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      C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

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      PLoS Genetics
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          Abstract

          The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes.

          Author summary

          The precursor cells that produce gametes—sperm and eggs in animals—have two copies of each chromosome, one from each parent. These precursors undergo specialized cell divisions that leave each gamete with only one copy of each chromosome; defects that produce incorrect chromosome number cause severe developmental abnormalities. In oocytes, these cell divisions are highly asymmetric, with extra chromosomes discarded into small membrane bound polar bodies, leaving one chromosome set within the much larger oocyte. How oocytes assemble the contractile apparatus that pinches off polar bodies remains poorly understood. To better understand this process, we have used the nematode Caenorhabditis elegans to investigate the relationship between the bipolar structure that separates oocyte chromosomes, called the spindle, and assembly of the contractile apparatus that pinches off polar bodies. We used a comparative approach, examining this relationship in three spindle assembly defective mutants. Bipolar spindle assembly and chromosome separation were not required for polar body extrusion, as it occurred normally in mutants lacking a protein called KLP-18. However, mutants lacking the protein CLS-2 failed to assemble the contractile apparatus, while mutants lacking the protein MEI-1 assembled a contractile apparatus that failed to fully constrict. We also found that CLS-2 down-regulates membrane ingression throughout the oocyte surface, and we discuss the relationship between oocyte membrane stiffness and polar body extrusion.

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          Most cited references67

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          Fiji: an open-source platform for biological-image analysis.

          Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
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            Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.

            Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.
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              Cytokinesis in animal cells.

              Cytokinesis, the final step in cell division, partitions the contents of a single cell into two. In animal cells, cytokinesis occurs through cortical remodeling orchestrated by the anaphase spindle. Cytokinesis relies on a tight interplay between signaling and cellular mechanics and has attracted the attention of both biologists and physicists for more than a century. In this review, we provide an overview of four topics in animal cell cytokinesis: (a) signaling between the anaphase spindle and cortex, (b) the mechanics of cortical remodeling, (c) abscission, and (d) regulation of cytokinesis by the cell cycle machinery. We report on recent progress in these areas and highlight some of the outstanding questions that these findings bring into focus.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                7 October 2020
                October 2020
                : 16
                : 10
                : e1008751
                Affiliations
                [001]Institute of Molecular Biology, University of Oregon, Eugene, OR, United States of America
                Harvard Medical School, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-7281-8851
                Article
                PGENETICS-D-20-00475
                10.1371/journal.pgen.1008751
                7571700
                33027250
                d73817f3-a0a1-4ce7-b3ad-111c0a5badc6
                © 2020 Schlientz, Bowerman

                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.

                History
                : 28 March 2020
                : 10 August 2020
                Page count
                Figures: 7, Tables: 0, Pages: 30
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R35GM131749
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01GM049869
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: T32GM007413
                Award Recipient :
                This work was supported by funding from the National Institutes of Health ( www.nih.gov) T32GM007413 (AJS) and R01GM049869 and R35GM131749 (AJS and BB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                OVA
                Oocytes
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Meiosis
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Meiosis
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cellular Extrusion
                Biology and Life Sciences
                Cell Biology
                Cell Physiology
                Cell Polarity
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cytoskeleton
                Microtubules
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Model Organisms
                Caenorhabditis Elegans
                Research and Analysis Methods
                Model Organisms
                Caenorhabditis Elegans
                Research and Analysis Methods
                Animal Studies
                Experimental Organism Systems
                Animal Models
                Caenorhabditis Elegans
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Nematoda
                Caenorhabditis
                Caenorhabditis Elegans
                Biology and Life Sciences
                Zoology
                Animals
                Invertebrates
                Nematoda
                Caenorhabditis
                Caenorhabditis Elegans
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Cell Membranes
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomes
                Chromosome Structure and Function
                Custom metadata
                vor-update-to-uncorrected-proof
                2020-10-19
                All relevant data are within the manuscript and its Supporting Information files.

                Genetics
                Genetics

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