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 G46 mutants contained extra micronuclei or abnormally sized nuclei, corresponding to CIN. Moreover, we observed that the so-called lagging chromosomes retained between chromosomal masses separated toward spindle poles at telophase I. Time-lapse recordings show that micronuclei were generated from lagging chromosomes, and the abnormal chromosomes in mxc G46 mutants lacked centromeres. In normal spermatocyte nuclei, the HLB component FLASH colocalized with Mxc, whereas FLASH was dispersed in mxc G46 spermatocyte nuclei. Furthermore, we observed genetic interactions between Mxc and other HLB components in meiotic chromosome segregation, which suggests that inhibition of HLB formation is responsible for aberrant chromosome segregation in mxc G46. Quantitative real-time PCR revealed that canonical histone mRNA levels were decreased in mxc G46. Lastly, similar meiotic phenotypes appeared in the spermatids of histone H4 mutants and in the spermatids in testes depleted for chromosome-construction factors. Considering these genetic data, we propose that abnormal chromosome segregation leading to CIN development results from a loss of chromosome integrity caused by diminished canonical histone levels in mxc mutants.
Background: The central channel of the nuclear pore complex plays an important role in the selective transport of proteins between the nucleus and cytoplasm. Previous studies have demonstrated that the depletion of the Nup62 complex, constructing the nuclear pore channel in premeiotic Drosophila cells, resulted in the absence of meiotic cells. We attempted to understand the mechanism underlying the cell cycle arrest before meiosis. Methods: We induced dsRNAs against the nucleoporin mRNAs using the Gal4/UAS system in Drosophila. Results: The cell cycle of the Nup62-depleted cells was arrested before meiosis without CDK1 activation. The ectopic over-expression of CycB, but not constitutively active CDK1, resulted in partial rescue from the arrest. CycB continued to exist in the nuclei of Nup62-depleted cells and cells depleted of exportin encoded by emb. Protein complexes containing CycB, Emb, and Nup62 were observed in premeiotic spermatocytes. CycB, which had temporally entered the nucleus, was associated with Emb, and the complex was transported back to the cytoplasm through the central channel, interacting with the Nup62 complex. Conclusion: We proposed that CycB is exported with Emb through the channel interacting with the Nup62 complex before the onset of meiosis. The nuclear export ensures the modification and formation of sufficient CycB-CDK1 in the cytoplasm.
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 the M phase. To identify genes required for this process and understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes, expressing fluorescently tagged centrioles. We demonstrated that an essential microtubule polymerisation factor, Orbit/CLASP, 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 for determining 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 harbouring Klp10A depletion. 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 are unpaired. Our findings contribute to understanding the mechanism of centriole integrity, leading to chromosome instability in cancer cells.
Background: The central channel of the nuclear pore complex (NPC) plays an important role in the selective transport of proteins between the nucleus and cytoplasm. Previous studies have demonstrated that depletion of the Nup62 complex, constructing the nuclear pore channel in premeiotic Drosophila cells, resulted in absence of meiotic cells. We attempted understanding the mechanism underlying the cell cycle arrest before meiosis. Methods: We induced dsRNAs against the nucleoporin mRNAs using the Gal4/UAS system in Drosophila. Results: The cell cycle of the Nup62-depleted cells was arrested before meiosis without CDK1 activation. Ectopic over-expression of CycB, but not constitutively active CDK1, resulted in partial rescue from the arrest. CycB accumulated precociously in the nuclei of Nup62-depleted cells and cells depleted of exportin (encoded by emb). Protein complexes containing CycB, Emb, and Nup62 complex components were observed in premeiotic spermatocytes. CycB, which had precociously migrated into the nucleus, was associated with Emb, and the complex was transported back to the cytoplasm through the central channel, interacting with the Nup62 complex. Conclusion: We proposed that CycB is exported with Emb through the channel interacting with the Nup62 complex before initiation of meiosis. The nuclear export ensures production of active CycB-CDK1 in the cytoplasm.
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