scholarly journals Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Aidan M Fenix ◽  
Abigail C Neininger ◽  
Nilay Taneja ◽  
Karren Hyde ◽  
Mike R Visetsouk ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Stress Fibers ◽  
Myosin Filament ◽  
Cardiac Myosin ◽  
Human Cardiomyocytes ◽  
Filament Assembly ◽  
Muscle Stress ◽  
Sarcomere Assembly ◽  
Muscle Myosin

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then ‘stitch’ together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

scholarly journals Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

2017 ◽  
Author(s):  
Aidan M. Feinx ◽  
Nilay Taneja ◽  
Abigail C. Neininger ◽  
Mike R. Visetsouk ◽  
Benjamin R. Nixon ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Muscle Cells ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Myosin Filament ◽  
Human Cardiomyocytes ◽  
Fiber Assembly ◽  
Filament Assembly ◽  
Sarcomere Assembly

AbstractThe sarcomere is the basic contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating thin (i.e., actin) and thick (i.e., myosin) filament assembly during sarcomere formation. Thus, we developed an assay using human cardiomyocytes to test de novo sarcomere assembly. Using this assay, we report a population of muscle-specific stress fibers are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from these muscle stress fibers. This process requires formin-mediated but not Arp2/3-mediated actin polymerization and nonmuscle myosin IIB but not non-muscle myosin IIA. Furthermore, we show a short species of β cardiac myosin II filaments grows to form ~1.5 long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., A-band). Interestingly, these are different from mechanisms that have previously been reported during stress fiber assembly in non-muscle cells. Thus, we provide a new model of cardiac sarcomere assembly based on distinct mechanisms of stress fiber regulation between non-muscle and muscle cells.

scholarly journals Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms

2003 ◽  
Vol 14 (3) ◽  
pp. 1002-1016 ◽  
Author(s):  
Nicole S. Bryce ◽  
Galina Schevzov ◽  
Vicki Ferguson ◽  
Justin M. Percival ◽  
Jim J.-C. Lin ◽  
...  
Keyword(s):  
Cell Size ◽  
Functional Properties ◽  
Actin Filament ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Molecular Composition ◽  
Cellular Migration ◽  
Stress Fibers ◽  
Human Homologue ◽  
Tropomyosin Isoforms

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.

scholarly journals Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts

2018 ◽  
Vol 29 (8) ◽  
pp. 911-922 ◽  
Author(s):  
Masahiro Kuragano ◽  
Taro Q. P. Uyeda ◽  
Keiju Kamijo ◽  
Yota Murakami ◽  
Masayuki Takahashi
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Contractile Force ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Proper Function ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Exogenous Expression ◽  
Main Component

Stress fibers (SFs) are contractile, force-generating bundled structures that can be classified into three subtypes, namely ventral SFs (vSFs), transverse arcs (TAs), and dorsal SFs. Nonmuscle myosin II (NMII) is the main component of SFs. This study examined the roles of the NMII isoforms NMIIA and NMIIB in the organization of each SF subtype in immortalized fibroblasts. Knockdown (KD) of NMIIA (a major isoform) resulted in loss of TAs from the lamella and caused the lamella to lose its flattened shape. Exogenous expression of NMIIB rescued this defect in TA formation. However, the TAs that formed on exogenous NMIIB expression in NMIIA-KD cells and the remaining TAs in NMIIB-KD cells, which mainly consisted of NMIIB and NMIIA, respectively, failed to rescue the defect in lamellar flattening. These results indicate that both isoforms are required for the proper function of TAs in lamellar flattening. KD of NMIIB resulted in loss of vSFs from the central region of the cell body, and this defect was not rescued by exogenous expression of NMIIA, indicating that NMIIA cannot replace the function of NMIIB in vSF formation. Moreover, we raised the possibility that actin filaments in vSFs are in a stretched conformation.

scholarly journals T-Plastin reinforces membrane protrusions to bridge matrix gaps during cell migration

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Damien Garbett ◽  
Anjali Bisaria ◽  
Changsong Yang ◽  
Dannielle G. McCarthy ◽  
Arnold Hayer ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Adhesion Sites ◽  
Membrane Protrusions ◽  
And Migration ◽  
Muscle Myosin ◽  
Migrating Cells

Abstract Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.

scholarly journals Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation

2011 ◽  
Vol 1 (5) ◽  
pp. 754-766 ◽  
Author(s):  
Tsubasa S. Matsui ◽  
Roland Kaunas ◽  
Makoto Kanzaki ◽  
Masaaki Sato ◽  
Shinji Deguchi
Keyword(s):  
Light Chain ◽  
Actin Filaments ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Physiological Level ◽  
Cell Shortening ◽  
Applied Forces ◽  
Selective Disassembly ◽  
The Individual ◽  
Muscle Myosin

Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin–myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin–myosin crossbridges. Actin–myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin–myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

scholarly journals Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Sari Tojkander ◽  
Gergana Gateva ◽  
Amjad Husain ◽  
Ramaswamy Krishnan ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Physical Mechanisms ◽  
Filament Assembly ◽  
Contractile Stress ◽  
Lateral Fusion

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

scholarly journals Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

2008 ◽  
Vol 19 (11) ◽  
pp. 5006-5018 ◽  
Author(s):  
Tom W. Anderson ◽  
Andrew N. Vaughan ◽  
Louise P. Cramer
Keyword(s):  
Actin Filaments ◽  
Myosin Ii ◽  
Stress Fibers ◽  
Live Cells ◽  
Retrograde Flow ◽  
Mechanism Of Formation ◽  
Unknown Mechanism ◽  
Cell Edge ◽  
Cell Protrusion ◽  
Filament Bundles

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.

scholarly journals The Kinetics Underlying the Velocity of Smooth Muscle Myosin Filament Sliding on Actin Filaments in vitro

2015 ◽  
Vol 108 (2) ◽  
pp. 9a
Author(s):  
Brian D. Haldeman ◽  
Richard K. Brizendine ◽  
Diego Alcala ◽  
Kevin C. Facemyer ◽  
Josh E. Baker ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Smooth Muscle Myosin ◽  
Myosin Filament ◽  
Muscle Myosin

scholarly journals Cardiomyopathy Mutations Impact the Power Stroke of Human Cardiac Myosin

2020 ◽  
Author(s):  
W. Tang ◽  
J. Ge ◽  
W.C. Unrath ◽  
R. Desetty ◽  
C. M. Yengo
Keyword(s):  
Cardiac Muscle ◽  
Actin Filaments ◽  
Structural Changes ◽  
Power Stroke ◽  
Contractile Dysfunction ◽  
Cardiac Myosin ◽  
Phosphate Release ◽  
Force Development ◽  
Lever Arm ◽  
Muscle Myosin

AbstractCardiac muscle contraction is driven by the molecular motor myosin that uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, while it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a Förster resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human β-cardiac myosin subfragment 1 (M2β-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2β-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic stopped-flow measurements. We proposed that the fast phase measured with our FRET probes represents the structural change associated with rotation of the lever arm during the power stroke in M2β-S1. Our results demonstrated human cardiac muscle myosin has a slower power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the power stroke and phosphate release revealed that the power stroke occurs before phosphate release, and the two steps are tightly coupled. We speculate that the slower power stroke rate constant in cardiac myosin may correlate with the slower force development and/or unique thin filament activation properties in cardiac muscle. Additionally, we demonstrated that HCM (R723G) and DCM (F764L) associated mutations both reduced actin-activation of the power stroke in M2β-S1. We also demonstrate that both mutations decrease ensemble force development in the loaded in vitro motility assay. Thus, examining the structural kinetics of the power stroke in M2β-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction.SignificanceMutations in human beta-cardiac myosin are known to cause various forms of heart disease, while it is unclear how the mutations lead to contractile dysfunction and pathogenic remodeling of the heart. In this study, we investigated two mutations with opposing phenotypes and examined their impact on ATPase cycle kinetics, structural changes associated with the myosin power stroke, and ability to slide actin filaments in the presence of load. We found that both mutations impair the myosin power stroke and other key kinetic steps as well as the ability to produce ensemble force. Thus, our results provide a structural basis for how mutations disrupt molecular level contractile dysfunction.

scholarly journals Generation of stress fibers through myosin-driven re-organization of the actin cortex

2020 ◽  
Author(s):  
JI Lehtimäki ◽  
EK Rajakylä ◽  
S Tojkander ◽  
P Lappalainen
Keyword(s):  
De Novo ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Cellular Processes ◽  
Actin Cortex ◽  
Muscle Myosin ◽  
Subsequent Stabilization

SummaryContractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex, and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.

Sign in / Sign up

Export Citation Format

Share Document