terminal Oocytes Oogenesis Organelles Organism Paracrine Communication Phosphotransferases Play Process Proteins Proteomics Publishing Recruitment Activity Reproduction Reproductive system Role Signal Transduction Spermatids Spermatogenesis Spermatogonia Sterility Structure Testis Yeasts base chemical genetics granulosa cell high throughput screening hypothalamic pituitary gonadal axis in vivo inhibitor/antagonist intercellular communication kinesin-like protein 1 male mgcRacGAP novel prevent protein protein interaction sertoli cell small molecule sperm cell

项目摘要

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.

细胞间的通讯对所有多细胞生物来说都是必不可少的。一种独特的细胞间形式生殖细胞之间发生通讯；在进化过程中，0.5-3毫米的“通道”，称为细胞间桥在基本上所有多细胞生物的性腺中相互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，但在我们敲除睾丸表达基因14(TEX14)，尚未发现哺乳动物或脊椎动物所必需的蛋白质细胞间的桥梁。我们已经证明TEX14是一个162.5 kD的蛋白，含有3个N-末端的Ankyrin重复序列和定位于雄性和雌性生殖细胞细胞间桥的一种类似于激酶的结构域。TEX14-阳性一旦精原细胞开始连接人类和小鼠的精原细胞，细胞间桥就会相互连接分化并继续通过形成成熟的精子向上连接雄性生殖细胞。 TEX14基因敲除会破坏雄性和雌性小鼠的细胞间桥，并导致雄性不育，但不会女性。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TEX14不起关键作用是第一种脊椎动物蛋白质，对细胞间桥是必不可少的。通过使用TEX14作为必要的细胞间桥的标记，我们能够对细胞间的然后进行蛋白质组学分析。在这个过程中，我们鉴定了两种中体基质蛋白，有丝分裂激动素样蛋白1(MKLP1)和雄性生殖细胞Rac GTP酶激活蛋白(MgcracGap)，分别是既是胞质分裂所必需的，也是细胞间桥的组成部分；TEX14将这些中体转化为蛋白质转化为稳定的细胞间桥梁成分。我们鉴定出了其他桥蛋白，包括中心体蛋白55(CEP55)和一种新的睾丸富集蛋白--细胞间桥蛋白2 (IBP2)。通过酵母双杂交分析，我们发现全长TEX14与自身、MKLP1、 CEP55和IBP2。因此，TEX14在细胞间桥相互作用组中起着核心作用。整体而言这些研究的假设是TEX14直接将IBP2招募到细胞间桥，并且 TEX14：CEP55相互作用是形成细胞间桥和阻止完井所必需的胞质分裂。根据我们的发现，这项为期两年的提案的具体目标如下：1)产生一个敲除细胞间桥蛋白2(IBP2)；和2)定义蛋白质：蛋白质相互作用网络，阻止细胞质分裂的完成，并导致稳定的细胞间桥。我们的研究是他们的第一个类分子定义哺乳动物细胞间桥相互作用组，确定其基本功能活体细胞间桥的各个组成部分，并了解桥如何保持稳定实体通过多个单元分裂。我们提出的具体目标也将帮助我们发展更高的通过筛选鉴定细胞间桥的小分子干扰物。