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Drosophila spermatocytes grow up to 25 times their original volume before the onset of male meiosis. Several insulin-like peptides and their cognate receptors (InR) are essential for the cell growth process in Drosophila. Here, we aimed to identify additional signaling pathways and other regulatory factors required for germline cell growth in Drosophila males. Spermatocyte-specific expression of the dominant-negative form of InR inhibits cell growth. Conversely, constitutively active forms of signaling factors downstream of InR suppress growth inhibition. Furthermore, hypomorphic mutations in the target of rapamycin (Tor) inhibit spermatocyte growth. These data indicate that the insulin/TOR pathway is essential for the growth of premeiotic spermatocytes. RNA interference (RNAi) screening for the identification of other novel genes associated with cell growth showed that the silencing of each of the five members of heat shock cognate 70 (Hsc70) genes significantly inhibited the process. Hsc70-silenced spermatocytes showed Akt inhibition downstream of the insulin signaling pathway. Our pleckstrin homology domain-​green fluorescent protein (PH-GFP) reporter studies indicated that PI3K remained activated in Hsc70-4-silenced cells, suggesting that the Hsc70-4 protein possibly targets Akt or Pdk1 acting downstream of PI3K. Moreover, each of the Hsc70 proteins showed different subcellular localizations. Hsc70-2 exhibited cytoplasmic colocalization with Akt in spermatocytes before nuclear entry of the kinase during the growth phase. These results indicated the involvement of Hsc70 proteins in the activation of various steps in the insulin signaling pathway, which is essential for spermatocyte growth. Our findings provide insights into the mechanism(s) that enhance signal transduction to stimulate the growth of Drosophila spermatocytes.

After centrosome duplication, centrioles elongate before M phase. To identify genes required for this process and to understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes expressing fluorescently tagged centriolar proteins. We demonstrated that an essential microtubule polymerisation factor, Orbit (the Drosophila CLASP orthologue, encoded by chb), accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule-severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically to determine centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. We observed loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs, in Cp110-null mutant cells either overexpressing Orbit or depleted of Klp10A. Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they were unpaired. Our findings contribute to understanding the mechanism of centriole integrity, disruption of which leads to chromosome instability in cancer cells., Summary: A microtubule polymerising factor, Orbit, localises on centrioles and determines centriole length by antagonising Klp10A in Drosophila spermatocytes.

Drosophila Mxc protein is a component of the histone locus body (HLB), which is required for the expression of canonical histone genes, and severe mxc mutations generate tumors in larval hematopoietic tissues. A common characteristic of cancer cells is chromosomal instability (CIN), but whether mxc mutants exhibit this feature is unknown. Here, examination of post-meiotic spermatids created after male meiosis revealed that a fraction of the spermatids in hypomorphic mxc