From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM–ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM–ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

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Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0661 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1263-1273 ◽

Cited By ~ 42

Author(s):

Christopher D. Lynch ◽

Nils C. Gauthier ◽

Nicolas Biais ◽

Andre M. Lazar ◽

Pere Roca-Cusachs ◽

...

Keyword(s):

Three Dimensional ◽

Focal Adhesions ◽

Stress Fibers ◽

Cortical Actin ◽

Compensatory Mechanisms ◽

Embryonic Fibroblasts ◽

Traction Forces ◽

Actin Networks ◽

Cellular Integrity ◽

Adhesion Stability

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB–/– mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA–/– MEFs, but not FlnB–/– MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

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Supervillin modulation of focal adhesions involving TRIP6/ZRP-1

The Journal of Cell Biology ◽

10.1083/jcb.200512051 ◽

2006 ◽

Vol 174 (3) ◽

pp. 447-458 ◽

Cited By ~ 36

Author(s):

Norio Takizawa ◽

Tara C. Smith ◽

Thomas Nebl ◽

Jessica L. Crowley ◽

Stephen J. Palmieri ◽

...

Keyword(s):

Focal Adhesions ◽

Stress Fibers ◽

Regulatory Sequence ◽

Interacting Protein ◽

Substrate Adhesion ◽

Lim Domains ◽

Cell Substrate ◽

Thyroid Receptor ◽

And Function ◽

Cell Substrate Adhesion

Cell–substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)—a peripheral membrane protein that binds myosin II and F-actin in such cells—negatively regulates stress fibers, FAs, and cell–substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor–interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV–TRIP6 interaction may regulate FA maturation and/or disassembly.

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Costameres, focal adhesions, and cardiomyocyte mechanotransduction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00749.2005 ◽

2005 ◽

Vol 289 (6) ◽

pp. H2291-H2301 ◽

Cited By ~ 174

Author(s):

Allen M. Samarel

Keyword(s):

Growth Factor ◽

Focal Adhesions ◽

Growth Factor Signaling ◽

Cellular Mechanisms ◽

Growth And Survival ◽

Specific Proteins ◽

And Function ◽

Cytoskeletal Elements ◽

Biochemical Signals ◽

Cellular Components

Mechanotransduction refers to the cellular mechanisms by which load-bearing cells sense physical forces, transduce the forces into biochemical signals, and generate appropriate responses leading to alterations in cellular structure and function. This process affects the beat-to-beat regulation of cardiac performance but also affects the proliferation, differentiation, growth, and survival of the cellular components that comprise the human myocardium. This review focuses on the experimental evidence indicating that the costamere and its structurally related structure the focal adhesion complex are critical cytoskeletal elements involved in cardiomyocyte mechanotransduction. Biochemical signals originating from the extracellular matrix-integrin-costameric protein complex share many common features with those signals generated by growth factor receptors. The roles of key regulatory kinases and other muscle-specific proteins involved in mechanotransduction and growth factor signaling are discussed, and issues requiring further study in this field are outlined.

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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

PLoS ONE ◽

10.1371/journal.pone.0250749 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0250749

Author(s):

Lara Hauke ◽

Shwetha Narasimhan ◽

Andreas Primeßnig ◽

Irina Kaverina ◽

Florian Rehfeldt

Keyword(s):

Focal Adhesion ◽

Cross Correlation ◽

Focal Adhesions ◽

Force Production ◽

Traction Force ◽

Stress Fibers ◽

Coupled Analysis ◽

Matrix Remodeling ◽

Fiber System ◽

Actin Stress Fibers

Focal 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.

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

10.1101/2021.08.15.456175 ◽

2021 ◽

Author(s):

Yukako Nishimura ◽

Shidong Shi ◽

Qingsen Li ◽

Alexander D. Bershadsky ◽

Virgile Viasnoff

Keyword(s):

Contractile Activity ◽

Myosin Ii ◽

Focal Adhesions ◽

Traction Force ◽

Stress Fiber ◽

Force Generation ◽

Stress Fibers ◽

Force Microscopy ◽

Fiber Dynamics ◽

The Relationship

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

10.1101/2020.06.30.179283 ◽

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.

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Generation of stress fibers through myosin-driven reorganization of the actin cortex

eLife ◽

10.7554/elife.60710 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jaakko I Lehtimäki ◽

Eeva Kaisa Rajakylä ◽

Sari Tojkander ◽

Pekka Lappalainen

Keyword(s):

De Novo ◽

Myosin Ii ◽

Focal Adhesions ◽

Stress Fibers ◽

Cell Cortex ◽

Cortical Actin ◽

Cellular Processes ◽

Actin Cortex ◽

Muscle Myosin ◽

Subsequent Stabilization

Contractile 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.

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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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