Isoform and domain dependence of nonmuscle myosin II in vivo and in vitro

To better understand the distinct functional roles of the 220- and 130-kDa forms of myosin light chain kinase (MLCK), expression and intracellular localization were determined during development and in adult mouse tissues. Northern blot, Western blot, and histochemical studies show that the 220-kDa MLCK is widely expressed during development as well as in several adult smooth muscle and nonmuscle tissues. The 130-kDa MLCK is highly expressed in all adult tissues examined and is also detectable during embryonic development. Colocalization studies examining the distribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDa MLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB or F-actin. In contrast, the 220-kDa MLCK did not colocalize with either nonmuscle myosin II isoform but instead colocalizes with thick interconnected bundles of F-actin. These results suggest that in vivo, the physiological functions of the 220- and 130-kDa MLCKs are likely to be regulated by their intracellular trafficking and distribution.

Download Full-text

In vivo studies on nonmuscle myosin II expression and function in heart development

Frontiers in Bioscience ◽

10.2741/3942 ◽

2012 ◽

Vol 17 (1) ◽

pp. 545 ◽

Cited By ~ 16

Author(s):

Xuefei Ma

Keyword(s):

Heart Development ◽

Myosin Ii ◽

In Vivo Studies ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

And Function

Download Full-text

Two Regions of the Tail Are Necessary for the Isoform-specific Functions of Nonmuscle Myosin IIB

Molecular Biology of the Cell ◽

10.1091/mbc.e06-08-0706 ◽

2007 ◽

Vol 18 (3) ◽

pp. 1009-1017 ◽

Cited By ~ 24

Author(s):

Masaaki K. Sato ◽

Masayuki Takahashi ◽

Michio Yazawa

Keyword(s):

Heavy Chain ◽

Myosin Ii ◽

Nonmuscle Myosin ◽

Recognition Sites ◽

Self Recognition ◽

Nonmuscle Myosin Ii ◽

Exogenous Expression ◽

Mhc Iib ◽

C 63

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB−/− fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament.

Download Full-text

Myosin II Is Involved in the Production of Constitutive Transport Vesicles from the TGN

The Journal of Cell Biology ◽

10.1083/jcb.138.2.291 ◽

1997 ◽

Vol 138 (2) ◽

pp. 291-306 ◽

Cited By ~ 139

Author(s):

Anne Müsch ◽

David Cohen ◽

Enrique Rodriguez-Boulan

Keyword(s):

Mdck Cells ◽

Myosin Ii ◽

Nonmuscle Myosin ◽

Vesicle Release ◽

Golgi Membranes ◽

Transport Vesicles ◽

Nonmuscle Myosin Ii ◽

Vesicular Carriers ◽

Vesicular Stomatitis

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. Here we show that the putative TGN coat protein p200 (Narula, N., I. McMorrow, G. Plopper, J. Doherty, K.S. Matlin, B. Burke, and J.L. Stow. 1992. J. Cell Biol. 114: 1113–1124) is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, we provide functional evidence that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II could be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibited the release of VSVG-containing vesicles from the TGN.

Download Full-text

Understanding the function of nonmuscle myosin II-A (NM II-A) in vivo

Developmental Biology ◽

10.1016/j.ydbio.2008.05.462 ◽

2008 ◽

Vol 319 (2) ◽

pp. 609

Author(s):

Aibing Wang ◽

X. Ma ◽

S. Kawamoto ◽

R.S. Adelstein

Keyword(s):

Myosin Ii ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii

Download Full-text

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.

Download Full-text

Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.270.2.g355 ◽

1996 ◽

Vol 270 (2) ◽

pp. G355-G362 ◽

Cited By ~ 11

Author(s):

J. Y. Wang ◽

S. A. McCormack ◽

L. R. Johnson

Keyword(s):

Cell Migration ◽

Myosin Ii ◽

Specific Inhibitor ◽

Intestinal Epithelial ◽

Nonmuscle Myosin ◽

Nonmuscle Myosin Ii ◽

Epithelial Cell Migration ◽

Vitro Model

The current study determines whether nonmuscle myosin II is involved in the process requiring polyamines for the stimulation of cell migration in an in vitro model that mimics the early stages of epithelial restitution. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the IEC-6 cells. Nonmuscle myosin II concentrations in DFMO-treated cells were decreased by 75%, and stress fibers were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous putrescine not only returned nonmuscle myosin II levels and distribution toward normal but also restored cell migration to control levels. The administration of wortmannin, an inhibitor of myosin light chain kinase, significantly inhibited cell migration over the denuded area in control cells and in those treated with DFMO + polyamines. These results indicate that 1) polyamine depletion by DFMO is associated with decreased concentration and reorganization of nonmuscle myosin II in IEC-6 cells and 2) exogenous spermidine reverses the inhibitory effects of DFMO.

Download Full-text

A serine-kinase associated with the p127-l(2)gl tumour suppressor of Drosophila may regulate the binding of p127 to nonmuscle myosin II heavy chain and the attachment of p127 to the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.109.6.1359 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1359-1368 ◽

Cited By ~ 3

Author(s):

A. Kalmes ◽

G. Merdes ◽

B. Neumann ◽

D. Strand ◽

B.M. Mechler

Keyword(s):

Plasma Membrane ◽

Heavy Chain ◽

Posttranslational Modifications ◽

Myosin Ii ◽

Gel Filtration ◽

Tumour Suppressor ◽

Nonmuscle Myosin ◽

Serine Kinase ◽

Nonmuscle Myosin Ii

The p127 tumour suppressor protein encoded by the lethal(2)giant larvae, [l(2)gl], gene of Drosophila melanogaster is a component of a cytoskeletal network distributed in both the cytoplasm and on the inner face of the plasma membrane. The p127 protein forms high molecular mass complexes consisting mainly of homo-oligomerized p127 molecules and at least ten additional proteins. One of these proteins has been recently identified as nonmuscle myosin type II heavy chain. To determine the functional interactions between p127 and other proteins present in the p127 complexes, we analyzed p127 for posttranslational modifications and found that p127 can be phosphorylated at serine residues. In this report we describe the characteristics of a serine kinase which is associated with p127, as judged by its recovery in p127 complexes purified by either gel filtration or immuno-affinity chromatography. This kinase phosphorylates p127 in vitro and its activation by supplementing ATP results in the release of p127 from the plasma membrane. Moreover, similar activation of the kinase present in immuno-purified p127 complexes dissociates nonmuscle myosin II from p127 without affecting the homo-oligomerization of p127. This dissociation can be inhibited by staurosporine and a 26mer peptide covering amino acid positions 651 to 676 of p127 and containing five serine residues which are evolutionarily conserved from Drosophila to humans. These results indicate that a serine-kinase tightly associated with p127 regulates p127 binding with components of the cytoskeleton present in both the cytoplasm and on the plasma membrane.

Download Full-text