Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells

Abstract

Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55-TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx 3 Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx 3 YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx 3 Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.TEX14 | intercellular bridges | CEP55 | germ cells | cytokinesis I ntercellular bridges are a distinct feature of spermatogenesis in mammalian germ cells. Although observations of intercellular bridges were reported more than 100 y ago, their molecular function is largely unknown and we have only recently begun to learn how they form at the molecular level. Interestingly, stable bridges have recently been recognized as providing a unique means of intercellular communication, because cytoplasmic molecules can pass through them (1). The loss of germ cell intercellular bridges disrupts spermatogenesis and causes sterility (2).The most direct method of cell-to-cell communication is to connect the separate cytosols of cells using a tunnel that allows macromolecules to pass from one cell to another. Various organisms achieve this type of direct intercellular transfer using tunneling nanotubes (3), intercellular bridges (also called ring canals) (1), and bacterial intercellular nanotubes (4). Somatic ring canals have also been found to equilibrate the levels of some proteins between connected cells in invertebrates such as Drosophila (5). Among these mechanisms, it has been shown that intercellular bridges having channels that are 0.5-3 μm in diameter are formed by the arrest of cell abscission at the final stage of cytokinesis in the germ cells of vertebrates (1).Whether the process of cell abscission is completed or not depends on the cell type. In the somatic cells of vertebrates, cell abscission occurs at the midbody (6), a structure that tethers two...

Zebrafishdazlregulates cystogenesis upstream of the meiotic transition and germline stem cell specification and independent of meiotic checkpoints

“…Our study examines the earliest stages of gonadogenesis and provides evidence that the conserved RBP, Dazl, is required for germline cyst formation and plays critical roles in germ cell amplification, and acts upstream of meiosis and establishment of germline stem cells to promote fertility. Immunoaffinity screens for Dazl targets have identified regulators of incomplete cytokinesis (Kim et al, 2015;Reynolds et al, 2005;Rosario et al, 2019;Rosario et al, 2017;Zagore et al, 2018). This work provides genetic evidence that Dazl is required to form germline cysts interconnected by ring canals.…”

“…Arrest of the cytokinetic furrow and maintenance of the midbody involves a complex regulatory network that stabilizes the actomyosin meshwork to arrest abscission and maintain the contractile ring, forming ring canals instead of dividing (Greenbaum et al, 2011;Haglund et al, 2011;Hime et al, 1996;Robinson and Cooley, 1996). In mouse, interaction between the inactive serine-threonine kinase TEX14 and CEP55 regulates intercellular bridge stability in part by blocking abscission factors (Alix, Escrt complex) (Greenbaum et al, 2011;Greenbaum et al, 2006;Kim et al, 2015;Morita et al, 2007). Intercellular bridge and germline cyst formation is a conserved feature of germ cell biology and is crucial for fertility (e.g.…”

Genome‐wide association study for age at puberty in young Nelore bulls

“…The TEX14 (testis‐expressed gene 14), a testis‐expressed gene and germ cell‐specific component, encodes a protein necessary for intercellular bridges in germ cells converting midbodies into stable intercellular bridges, which are crucial for successful spermatogenesis. The loss of germ cell intercellular bridges causes sterility through spermatogenesis disruption (Greenbaum, Iwamori, Agno, & Matzuk, ; Kim et al, ).…”

Altered germline cyst and oocyte differentiation inTex14mutant mice reveal a new mechanism underlying female reproductive life-span

“…During mammalian gametogenesis, intercellular bridges are found in fetal ovaries, fetal testes, and adult testes (9) (10) (11) (12) (13). Studies in adult mouse testes demonstrate that the Testis-expressed 14 (TEX14) protein is essential for blocking cytokinesis and converting transient midbody rings into stable intercellular bridges (14) (15) (16). Tex14 mutant testes are smaller in size but contain normal spermatogonia stem cells.…”