Characterization of the chondrocyte actin cytoskeleton in living three-dimensional culture: Response to anabolic and catabolic stimuli

Division of Arthritis Research, Department of Molecular and Experimental Medicine, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla CA 92037, USA.
Molecular & cellular biomechanics: MCB 10/2009; 6(3):135-44.
Source: PubMed


The actin cytoskeleton is a dynamic network required for intracellular transport, signal transduction, movement, attachment to the extracellular matrix, cellular stiffness and cell shape. Cell shape and the actin cytoskeletal configuration are linked to chondrocyte phenotype with regard to gene expression and matrix synthesis. Historically, the chondrocyte actin cytoskeleton has been studied after formaldehyde fixation--precluding real-time measurements of actin dynamics, or in monolayer cultured cells. Here we characterize the actin cytoskeleton of living low-passage human chondrocytes grown in three-dimensional culture using a stably expressed actin-GFP construct. GFP-actin expression does not substantially alter the production of endogenous actin at the protein level. GFP-actin incorporates into all actin structures stained by fluorescent phalloidin, and does not affect the actin cytoskeleton as seen by fluorescence microscopy. GFP-actin expression does not significantly change the chondrocyte cytosolic stiffness. GFP-actin does not alter the gene expression response to cytokines and growth factors such as IL-1beta and TGF-beta. Finally, GFP-actin does not alter production of extracellular matrix as measured by radiosulfate incorporation. Having established that GFP-actin does not measurably affect the chondrocyte phenotype, we tested the hypothesis that IL-1beta and TGF-beta differentially alter the actin cytoskeleton using time-lapse microscopy. TGF-beta increases actin extensions, and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1beta causes cellular contraction indicative of RhoA activation. The ability to visualize GFP-actin in living chondrocytes in 3D culture without disrupting the organization or function of the cytoskeleton is an advance in chondrocyte cell biology and provides a powerful tool for future studies in actin-dependent chondrocyte differentiation and mechanotransduction pathways.

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Available from: Dominik R Haudenschild
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    • "The cytoskeleton is composed of three interconnected filament systems: actin, microtubules and intermediate filaments (IFs) that contribute to a cell’s shape, structural integrity and movement. Among the components of the cytoskeleton, both actin and IFs [20]–[22], have been postulated to contribute to the mechanical properties of cells. The contribution of the actin cytoskeleton to the mechanical properties of chondrocytes has been widely studied [23]–[25]. "
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    ABSTRACT: Articular cartilage chondrocytes are responsible for the synthesis, maintenance, and turnover of the extracellular matrix, metabolic processes that contribute to the mechanical properties of these cells. Here, we systematically evaluated the effect of age and cytoskeletal disruptors on the mechanical properties of chondrocytes as a function of deformation. We quantified the indentation-dependent mechanical properties of chondrocytes isolated from neonatal (1-day), adult (5-year) and geriatric (12-year) bovine knees using atomic force microscopy (AFM). We also measured the contribution of the actin and intermediate filaments to the indentation-dependent mechanical properties of chondrocytes. By integrating AFM with confocal fluorescent microscopy, we monitored cytoskeletal and biomechanical deformation in transgenic cells (GFP-vimentin and mCherry-actin) under compression. We found that the elastic modulus of chondrocytes in all age groups decreased with increased indentation (15-2000 nm). The elastic modulus of adult chondrocytes was significantly greater than neonatal cells at indentations greater than 500 nm. Viscoelastic moduli (instantaneous and equilibrium) were comparable in all age groups examined; however, the intrinsic viscosity was lower in geriatric chondrocytes than neonatal. Disrupting the actin or the intermediate filament structures altered the mechanical properties of chondrocytes by decreasing the elastic modulus and viscoelastic properties, resulting in a dramatic loss of indentation-dependent response with treatment. Actin and vimentin cytoskeletal structures were monitored using confocal fluorescent microscopy in transgenic cells treated with disruptors, and both treatments had a profound disruptive effect on the actin filaments. Here we show that disrupting the structure of intermediate filaments indirectly altered the configuration of the actin cytoskeleton. These findings underscore the importance of the cytoskeletal elements in the overall mechanical response of chondrocytes, indicating that intermediate filament integrity is key to the non-linear elastic properties of chondrocytes. This study improves our understanding of the mechanical properties of articular cartilage at the single cell level.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "For instance, RhoA is important for thrombin-induced ICAM-1 expression, but not for the induction of ICAM-1 expression by TNFα, in endothelial cells [30]. In chondrocytes, RhoA regulates TGF-β- but not IL-1-induced actin cytoskeleton reorganization [31]. Furthermore, RhoA mediates TNFα- but not UV-induced NF-κB activation in COS-7 cells [32]. "
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    ABSTRACT: RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.
    Full-text · Article · Mar 2012 · PLoS ONE
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    • "Similar to our results with TGF-β1 treatment, in other studies using isolated rabbit meniscal cells cultured in 10% FBS and equivalent concentrations of TGF-β1, there was no effect of TGFβ1on cell proliferation at 48 hours [59]. TGF-β1 has been shown to increase F-actin levels in isolated chondrocytes [60] and increase actin extensions and lamellar ruffling in agarose embedded chondrocytes [61]. In other studies, 3T3 fibroblasts treated with TGF-β1 did not migrate or proliferate and contained stabilized microtubules [62], consistent with the overall effects observed in this study. "
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