nonmuscle myosin ii
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2021 ◽  
Author(s):  
Kai Weissenbruch ◽  
Magdalena Fladung ◽  
Justin Grewe ◽  
Laurent Baulesch ◽  
Ulrich Sebastian Schwarz ◽  
...  

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.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kei Yamamoto ◽  
Haruko Miura ◽  
Motohiko Ishida ◽  
Yusuke Mii ◽  
Noriyuki Kinoshita ◽  
...  

AbstractActomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kai Weißenbruch ◽  
Justin Grewe ◽  
Marc Hippler ◽  
Magdalena Fladung ◽  
Moritz Tremmel ◽  
...  

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.


2021 ◽  
Author(s):  
Daniel Flormann ◽  
Kevin Kaub ◽  
Doriane Vesperini ◽  
Moritz Schu ◽  
Christoph Anton ◽  
...  

Adhesion induces dramatic morphological and mechanical changes to cells, which are reflected by changes to the actin cortex. Among the many different proteins involved in this sub-membranous layer, motor proteins (e.g., nonmuscle myosin II [NMII]) and actin nucleators (e.g., Arp2/3, formins) are known to have significant influences on its dynamics and structure. The different roles of NMII, Arp2/3, and formins in the dynamics, structure, and mechanics of the actin cortex depend on the adhesion state of the cell. In this study, we unravel the interplay between the dynamics, structure, and mechanics of the actin cortex in adhered cells and in cells in suspension. We show that treatments with extrinsic cellular perturbants lead to alterations of all three properties that are correlated. However, intrinsic actin cortex variations between different cell adhesion states lead to unexpected correlations. Surprisingly, we find that NMII minifilaments have a minor influence on the actin cortex. Using new microscopy techniques, we show that NMII minifilaments are not localized within the actin cortex, as previously thought, but concentrated in a layer beneath it. Our treatments affecting Arp2/3 and formin reveal correlations between the actin cortex characteristics. Our data build towards a comprehensive understanding of the actin cortex. This understanding allows the prediction and control of cortical changes, which is essential for the study of general cellular processes, such as cell migration, metastasis, and differentiation.


Author(s):  
Marina Garrido-Casado ◽  
Gloria Asensio-Juárez ◽  
Miguel Vicente-Manzanares

Nonmuscle myosin II (NMII) is a multimeric protein complex that generates most mechanical force in eukaryotic cells. NMII function is controlled at three main levels. The first level includes events that trigger conformational changes that extend the complex to enable its assembly into filaments. The second level controls the ATPase activity of the complex and its binding to microfilaments in extended NMII filaments. The third level includes events that modulate the stability and contractility of the filaments. They all work in concert to finely control force generation inside cells. NMII is a common endpoint of mechanochemical signaling pathways that control cellular responses to physical and chemical extracellular cues. Specific phosphorylations modulate NMII activation in a context-dependent manner. A few kinases control these phosphorylations in a spatially, temporally, and lineage-restricted fashion, enabling functional adaptability to the cellular microenvironment. Here, we review mechanisms that control NMII activity in the context of cell migration and division. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.


2021 ◽  
Author(s):  
Kei Yamamoto ◽  
Haruko Miura ◽  
Motohiko Ishida ◽  
Satoshi Sawai ◽  
Yohei Kondo ◽  
...  

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, it is still unknown how actomyosin contractility generates force and maintains cellular morphology. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility. The system, named OptoMYPT, combines a catalytic subunit of the type I phosphatase-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination was sufficient to induce dephosphorylation of myosin regulatory light chains and decrease in traction force at the subcellular level. The OptoMYPT system was further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We found that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system will provide new opportunities to understand cellular and tissue mechanics.


2021 ◽  
Author(s):  
Allison M Gabbert ◽  
James Mondo ◽  
Joseph P Campanale ◽  
Denise J Montell

Collective cell migration is prevalent throughout development and common in metastatic tumors, yet this process is not fully understood. In this study, we explore the role of septins (Sep) in collective cell migration, using the Drosophila border cell model. We show that Sep2 and Pnut are expressed in migrating border cells and Sep1, 2, 4, and Peanut (Pnut) are required for migration. Pnut stability depends on the expression of Sep1 and Sep2 in epithelial follicle cells and migratory border cells. We show that knockdown of septins prevents normal protrusion and detachment behaviors. High resolution Airyscan imaging reveals Pnut localization in rings at the base of protrusions. While septins function independently of Cdc42, they colocalize dynamically with nonmuscle myosin II. We suggest that septin polymers may stabilize growing protrusions until sufficient myosin is recruited to retract them.


2020 ◽  
pp. jcs.248732
Author(s):  
Indranil Ghosh ◽  
Raman K. Singh ◽  
Manjari Mishra ◽  
Shobhna Kapoor ◽  
Siddhartha S. Jana

Cells can adopt both mesenchymal and amoeboid mode of migration through membrane protrusive activities, namely lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebbing is yet to be explored. Here, we show that addition of ROCK inhibitor, Y27632 or lower doses of (-) blebbistatin, an inhibitor of NMII ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of lower doses of ML7, an inhibitor of MLCK, induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebbing and lamellipodia membrane protrusion are able to maintain pRLC/RLC ratio at cortices when MLCK and ROCK are inhibited, respectively, either pharmacologically or genetically, suggesting that they are interlinked in BLC and LBC. Such BLC and LBC are also inducible in other cells like MCF7 and MCF10A. These studies reveal that relative activity of ROCK and MLCK, which controls both NMII's ATPase activity and filamentous property is a determining factor for a cell to exhibit blebbing or lamellipodia.


2020 ◽  
Author(s):  
Kai Weißenbruch ◽  
Justin Grewe ◽  
Kathrin Stricker ◽  
Laurent Baulesch ◽  
Ulrich S. Schwarz ◽  
...  

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.


2020 ◽  
Vol 31 (21) ◽  
pp. 2379-2397
Author(s):  
Amy Platenkamp ◽  
Elizabeth Detmar ◽  
Liz Sepulveda ◽  
Anna Ritz ◽  
Stephen L. Rogers ◽  
...  

The Rab GAP RN-tre regulates the activity, coalescence, and function of nonmuscle myosin II in Drosophila melanogaster cells through cross-talk with the Rho1 signaling pathway. This regulation is partially independent of RN-tre’s GAP activity.


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