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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    • "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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    PLoS ONE 04/2013; 8(4):e61651. DOI:10.1371/journal.pone.0061651 · 3.23 Impact Factor
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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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    PLoS ONE 03/2012; 7(3):e33773. DOI:10.1371/journal.pone.0033773 · 3.23 Impact Factor
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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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