Three-dimensional in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion

During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild-type data demonstrate that cell sorting can occur in two steps, suggesting that, in this Ax3 strain, spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip.

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In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis

Cell Motility and the Cytoskeleton ◽

10.1002/cm.20246 ◽

2008 ◽

Vol 65 (2) ◽

pp. 100-115 ◽

Cited By ~ 18

Author(s):

Ryota Uehara ◽

Hiroshi Hosoya ◽

Issei Mabuchi

Keyword(s):

Sea Urchin ◽

Light Chain ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Sea Urchin Eggs ◽

And Function

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In vivo phosphorylation of regulatory light chain of myosin II during mitosis of cultured cells

The Journal of Cell Biology ◽

10.1083/jcb.124.1.129 ◽

1994 ◽

Vol 124 (1) ◽

pp. 129-137 ◽

Cited By ~ 98

Author(s):

Y Yamakita ◽

S Yamashiro ◽

F Matsumura

Keyword(s):

Light Chain ◽

Myosin Ii ◽

Mitotic Arrest ◽

Regulatory Light Chain ◽

The Other ◽

Other Hand ◽

Mlc Phosphorylation ◽

Phosphate Incorporation ◽

Mitotic Cells

Phosphorylation of the regulatory light chain of myosin II (MLC) controls the contractility of actomyosin in nonmuscle and muscle cells. It has been reported that cdc2 phosphorylates MLC in vitro at Ser-1 or Ser-2 and Thr-9 which protein kinase C phosphorylates (Satterwhite, L. L., M. J. Lohka, K. L. Wilson, T. Y. Scherson, L. K. Cisek, J. L. Corden, and T. D. Pollard. 1992 J. Cell Biol. 118:595-605). We have examined in vivo phosphorylation of MLC during mitosis and after the release of mitotic arrest. Phosphate incorporation of MLC in mitotic cells is found to be 6-12 times greater than that in nonmitotic cells. Phosphopeptide maps have revealed that the MLC from mitotic cells is phosphorylated at Ser-1 and/or Ser-2 (Ser-1/2), but not at Thr-9. MLC is also phosphorylated to a much lesser extent at Ser-19 which myosin light chain kinase phosphorylates. On the other hand, MLC of nonmitotic cells is phosphorylated at Ser-19 but not at Ser-1/2. The extent of phosphate incorporation is doubled at 30 min after the release of mitotic arrest when some cells start cytokinesis. Phosphopeptide analyses have revealed that the phosphorylation at Ser-19 is increased 20 times, while the phosphorylation at Ser-1/2 is decreased by half. This high extent of MLC phosphorylation at Ser-19 is maintained for another 30 min and gradually decreased to near the level of interphase cells as cells complete spreading at 180 min. On the other hand, phosphorylation at Ser-1/2 is decreased to 18% at 60 min, and is practically undetectable at 180 min after the release of mitotic arrest. The stoichiometry of MLC phosphorylation has been determined by quantitation of phosphorylated and unphosphorylated forms of MLC separated on 2D gels. The molar ratio of phosphorylated MLC to total MLC is found to be 0.16 +/- 0.06 and 0.31 +/- 0.05 in interphase and mitotic cells, respectively. The ratio is increased to 0.49 +/- 0.05 at 30 min after the release of mitotic arrest. These results suggest that the change in the phosphorylation site from Ser-1/2 to Ser-19 plays an important role in signaling cytokinesis.

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Schizosaccharomyces pombe Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.200806127 ◽

2008 ◽

Vol 183 (5) ◽

pp. 785-793 ◽

Cited By ~ 22

Author(s):

Tsui-Han Loo ◽

Mohan Balasubramanian

Keyword(s):

Actin Cytoskeleton ◽

Schizosaccharomyces Pombe ◽

Light Chain ◽

Myosin Ii ◽

Regulatory Light Chain ◽

Eukaryotic Cells ◽

Myosin Regulatory Light Chain ◽

Related Protein ◽

Filamentous Actin

p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.

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Citron Kinase, a Rho-dependent Kinase, Induces Di-phosphorylation of Regulatory Light Chain of Myosin II

Molecular Biology of the Cell ◽

10.1091/mbc.e02-07-0427 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1745-1756 ◽

Cited By ~ 143

Author(s):

Shigeko Yamashiro ◽

Go Totsukawa ◽

Yoshihiko Yamakita ◽

Yasuharu Sasaki ◽

Pascal Madaule ◽

...

Keyword(s):

Light Chain ◽

Myosin Ii ◽

Specific Inhibitor ◽

Kinase Domain ◽

Regulatory Light Chain ◽

Myosin Phosphatase ◽

Fiber Assembly ◽

Citron Kinase

Citron kinase is a Rho-effector protein kinase that is related to Rho-associated kinases of ROCK/ROK/Rho-kinase family. Both ROCK and citron kinase are suggested to play a role in cytokinesis. However, no substrates are known for citron kinase. We found that citron kinase phosphorylated regulatory light chain (MLC) of myosin II at both Ser-19 and Thr-18 in vitro. Unlike ROCK, however, citron kinase did not phosphorylate the myosin binding subunit of myosin phosphatase, indicating that it does not inhibit myosin phosphatase. We found that the expression of the kinase domain of citron kinase resulted in an increase in MLC di-phosphorylation. Furthermore, the kinase domain was able to increase di-phosphorylation and restore stress fiber assembly even when ROCK was inhibited with a specific inhibitor, Y-27632. The expression of full-length citron kinase also increased di-phosphorylation during cytokinesis. These observations suggest that citron kinase phosphorylates MLC to generate di-phosphorylated MLC in vivo. Although both mono- and di-phosphorylated MLC were found in cleavage furrows, di-phosphorylated MLC showed more constrained localization than did mono-phosphorylated MLC. Because citron kinase is localized in cleavage furrows, citron kinase may be involved in regulating di-phosphorylation of MLC during cytokinesis.

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Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1702375114 ◽

2017 ◽

Vol 114 (32) ◽

pp. E6516-E6525 ◽

Cited By ~ 18

Author(s):

Xiong Liu ◽

Neil Billington ◽

Shi Shu ◽

Shu-Hua Yu ◽

Grzegorz Piszczek ◽

...

Keyword(s):

Light Scattering ◽

Light Chain ◽

Myosin Ii ◽

Monomer Concentration ◽

Regulatory Light Chain ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

New Location ◽

Regulatory Light Chain Phosphorylation

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.

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Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge

Biochemical Journal ◽

10.1042/bj20021559 ◽

2003 ◽

Vol 370 (2) ◽

pp. 551-556 ◽

Cited By ~ 24

Author(s):

Katsumi FUMOTO ◽

Takashi UCHIMURA ◽

Takahiro IWASAKI ◽

Kozue UEDA ◽

Hiroshi HOSOYA

Keyword(s):

Light Chain ◽

Hela Cells ◽

Myosin Ii ◽

Leading Edge ◽

Regulatory Light Chain ◽

Wild Type ◽

Wound Healing Assay ◽

Cell Monolayers ◽

Indirect Immunofluorescence Staining

To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, these mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable form, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cells, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving force in the migration of the cells but not necessary for localization of myosin II at the leading edge.

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