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.
Intercellular bridges are unique and evolutionary-conserved channels that interconnect the germ cells of all multicellular organisms from fruit flies and hydra to mice and humans. Using genomic, proteomic and genetic studies, we identified Testis EXpressed gene 14 (TEX14) as the first essential protein in the mammalian intercellular bridge, showed that absence of the bridge results in male sterility, demonstrated how the bridge forms from intermediate structures during the process of cytokinesis, and identified novel components of the intercellular bridge including Intercellular Bridge Protein 2 (IBP2). In this proposal, we will focus on determining how the proteins molecularly interact to form the intercellular bridge, define the functions of IBP2 in bridge formation and/or stability, and identify small molecules that disrupt the bridge in vitro and in vivo, with the translational goal of defining the relevance of the intercellular bridge components to male infertility and contraception.
