Functional midbody assembly in the absence of a central spindle

2022 ◽  
Vol 221 (3) ◽  
Author(s):  
Sophia M. Hirsch ◽  
Frances Edwards ◽  
Mimi Shirasu-Hiza ◽  
Julien Dumont ◽  
Julie C. Canman
Keyword(s):  
Microtubule Assembly ◽  
Primary Role ◽  
Intercellular Bridge ◽  
Contractile Ring ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Family Protein ◽  
Spindle Midzone ◽  
Ring Constriction ◽  
Spindle Microtubules

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

scholarly journals Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

2008 ◽  
Vol 19 (11) ◽  
pp. 4900-4908 ◽  
Author(s):  
Claudia M. Casanova ◽  
Sofia Rybina ◽  
Hideki Yokoyama ◽  
Eric Karsenti ◽  
Iain W. Mattaj
Keyword(s):  
Xenopus Laevis ◽  
Detailed Analysis ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
Spindle Midzone ◽  
Microtubule Growth ◽  
Spindle Microtubules

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

scholarly journals Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Open Biology ◽  
2013 ◽  
Vol 3 (8) ◽  
pp. 130081 ◽  
Author(s):  
Tetsuya Takeda ◽  
Iain M. Robinson ◽  
Matthew M. Savoian ◽  
John R. Griffiths ◽  
Anthony D. Whetton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Curvature ◽  
Cellular Process ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Functional Interaction ◽  
Cortical Dynamics ◽  
Central Spindle ◽  
Bar Domain ◽  
Spindle Microtubules

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

scholarly journals The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly

2011 ◽  
Vol 193 (1) ◽  
pp. 155-169 ◽  
Author(s):  
Lindsay Lewellyn ◽  
Ana Carvalho ◽  
Arshad Desai ◽  
Amy S. Maddox ◽  
Karen Oegema
Keyword(s):  
Contractile Ring ◽  
Chromosomal Passenger Complex ◽  
Ring Assembly ◽  
Assembly Pathway ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Ring Constriction ◽  
Simultaneous Inhibition ◽  
Control Transformation ◽  
Equatorial Band

The chromosomal passenger complex (CPC) and centralspindlin are conserved cytokinesis regulators that localize to the spindle midzone, which forms between the separating chromosomes. Previous work placed the CPC and centralspindlin in a linear pathway that governs midzone formation. Using Caenorhabditis elegans embryos, we test whether there is a similar linear relationship between centralspindlin and the CPC in contractile ring constriction during cytokinesis. We show that simultaneous inhibition of the CPC kinase Aurora BAIR-2 and the centralspindlin component MKLP1ZEN-4 causes an additive constriction defect. Consistent with distinct roles for the proteins, inhibition of filamentous septin guanosine triphosphatases alleviates constriction defects in Aurora BAIR-2–inhibited embryos, whereas inhibition of Rac does so in MKLP1ZEN-4-inhibited embryos. Centralspindlin and the CPC are not required to enrich ring proteins at the cell equator but instead regulate formation of a compact mature ring. Therefore, in contrast to the linear midzone assembly pathway, centralspindlin and the CPC make independent contributions to control transformation of the sheet-like equatorial band into a ribbon-like contractile ring at the furrow tip.

scholarly journals Fission yeast Cyk3p is a transglutaminase-like protein that participates in cytokinesis and cell morphogenesis

2012 ◽  
Vol 23 (13) ◽  
pp. 2433-2444 ◽  
Author(s):  
Luther W. Pollard ◽  
Masayuki Onishi ◽  
John R. Pringle ◽  
Matthew Lord
Keyword(s):  
Fission Yeast ◽  
Primary Role ◽  
Cell Integrity ◽  
Cell Morphogenesis ◽  
Contractile Ring ◽  
Physiological Importance ◽  
Complex Process ◽  
Catalytic Active Site ◽  
Thiol Proteases ◽  
Ring Constriction

Cell morphogenesis is a complex process that relies on a diverse array of proteins and pathways. We have identified a transglutaminase-like protein (Cyk3p) that functions in fission yeast morphogenesis. The phenotype of a cyk3 knockout strain indicates a primary role for Cyk3p in cytokinesis. Correspondingly, Cyk3p localizes both to the actomyosin contractile ring and the division septum, promoting ring constriction, septation, and subsequent cell separation following ring disassembly. In addition, Cyk3p localizes to polarized growth sites and plays a role in cell shape determination, and it also appears to contribute to cell integrity during stationary phase, given its accumulation as dynamic puncta at the cortex of such cells. Our results and the conservation of Cyk3p across fungi point to a role in cell wall synthesis and remodeling. Cyk3p possesses a transglutaminase domain that is essential for function, even though it lacks the catalytic active site. In a wider sense, our work illustrates the physiological importance of inactive members of the transglutaminase family, which are found throughout eukaryotes. We suggest that the proposed evolution of animal transglutaminase cross-linking activity from ancestral bacterial thiol proteases was accompanied by the emergence of a subclass whose function does not depend on enzymatic activity.

scholarly journals Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

1985 ◽  
Vol 101 (5) ◽  
pp. 1966-1976 ◽  
Author(s):  
J B Tucker ◽  
S A Mathews ◽  
K A Hendry ◽  
J B Mackie ◽  
D L Roche
Keyword(s):  
Nuclear Envelope ◽  
Early Stage ◽  
Microtubule Assembly ◽  
Membrane Association ◽  
Paramecium Tetraurelia ◽  
Spindle Microtubule ◽  
Spatial Control ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Anaphase B

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

scholarly journals Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

2008 ◽  
Vol 181 (4) ◽  
pp. 595-603 ◽  
Author(s):  
Thomas M. Durcan ◽  
Elizabeth S. Halpin ◽  
Trisha Rao ◽  
Nicholas S. Collins ◽  
Emily K. Tribble ◽  
...  
Keyword(s):  
Cell Formation ◽  
Binucleate Cell ◽  
Aurora B ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Interfering Rna

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

scholarly journals Mechanical and genetic separation of aster- and midzone-positioned cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 381-383 ◽  
Author(s):  
Henrik Bringmann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mitotic Spindle ◽  
Heterotrimeric G Proteins ◽  
Contractile Ring ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
Two Populations ◽  
Provided Examples

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

scholarly journals Cep57 Protein Is Required for Cytokinesis by Facilitating Central Spindle Microtubule Organization

2013 ◽  
Vol 288 (20) ◽  
pp. 14384-14390 ◽  
Author(s):  
Runsheng He ◽  
Qixi Wu ◽  
Haining Zhou ◽  
Ning Huang ◽  
Jianguo Chen ◽  
...  
Keyword(s):  
Genetic Material ◽  
Microtubule Assembly ◽  
Aurora B ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Daughter Cells ◽  
Binuclear Cells ◽  
A Cell ◽  
Centrosome Protein

Cytokinesis is the final stage of cell division in which the cytoplasm of a cell is divided into two daughter cells after the segregation of genetic material, and the central spindle and midbody are considered to be the essential structures required for the initiation and completion of cytokinesis. Here, we determined that the centrosome protein Cep57, which is localized to the central spindle and midbody, acts as a spindle organizer and is required for cytokinesis. Depletion of Cep57 disrupted microtubule assembly of the central spindle and further led to abnormal midbody localization of MKLP1, Plk1, and Aurora B, which resulted in cytokinesis failure and the formation of binuclear cells. Furthermore, we found that Cep57 directly recruited Tektin 1 to the midbody matrix to regulate microtubule organization. Thus, our data reveal that Cep57 is essential for cytokinesis via regulation of central spindle assembly and formation of the midbody.

scholarly journals NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS

1973 ◽  
Vol 58 (1) ◽  
pp. 96-106 ◽  
Author(s):  
B. R. Brinkley ◽  
Potu N. Rao
Keyword(s):  
Nitrous Oxide ◽  
Hela Cells ◽  
Metaphase Plate ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Microtubule ◽  
Mitotic Apparatus ◽  
Ultrastructural Studies ◽  
Multipolar Spindles ◽  
Spindle Microtubules

When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously. Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles.

scholarly journals Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

2008 ◽  
Vol 19 (10) ◽  
pp. 4534-4544 ◽  
Author(s):  
Jérémie Gaillard ◽  
Emmanuelle Neumann ◽  
Daniel Van Damme ◽  
Virginie Stoppin-Mellet ◽  
Christine Ebel ◽  
...  
Keyword(s):  
Preprophase Band ◽  
Spindle Microtubule ◽  
Microtubule Associated Proteins ◽  
Late Anaphase ◽  
Associated Proteins ◽  
Planar Network ◽  
Spindle Midzone ◽  
Spindle Microtubules

The Arabidopsis MAP65s are a protein family with similarity to the microtubule-associated proteins PRC1/Ase1p that accumulate in the spindle midzone during late anaphase in mammals and yeast, respectively. Here we investigate the molecular and functional properties of AtMAP65-5 and improve our understanding of AtMAP65-1 properties. We demonstrate that, in vitro, both proteins promote the formation of a planar network of antiparallel microtubules. In vivo, we show that AtMAP65-5 selectively binds the preprophase band and the prophase spindle microtubule during prophase, whereas AtMAP65-1-GFP selectively binds the preprophase band but does not accumulate at the prophase spindle microtubules that coexists within the same cell. At later stages of mitosis, AtMAP65-1 and AtMAP65-5 differentially label the late spindle and phragmoplast. We present evidence for a mode of action for both proteins that involves the binding of monomeric units to microtubules that “zipper up” antiparallel arranged microtubules through the homodimerization of the N-terminal halves when adjacent microtubules encounter.

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