scholarly journals Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells

2009 ◽  
Vol 20 (1) ◽  
pp. 338-347 ◽  
Author(s):  
Mark T. Breckenridge ◽  
Natalya G. Dulyaninova ◽  
Thomas T. Egelhoff
Keyword(s):  
Conformational Change ◽  
Mammalian Cells ◽  
Myosin Ii ◽  
Phosphorylation Site ◽  
Leading Edge ◽  
Live Cells ◽  
Nonmuscle Myosin ◽  
Related Disorder ◽  
Filament Assembly ◽  
Nonmuscle Myosin Ii

To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (ΔIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Δtailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Δtailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

scholarly journals Myosin IIA drives membrane bleb retraction

2019 ◽  
Vol 30 (9) ◽  
pp. 1051-1059 ◽  
Author(s):  
Nilay Taneja ◽  
Dylan T. Burnette
Keyword(s):  
Mammalian Cells ◽  
Myosin Ii ◽  
Cross Linking ◽  
Nonmuscle Myosin ◽  
Final Phase ◽  
Biophysical Properties ◽  
Actin Cortex ◽  
Nonmuscle Myosin Ii ◽  
Membrane Blebs ◽  
Ability To Drive

Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.

scholarly journals Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kai Weißenbruch ◽  
Justin Grewe ◽  
Marc Hippler ◽  
Magdalena Fladung ◽  
Moritz Tremmel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Myosin Ii ◽  
Three Dimensional ◽  
Nonmuscle Myosin ◽  
Collagen Gels ◽  
Nonmuscle Myosin Ii ◽  
Dynamic Cell ◽  
Tensional Homeostasis ◽  
And Migration ◽  
Coated Surfaces

Nonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogenously-coated surfaces, in collagen gels, and on micropatterned substrates. In contrast to homogenously-coated surfaces, a structured environment supports a cellular phenotype with invaginated actin arcs even in the absence of NM IIA-induced contractility. A quantitative shape analysis of cells on micropatterns combined with a scale-bridging mathematical model reveals that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. A dynamic cell stretch/release experiment in a three-dimensional scaffold confirms these conclusions and in addition reveals a novel role for NM IIC, namely the ability to establish tensional homeostasis.

scholarly journals Distinct roles of nonmuscle myosin II isoforms for establishing tension and elasticity during cell morphodynamics

2020 ◽  
Author(s):  
Kai Weißenbruch ◽  
Justin Grewe ◽  
Kathrin Stricker ◽  
Laurent Baulesch ◽  
Ulrich S. Schwarz ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Myosin Ii ◽  
Force Generation ◽  
Nonmuscle Myosin ◽  
Cellular Processes ◽  
Scale Bridging ◽  
Crossbridge Cycle ◽  
Nonmuscle Myosin Ii ◽  
Actin Fiber ◽  
And Migration

AbstractNonmuscle myosin II (NM II) is an integral part of essential cellular processes, including adhesion and migration. Mammalian cells express up to three isoforms termed NM IIA, B, and C. We used U2OS cells to create CRISPR/Cas9-based knockouts of all three isoforms and analyzed the phenotypes on homogeneous and micropatterned substrates. We find that NM IIA is essential to build up cellular tension during initial stages of force generation, while NM IIB is necessary to elastically stabilize NM IIA-generated tension. The knockout of NM IIC has no detectable effects. A scale-bridging mathematical model explains our observations by relating actin fiber stability to the molecular rates of the myosin crossbridge cycle. We also find that NM IIA initiates and guides co-assembly of NM IIB into heterotypic minifilaments. We finally use mathematical modeling to explain the different exchange dynamics of NM IIA and B in minifilaments, as measured in FRAP experiments.

scholarly journals Electrostatic and bending energies predict staggering and splaying in nonmuscle myosin II minifilaments

2020 ◽  
Author(s):  
Tom Kaufmann ◽  
Ulrich S. Schwarz
Keyword(s):  
Elasticity Theory ◽  
Stochastic Dynamics ◽  
Molecular Motor ◽  
Myosin Ii ◽  
Super Resolution ◽  
Live Cells ◽  
Sequence Information ◽  
Nonmuscle Myosin ◽  
Resolution Limit ◽  
Nonmuscle Myosin Ii

AbstractRecent experiments with super-resolution live cell microscopy revealed that nonmuscle myosin II minifilaments are much more dynamic than formerly appreciated, often showing plastic processes such as splitting, concatenation and stacking. Here we combine sequence information, electrostatics and elasticity theory to demonstrate that the parallel staggers at 14.3, 43.2 and 72 nm have a strong tendency to splay their heads away from the minifilament, thus potentially initiating the diverse processes seen in live cells. In contrast, the straight antiparallel stagger with an overlap of 43 nm is very stable and likely initiates minifilament nucleation. Using stochastic dynamics in a newly defined energy landscape, we predict that the optimal parallel staggers between the myosin rods are obtained by a trial-and-error process in which two rods attach and re-attach at different staggers by rolling and zipping motion. The experimentally observed staggers emerge as the configurations with the largest contact times. We find that contact times increase from isoforms C to B to A, that A-B-heterodimers are surprisingly stable and that myosin 18A should incorporate into mixed filaments with a small stagger. Our findings suggest that nonmuscle myosin II minifilaments in the cell are first formed by isoform A and then convert to mixed A-B-filaments, as observed experimentally.Author summaryNonmuscle myosin II (NM2) is a non-processive molecular motor that assembles into minifilaments with a typical size of 300 nm to generate force and motion in the actin cytoskeleton. This process is essential for many cellular processes such as adhesion, migration, division and mechanosensing. Due to their small size below the resolution limit, minifilaments are a challenge for imaging with traditional light microscopy. With the advent of super-resolution microscopy, however, it has become apparent that the formation of NM2-minifilaments is much more dynamic than formerly appreciated. Modeling the electrostatic interaction between the rigid rods of the myosin monomers has confirmed the main staggers observed in experiments, but cannot explain these high dynamics. Here we complement electrostatics by elasticity theory and stochastic dynamics to show that the parallel staggers are likely to splay away from the main axis of the minifilament and that monomers attach and deattach with rolling and zipping motions. Based on the sequences of the different NM2-isoforms, we predict that isoform A forms the most stable homofilaments and that A-B-heterofilaments are also very stable.

scholarly journals Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

2021 ◽  
Author(s):  
Kai Weissenbruch ◽  
Magdalena Fladung ◽  
Justin Grewe ◽  
Laurent Baulesch ◽  
Ulrich Sebastian Schwarz ◽  
...  
Keyword(s):  
Light Chain ◽  
Mammalian Cells ◽  
Myosin Ii ◽  
Regulatory Light Chain ◽  
Force Generation ◽  
Nonmuscle Myosin ◽  
Force Output ◽  
Nonmuscle Myosin Ii ◽  
Phosphorylation Pattern ◽  
Regulatory Light Chain Phosphorylation

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

Drosophila myosin phosphatase and its role in dorsal closure

Development ◽  
2002 ◽  
Vol 129 (5) ◽  
pp. 1215-1223 ◽  
Author(s):  
Tomoaki Mizuno ◽  
Kyoko Tsutsui ◽  
Yasuyoshi Nishida
Keyword(s):  
Genetic Interaction ◽  
Shape Change ◽  
Myosin Ii ◽  
Rho Kinase ◽  
Leading Edge ◽  
Subcellular Location ◽  
Dorsal Closure ◽  
Nonmuscle Myosin ◽  
Myosin Phosphatase ◽  
Nonmuscle Myosin Ii

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.

scholarly journals Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly

2006 ◽  
Vol 17 (3) ◽  
pp. 1364-1374 ◽  
Author(s):  
Michael Rosenberg ◽  
Shoshana Ravid
Keyword(s):  
Cellular Localization ◽  
Myosin Ii ◽  
Dominant Negative ◽  
Phosphorylation Sites ◽  
Nonmuscle Myosin ◽  
Wild Type ◽  
Filament Assembly ◽  
Nonmuscle Myosin Ii ◽  
Pkc Isoforms ◽  
Epidermal Growth

Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKCβII and PKCγ, PKCγ directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKCγ result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKCγ on NMHC-IIB. Conversion of the PKCγ phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKCγ regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation.

scholarly journals A regulatory motif in nonmuscle myosin II-B regulates its role in migratory front–back polarity

2015 ◽  
Vol 209 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Alba Juanes-Garcia ◽  
Jessica R. Chapman ◽  
Rocio Aguilar-Cuenca ◽  
Cristina Delgado-Arevalo ◽  
Jennifer Hodges ◽  
...  
Keyword(s):  
Myosin Ii ◽  
Live Cells ◽  
Spectrometric Analysis ◽  
Nonmuscle Myosin ◽  
Molecular Determinant ◽  
C Terminus ◽  
Nonmuscle Myosin Ii ◽  
Site Mutagenesis ◽  
Migratory Cell

In this study, we show that the role of nonmuscle myosin II (NMII)-B in front–back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935–1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B–like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front–back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.

scholarly journals Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility

2017 ◽  
Vol 216 (9) ◽  
pp. 2877-2889 ◽  
Author(s):  
Maria S. Shutova ◽  
Sreeja B. Asokan ◽  
Shefali Talwar ◽  
Richard K. Assoian ◽  
James E. Bear ◽  
...  
Keyword(s):  
Cell Motility ◽  
Cell Polarity ◽  
Myosin Ii ◽  
Leading Edge ◽  
Stress Fibers ◽  
Cell Behavior ◽  
Retrograde Flow ◽  
Nonmuscle Myosin ◽  
Cell Contractility ◽  
Nonmuscle Myosin Ii

Nonmuscle myosin II (NMII) is uniquely responsible for cell contractility and thus defines multiple aspects of cell behavior. To generate contraction, NMII molecules polymerize into bipolar minifilaments. Different NMII paralogs are often coexpressed in cells and can copolymerize, suggesting that they may cooperate to facilitate cell motility. However, whether such cooperation exists and how it may work remain unknown. We show that copolymerization of NMIIA and NMIIB followed by their differential turnover leads to self-sorting of NMIIA and NMIIB along the front–rear axis, thus producing a polarized actin–NMII cytoskeleton. Stress fibers newly formed near the leading edge are enriched in NMIIA, but over time, they become progressively enriched with NMIIB because of faster NMIIA turnover. In combination with retrograde flow, this process results in posterior accumulation of more stable NMIIB-rich stress fibers, thus strengthening cell polarity. By copolymerizing with NMIIB, NMIIA accelerates the intrinsically slow NMIIB dynamics, thus increasing cell motility and traction and enabling chemotaxis.

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