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.

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.

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.

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.

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.

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.

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.

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.

Polarity and endocytosis: reciprocal regulation.

The establishment and maintenance of polarized plasma membrane domains is essential for cellular function and proper development of organisms. The molecules and pathways involved in determining cell polarity are remarkably well conserved between animal species. Historically, exocytic mechanisms have received primary emphasis among trafficking routes responsible for cell polarization. Accumulating evidence now reveals that endocytosis plays an equally important role in the proper localization of key polarity proteins. Intriguingly, some polarity proteins can also regulate the endocytic machinery. Here, we review emerging evidence for the reciprocal regulation between polarity proteins and endocytic pathways, and discuss possible models for how these distinct processes could interact to create separate cellular domains.

Endosomal recycling regulation during cytokinesis.

Successful cytokinesis is critical for cell proliferation and development. In animal cells, cytokinesis relies on temporally and spatially regulated membrane addition to the cleavage site. An important source for the new membrane is recycling endosomes. Yet how these endocytic vesicles are transported and regulated remains unclear. Several potential factors have been recently identified that regulate the trafficking of recycling endosomes during cytokinesis. Dynein and dynactin are required for the retrograde transport of recycling endosomes, while Kinesin-1 is responsible for endosome delivery to the furrow and midbody. Other regulators of recycling endosome trafficking have been identified, including RACK1, JIP3/4 and ECT2, which target recycling endosomes during the cell cycle. Here, we provide insights into the mechanisms controlling endosomal trafficking during cytokinesis.

Dynamin participates in the maintenance of anterior polarity in the Caenorhabditis elegans embryo.

Cell polarity is crucial for the generation of cell diversity. Recent evidence suggests that the actin cytoskeleton plays a key role in establishment of embryonic polarity, yet the mechanisms that maintain polarity cues in particular membrane domains during development remain unclear. Dynamin, a large GTPase, functions in both endocytosis and actin dynamics. Here, the Caenorhabditis elegans dynamin ortholog, DYN-1, maintains anterior polarity cues. DYN-1-GFP foci are enriched in the anterior cortex in a manner dependent on the anterior polarity proteins, PAR-6 and PKC-3. Membrane internalization and actin comet formation are enriched in the anterior, and are dependent on DYN-1. PAR-6-labeled puncta are also internalized from cortical accumulations of DYN-1-GFP. Our results demonstrate a mechanism for the spatial and temporal regulation of endocytosis in the anterior of the embryo, contributing to the precise localization and maintenance of polarity factors within a dynamic plasma membrane.

RACK-1 directs dynactin-dependent RAB-11 endosomal recycling during mitosis in Caenorhabditis elegans.

Membrane trafficking pathways are necessary for the addition and removal of membrane during cytokinesis. In animal cells, recycling endosomes act as a major source of the additional membranes during furrow progression and abscission. However, the mechanisms and factors that regulate recycling endosomes during the cell cycle remain poorly understood. Here, we show that the Caenorhabditis elegans Receptor of Activated C Kinase 1 (RACK-1) is required for cytokinesis, germline membrane organization, and the recruitment of RAB-11-labeled recycling endosomes to the pericentrosomal region and spindle. RACK-1 is also required for proper chromosome separation and astral microtubule length. RACK-1 localizes to the centrosomes, kinetochores, the midbody, and nuclear envelopes during the cell cycle. We found that RACK-1 directly binds to DNC-2, the C. elegans p50/dynamitin subunit of the dynactin complex. Last, RACK-1 may facilitate the sequestration of recycling endosomes by targeting DNC-2 to centrosomes and the spindle. Our findings suggest a mechanism by which RACK-1 directs the dynactin-dependent redistribution of recycling endosomes during the cell cycle, thus ensuring proper membrane trafficking events during cytokinesis.

Cell division screens and dynamin.

Cell division is the most fundamental process in the development of all living organisms. The generation of cell diversity throughout development, the multiplication of cells during wound repair and the maintenance of stem cells in several tissues and organs all rely on proper progress through cell division. Historically, biochemical studies of cell division proved to be difficult, since mitosis is a moving target. The rapid and dynamic nature of mitosis means necessary proteins often exist in multiple isoforms and some for only brief moments during a particular stage in the cell cycle. The advent of proteomics and the introduction of stage-specific inhibitors have enabled the field to identify numerous factors required at distinct steps in the cell cycle. One such factor identified in many of these screens was the highly conserved protein dynamin. Dynamin, long known for its role in endocytosis, is also necessary for co-ordinating actin assembly at membranes. Our knowledge of its precise cell cycle function and upstream/downstream targets, however, is unclear. Our review will describe current knowledge regarding the impacts of several cell division screens and the multiple roles that dynamin may play during mitosis.

Src and Wnt signaling regulate dynactin accumulation to the P2-EMS cell border in C. elegans embryos.

In many organisms, the dynein-dynactin complex is required for the alignment of the mitotic spindle onto the axis of polarity of a cell undergoing asymmetric cell division. How this complex transduces polarity cues, either intrinsic or extrinsic, and rotationally aligns the spindle accordingly is not well understood. The Caenorhabditis elegans blastomere P2 polarizes the neighboring EMS blastomere, which causes the EMS spindle to rotationally align along the defined axis of polarity via two redundant signaling pathways: Wnt and Src. Here, we describe how components of the dynactin complex became locally enriched at the P2-EMS border prior to and during rotational alignment of their spindles. Wnt and Src signaling were required for both localized dynactin enrichment, and for rotational alignment of the P2 and EMS spindles. Depleting the trimeric G-protein subunit G alpha did not abolish dynactin accumulation to the P2-EMS border, yet both EMS and P2 spindles failed to rotationally align, indicating that G alpha might act to regulate dynein/dynactin motor activity. By RNAi of a weak dnc-1(ts) allele, we showed that dynactin activity was required at least for EMS spindle rotational alignment.

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.