Crosstalk between myosin II and formin functions in the regulation of force generation and actomyosin dynamics in stress fibers

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation.

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LIMCH1 regulates nonmuscle myosin-II activity and suppresses cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e15-04-0218 ◽

2017 ◽

Vol 28 (8) ◽

pp. 1054-1065 ◽

Cited By ~ 11

Author(s):

Yu-Hung Lin ◽

Yen-Yi Zhen ◽

Kun-Yi Chien ◽

I-Ching Lee ◽

Wei-Chi Lin ◽

...

Keyword(s):

Cell Migration ◽

Hela Cells ◽

Myosin Ii ◽

Focal Adhesions ◽

Stress Fiber ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Nonmuscle Myosin ◽

Head Region ◽

Nonmuscle Myosin Ii

Nonmuscle myosin II (NM-II) is an important motor protein involved in cell migration. Incorporation of NM-II into actin stress fiber provides a traction force to promote actin retrograde flow and focal adhesion assembly. However, the components involved in regulation of NM-II activity are not well understood. Here we identified a novel actin stress fiber–associated protein, LIM and calponin-homology domains 1 (LIMCH1), which regulates NM-II activity. The recruitment of LIMCH1 into contractile stress fibers revealed its localization complementary to actinin-1. LIMCH1 interacted with NM-IIA, but not NM-IIB, independent of the inhibition of myosin ATPase activity with blebbistatin. Moreover, the N-terminus of LIMCH1 binds to the head region of NM-IIA. Depletion of LIMCH1 attenuated myosin regulatory light chain (MRLC) diphosphorylation in HeLa cells, which was restored by reexpression of small interfering RNA–resistant LIMCH1. In addition, LIMCH1-depleted HeLa cells exhibited a decrease in the number of actin stress fibers and focal adhesions, leading to enhanced cell migration. Collectively, our data suggest that LIMCH1 plays a positive role in regulation of NM-II activity through effects on MRLC during cell migration.

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

eLife ◽

10.7554/elife.06126 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 74

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.

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Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

eLife ◽

10.7554/elife.68627 ◽

2021 ◽

Vol 10 ◽

Author(s):

Daan Vorselen ◽

Sarah R Barger ◽

Yifan Wang ◽

Wei Cai ◽

Julie A Theriot ◽

...

Keyword(s):

Myosin Ii ◽

Traction Force ◽

Light Sheet ◽

Force Generation ◽

Actin Dynamics ◽

Force Microscopy ◽

Light Sheet Microscopy ◽

Macrophage Phagocytosis ◽

Actin Protrusions

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely driven by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that appear to be interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, supporting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings present a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

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Phagocytic 'teeth' and myosin-II 'jaw' power target constriction during phagocytosis

10.1101/2021.03.14.435346 ◽

2021 ◽

Author(s):

Daan Vorselen ◽

Sarah R. Barger ◽

Yifan Wang ◽

Wei Cai ◽

Julie A. Theriot ◽

...

Keyword(s):

Myosin Ii ◽

Traction Force ◽

Light Sheet ◽

Force Generation ◽

Actin Dynamics ◽

Force Microscopy ◽

Light Sheet Microscopy ◽

Macrophage Phagocytosis ◽

Actin Protrusions

Phagocytosis requires rapid actin reorganization and spatially controlled force generation to ingest targets ranging from pathogens to apoptotic cells. How actomyosin activity directs membrane extensions to engulf such diverse targets remains unclear. Here, we combine lattice light-sheet microscopy (LLSM) with microparticle traction force microscopy (MP-TFM) to quantify actin dynamics and subcellular forces during macrophage phagocytosis. We show that spatially localized forces leading to target constriction are prominent during phagocytosis of antibody-opsonized targets. This constriction is largely mediated by Arp2/3-mediated assembly of discrete actin protrusions containing myosin 1e and 1f ('teeth') that are interconnected in a ring-like organization. Contractile myosin-II activity contributes to late-stage phagocytic force generation and progression, suggesting a specific role in phagocytic cup closure. Observations of partial target eating attempts and sudden target release via a popping mechanism suggest that constriction may be critical for resolving complex in vivo target encounters. Overall, our findings suggest a phagocytic cup-shaping mechanism that is distinct from cytoskeletal remodeling in 2D cell motility and may contribute to mechanosensing and phagocytic plasticity.

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ATP allosterically stabilizes Integrin-linked kinase for efficient force generation

10.1101/2021.03.30.437490 ◽

2021 ◽

Author(s):

Isabel M. Martin ◽

Michele M. Nava ◽

Sara A. Wickstroem ◽

Frauke Graeter

Keyword(s):

Focal Adhesion ◽

Protein Unfolding ◽

Focal Adhesions ◽

Traction Force ◽

Force Generation ◽

Force Microscopy ◽

Integrin Linked Kinase ◽

And Migration ◽

Dynamics Simulations ◽

Propagation Network

Focal adhesions link the actomyosin cytoskeleton to the extracellular matrix regulating cell adhesion, shape, and migration. Adhesions are dynamically assembled and disassembled in response to extrinsic and intrinsic forces, but how the essential adhesion component intergrin-linked kinase (ILK) dynamically responds to mechanical force and what role ATP bound to this pseudokinase plays remains elusive. Here, we apply force-probe molecular dynamics simulations of ILK:alpha-parvin coupled to traction force microscopy to explore ILK mechanotransducing functions. We identify two key saltbridge-forming arginines within the allosteric, ATP-dependent force-propagation network of ILK. Disrupting this network by mutation impedes parvin binding, focal adhesion stabilization, force generation, and thus migration. Under tension, ATP shifts the balance from rupture of the complex to protein unfolding, indicating that ATP increases the force threshold required for focal adhesion disassembly. Our study proposes a new role of ATP as an obligatory binding partner for structural and mechanical integrity of the pseudokinase ILK, ensuring efficient cellular force generation and migration.

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Actomyosin stress fiber mechanosensing in 2D and 3D

F1000Research ◽

10.12688/f1000research.8800.1 ◽

2016 ◽

Vol 5 ◽

pp. 2261 ◽

Cited By ~ 23

Author(s):

Stacey Lee ◽

Sanjay Kumar

Keyword(s):

Extracellular Matrix ◽

Myosin Ii ◽

Three Dimensional ◽

Focal Adhesions ◽

Stress Fiber ◽

Stress Fibers ◽

Myosin Isoforms ◽

Matrix Mechanics ◽

Tensile Forces ◽

Muscle Myosin

Mechanotransduction is the process through which cells survey the mechanical properties of their environment, convert these mechanical inputs into biochemical signals, and modulate their phenotype in response. These mechanical inputs, which may be encoded in the form of extracellular matrix stiffness, dimensionality, and adhesion, all strongly influence cell morphology, migration, and fate decisions. One mechanism through which cells on planar or pseudo-planar matrices exert tensile forces and interrogate microenvironmental mechanics is through stress fibers, which are bundles composed of actin filaments and, in most cases, non-muscle myosin II filaments. Stress fibers form a continuous structural network that is mechanically coupled to the extracellular matrix through focal adhesions. Furthermore, myosin-driven contractility plays a central role in the ability of stress fibers to sense matrix mechanics and generate tension. Here, we review the distinct roles that non-muscle myosin II plays in driving mechanosensing and focus specifically on motility. In a closely related discussion, we also describe stress fiber classification schemes and the differing roles of various myosin isoforms in each category. Finally, we briefly highlight recent studies exploring mechanosensing in three-dimensional environments, in which matrix content, structure, and mechanics are often tightly interrelated. Stress fibers and the myosin motors therein represent an intriguing and functionally important biological system in which mechanics, biochemistry, and architecture all converge.

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A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

10.1101/2021.04.14.439781 ◽

2021 ◽

Author(s):

Lara Hauke ◽

Shwetha Narasimhan ◽

Andreas Primeßnig ◽

Irina Kaverina ◽

Florian Rehfeldt

Keyword(s):

Extracellular Matrix ◽

Quantitative Analysis ◽

Focal Adhesion ◽

Cross Correlation ◽

Focal Adhesions ◽

Traction Force ◽

Stress Fibers ◽

Traction Force Microscopy ◽

Force Microscopy ◽

Actin Stress Fibers

AbstractFocal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.Author summaryOur novel Focal Adhesion Filament Cross-correlation Kit (FAFCK) allows for fast, reliable, unbiased, and systematic detection of focal adhesions and actin stress fibers in cells and their mutual correlation. Detailed analysis of these structures which are both key elements in mechano-sensing and force transduction will help tremendously to improve quantitative analysis of mechanocellular experiments, key to understanding the complex interplay between cells and the extracellular matrix. In particular, sophisticated analysis methods such as model-based traction force microscopy will benefit from correlating the detailed datasets of stress fibers and focal adhesions.

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Slipping or Gripping? Fluorescent Speckle Microscopy in Fish Keratocytes Reveals Two Different Mechanisms for Generating a Retrograde Flow of Actin

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0860 ◽

2005 ◽

Vol 16 (2) ◽

pp. 507-518 ◽

Cited By ~ 146

Author(s):

Carlos Jurado ◽

John R. Haserick ◽

Juliet Lee

Keyword(s):

Flow Rate ◽

Potent Inhibitor ◽

Myosin Ii ◽

Force Production ◽

Traction Force ◽

The Other ◽

Force Generation ◽

Retrograde Flow ◽

Transfected Cells ◽

The Relationship

Fish keratocytes can generate rearward directed traction forces within front portions of the lamellipodium, suggesting that a retrograde flow of actin may also occur here but this was not detected by previous photoactivation experiments. To investigate the relationship between retrograde flow and traction force generation, we have transfected keratocytes with GFP-actin and used fluorescent speckle microscopy, to observe speckle flow. We detected a retrograde flow of actin within the leading lamellipodium that is inversely proportional to both protrusion rate and cell speed. To observe the effect of reducing contractility, we treated transfected cells with ML7, a potent inhibitor of myosin II. Surprisingly, ML7 treatment led to an increase in retrograde flow rate, together with a decrease in protrusion and cell speed, but only in rapidly moving cells. In slower moving cells, retrograde flow decreased, whereas protrusion rate and cell speed increased. These results suggest that there are two mechanisms for producing retrograde flow. One involves slippage between the cytoskeleton and adhesions, that decreases traction force production. The other involves slippage between adhesions and the substratum, which increases traction force production. We conclude that a biphasic relationship exists between retrograde actin flow and adhesiveness in moving keratocytes.

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Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

Cellular Physiology and Biochemistry ◽

10.1159/000484064 ◽

2017 ◽

Vol 43 (5) ◽

pp. 1777-1789 ◽

Cited By ~ 7

Author(s):

Lei Zhang ◽

Tianrong Ji ◽

Qin Wang ◽

Kexin Meng ◽

Rui Zhang ◽

...

Keyword(s):

Protective Action ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Dependent Manner ◽

Calcium Sensing Receptor ◽

Rhoa Activity ◽

Rhoa Activation ◽

Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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