Nuclear Export of Cyclin B Mediated by the Nup62 Complex Is Required for Meiotic Initiation in Drosophila Males

Abstract

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.

Heat shock cognate 70 genes contribute to Drosophila spermatocyte growth progression possibly through the insulin signaling pathway

“…Two cysts contained spermatids that each harbored a single Nebenkern and a larger single nucleus (Table 2). As mentioned above, these phenotypic characteristics were similar to those of meiotic mutants with failure of meiosis initiation (Inoue et al., 2012; Okazaki et al., 2020; White‐Cooper et al., 1993, 1998), suggesting that Hsc70‐4 is crucial for meiosis initiation. Our investigation revealed that in Drosophila testes, the silencing of either Hsc70‐3 or Hsc70‐4 led to cell growth arrest in the phases S3 and S4, respectively.…”

“…We used anti‐HA and anti‐Akt antibodies (C67E7, #4691, Cell Signaling, Danvers, MA, USA) to identify complexes containing Hsc70‐2‐HA and Akt. PLA in situ (Sigma‐Aldrich Co., MO, USA) was performed according to a method described previously (Okazaki et al., 2020). The specimens were stained with DAPI and then mounted using VECTASHIELD Mounting Medium (#H‐1200, Vector Laboratories).…”

“…In previous studies, RNAi screening has been used to identify the genes required for spermatocyte growth and male meiosis progression (Hayashi et al., 2016; Okazaki et al., 2020). Individual double‐stranded RNAs (dsRNAs) against the mRNAs of approximately 300 genes related to cell growth and proliferation were introduced in the premeiotic spermatocytes to evaluate the contribution of each gene in the cell growth process (Hayashi et al., 2016).…”

“…Individual double‐stranded RNAs (dsRNAs) against the mRNAs of approximately 300 genes related to cell growth and proliferation were introduced in the premeiotic spermatocytes to evaluate the contribution of each gene in the cell growth process (Hayashi et al., 2016). Candidate genes potentially involved in the processes described above were selected by observing postmeiotic cells called spermatids at the onion stage (Hayashi et al., 2016; Okazaki et al., 2020). Among them, genes encoding proteins of the heat shock cognate protein 70 (Hsc70) family were first identified to significantly affect the phenotypes in the previous screening.…”

Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes

“…The proximity ligation assay (PLA), which enables detection of protein interactions within a cell, was performed according to the Duolink kit method (Nacalai Inc., Kyoto, Japan) as described previously ( Okazaki et al, 2020 ). We applied the PLA method to examine a close association between Cp110 and GFP–Orbit.…”