Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans

In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

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PAR-3 and PAR-1 Inhibit LET-99 Localization to Generate a Cortical Band Important for Spindle Positioning in Caenorhabditis elegans Embryos

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0105 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4470-4482 ◽

Cited By ~ 25

Author(s):

Jui-Ching Wu ◽

Lesilee S. Rose

Keyword(s):

Caenorhabditis Elegans ◽

Cell Fate ◽

Protein Signaling ◽

Posterior Cortex ◽

Par Proteins ◽

Anterior Cortex ◽

Spindle Positioning ◽

High Let ◽

Fate Determinants ◽

Cell Fate Determinants

The conserved PAR proteins are localized in asymmetric cortical domains and are required for the polarized localization of cell fate determinants in many organisms. In Caenorhabditis elegans embryos, LET-99 and G protein signaling act downstream of the PARs to regulate spindle positioning and ensure asymmetric division. PAR-3 and PAR-2 localize LET-99 to a posterior cortical band through an unknown mechanism. Here we report that LET-99 asymmetry depends on cortically localized PAR-1 and PAR-4 but not on cytoplasmic polarity effectors. In par-1 and par-4 embryos, LET-99 accumulates at the entire posterior cortex, but remains at low levels at the anterior cortex occupied by PAR-3. Further, PAR-3 and PAR-1 have graded cortical distributions with the highest levels at the anterior and posterior poles, respectively, and the lowest levels of these proteins correlate with high LET-99 accumulation. These results suggest that PAR-3 and PAR-1 inhibit the localization of LET-99 to generate a band pattern. In addition, PAR-1 kinase activity is required for the inhibition of LET-99 localization, and PAR-1 associates with LET-99. Finally, examination of par-1 embryos suggests that the banded pattern of LET-99 is critical for normal posterior spindle displacement and to prevent spindle misorientation caused by cell shape constraints.

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A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0712 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1056-1070 ◽

Cited By ~ 56

Author(s):

Sandra E. Encalada ◽

John Willis ◽

Rebecca Lyczak ◽

Bruce Bowerman

Keyword(s):

Caenorhabditis Elegans ◽

Chromosome Segregation ◽

Spindle Checkpoint ◽

Metaphase Plate ◽

Time Lapse ◽

Cell Types ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Animal Cells ◽

Dividing Cells

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.

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Two distinct myosin II populations coordinate ovulatory contraction of the myoepithelial sheath in the Caenorhabditis elegans somatic gonad

Molecular Biology of the Cell ◽

10.1091/mbc.e15-09-0648 ◽

2016 ◽

Vol 27 (7) ◽

pp. 1131-1142 ◽

Cited By ~ 16

Author(s):

Kanako Ono ◽

Shoichiro Ono

Keyword(s):

Caenorhabditis Elegans ◽

Striated Muscle ◽

Myosin Ii ◽

Coiled Coil ◽

The Body ◽

Nonmuscle Myosin ◽

Heavy Chains ◽

Somatic Gonad ◽

Nonmuscle Myosin Ii ◽

Muscle Myosin

The myoepithelial sheath in the somatic gonad of the nematode Caenorhabditis elegans has nonstriated contractile actomyosin networks that produce highly coordinated contractility for ovulation of mature oocytes. Two myosin heavy chains are expressed in the myoepithelial sheath, which are also expressed in the body-wall striated muscle. The troponin/tropomyosin system is also present and essential for ovulation. Therefore, although the myoepithelial sheath has smooth muscle–like contractile apparatuses, it has a striated muscle–like regulatory mechanism through troponin/tropomyosin. Here we report that the myoepithelial sheath has a distinct myosin population containing nonmuscle myosin II isoforms, which is regulated by phosphorylation and essential for ovulation. MLC-4, a nonmuscle myosin regulatory light chain, localizes to small punctate structures and does not colocalize with large, needle-like myosin filaments containing MYO-3, a striated-muscle myosin isoform. RNA interference of MLC-4, as well as of its upstream regulators, LET-502 (Rho-associated coiled-coil forming kinase) and MEL-11 (a myosin-binding subunit of myosin phosphatase), impairs ovulation. Expression of a phosphomimetic MLC-4 mutant mimicking a constitutively active state also impairs ovulation. A striated-muscle myosin (UNC-54) appears to provide partially compensatory contractility. Thus the results indicate that the two spatially distinct myosin II populations coordinately regulate ovulatory contraction of the myoepithelial sheath.

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Genetic Analysis Demonstrates a Direct Link Between Rho Signaling and Nonmuscle Myosin Function During Drosophila Morphogenesis

Genetics ◽

10.1093/genetics/155.3.1253 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1253-1265 ◽

Cited By ~ 8

Author(s):

Susan R Halsell ◽

Benjamin I Chu ◽

Daniel P Kiehart

Keyword(s):

Signal Transduction ◽

Myosin Ii ◽

Signal Transduction Pathway ◽

P Element ◽

Nonmuscle Myosin ◽

Genetic Screens ◽

Induced Mutations ◽

Nonmuscle Myosin Ii ◽

Carboxyl Terminal ◽

Actomyosin Cytoskeleton

Abstract A dynamic actomyosin cytoskeleton drives many morphogenetic events. Conventional nonmuscle myosin-II (myosin) is a key chemomechanical motor that drives contraction of the actin cytoskeleton. We have explored the regulation of myosin activity by performing genetic screens to identify gene products that collaborate with myosin during Drosophila morphogenesis. Specifically, we screened for second-site noncomplementors of a mutation in the zipper gene that encodes the nonmuscle myosin-II heavy chain. We determined that a single missense mutation in the zipperEbr allele gives rise to its sensitivity to second-site noncomplementation. We then identify the Rho signal transduction pathway as necessary for proper myosin function. First we show that a lethal P-element insertion interacts genetically with zipper. Subsequently we show that this second-site noncomplementing mutation disrupts the RhoGEF2 locus. Next, we show that two EMS-induced mutations, previously shown to interact genetically with zipperEbr, disrupt the RhoA locus. Further, we have identified their molecular lesions and determined that disruption of the carboxyl-terminal CaaX box gives rise to their mutant phenotype. Finally, we show that RhoA mutations themselves can be utilized in genetic screens. Biochemical and cell culture analyses suggest that Rho signal transduction regulates the activity of myosin. Our studies provide direct genetic proof of the biological relevance of regulation of myosin by Rho signal transduction in an intact metazoan.

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Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals

The Journal of Cell Biology ◽

10.1083/jcb.201612126 ◽

2017 ◽

Vol 216 (7) ◽

pp. 1925-1936 ◽

Cited By ~ 29

Author(s):

Oleg Milberg ◽

Akiko Shitara ◽

Seham Ebrahim ◽

Andrius Masedunskas ◽

Muhibullah Tora ◽

...

Keyword(s):

Molecular Mechanisms ◽

Secretory Granules ◽

Myosin Ii ◽

Membrane Remodeling ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Mammalian Tissues ◽

Actomyosin Cytoskeleton

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

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The Nonmuscle Myosin Regulatory Light Chain Gene mlc-4 Is Required for Cytokinesis, Anterior-Posterior Polarity, and Body Morphology during Caenorhabditis elegans Embryogenesis

The Journal of Cell Biology ◽

10.1083/jcb.146.2.439 ◽

1999 ◽

Vol 146 (2) ◽

pp. 439-451 ◽

Cited By ~ 135

Author(s):

Christopher A. Shelton ◽

J. Clayton Carter ◽

Gregory C. Ellis ◽

Bruce Bowerman

Keyword(s):

Caenorhabditis Elegans ◽

Light Chain ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Chain Gene ◽

Nonmuscle Myosin ◽

Light Chain Gene ◽

C Elegans ◽

Nonmuscle Myosin Ii ◽

Anterior Posterior

Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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