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• W2983251036 abstract "•DNA segregation defects due to furrow mispositioning can be corrected•Correction involves opposite nuclear and furrow movements•Cortical relaxation and cytoplasmic flow due to blebbing reposition the furrow•Microtubules and cytoplasmic flow both contribute to nuclear movement Coordinating mitotic spindle and cytokinetic furrow positioning is essential to ensure proper DNA segregation. Here, we present a novel mechanism, which corrects DNA segregation defects due to cytokinetic furrow mispositioning during the first division of C. elegans embryos. Correction of DNA segregation defects due to an abnormally anterior cytokinetic furrow relies on the concomitant and opposite displacements of the furrow and of the anterior nucleus toward the posterior and anterior poles of the embryo, respectively. It also coincides with cortical blebbing and an anteriorly directed cytoplasmic flow. Although microtubules contribute to nuclear displacement, relaxation of an excessive tension at the anterior cortex plays a central role in the correction process and simultaneously regulates cytoplasmic flow as well as nuclear and furrow displacements. This work thus reveals the existence of a so-far uncharacterized correction mechanism, which is critical to correct DNA segregation defects due to cytokinetic furrow mispositioning. Coordinating mitotic spindle and cytokinetic furrow positioning is essential to ensure proper DNA segregation. Here, we present a novel mechanism, which corrects DNA segregation defects due to cytokinetic furrow mispositioning during the first division of C. elegans embryos. Correction of DNA segregation defects due to an abnormally anterior cytokinetic furrow relies on the concomitant and opposite displacements of the furrow and of the anterior nucleus toward the posterior and anterior poles of the embryo, respectively. It also coincides with cortical blebbing and an anteriorly directed cytoplasmic flow. Although microtubules contribute to nuclear displacement, relaxation of an excessive tension at the anterior cortex plays a central role in the correction process and simultaneously regulates cytoplasmic flow as well as nuclear and furrow displacements. This work thus reveals the existence of a so-far uncharacterized correction mechanism, which is critical to correct DNA segregation defects due to cytokinetic furrow mispositioning. Ensuring equal DNA segregation is a critical feature of cell division. The correct assembly of the mitotic spindle at metaphase followed by the separation of sister chromatids at anaphase are essential steps in this process. Ingression of the cytokinetic furrow between the separated chromatids then ensures that the genetic material is equally inherited by the two daughter cells. It is therefore crucial that the positions of the cytokinetic furrow and the mitotic spindle are well coordinated. Two main signaling pathways emanating from the mitotic spindle control furrow position by restricting the localization of myosin contractile ring components to the vicinity of the central spindle [1Bringmann H. Hyman A.A. A cytokinesis furrow is positioned by two consecutive signals.Nature. 2005; 436: 731-734Crossref PubMed Scopus (175) Google Scholar, 2Dechant R. Glotzer M. Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation.Dev. Cell. 2003; 4: 333-344Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar]. First, the centralspindlin complex, which localizes both at the central spindle and at the adjacent equatorial cortex, activates myosin contractility through the regulation of the small guanosine triphosphatase (GTPase) Rho [3Basant A. Lekomtsev S. Tse Y.C. Zhang D. Longhini K.M. Petronczki M. Glotzer M. Aurora B kinase promotes cytokinesis by inducing centralspindlin oligomers that associate with the plasma membrane.Dev. Cell. 2015; 33: 204-215Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Nishimura Y. Yonemura S. Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis.J. Cell Sci. 2006; 119: 104-114Crossref PubMed Scopus (197) Google Scholar, 5Yüce O. Piekny A. Glotzer M. An ECT2-centralspindlin complex regulates the localization and function of RhoA.J. Cell Biol. 2005; 170: 571-582Crossref PubMed Scopus (362) Google Scholar]. Second, astral microtubules prevent myosin activity at the pole of dividing cells through mechanisms that remain to be precisely determined [6Bringmann H. Cowan C.R. Kong J. Hyman A.A. LET-99, GOA-1/GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis.Curr. Biol. 2007; 17: 185-191Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Lewellyn L. Dumont J. Desai A. Oegema K. Analyzing the effects of delaying aster separation on furrow formation during cytokinesis in the Caenorhabditis elegans embryo.Mol. Biol. Cell. 2010; 21: 50-62Crossref PubMed Scopus (33) Google Scholar, 8Mangal S. Sacher J. Kim T. Osório D.S. Motegi F. Carvalho A.X. Oegema K. Zanin E. TPXL-1 activates Aurora A to clear contractile ring components from the polar cortex during cytokinesis.J. Cell Biol. 2018; 217: 837-848Crossref PubMed Scopus (40) Google Scholar, 9Price K.L. Rose L.S. LET-99 functions in the astral furrowing pathway, where it is required for myosin enrichment in the contractile ring.Mol. Biol. Cell. 2017; 28: 2360-2373Crossref PubMed Google Scholar, 10Werner M. Munro E. Glotzer M. Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis.Curr. Biol. 2007; 17: 1286-1297Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar]. However, asymmetrically localized myosin also contributes to furrow localization [11Cabernard C. Prehoda K.E. Doe C.Q. A spindle-independent cleavage furrow positioning pathway.Nature. 2010; 467: 91-94Crossref PubMed Scopus (127) Google Scholar, 12Ou G. Stuurman N. D’Ambrosio M. Vale R.D. Polarized myosin produces unequal-size daughters during asymmetric cell division.Science. 2010; 330: 677-680Crossref PubMed Scopus (124) Google Scholar, 13Pacquelet A. Uhart P. Tassan J.-P. Michaux G. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.J. Cell Biol. 2015; 210: 1085-1099Crossref PubMed Scopus (27) Google Scholar]. During the first division of the one-cell C. elegans embryo, myosin transiently accumulates at the anterior cortex at early anaphase before being restricted to the equatorial cortex at the presumptive furrow ingression site [13Pacquelet A. Uhart P. Tassan J.-P. Michaux G. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.J. Cell Biol. 2015; 210: 1085-1099Crossref PubMed Scopus (27) Google Scholar]. This dynamic localization of myosin is tightly regulated. In particular, in embryos lacking both the kinase PIG-1 and the anillin ANI-1, myosin abnormally accumulates at the anterior cortex and leads to the displacement of the cytokinetic furrow, which position is thus no longer coordinated with the position of the mitotic spindle [13Pacquelet A. Uhart P. Tassan J.-P. Michaux G. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.J. Cell Biol. 2015; 210: 1085-1099Crossref PubMed Scopus (27) Google Scholar]. Mistakes occurring at any of the successive steps of mitosis may impair DNA segregation. However, several mechanisms, such as the spindle assembly [14Lara-Gonzalez P. Westhorpe F.G. Taylor S.S. The spindle assembly checkpoint.Curr. Biol. 2012; 22: R966-R980Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar] or the abscission checkpoints [15Norden C. Mendoza M. Dobbelaere J. Kotwaliwale C.V. Biggins S. Barral Y. The NoCut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage.Cell. 2006; 125: 85-98Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 16Steigemann P. Wurzenberger C. Schmitz M.H.A. Held M. Guizetti J. Maar S. Gerlich D.W. Aurora B-mediated abscission checkpoint protects against tetraploidization.Cell. 2009; 136: 473-484Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar], limit the occurrence of detrimental DNA segregation defects. In this study, we identify and characterize a new type of correction mechanism that prevents DNA segregation defects resulting from the lack of coordination between cytokinetic furrow and mitotic spindle positions. In ani-1(RNAi);pig-1(gm344) embryos (further referred to as ani-1;pig-1), excessive myosin accumulation at the anterior cortex displaces the furrow toward the anterior of the embryo. As a result, the two nuclei are often both located on the posterior side of the furrow (17/20 embryos; Figure 1A; t = 300 or 380 s; Video S1) [13Pacquelet A. Uhart P. Tassan J.-P. Michaux G. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.J. Cell Biol. 2015; 210: 1085-1099Crossref PubMed Scopus (27) Google Scholar]. However, most of those DNA segregation defects are corrected, with the anterior nucleus finally being located on the anterior side of the furrow (16/17 embryos; Figure 1A; t = 540 s; Video S1). After correction, furrow ingression completes in 13/16 embryos and persists until the beginning of the second division in 8/16 embryos. Moreover, the anterior nucleus contained the expected number (i.e., 12) of chromosomes (n = 10 embryos; Video S2). Hence, the correction process ensures that each daughter cell inherits the correct number of chromosomes. https://www.cell.com/cms/asset/e7dd9df7-843c-4828-8fd2-f129bc696cbf/mmc2.mp4Loading ... Download .mp4 (5.05 MB) Help with .mp4 files Video S1. Correction of DNA Segregation Defects Resulting from Furrow Mispositioning in ani-1;pig-1 Embryos, Related to Figure 1Control (left) and ani-1(RNAi);pig-1(gm344) (right) embryos expressing NMY-2::GFP (magenta) and mCherry::HIS (yellow). Increased myosin levels at the anterior cortex of ani-1(RNAi);pig-1(gm344) embryos lead to furrow mispositioning and DNA segregation defects which are corrected late during mitosis. In control embryos the anterior nucleus (yellow asterisk) is slightly displaced during cytokinesis and furrow position remains stable (arrow). Correction in ani-1(RNAi);pig-1(gm344) embryos involves the strong displacement of the anterior nucleus (yellow asterisk) to the anterior and the displacement of the furrow (arrow) to the posterior. t0 = anaphase onset. Images were recorded at 4 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2ZWJiMzYxNjQ4NzQxN2E2ODUwNjdhZTZhNWVhZGI1MCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4MjczNDU5fQ.WqpODMiXrZZebQfpQLOM2iAh73-Fk-povN2xOLDEKjxN77xFTr4d7AFJADtQM1KgU-KyV7orxYhJvGd1rK3dBfig0In2FjHRqtwxS_JureB9eg6WY-QD1Zzc0mnXSiKXG1OGlnKhi4BKIFqF62178J74qHEH07FHa_WsBt5zbaZ9zVyQLG0JsbEP1RzIJ_C9oEdvn12LULezbsl4Ov1k9bJBRuPydj5PythwJASDjRRmCQ4Oav3eDD9gQI_6Vod7Q87lQLav4F_62Lx7NCbOpH0wwdUB0fW4Df8eTfNIHWimzNE2QhdcZ122dg1NPeL1dV4ho6CyuTReh5iaCnxSAw Download .mp4 (1.56 MB) Help with .mp4 files Video S2. 3D Reconstruction Image of Chromosomes in the Anterior Nucleus of a ani-1;pig-1 Embryo, Related to Figure 1ani-1(RNAi);pig-1(gm344) embryo expressing NMY-2::GFP (not shown) and mCherry::HIS (red, to label chromosomes). Scale bar: 2 μm. As cytokinesis is delayed in ani-1;pig-1 embryos (e.g., Figure 1A), we asked when the correction of DNA segregation defects occurs relative to other cell cycle events. We first monitored nuclear envelope reformation. Both in control and ani-1;pig-1 embryos, nuclear envelope reassembles during cytokinesis (Figure 1B; Video S3). In ani-1;pig-1 embryos, this always precedes the correction of DNA segregation defects (n = 18/18). We next examined mitotic and central spindle disassembly. Although spindle disassembly coincides with the end of furrow ingression in control embryos, it precedes the correction of DNA segregation defects in ani-1;pig-1 embryos (n = 11/12; Video S4). In control embryos, the central spindle starts to lose its compact structure at the end of furrow ingression and central spindle remnants associate with the ingressing furrow to form the midbody (Figure 1C; Video S5A). In ani-1;pig-1 embryos, the central spindle starts to disassemble before the correction of DNA segregation defects (n = 21/22; Figure 1C; Video S5B). Some central spindle remnants associate with the closing cytokinetic ring (Video S5C and legend), but others become fainter and cannot be detected anymore at the end of cytokinesis (Video S5D). In summary, the correction of DNA segregation defects in ani-1;pig-1 embryos occurs late during the cell cycle, when the nuclear envelope has already reformed and the mitotic and central spindle have disassembled. Importantly, this correction mechanism is not specific to ani-1;pig-1 embryos. Similarly, increased myosin activity following depletion of the Rho GAPs RGA-3/4 results in furrow displacement and DNA segregation defects, which are then corrected (Figure 1D and legend; Video S6). Laser ablation of anaphase centrosomes also leads to the relative mispositioning of the mitotic spindle and cytokinetic furrow. Notably, although DNA segregation defects resulting from anterior and posterior centrosome ablations occur in the opposite direction, both can be corrected (Figures 1E and 1F and legend; Video S7). Altogether, those results show that DNA segregation defects due to furrow and/or spindle mispositioning can be corrected in different contexts. https://www.cell.com/cms/asset/0c644d6e-a0e7-48c8-bf8a-1ae7cf5bd63e/mmc4.mp4Loading ... Download .mp4 (4.55 MB) Help with .mp4 files Video S3. Correction of DNA Segregation Defects in ani-1;pig-1 Embryos Occurs after Nuclear Envelope Reformation, Related to Figure 1Control (left) and ani-1(RNAi);pig-1(gm344) (right) embryos expressing NMY-2::GFP (magenta) and EMR-1::mCherry (yellow). Nuclear envelope reassembles before the correction of DNA segregation defects. t0 = nuclear envelope breakdown. Images were recorded at 5 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJmOGJhYzYwOTk1YjllNmYzZmNiMzVjYmFmMjgwOWI1ZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4MjczNDU5fQ.ZNqKUMwGR9qoNRZuqKcMRNHAg01WGS2QYa6aifmvEFKBxgRox0V3rvcMKu8hfx4WoQhRFuJXxp4A0Hb3EA5FAO59QBNND8DVNQeRUIgwq9BajT3nLqIEBC02l-F2Hqh7gzM6kyIHjMdm1dlEW3Xdlqj8UCkaZNQgg5WRU8K3pcA_Sqc9Bvkfvnv8cYLQA0q3yTENvB2aKM7SeRbqRYaL9vXqtSkTh6KikrIdE52nX4yy-2bV-xw6fxJjiBBYEoJAFsKanjLpaUziK9zgrEg1eZTkul9riDQN9H2r43E8e4dplJHJ6uEMPiZnVl5muBPUK4PypLtz8tSuisX4TXuHzw Download .mp4 (7.32 MB) Help with .mp4 files Video S4. Correction of DNA Segregation Defects in ani-1;pig-1 Embryos Occurs after Mitotic Spindle Disassembly, Related to Figure 1Control (left) and ani-1(RNAi);pig-1(gm344) (right) embryos expressing α-tubulin::YFP (magenta in upper images, gray in lower images) and mCherry::HIS (yellow in upper images). The mitotic spindle starts disassembling before the correction of DNA segregation defects. t0 = anaphase onset. Images were recorded at 5 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. https://www.cell.com/cms/asset/9f7ecd0f-9748-49f3-a8a1-74e8f47f9763/mmc6.mp4Loading ... Download .mp4 (5.66 MB) Help with .mp4 files Video S5. Correction of DNA Segregation Defects in ani-1;pig-1 Embryos Occurs after Central Spindle Disassembly, Related to Figure 1A-D. Control (A) and ani-1(RNAi);pig-1(gm344) (B-D) embryos expressing SPD-1::GFP (magenta, labels the central spindle, centrosomes and the nuclei after division), PH::GFP (magenta, membrane labeling) and mCherry::HIS (yellow, not shown in C-D). The central spindle starts disassembling before the correction of DNA segregation defects (B). Note that central spindle remnants can be displaced toward the anterior by the cytoplasmic flow (B, 13/24 embryos) or stay in the posterior cytoplasm (D, 11/24 embryos). In some embryos, we observed that some central spindle remnants associate with the closing cytokinetic ring (C, arrowhead, 8/24 embryos). In others, no central spindle remnants could be observed at the time of ring closure (D, 14/24 embryos). In A-B, the SPD-1::GFP signal was imaged during the whole division process. t0 = anaphase onset. Images were recorded at 5 s intervals. In C-D, the SPD-1::GFP signal was imaged only at the end of the correction process (see STAR method for details). t0 = cytokinetic ring closure. Images were recorded at 10 s intervals. All videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. https://www.cell.com/cms/asset/a096ab99-46c6-44b3-839d-8559d9879e9d/mmc7.mp4Loading ... Download .mp4 (3.89 MB) Help with .mp4 files Video S6. Correction of DNA Segregation Defects Resulting from Furrow Mispositioning in rga-3/4 Embryos, Related to Figure 1Control (left) and rga-3/4(RNAi) (right) embryos expressing NMY-2::GFP (magenta) and mCherry::HIS (yellow). Increased myosin levels at the anterior cortex of rga-3/4(RNAi) embryos lead to furrow mispositioning and DNA segregation defects, which are corrected late during mitosis. Correction involves the opposite displacement of the anterior nucleus (yellow asterisk) and the furrow (white arrow) toward the anterior and posterior pole of the embryo, respectively. Cortical blebbing is first observed at the pole of the embryo (magenta arrow, 11/13 embryos) then on the anterior side of the furrow (magenta arrowheads, 12/13 embryos). Cytoplasmic flow is directed toward the anterior pole of the embryo (white arrowhead, 13/13 embryos). t0 = anaphase onset. Images were recorded at 2 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. https://www.cell.com/cms/asset/78d48a96-cc67-4792-8d6e-1d0141af53f3/mmc8.mp4Loading ... Download .mp4 (9.52 MB) Help with .mp4 files Video S7. Correction of DNA Segregation Defects following Centrosome Ablations, Related to Figure 1Ablation of anterior (left) or posterior (right) centrosome (white circle) in embryos expressing NMY-2::GFP (green, cortical and furrow signals), GFP::α-tubulin (green, labels the centrosome and to a lesser extent microtubules) and GFP::HIS (green, labels the DNA). The lower panel shows DIC recordings of the same embryos. Centrosome ablation leads to spindle and/or furrow mispositioning. Correction of the resulting DNA segregation defects involves both nucleus (asterisk) and furrow (arrow) displacement. In the case of anterior centrosome ablation (left), the anterior nucleus moves to the anterior and the furrow to the posterior of the embryo. Reversely, in the case of posterior centrosome ablation (right), the posterior nucleus moves to the posterior and the furrow to the anterior of the embryo. Cortical blebbing (arrowheads) and cytoplasmic flow (arrows) can be observed on DIC images. In the case of anterior centrosome ablation (left), cortical blebs form on the anterior side of the furrow (13/15 embryos) and an anteriorly directed flow is observed (15/15 embryos). In the case of posterior centrosome ablation (right), cortical blebs form on the posterior side of the furrow (14/15 embryos) and an anteriorly directed flow is observed (14/15 embryos). t0 = anaphase onset. Images were recorded at 2 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. To elucidate the mechanisms underlying this correction process, we first tracked nuclei and cytokinetic furrow positions. In control embryos, the two nuclei are displaced toward the poles of the embryo during and after cytokinesis and furrow position remains stable during ring closure (Figures 1A and 2A–2C ; Video S1). In ani-1;pig-1 embryos, posterior nucleus displacement is similar to control embryos (Figure 2B), but the anterior nucleus moves faster and further (Figures 1A and 2A; Video S1). Furthermore, although the furrow initially moves toward the anterior, it is then displaced toward the posterior during the correction process (Figures 1A and 2C; Video S1). Notably, the opposite displacements of the anterior nucleus and the cytokinetic furrow occur concomitantly (Figure 2D). Similarly, opposite anterior nucleus and furrow movements occur when DNA segregation defects due to RGA-3/4 depletion or anterior centrosome ablations are corrected (Figures S1A–S1C and S1E; Videos S6 and S7). Reverse displacements are observed following posterior centrosome ablation (Figures S1D and S1E; Video S7). Opposite DNA and furrow movements thus correct DNA segregation defects due to furrow mispositioning. We next searched for the mechanisms involved in regulating nuclear displacement. We first tested the role of microtubules and used a thermosensitive allele (zyg-12(or577)) to inactivate the KASH protein ZYG-12 and prevent nuclei/microtubule interactions during the last steps of mitosis (Figure 3A and legend). This strongly inhibits anterior nuclear displacement in control embryos (Figure 3B) but only moderately affects anterior nuclear displacement in ani-1;pig-1 embryos (Figure 3C). Moreover, it does not prevent DNA segregation defect correction (16/16 ani-1;pig-1 and 13/14 zyg-12;ani-1;pig-1 embryos with corrected DNA segregation defects). Hence, although nuclear/microtubule interactions are critical to move nuclei away from the cytokinetic furrow in control embryos, they are involved, but not essential for anterior nuclear displacement in ani-1;pig-1 embryos. We next looked for an additional mechanism that could participate in anterior nucleus displacement and tested the possible involvement of myosin by using a thermosensitive allele of non-muscle myosin-2 (nmy-2(1490)). nmy-2;ani-1;pig-1 embryos were initially grown at permissive temperature to ensure that the increase in myosin activity due to the lack of PIG-1 and ANI-1 is sufficient to induce furrow mispositioning. They were then shifted at restrictive temperature 220 s after anaphase onset. In control embryos, myosin inactivation moderately reduces nuclear displacement (Figure 4A) and does not enhance the effect of ZYG-12 inactivation (Figure 4C). Thus, contrary to nuclear/microtubule interactions, myosin has a minor role in the regulation of nuclear position in control embryos. Myosin inactivation also moderately inhibits anterior nuclear displacement in ani-1;pig-1 embryos (Figure 4B). By contrast, simultaneous inactivation of ZYG-12 and myosin severely reduces the efficiency of DNA segregation defect correction (correction in 12/12 nmy-2;ani-1;pig-1 and 11/20 nmy-2;zyg-12;ani-1;pig-1 embryos; Figure 4D). Furthermore, anterior nucleus displacement is strongly impaired, both in rescued and non-rescued nmy-2;zyg-12;ani-1;pig-1 embryos (Figure 4D). Altogether, our results demonstrate that microtubules and myosin independently contribute to the positioning of the anterior nucleus during the correction of DNA segregation defects in ani-1;pig-1 embryos. Importantly, we also found that myosin inactivation prevents the furrow from moving back toward the posterior in nmy-2;ani-1;pig-1 embryos (Figures 4E and 4F; Video S8). Myosin therefore plays a crucial role in the correction process by concomitantly regulating nuclear and furrow displacements. https://www.cell.com/cms/asset/4d2e01f2-fac5-4433-ba15-288cd6a952f9/mmc9.mp4Loading ... Download .mp4 (11.23 MB) Help with .mp4 files Video S8. Myosin Activity Controls Furrow Displacement, Related to Figure 4ani-1(RNAi);pig-1(gm344) (left) and nmy-2(ne1490);ani-1(RNAi);pig-1(gm344) (right) embryos expressing GFP::PH (magenta) and mCherry::HIS (yellow, anterior nucleus also marked with asterisk). During correction, the furrow is displaced toward the posterior in ani-1(RNAi);pig-1(gm344) embryos but not in nmy-2(ne1490);ani-1(RNAi);pig-1(gm344) embryos. In ani-1(RNAi);pig-1(gm344) embryos, furrow displacement is associated with cortical blebbing on the anterior side of the furrow (white arrowheads). Formation of those cortical blebs locally displaces the base of the furrow and leads to furrow inclination. t0 = anaphase onset. Images were recorded at 2 s intervals and videos are played at 10 frames per second. Embryos are oriented with the anterior to the left. Scale bar: 10 μm. Considering the importance of myosin in the correction process, we carefully examined myosin cortical levels. Anterior myosin cortical level briefly increases in control anaphase embryos whereas an excessive and long-lasting accumulation of myosin is observed in ani-1;pig-1 embryos (Figure 5A, left) [13Pacquelet A. Uhart P. Tassan J.-P. Michaux G. PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.J. Cell Biol. 2015; 210: 1085-1099Crossref PubMed Scopus (27) Google Scholar]. This accumulation slowly drops off during the correction process (Figure 5A, left). Those changes are specific to the anterior cortex, as myosin posterior cortical levels only weakly and briefly increase in control and ani-1;pig-1 embryos (Figure 5A, right). Those observations prompted us to assess possible changes in cortical tension by performing laser-induced cortical ablation [18Mayer M. Depken M. Bois J.S. Jülicher F. Grill S.W. Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows.Nature. 2010; 467: 617-621Crossref PubMed Scopus (336) Google Scholar, 19Saha A. Nishikawa M. Behrndt M. Heisenberg C.-P. Jülicher F. Grill S.W. Determining physical properties of the cell cortex.Biophys. J. 2016; 110: 1421-1429Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar] (Figure 5B; Video S9). In control embryos, cortical tension during anaphase is slightly higher at the anterior than at the posterior cortex (Figure 5C). Although posterior cortical tension does not change significantly in ani-1;pig-1 embryos, anterior cortical tension increases during anaphase before declining at the end of the rescue process (Figure 5C). All these variations in cortical tension are consistent with the changes in myosin levels that we measured (Figure 5A). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIxOTI0MjNiODc3MmUyNzVhMmQwOWQ4OWM3MjAwNDQ0YiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4MjczNDYwfQ.ffNbRPpKYCTUP9wv2TL5ZM2QfzX-5RHpd1F5RLqZJtydTkpG4L8We0sRXMzon6HOADYeaD4v_1fdKUhtpl5vfWctiC01xrWXfRv7VVlro3v1LRTZDRgEvYDJ3JqzzidNbT3jqfT1OGDPm5m79TLvV6qGbTnBv6wgbtRIWuMrAT-3eZJyqCc4gyPHCTVaKtP8-eEcv7NfwV3-Oy5Dj3PpL8weC75txO9Mil7xLF3hz7yxzTsC56lyitoR6jfjIRc0O_xK_GLvvGigJ5Qd9DSnVnmGexETWBpBVCVaqRtf7bNf_S16D9pqObDKvZv1pXeTuzUm2PIeYoGr9YJ_sxAlXg Download .mp4 (0.47 MB) Help with .mp4 files Video S9. Laser-Induced Cortical Ablation, Related to Figure 5Ablation of the anterior cortex of a ani-1(RNAi);pig-1(gm344) embryo was performed during anaphase. The embryo expresses NMY-2::GFP (green) and mCherry::HIS (not shown). White bar indicates the area targeted for laser ablation. Video starts 3 s before laser ablation. Images were recorded at 0.5 s intervals and video is played at 10 frames per second. Scale bar: 5 μm. Cortical tension imbalance and asymmetric myosin-driven contraction of the cortex can alter daughter cell size in dividing HeLa cells [20Sedzinski J. Biro M. Oswald A. Tinevez J.-Y. Salbreux G. Paluch E. Polar actomyosin contractility destabilizes the position of the cytokinetic furrow.Nature. 2011; 476: 462-466Crossref PubMed Scopus (247) Google Scholar]. Variations in myosin level and cortical tension could thus explain furrow movements in ani-1;pig-1 embryos. Consistent with this hypothesis, movement of the nascent furrow (ring diameter > 15 μm) is correlated with cortical myosin levels: large differences of myosin levels between the anterior and posterior cortex (Δmyosin > 50 a.u.) are associated with the initial anterior displacement of the nascent furrow and weaker differences (Δmyosin < 30 a.u.) coincide with the furrow starting to move back toward the posterior pole (Figure 6A). If this correlation results from the mechanical action of cortical myosin on the furrow, we reasoned that furrow speed must be affected by factors that influence the mechanical resistance to furrow movements. Forces resisting furrow displacement include the resistance of the cortex itself, Fcort, and the drag force exerted by the cytoplasm, Fcyt (Figure 6B). As the embryo is confined in its eggshell, the cytopl" @default.
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• W2983251036 title "Simultaneous Regulation of Cytokinetic Furrow and Nucleus Positions by Cortical Tension Contributes to Proper DNA Segregation during Late Mitosis" @default.
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