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Spermatozoa in animal species are usually highly elongated cells with a long motile tail attached to a head that contains the haploid genome in a compact and often elongated nucleus. In Drosophila melanogaster, the nucleus is compacted two hundred-fold in volume during spermiogenesis and re-modeled into a needle that is thirty-fold longer than its diameter. Nuclear elongation is preceded by a striking relocalization of nuclear pore complexes (NPCs). While NPCs are initially located throughout the nuclear envelope (NE) around the spherical nucleus of early round spermatids, they are later confined to one hemisphere. In the cytoplasm adjacent to this NPC-containing NE, the so-called dense complex with a strong bundle of microtubules is assembled. While this conspicuous proximity argued for functional significance of NPC-NE and microtubule bundle, experimental confirmation of their contributions to nuclear elongation has not yet been reported. Our functional characterization of the spermatid specific Mst27D protein now resolves this deficit. We demonstrate that Mst27D establishes physical linkage between NPC-NE and dense complex. The C-terminal region of Mst27D binds to the nuclear pore protein Nup358. The N-terminal CH domain of Mst27D, which is similar to that of EB1 family proteins, binds to microtubules. At high expression levels, Mst27D promotes bundling of microtubules in cultured cells. Microscopic analyses indicated co-localization of Mst27D with Nup358 and with the microtubule bundles of the dense complex. Time-lapse imaging revealed that nuclear elongation is accompanied by a progressive bundling of microtubules into a single elongated bundle. In Mst27D null mutants, this bundling process does not occur and nuclear elongation is abnormal. Thus, we propose that Mst27D permits normal nuclear elongation by promoting the attachment of the NPC-NE to the microtubules of the dense complex, as well as the progressive bundling of these microtubules., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A change in ambient temperature is predicted to disrupt cellular homeostasis by affecting all cellular processes in an albeit non-uniform manner. Diffusion is generally less temperature-sensitive than enzymes, for example, and each enzyme has a characteristic individual temperature profile. The actual effects of temperature variation on cells are still poorly understood at the molecular level. Towards an improved understanding, we have performed a genome-wide RNA interference screen with S2R + cells. This Drosophila cell line proliferates over a temperature range comparable to that tolerated by the parental ectothermic organism. Based on effects on cell counts and cell cycle profile after knockdown at 27 and 17 °C, respectively, genes were identified with an apparent greater physiological significance at one or the other temperature. While 27 °C is close to the temperature optimum, the substantially lower 17 °C was chosen to identify genes important at low temperatures, which have received less attention compared to the heat shock response. Among a substantial number of screen hits, we validated a set successfully in cell culture and selected ballchen for further evaluation in the organism. This gene encodes the conserved metazoan VRK protein kinase that is crucial for the release of chromosomes from the nuclear envelope during mitosis. Our analyses in early embryos and larval wing imaginal discs confirmed a higher requirement for ballchen function at temperatures below the optimum. Overall, our experiments validate the genome-wide screen as a basis for future characterizations of genes with increased physiological significance at the lower end of the readily tolerated temperature range., (© 2023. The Author(s).)

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster. The proteins SNM, UNO and MNM are likely constituents of a physical linkage that conjoins homologs in D. melanogaster spermatocytes. Here, we report that SNM binds tightly to the C-terminal region of UNO. This interaction is homologous to that of the cohesin subunits stromalin/Scc3/STAG and α-kleisin, as revealed by sequence similarities, structure modeling and cross-link mass spectrometry. Importantly, purified SU_C, the heterodimeric complex of SNM and the C-terminal region of UNO, displayed DNA-binding in vitro. DNA-binding was severely impaired by mutational elimination of positively charged residues from the C-terminal helix of UNO. Phenotypic analyses in flies fully confirmed the physiological relevance of this basic helix for chromosome-binding and homolog conjunction during male meiosis. Beyond DNA, SU_C also bound MNM, one of many isoforms expressed from the complex mod(mdg4) locus. This binding of MNM to SU_C was mediated by the MNM-specific C-terminal region, while the purified N-terminal part common to all Mod(mdg4) isoforms multimerized into hexamers in vitro. Similarly, the UNO N-terminal domain formed tetramers in vitro. Thus, we suggest that multimerization confers to SUM, the assemblies composed of SNM, UNO and MNM, the capacity to conjoin homologous chromosomes stably by the resultant multivalent DNA-binding. Moreover, to permit homolog separation during anaphase I, SUM is dissociated by separase, since UNO, the α-kleisin-related protein, includes a separase cleavage site. In support of this proposal, we demonstrate that UNO cleavage by tobacco etch virus protease is sufficient to release homolog conjunction in vivo after mutational exchange of the separase cleavage site with that of the bio-orthogonal protease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Kabakci et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes., Competing Interests: The authors have declared that no competing interests exist.

The bivalent chromosomes that are generated during prophase of meiosis I comprise a pair of homologous chromosomes. Homolog pairing during prophase I must include mechanisms that avoid or eliminate entanglements between non-homologous chromosomes. In Drosophila spermatocytes, non-homologous associations are disrupted by chromosome territory formation, while linkages between homologous chromosomes are maintained by special conjunction proteins. These proteins function as alternative for crossovers that link homologs during canonical meiosis but are absent during the achiasmate Drosophila male meiosis. How and where within bivalents the alternative homolog conjunction proteins function is still poorly understood. To clarify the rules that govern territory formation and alternative homolog conjunction, we have analyzed spermatocytes with chromosomal aberrations. We examined territory formation after acute chromosome cleavage by Cas9, targeted to the dodeca satellite adjacent to the centromere of chromosome 3 specifically in spermatocytes. Moreover, we studied territory organization, as well as the eventual orientation of chromosomes during meiosis I, in spermatocytes with stable structural aberrations, including heterozygous reciprocal autosomal translocations. Our observations indicate that alternative homolog conjunction is applied in a spatially confined manner. Comparable to crossovers, only a single conjunction spot per chromosome arm appears to be applied usually. These conjunction spots resist separation by the dispersing forces that drive apart homologous pericentromeric heterochromatin and embedded centromeres within territories, as well as the distinct chromosomal entities into peripheral, maximally separated territories within the spermatocyte nucleus., Competing Interests: The authors have declared that no competing interests exist.

In many species, centromere identity is specified epigenetically by special nucleosomes containing a centromere-specific histone H3 variant, designated as CENP-A in humans and CID in Drosophila melanogaster. After partitioning of centromere-specific nucleosomes onto newly replicated sister centromeres, loading of additional CENP-A/CID into centromeric chromatin is required for centromere maintenance in proliferating cells. Analyses with cultured cells have indicated that transcription of centromeric DNA by RNA polymerase II is required for deposition of new CID into centromere chromatin. However, a dependence of centromeric CID loading on transcription is difficult to reconcile with the notion that the initial embryonic stages appear to proceed in the absence of transcription in Drosophila, as also in many other animal species. To address the role of RNA polymerase II-mediated transcription for CID loading in early Drosophila embryos, we have quantified the effects of alpha-amanitin and triptolide on centromeric CID-EGFP levels. Our analyses demonstrate that microinjection of these two potent inhibitors of RNA polymerase II-mediated transcription has at most a marginal effect on centromeric CID deposition during progression through the early embryonic cleavage cycles. Thus, we conclude that at least during early Drosophila embryogenesis, incorporation of CID into centromeres does not depend on RNA polymerase II-mediated transcription., (© 2022. The Author(s).)