Reorganization of actin cytoskeleton at the growing end of the cleavage furrow of Xenopus egg during cytokinesis

2001 ◽  
Vol 114 (2) ◽  
pp. 401-412 ◽  
Author(s):  
T. Noguchi ◽  
I. Mabuchi
Keyword(s):  
Rapid Growth ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Laser Scanning Microscopy ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Rhodamine Phalloidin ◽  
Actin Bundles ◽  
Wheatgerm Agglutinin

We studied reorganization of actin-myosin cytoskeleton at the growing ends of the cleavage furrow of Xenopus eggs in order to understand how the contractile ring is formed during cytokinesis. Reorganization of F-actin structures during the furrow formation was demonstrated by rhodamine-phalloidin staining of the cleavage furrow and by time-lapse scanning with laser scanning microscopy of F-actin structures in the cleavage furrow of live eggs to which rhodamine-G-actin had been injected. Actin filaments assemble to form small clusters that we call ‘F-actin patches’ at the growing end of the furrow. In live recordings, we observed emergence and rapid growth of F-actin patches in the furrow region. These patches then align in tandem, elongate and fuse with each other to form short F-actin bundles. The short bundles then form long F-actin bundles that compose the contractile ring. During the furrow formation, a cortical movement towards the division plane occurs at the growing ends of the furrow, as shown by monitoring wheatgerm agglutinin-conjugated fluorescent beads attached to the egg surface. As a result, wheatgerm agglutinin-binding sites accumulate and form ‘bleb-like’ structures on the surface of the furrow region. The F-actin patch forms and grows underneath this structure. The slope of F-actin accumulation in the interior region of the furrow exceeds that of accumulation of the cortex transported by the cortical movement. In addition, rhodamine-G-actin microinjected at the growing end is immediately incorporated into the F-actin patches. These data, together with the rapid growth of F-actin patches in the live image, suggest that actin polymerization occurs in the contractile ring formation. Distribution of myosin II in the cleavage furrow was also examined by immunofluorescence microscopy. Myosin II assembles as spots at the growing end underneath the bleb-like structure. It was suggested that myosin is transported and accumulates as spots by way of the cortical movement. F-actin accumulates at the position of the myosin spot a little later as the F-actin patches. The myosin spots and the F-actin patches are then simultaneously reorganized to form the contractile ring bundles

scholarly journals Polar relaxation by dynein-mediated removal of cortical myosin II

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Bernardo Chapa-y-Lazo ◽  
Motonari Hamanaka ◽  
Alexander Wray ◽  
Mohan K. Balasubramanian ◽  
Masanori Mishima
Keyword(s):  
Recent Work ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Myosin Ii ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
C Elegans ◽  
Polar Cell

Nearly six decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction, whereas suppression of dynein activity delayed furrowing. We show that dynein-mediated removal of myosin II from the polar cortexes triggers a bidirectional cortical flow toward the cell equator, which induces the assembly of the actomyosin contractile ring. These results provide a molecular mechanism for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

scholarly journals Macropinocytosis as a Mechanism of Entry into Primary Human Urethral Epithelial Cells by Neisseria gonorrhoeae

2000 ◽  
Vol 68 (3) ◽  
pp. 1696-1699 ◽  
Author(s):  
Michael K. Zenni ◽  
Peter C. Giardina ◽  
Hillery A. Harvey ◽  
Jianqiang Shao ◽  
Margaret R. Ketterer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Laser Scanning ◽  
Actin Polymerization ◽  
Kinase Inhibitors ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Transmission Electron ◽  
Microscopy Analysis ◽  
Phosphoinositide 3 Kinase

ABSTRACT Gonococcal entry into primary human urethral epithelial cells (HUEC) can occur by macropinocytosis. Scanning and transmission electron microscopy revealed lamellipodia surrounding gonococci, and confocal laser scanning microscopy analysis showed organisms colocalized with M r 70,000 fluorescein isothiocyanate-labeled dextran within the cells. Phosphoinositide 3-kinase inhibitors and an actin polymerization inhibitor prevented macropinocytic entry of gonococci into HUEC.

scholarly journals Contractile Ring-independent Localization of DdINCENP, a Protein Important for Spindle Stability and Cytokinesis

2006 ◽  
Vol 17 (2) ◽  
pp. 779-788 ◽  
Author(s):  
Qian Chen ◽  
Hui Li ◽  
Arturo De Lozanne
Keyword(s):  
Myosin Ii ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Subsequent Formation ◽  
Daughter Cells ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Spindle Stability ◽  
Passenger Protein

Dictyostelium DdINCENP is a chromosomal passenger protein associated with centromeres, the spindle midzone, and poles during mitosis and the cleavage furrow during cytokinesis. Disruption of the single DdINCENP gene revealed important roles for this protein in mitosis and cytokinesis. DdINCENP null cells lack a robust spindle midzone and are hypersensitive to microtubule-depolymerizing drugs, suggesting that their spindles may not be stable. Furthermore DdCP224, a protein homologous to the microtubule-stabilizing protein TOGp/XMAP215, was absent from the spindle midzone of DdINCENP null cells. Overexpression of DdCP224 rescued the weak spindle midzone defect of DdINCENP null cells. Although not required for the localization of the myosin II contractile ring and subsequent formation of a cleavage furrow, DdINCENP is important for the abscission of daughter cells at the end of cytokinesis. Finally, we show that the localization of DdINCENP at the cleavage furrow is modulated by myosin II but it occurs by a mechanism different from that controlling the formation of the contractile ring.

scholarly journals SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Anne McIntosh ◽  
Lynsey M. Meikle ◽  
Michael J. Ormsby ◽  
Beth A. McCormick ◽  
John M. Christie ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Actin Polymerization ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Limiting Factor ◽  
Dual Function ◽  
Content Type

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias. Studies on living cells

1994 ◽  
Vol 107 (7) ◽  
pp. 1929-1934 ◽  
Author(s):  
U. Meindl ◽  
D. Zhang ◽  
P.K. Hepler
Keyword(s):  
Laser Scanning ◽  
Nuclear Migration ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Cortex ◽  
Actin Network ◽  
Cortical Actin ◽  
Electron Microscopic ◽  
Rhodamine Phalloidin ◽  
Growing Cell

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.

scholarly journals Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

2017 ◽  
Vol 216 (9) ◽  
pp. 2657-2667 ◽  
Author(s):  
Ting Gang Chew ◽  
Junqi Huang ◽  
Saravanan Palani ◽  
Ruth Sommese ◽  
Anton Kamnev ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Contractile Ring ◽  
Ring Contraction ◽  
Actin Turnover ◽  
Exogenous Addition ◽  
Turn Over

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.

scholarly journals Classical and Emerging Regulatory Mechanisms of Cytokinesis in Animal Cells

Biology ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 55 ◽  
Author(s):  
Vikash Verma ◽  
Alex Mogilner ◽  
Thomas J. Maresca
Keyword(s):  
Molecular Mechanisms ◽  
Myosin Ii ◽  
Dynamic Structure ◽  
Regulatory Proteins ◽  
Cleavage Furrow ◽  
Filamentous Actin ◽  
Animal Cells ◽  
Contractile Ring ◽  
Daughter Cells ◽  
Sister Chromatids

The primary goal of cytokinesis is to produce two daughter cells, each having a full set of chromosomes. To achieve this, cells assemble a dynamic structure between segregated sister chromatids called the contractile ring, which is made up of filamentous actin, myosin-II, and other regulatory proteins. Constriction of the actomyosin ring generates a cleavage furrow that divides the cytoplasm to produce two daughter cells. Decades of research have identified key regulators and underlying molecular mechanisms; however, many fundamental questions remain unanswered and are still being actively investigated. This review summarizes the key findings, computational modeling, and recent advances in understanding of the molecular mechanisms that control the formation of the cleavage furrow and cytokinesis.

Microtubule-mediated centrosome motility and the positioning of cleavage furrows in multinucleate myosin II-null cells

1998 ◽  
Vol 111 (9) ◽  
pp. 1227-1240 ◽  
Author(s):  
R. Neujahr ◽  
R. Albrecht ◽  
J. Kohler ◽  
M. Matzner ◽  
J.M. Schwartz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Myosin Ii ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Mitotic Apparatus ◽  
Alpha Tubulin ◽  
Multinucleate Cells ◽  
Green Fluorescent

To study centrosome motility and the interaction of microtubules with the cell cortex in mitotic, post-mitotic and interphase cells, (alpha)-tubulin was tagged in Dictyostelium discoideum with green fluorescent protein. Multinucleate cells formed by myosin II-null mutants proved to be especially suited for the analysis of the control of cleavage furrow formation by the microtubule system. After docking of the mitotic apparatus onto the cell cortex during anaphase, the cell surface is activated to form ruffles on top of the asters of microtubules that emanate from the centrosomes. Cleavage furrows are initiated at spaces between the asters independently of the positions of spindles. Once initiated, the furrows expand as deep folds without a continued connection to the microtubule system. Occurrence of unilateral furrows indicates that a closed contractile ring is dispensable for cytokinesis in Dictyostelium. The progression of cytokinesis in the multinucleate cells underlines the importance of proteins other than myosin II in specifying a cleavage furrow. The analysis of centrosome motility suggests a major role for a minus-end directed motor protein, probably cytoplasmic dynein, in applying traction forces on guiding microtubules that connect the centrosome with the cell cortex.

scholarly journals Chorein Sensitive Arrangement of Cytoskeletal Architecture

2015 ◽  
Vol 37 (1) ◽  
pp. 399-408 ◽  
Author(s):  
Sabina Honisch ◽  
Shuchen Gu ◽  
Jennifer Müller vom Hagen ◽  
Saad Alkahtani ◽  
Abdullah A. Al Kahtane ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Actin Polymerization ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Pcr Analysis ◽  
Loss Of Function ◽  
Rt Pcr ◽  
Behavioral Abnormalities

Background/Aims: Chorein is a protein expressed in various cell types. Loss of function mutations of the chorein encoding gene VPS13A lead to chorea-acanthocytosis, an autosomal recessive genetic disease characterized by movement disorder and behavioral abnormalities. Recent observations revealed that chorein is a powerful regulator of actin cytoskeleton in erythrocytes, platelets, K562 and endothelial HUVEC cells. Methods: In the present study we have used Western blotting to study actin polymerization dynamics, laser scanning microscopy to evaluate in detail the role of chorein in microfilaments, microtubules and intermediate filaments cytoskeleton architecture and RT-PCR to assess gene transcription of the cytoskeletal proteins. Results: We report here powerful depolymerization of actin microfilaments both, in erythrocytes and fibroblasts isolated from chorea-acanthocytosis patients. Along those lines, morphological analysis of fibroblasts from chorea-acanthocytosis patients showed disarranged microtubular network, when compared to fibroblasts from healthy donors. Similarly, the intermediate filament networks of desmin and cytokeratins showed significantly disordered organization with clearly diminished staining in patient's fibroblasts. In line with this, RT-PCR analysis revealed significant downregulation of desmin and cytokeratin gene transcripts. Conclusion: Our results provide for the first time evidence that defective chorein is accompanied by significant structural disorganization of all cytoskeletal structures in human fibroblasts from chorea-acanthocytosis patients.

scholarly journals An Essential Role for a Membrane Lipid in Cytokinesis

2000 ◽  
Vol 149 (6) ◽  
pp. 1215-1224 ◽  
Author(s):  
Kazuo Emoto ◽  
Masato Umeda
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Mutant Cell ◽  
Myosin Ii ◽  
Membrane Lipid ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Cytoplasmic Bridge ◽  
Daughter Cells

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is mainly localized in the inner leaflet of the plasma membrane. We previously demonstrated that PE was exposed on the cell surface of the cleavage furrow during cytokinesis. Immobilization of cell surface PE by a PE-binding peptide inhibited disassembly of the contractile ring components, including myosin II and radixin, resulting in formation of a long cytoplasmic bridge between the daughter cells. This blockade of contractile ring disassembly was reversed by removal of the surface-bound peptide, suggesting that the PE exposure plays a crucial role in cytokinesis. To further examine the role of PE in cytokinesis, we established a mutant cell line with a specific decrease in the cellular PE level. On the culture condition in which the cell surface PE level was significantly reduced, the mutant ceased cell growth in cytokinesis, and the contractile ring remained in the cleavage furrow. Addition of PE or ethanolamine, a precursor of PE synthesis, restored the cell surface PE on the cleavage furrow and normal cytokinesis. These findings provide the first evidence that PE is required for completion of cytokinesis in mammalian cells, and suggest that redistribution of PE on the cleavage furrow may contribute to regulation of contractile ring disassembly.

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