Direct Observations of the Mechanical Behaviors of the Cytoskeleton in Living Fibroblasts

Heidemann, Steven R.; Kaech, Stefanie; Buxbaum, Robert E.; Matus, Andrew

doi:10.1083/jcb.145.1.109

Cited by 185 publications

(143 citation statements)

References 49 publications

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“…Our data suggest that, in the absence of myosin contractility, MT bending and breaking is reduced, and fewer free minus ends are generated, resulting in slower overall turnover. These results provide direct evidence for myosingenerated forces modulating the kinetic behavior of MTs (7,17).…”

Section: Mt Movement Requiressupporting

confidence: 49%

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Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

Yvon

Gross

Wadsworth

2001

Proc. Natl. Acad. Sci. U.S.A.

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Photoactivation of caged fluorescent tubulin was used mark the microtubule (MT) lattice and monitor MT behavior in interphase cells. A broadening of the photoactivated region occurred as MTs moved bidirectionally. MT movement was not inhibited when MT assembly was suppressed with nocodazole or Taxol; MT movement was suppressed by inhibition of myosin light chain kinase with ML7 or by a peptide inhibitor. Conversely, MT movement was increased after inhibition of cytoplasmic dynein with the antibody 70.1. In addition, the half-time for MT turnover was decreased in cells treated with ML7. These results demonstrate that myosin II and cytoplasmic dynein contribute to a balance of forces that regulates MT organization, movement, and turnover in interphase cells. microtubule motion T he capacity for microtubules (MTs) to undergo dynamic rearrangement is critical to the various vital cellular activities to which they contribute, including the assembly and function of the mitotic spindle, intracellular transport of organelles and vesicles, and the establishment and maintenance of cell shape and polarity. MTs are nucleated at the centrosome and maintain a generally radial organization, with plus-ends at the cell periphery. Direct observations show that peripheral plus-ends undergo dynamic instability behavior, characterized by the stochastic switching between phases of elongation and rapid shortening (1). Dynamic instability is tempered, such that turnover is more rapid in the peripheral region of the cell (2). MT turnover in the more central cytoplasmic domain is thought to occur by subunit turnover at both plus-and minus-ends (3, 4).MTs modulate the behavior of the actin cytoskeleton in a temporally and spatially regulated manner. For example, MTs are required to establish the site of contractile ring formation in cytokinesis and to restrict ruffling behavior to the leading edge of motile fibroblasts (5). The nature of the cross-talk between the MT and actin cytoskeletons is not well understood, but recent work indicates that a feedback mechanism involving Rho family members may contribute to these regulatory interactions (6). In addition, molecular motors may integrate the MT and actin systems. Actomyosin moves MTs rearward at the cell periphery (3), and myosin-generated forces are responsible for MT transport in motile cells and for axon retraction in cultured neurons (7,8). Cytoplasmic dynein contributes to MT organization in interphase cells, to the assembly and function of the mitotic spindle, and to MT transport in neurons (9, 10). These observations led us to test the hypothesis that molecular motors modulate the organization, rearrangement, and turnover of MTs in interphase cells. Materials and MethodsMaterials. All materials for cell culture were obtained from Life Technologies (Gaithersburg, MD), with the exception of FCS, which was obtained from Atlanta Biologicals (Norcross, GA). Unless otherwise noted, all other chemicals were obtained from Sigma.Cell Culture, Cell Staining, and Microinjection. Cell culture...

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Section: Mt Movement Requiressupporting

confidence: 49%

“…Individual MTs behaved independently: adjacent MTs could move in opposite directions, indicating that the mechanism for movement is highly local (Fig. 1d) (17). The apparent lengthening of the fluorescent mark on some MTs was likely to be the result of closely aligned MTs moving at different rates or in opposite directions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

Yvon

Gross

Wadsworth

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…2A), as previously observed in other cells (23). Buckling observed in an end-on loading configuration is indicative of compressive loading; however, some investigators have suggested that this form of microtubule buckling could result from fluid flow in the surrounding cytosol (24). To directly determine the relative importance of fluid flow for structural reorganization in the cytosol, displacements of EYFPmitochondria and GFP-microtubules were analyzed in live endothelial cells adherent to deformable polyacrylamide gels (17).…”

Section: Resultsmentioning

confidence: 99%

Mechanical behavior in living cells consistent with the tensegrity model

Wang

Naruse

Stamenović

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

612

525

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Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.cytoskeleton ͉ microtubules ͉ cell mechanics ͉ myosin light chain phosphorylation ͉ mechanotransduction M echanical stress-induced alterations in cell shape and structure are critical for control of many cell functions, including growth, motility, contraction, and mechanotransduction (1). These functional alterations are mediated through changes in the internal cytoskeleton (CSK), which is composed of an interconnected network of microfilaments, microtubules, and intermediate filaments that links the nucleus to surface adhesion receptors. Advances in cell biology have resulted in better understanding of the polymerization behavior and physical properties of individual CSK filaments as well as of gels composed of combinations of filaments. Yet, the material properties measured in vitro neither explain nor predict complex mechanical behaviors that are observed in living cells (2, 3). At the same time, engineers have approached the problem of how cells stabilize their shape by developing mechanical models, without considering molecular specificity. For example, the living cell is often modeled as a continuum that contains an elastic cortex that surrounds a viscous (4) or viscoelastic (5) fluid; a more complex variation includes an elastic nucleus within a viscous cytoplasm (6). These models provide reasonable empirical fits to measured elastic moduli and viscosity in cells under specific experimental conditions (4-6), but they cannot predict from mechanistic principles how these properties alter under different challenges to the cell. Continuum models also assume that the load-bearing elements are infinitesimally small relative to the size of the cell and thus, they...

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“…Green fluorescent protein (GFP)-tagged fluorescent markers have been used to study the displacement of cellular organelles and the formation of a focal adhesion complex induced by mechanical stimuli [22][23][24][25] , but these inert fluorescence markers cannot monitor the dynamic signal transduction process. Our FRET-based Src reporter enables the visualization and quantification of the mechano-activated Src with high temporal and spatial resolution in live cells.…”

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confidence: 99%