The meiotic phosphatase GSP-2/PP1 promotes germline immortality and small RNA-mediated genome silencing.

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies (PB). During cytokinesis in somatic cells, the midbody (MB) and subsequent assembly of the midbody remnant (MBR), a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the MB and MBR in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic MBs (mMB) and mMB remnants (mMBRs) using mouse oocytes and demonstrate that mMBs have a specialized meiotic mMB cap structure that is orientated toward PBs. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

Traditionally, midbody remnants (MBRs) are isolated from cell culture medium using ultracentrifugation, which is expensive and time consuming. Here, we present a protocol for isolating MBRs or large extracellular vesicles (EVs) from mammalian cell culture using either 1.5% polyethylene glycol 6000 (PEG6000) or PEG5000-coated gold nanoparticles. We describe steps for growing cells, collecting media, and precipitating MBRs and EVs from cell culture medium. We then detail characterization of MBRs through immunofluorescent antibody staining and immunofluorescent imaging.

The mammalian midbody and midbody remnant are assembly sites for RNA and localized translation.

Long ignored as a vestigial remnant of cytokinesis, the mammalian midbody (MB) is released post-abscission inside large extracellular vesicles called MB remnants (MBRs). Recent evidence suggests that MBRs can modulate cell proliferation and cell fate decisions. Here, we demonstrate that the MB matrix is the site of ribonucleoprotein assembly and is enriched in mRNAs that encode proteins involved in cell fate, oncogenesis, and pluripotency, which we are calling the MB granule. Both MBs and post-abscission MBRs are sites of spatiotemporally regulated translation, which is initiated when nascent daughter cells re-enter G1 and continues after extracellular release. MKLP1 and ARC are necessary for the localization and translation of RNA in the MB dark zone, whereas ESCRT-III is necessary to maintain translation levels in the MB. Our work reveals a unique translation event that occurs during abscission and within a large extracellular vesicle.

Midbodies and phragmoplasts: analogous structures involved in cytokinesis.

Cytokinesis is an event common to all organisms that involves the precise coordination of independent pathways involved in cell-cycle regulation and microtubule, membrane, actin and organelle dynamics. In animal cells, the spindle midzone/midbody with associated endo-membrane system are required for late cytokinesis events, including furrow ingression and scission. In plants, cytokinesis is mediated by the phragmoplast, an array of microtubules, actin filaments and associated molecules that act as a framework for the future cell wall. In this article (which is part of the Cytokinesis series), we discuss recent studies that highlight the increasing number of similarities in the components and function of the spindle midzone/midbody in animals and the phragmoplast in plants, suggesting that they might be analogous structures.

SPD-3 is required for spindle alignment in Caenorhabditis elegans embryos and localizes to mitochondria.

During the development of multicellular organisms, cellular diversity is often achieved through asymmetric cell divisions that produce two daughter cells having different developmental potentials. Prior to an asymmetric cell division, cellular components segregate to opposite ends of the cell defining an axis of polarity. The mitotic spindle rotationally aligns along this axis of polarity, thereby ensuring that the cleavage plane is positioned such that segregated components end up in individual daughter cells. Here we report our characterization of a novel gene required for spindle alignment in Caenorhabditis elegans. During the first mitosis in spd-3(oj35) embryos the spindle failed to align along the anterior/posterior axis, leading to abnormal cleavage configurations. spd-3(oj35) embryos had additional defects reminiscent of dynein/dynactin loss-of-function possibly caused by the mislocalization of dynactin. Surprisingly, we found that SPD-3GFP localized to mitochondria. Consistent with this localization, spd-3(oj35) worms exhibited slow growth and increased ATP concentrations, which are phenotypes similar to those described for other mitochondrial mutants in C. elegans. To our knowledge, SPD-3 is the first example of a link between mitochondria and spindle alignment in C. elegans.

The entrance: how life experience shaped my passion for diversity and inclusion.

I am deeply honored to receive the American Society for Cell Biology (ASCB) Prize for Excellence in Inclusivity made possible through a grant from the Howard Hughes Medical Institute. This generous award of $5000 will go toward travel and registration support for underrepresented students from the University of Wisconsin-Madison to attend the ASCB and SACNAS (Society for Advancement of Chicanos/Hispanics and Native Americans in Science) conferences. In this essay, I have woven together a few stories on how my life experiences have shaped my passion for diversity and inclusion in STEM.

The large GTPase dynamin associates with the spindle midzone and is required for cytokinesis.

Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.

Correction: Profiling of the Mammalian Mitotic Spindle Proteome Reveals an ER Protein, OSTD-1, as Being Necessary for Cell Division and ER Morphology.

[This corrects the article DOI: 10.1371/journal.pone.0077051.].

The RNA-binding protein ATX-2 regulates cytokinesis through PAR-5 and ZEN-4.

The spindle midzone harbors both microtubules and proteins necessary for furrow formation and the completion of cytokinesis. However, the mechanisms that mediate the temporal and spatial recruitment of cell division factors to the spindle midzone and midbody remain unclear. Here we describe a mechanism governed by the conserved RNA-binding protein ATX-2/Ataxin-2, which targets and maintains ZEN-4 at the spindle midzone. ATX-2 does this by regulating the amount of PAR-5 at mitotic structures, particularly the spindle, centrosomes, and midbody. Preventing ATX-2 function leads to elevated levels of PAR-5, enhanced chromatin and centrosome localization of PAR-5-GFP, and ultimately a reduction of ZEN-4-GFP at the spindle midzone. Codepletion of ATX-2 and PAR-5 rescued the localization of ZEN-4 at the spindle midzone, indicating that ATX-2 mediates the localization of ZEN-4 upstream of PAR-5. We provide the first direct evidence that ATX-2 is necessary for cytokinesis and suggest a model in which ATX-2 facilitates the targeting of ZEN-4 to the spindle midzone by mediating the posttranscriptional regulation of PAR-5.

Spindlegate: the biological consequences of disrupting traffic.

The function of membrane trafficking during mitosis has become the focus of increasing interest. In this issue of Developmental Cell, Hehnly and Doxsey (2014) provide new insight into the role that endosomes play during spindle assembly.

Profiling of the mammalian mitotic spindle proteome reveals an ER protein, OSTD-1, as being necessary for cell division and ER morphology.

Cell division is important for many cellular processes including cell growth, reproduction, wound healing and stem cell renewal. Failures in cell division can often lead to tumors and birth defects. To identify factors necessary for this process, we implemented a comparative profiling strategy of the published mitotic spindle proteome from our laboratory. Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease. Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins. A secondary, visual screen to detect distinct defects in cell division revealed 21 genes that were necessary for cytokinesis. One of these candidates, OSTD-1, an ER resident protein, was further characterized due to the aberrant cleavage furrow placement and failures in division. We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle. In addition, 65% of all ostd-1 RNAi-treated embryos failed to correctly position cleavage furrows, suggesting that proper ER morphology plays a necessary function during animal cell division.

Arp2/3 mediates early endosome dynamics necessary for the maintenance of PAR asymmetry in Caenorhabditis elegans.

The widely conserved Arp2/3 complex regulates branched actin dynamics that are necessary for a variety of cellular processes. In Caenorhabditis elegans, the actin cytoskeleton has been extensively characterized in its role in establishing PAR asymmetry; however, the contributions of actin to the maintenance of polarity before the onset of mitosis are less clear. Endocytic recycling has emerged as a key mechanism in the dynamic stabilization of cellular polarity, and the large GTPase dynamin participates in the stabilization of cortical polarity during maintenance phase via endocytosis in C. elegans. Here we show that disruption of Arp2/3 function affects the formation and localization of short cortical actin filaments and foci, endocytic regulators, and polarity proteins during maintenance phase. We detect actin associated with events similar to early endosomal fission, movement of endosomes into the cytoplasm, and endosomal movement from the cytoplasm to the plasma membrane, suggesting the involvement of actin in regulating processes at the early endosome. We also observe aberrant accumulations of PAR-6 cytoplasmic puncta near the centrosome along with early endosomes. We propose a model in which Arp2/3 affects the efficiency of rapid endocytic recycling of polarity cues that ultimately contributes to their stable maintenance.

Anterior PAR proteins function during cytokinesis and maintain DYN-1 at the cleavage furrow in Caenorhabditis elegans.

PAR proteins are key regulators of cellular polarity and have links to the endocytic machinery and the actin cytoskeleton. Our data suggest a unique role for PAR proteins in cytokinesis. We have found that at the onset of cytokinesis, anterior PAR-6 and posterior PAR-2 proteins are redistributed to the furrow membrane in a temporal and spatial manner. PAR-6 and PAR-2 localize to the furrow membrane during ingression but PAR-2-GFP is distinct in that it is excluded from the extreme tip of the furrow. Once the midbody has formed, PAR-2-GFP becomes restricted to the midbody region (the midbody plus the membrane flanking it). Depletion of both anterior PAR proteins, PAR-3 and PAR-6, led to an increase in multinucleate embryos, suggesting that the anterior PAR proteins are necessary during cytokinesis and that PAR-3 and PAR-6 function in cytokinesis may be partially redundant. Lastly, anterior PAR proteins play a role in the maintenance of DYN-1 in the cleavage furrow. Our data indicate that the PAR proteins are involved in the events that occur during cytokinesis and may play a role in promoting the membrane trafficking and remodeling events that occur during this time.

Long astral microtubules and RACK-1 stabilize polarity domains during maintenance phase in Caenorhabditis elegans embryos.

Cell polarity is a very well conserved process important for cell differentiation, cell migration, and embryonic development. After the establishment of distinct cortical domains, polarity cues have to be stabilized and maintained within a fluid and dynamic membrane to achieve proper cell asymmetry. Microtubules have long been thought to deliver the signals required to polarize a cell. While previous studies suggest that microtubules play a key role in the establishment of polarity, the requirement of microtubules during maintenance phase remains unclear. In this study, we show that depletion of Caenorhabditis elegans RACK-1, which leads to short astral microtubules during prometaphase, specifically affects maintenance of cortical PAR domains and Dynamin localization. We then investigated the consequence of knocking down other factors that also abolish astral microtubule elongation during polarity maintenance phase. We found a correlation between short astral microtubules and the instability of PAR-6 and PAR-2 domains during maintenance phase. Our data support a necessary role for astral microtubules in the maintenance phase of cell polarity.

Mitotic spindle proteomics in Chinese hamster ovary cells.

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.