Open Access

ARTICLE

Characterization of the Chondrocyte Actin Cytoskeleton in Living Three-Dimensional Culture: Response to Anabolic and Catabolic Stimuli

Dominik R. Haudenschild^∗,†, Jianfen Chen^∗,†, Nikolai Steklov^†, Martin K. Lotz^*, Darryl D. D’Lima^∗,‡

∗ Division of Arthritis Research, Department of Molecular and Experimental Medicine, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla CA 92037
† Shiley Center for Orthopaedic Research and Education at Scripps Clinic, 11025 North Torrey Pines Road Suite 140, La Jolla CA 92037
‡ Corresponding Author. Shiley Center for Orthopaedic Research and Education at Scripps Clinic, 11025 North Torrey Pines Road Suite 140, La Jolla CA 92037. Tel: 858-332-0166; Fax: 858-332-0127; email: ddlima@scripps.edu

Molecular & Cellular Biomechanics 2009, 6(3), 135-144. https://doi.org/10.3970/mcb.2009.006.135

Abstract

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-1band TGF-b. 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-1band TGF-bdifferentially alter the actin cytoskeleton using time-lapse microscopy. TGF-bincreases actin extensions and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1bcauses 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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