Essential Role of COPII Proteins in Maintaining the Contractile Ring Anchoring to the Plasma Membrane during Cytokinesis in Drosophila Male Meiosis.

Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Here, we report that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. We explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila.

A possible pattern in the evolution of male meiotic cytokinesis in angiosperms.

Evolution of cellular characteristics is a fundamental aspect of evolutionary biology, but knowledge about evolution at the cellular level is very limited. In particular, whether a certain intracellular characteristic evolved in angiosperms, and what significance of such evolution is to angiosperms, if it exists, are important and yet unanswered questions. We have found that bidirectional cytokinesis occurs or likely occurs in male meiosis in extant basal and near-basal angiosperm lineages, which differs from the unidirectional cytokinesis in male meiosis in monocots and eudicots. This pattern of cytokinesis in angiosperms seems to align with the distribution pattern of angiosperms with the lineages basal to monocots and eudicots living in tropical, subtropical or temperate environments and monocots and eudicots in an expanded range of environments including tropical, subtropical, temperate, subarctic and arctic environments. These two cytokinetic modes seem to result from two phragmoplast types, respectively. A phragmoplast in the bidirectional cytokinesis dynamically associates with the leading edge of a growing cell plate whereas a phragmoplast in the unidirectional cytokinesis is localized to an entire division plane. The large assembly of microtubules in the phragmoplast in unidirectional cytokinesis may be indicative of increased microtubule stability compared with that of the small microtubule assembly in the phragmoplast in bidirectional cytokinesis. Microtubules could conceivably increase their stability from evolutionary changes in tubulins and/or microtubule-associated proteins. Microtubules are very sensitive to low temperatures, which should be a reason for plants to be sensitive to low temperatures. If monocots and eudicots have more stable microtubules than other angiosperms, they will be expected to deal with low temperatures better than other angiosperms. Future investigations into the male meiotic cytokinetic directions, microtubule stability at low temperatures, and proteins affecting microtubule stability in more species may shed light on how plants evolved to inhabit cold environments.

Live Imaging and Analysis of Meiotic Cytokinesis in Drosophila Testes.

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions. Key features • In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts. • In vitro method allows acute treatment with various pharmacological agents to study cytokinesis. • Ex vivo method to image male meiosis cytokinesis in intact adult testes. • Requires 15-60 min to set up and could be imaged up to 6-12 h.

Cyclin B Export to the Cytoplasm via the Nup62 Subcomplex and Subsequent Rapid Nuclear Import Are Required for the Initiation of Drosophila Male Meiosis.

The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. We investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin ß at the onset of meiosis. We propose a model regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis.

Knockdown of DOM/Tip60 Complex Subunits Impairs Male Meiosis of Drosophila melanogaster.

ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including the DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.

Unusual nuclear structures in male meiocytes of wild-type rye as revealed by volume microscopy.

BACKGROUND AND AIMS: During the analysis of plant male meiocytes coming from destroyed meiocyte columns (united multicellular structures formed by male meiocytes in each anther locule), a considerable amount of information becomes unavailable. Therefore, in this study intact meiocyte columns were studied by volume microscopy in wild-type rye for the most relevant presentation of 3-D structure of rye meiocytes throughout meiosis. METHODS: We used two types of volume light microscopy: confocal laser scanning microscopy and non-confocal bright-field scanning microscopy combined with alcohol and aldehyde fixation, as well as serial block-face scanning electron microscopy. KEY RESULTS: Unusual structures, called nuclear protuberances, were detected. At certain meiotic stages, nuclei formed protuberances that crossed the cell wall through intercellular channels and extended into the cytoplasm of neighbouring cells, while all other aspects of cell structure appeared to be normal. This phenomenon of intercellular nuclear migration (INM) was detected in most meiocytes at leptotene/zygotene. No cases of micronucleus formation or appearance of binucleated meiocytes were noticed. There were instances of direct contact between two nuclei during INM. No influence of fixation or of mechanical impact on the induction of INM was detected. CONCLUSIONS: Intercellular nuclear migration in rye may be a programmed process (a normal part of rye male meiosis) or a tricky artefact that cannot be avoided in any way no matter which approach to meiocyte imaging is used. In both cases, INM seems to be an obligatory phenomenon that has previously been hidden by limitations of common microscopic techniques and by 2-D perception of plant male meiocytes. Intercellular nuclear migration cannot be ignored in any studies involving manipulations of rye anthers.

Microtubule and Actin Cytoskeletal Dynamics in Male Meiotic Cells of Drosophila melanogaster.

Drosophila dividing spermatocytes offer a highly suitable cell system in which to investigate the coordinated reorganization of microtubule and actin cytoskeleton systems during cell division of animal cells. Like male germ cells of mammals, Drosophila spermatogonia and spermatocytes undergo cleavage furrow ingression during cytokinesis, but abscission does not take place. Thus, clusters of primary and secondary spermatocytes undergo meiotic divisions in synchrony, resulting in cysts of 32 secondary spermatocytes and then 64 spermatids connected by specialized structures called ring canals. The meiotic spindles in Drosophila males are substantially larger than the spindles of mammalian somatic cells and exhibit prominent central spindles and contractile rings during cytokinesis. These characteristics make male meiotic cells particularly amenable to immunofluorescence and live imaging analysis of the spindle microtubules and the actomyosin apparatus during meiotic divisions. Moreover, because the spindle assembly checkpoint is not robust in spermatocytes, Drosophila male meiosis allows investigating of whether gene products required for chromosome segregation play additional roles during cytokinesis. Here, we will review how the research studies on Drosophila male meiotic cells have contributed to our knowledge of the conserved molecular pathways that regulate spindle microtubules and cytokinesis with important implications for the comprehension of cancer and other diseases.

Corrigendum: Serial Block-Face Scanning Electron Microscopy Reveals That Intercellular Nuclear Migration Occurs in Most Normal Tobacco Male Meiocytes.

[This corrects the article DOI: 10.3389/fpls.2021.672642.].

RNA N6-methyladenosine modification promotes auxin biosynthesis required for male meiosis in rice.

N6-methyladenosine (m6A) RNA modification confers an essential layer of gene regulation in living organisms, including plants; yet, the underlying mechanisms of its deposition on specific target mRNAs involved in key plant developmental processes are so far unknown. Here, we show that a core component of the rice m6A methyltransferase complex, OsFIP37, is recruited by an RNA-binding protein, OsFIP37-associated protein 1 (OsFAP1), to mediate m6A RNA modification on an auxin biosynthesis gene, OsYUCCA3, during microsporogenesis. This stabilizes OsYUCCA3 mRNA and promotes local auxin biosynthesis in anthers during male meiosis, which is essential for meiotic division and subsequent pollen development in rice. Loss of function of OsFAP1 causes dissociation of OsFIP37 with OsYUCCA3 and the resulting abolished m6A deposition on OsYUCCA3. Our findings reveal that OsFAP1-dependent m6A deposition on OsYUCCA3 by OsFIP37 constitutes a hitherto unknown link between RNA modification and hormonal control of male meiosis in plant reproductive development.

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70 °C for 1 week. For cryosectioning, the frozen material was sectioned at - 20 °C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Identification of GOLPH3 Partners in Drosophila Unveils Potential Novel Roles in Tumorigenesis and Neural Disorders.

Golgi phosphoprotein 3 (GOLPH3) is a highly conserved peripheral membrane protein localized to the Golgi apparatus and the cytosol. GOLPH3 binding to Golgi membranes depends on phosphatidylinositol 4-phosphate [PI(4)P] and regulates Golgi architecture and vesicle trafficking. GOLPH3 overexpression has been correlated with poor prognosis in several cancers, but the molecular mechanisms that link GOLPH3 to malignant transformation are poorly understood. We recently showed that PI(4)P-GOLPH3 couples membrane trafficking with contractile ring assembly during cytokinesis in dividing Drosophila spermatocytes. Here, we use affinity purification coupled with mass spectrometry (AP-MS) to identify the protein-protein interaction network (interactome) of Drosophila GOLPH3 in testes. Analysis of the GOLPH3 interactome revealed enrichment for proteins involved in vesicle-mediated trafficking, cell proliferation and cytoskeleton dynamics. In particular, we found that dGOLPH3 interacts with the Drosophila orthologs of Fragile X mental retardation protein and Ataxin-2, suggesting a potential role in the pathophysiology of disorders of the nervous system. Our findings suggest novel molecular targets associated with GOLPH3 that might be relevant for therapeutic intervention in cancers and other human diseases.

Serial Block-Face Scanning Electron Microscopy Reveals That Intercellular Nuclear Migration Occurs in Most Normal Tobacco Male Meiocytes.

Serial block-face scanning electron microscopy (SBF-SEM) was used here to study tobacco male meiosis. Three-dimensional ultrastructural analyses revealed that intercellular nuclear migration (INM) occurs in 90-100% of tobacco meiocytes. At the very beginning of meiosis, every meiocyte connected with neighboring cells by more than 100 channels was capable of INM. At leptotene and zygotene, the nucleus in most tobacco meiocytes approached the cell wall and formed nuclear protuberances (NPs) that crossed the cell wall through the channels and extended into the cytoplasm of a neighboring cell. The separation of NPs from the migrating nuclei and micronuclei formation were not observed. In some cases, the NPs and nuclei of neighboring cells appeared apposed to each other, and the gap between their nuclear membranes became invisible. At pachytene, NPs retracted into their own cells. After that, the INM stopped. We consider INM a normal part of tobacco meiosis, but the reason for such behavior of nuclei is unclear. The results obtained by SBF-SEM suggest that there are still many unexplored features of plant meiosis hidden by limitations of common types of microscopy and that SBF-SEM can turn over a new leaf in plant meiosis research.

A simple and rapid method for imaging male meiotic cells in anthers of model and non-model plant species.

KEY MESSAGE: We describe a simple method to view meiotic cells in whole anthers from a range of plants. The method retains spatial organisation and enables simultaneous analysis of many meiotic cells. Understanding the process of male meiosis in flowering plants, and the role of genes involved in this process, offers potential for plant breeding, such as through increasing the level of genetic variation or the manipulation of ploidy levels in the gametes. A key to the characterisation of meiotic gene function and meiosis in non-model crop plants, is the analysis of cells undergoing meiosis, a task made difficult by the inaccessible nature of these cells. Here, we describe a simple and rapid method to analyse plant male meiosis in intact anthers in a range of plant species. This method allows analysis of numerous cells undergoing meiosis and, as meiotic cells stay within the anther, it retains information of the three-dimensional organisation and the location of organelles in meiotic cells. We show that the technique provides information on male meiosis by looking at the synchrony of meiotic progression between and within locules, and comparing wildtype and mutant plants through the chromosome separation stages in Arabidopsis thaliana. Additionally, we demonstrate that the protocol can be adopted to other plants with different floral morphology using Medicago truncatula as an example with small floral buds and the non-model plant kiwifruit (Actinidia chinensis) with larger buds and anthers.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila.

In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

Familial Infertility (Azoospermia and Cryptozoospermia) in Two Brothers-Carriers of t(1;7) Complex Chromosomal Rearrangement (CCR): Molecular Cytogenetic Analysis.

Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants: ASMT, GARNL3, and SESTD1, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.

Heat stress interferes with chromosome segregation and cytokinesis during male meiosis in Arabidopsis thaliana.

In higher plants, male meiosis is a key process of microsporogenesis and is crucial for plant fertility. Male meiosis programs are prone to be influenced by altered temperature conditions. Studies have reported that an increased temperature (28°C) within a fertile threshold can affect the frequency of meiotic recombination in Arabidopsis. However, not much has been known how male meiosis responses to an extremely high temperature beyond the fertile threshold. To understand the impact of extremely high temperature on male meiosis in Arabidopsis, we treated flowering Arabidopsis plants with 36-38°C and found that the high-temperature condition significantly reduced pollen shed and plant fertility, and led to formation of pollen grains with varied sizes. The heat stress-induced unbalanced tetrads, polyad and meiotic restitution, suggesting that male meiosis was interfered. Fluorescence in situ hybridization (FISH) assay confirmed that both homologous chromosome separation and sister chromatids cohesion were influenced. Aniline blue staining of tetrad-stage pollen mother cells (PMCs) revealed that meiotic cytokinesis was severely disrupted by the heat stress. Supportively, immunolocalization of ɑ-tubulin showed that the construction of spindle and phragmoplast at both meiosis I and II were interfered. Overall, our findings demonstrate that an extremely high-temperature stress over the fertile threshold affects both chromosome segregation and cytokinesis during male meiosis by disturbing microtubular cytoskeleton in Arabidopsis.

New data on karyotype, spermatogenesis and ovarian trophocyte ploidy in three aquatic bug species of the families Naucoridae, Notonectidae, and Belostomatidae (Nepomorpha, Heteroptera).

We report the karyotype, some aspects of spermatogenesis, and ovarian trophocytes ploidy in three aquatic bug species: Ilyocoris cimicoides (Linnaeus, 1758), Notonecta glauca Linnaeus, 1758, and Diplonychus rusticus Fabricius, 1871 from previously unexplored regions - South Europe (Bulgaria) and Southeast Asia (Vietnam). Our results add considerable support for the published karyotype data for these species. In I. cimicoides, we observed achiasmate male meiosis - the first report of achiasmy for the family Naucoridae. More comprehensive cytogenetic studies in other species of the Naucoridae are required to elucidate the role of achiasmy as a character in the systematics of the family. Our observations on the association between phases of spermatogenesis and developmental stages in I. cimicoides and N. glauca differ from the previously published data. In these species, we assume that the spermatogenesis phases are not strongly associated with certain developmental stages. For further cytogenetic studies (on the Balkan Peninsula), we recommend July as the most appropriate month for collection of I. cimicoides and N. glauca. In the ovaries of both species, we studied the level of ploidy in metaphase and interphase trophocytes. In I. cimicoides, diploid and tetraploid metaphase trophocytes were found. Heteropycnotic elements, observed in interphase trophocytes of this species, represented the X chromosomes. It allowed us to determine the trophocytes ploidy at interphase (2n was repeated up to 16 times). The situation with N. glauca was different. The metaphase trophocytes were diploid and we were not able to determine the ploidy of interphase trophocytes since such conspicuous heteropycnotic elements were not found. The scarce data available suggest a tendency for a low level of trophocyte ploidy in the basal infraorders (Nepomorpha and Gerromorpha) and for a high level in the more advanced Pentatomomorpha. Data about this character in species from other infraorders are needed to confirm that tendency.

Male meiotic spindle features that efficiently segregate paired and lagging chromosomes.

Chromosome segregation during male meiosis is tailored to rapidly generate multitudes of sperm. Little is known about mechanisms that efficiently partition chromosomes to produce sperm. Using live imaging and tomographic reconstructions of spermatocyte meiotic spindles in Caenorhabditis elegans, we find the lagging X chromosome, a distinctive feature of anaphase I in C. elegans males, is due to lack of chromosome pairing. The unpaired chromosome remains tethered to centrosomes by lengthening kinetochore microtubules, which are under tension, suggesting that a 'tug of war' reliably resolves lagging. We find spermatocytes exhibit simultaneous pole-to-chromosome shortening (anaphase A) and pole-to-pole elongation (anaphase B). Electron tomography unexpectedly revealed spermatocyte anaphase A does not stem solely from kinetochore microtubule shortening. Instead, movement of autosomes is largely driven by distance change between chromosomes, microtubules, and centrosomes upon tension release during anaphase. Overall, we define novel features that segregate both lagging and paired chromosomes for optimal sperm production.

The Drosophila Citrate Lyase Is Required for Cell Division during Spermatogenesis.

The Drosophila melanogasterDmATPCL gene encodes for the human ATP Citrate Lyase (ACL) ortholog, a metabolic enzyme that from citrate generates glucose-derived Acetyl-CoA, which fuels central biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine, and the acetylation of proteins and histones. We had previously reported that, although loss of Drosophila ATPCL reduced levels of Acetyl-CoA, unlike its human counterpart, it does not affect global histone acetylation and gene expression, suggesting that its role in histone acetylation is either partially redundant in Drosophila or compensated by alternative pathways. Here, we describe that depletion of DmATPCL affects spindle organization, cytokinesis, and fusome assembly during male meiosis, revealing an unanticipated role for DmATPCL during spermatogenesis. We also show that DmATPCL mutant meiotic phenotype is in part caused by a reduction of fatty acids, but not of triglycerides or cholesterol, indicating that DmATPCL-derived Acetyl-CoA is predominantly devoted to the biosynthesis of fatty acids during spermatogenesis. Collectively, our results unveil for the first time an involvement for DmATPCL in the regulation of meiotic cell division, which is likely conserved in human cells.