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

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.

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

eLife ◽

10.7554/elife.71888 ◽

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.

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

10.1101/2021.12.20.473479 ◽

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.

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Myosin IIA drives membrane bleb retraction

Molecular Biology of the Cell ◽

10.1091/mbc.e18-11-0752 ◽

2019 ◽

Vol 30 (9) ◽

pp. 1051-1059 ◽

Cited By ~ 7

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.

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GS4-1 ROLES OF NONMUSCLE MYOSIN II IN CONTRACTILE FORCE GENERATION(GS4: Molecular Biomechanics)

The Proceedings of the Asian Pacific Conference on Biomechanics emerging science and technology in biomechanics ◽

10.1299/jsmeapbio.2015.8.162 ◽

2015 ◽

Vol 2015.8 (0) ◽

pp. 162

Author(s):

Shinji Deguchi ◽

Sho Yokoyama ◽

Tsubasa S. Matsui ◽

Kagayaki Kato

Keyword(s):

Myosin Ii ◽

Contractile Force ◽

Force Generation ◽

Nonmuscle Myosin ◽

Molecular Biomechanics ◽

Nonmuscle Myosin Ii

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Rho Kinase Differentially Regulates Phosphorylation of Nonmuscle Myosin II Isoforms A and B during Cell Rounding and Migration

Journal of Biological Chemistry ◽

10.1074/jbc.m605343200 ◽

2006 ◽

Vol 281 (47) ◽

pp. 35873-35883 ◽

Cited By ~ 132

Author(s):

Joshua C. Sandquist ◽

Katherine I. Swenson ◽

Kris A. DeMali ◽

Keith Burridge ◽

Anthony R. Means

Keyword(s):

Myosin Ii ◽

Rho Kinase ◽

Nonmuscle Myosin ◽

Cell Rounding ◽

Nonmuscle Myosin Ii ◽

And Migration

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

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0372 ◽

2009 ◽

Vol 20 (1) ◽

pp. 338-347 ◽

Cited By ~ 64

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.

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