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

Research output: Contribution to journalArticle

23 Scopus citations

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-1β and TGF-β. 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-1β and TGF-β differentially alter the actin cytoskeleton using time-lapse microscopy. TGF-β increases actin extensions and lamellar ruffling indicative of Rac/CDC42 activation, while IL-1β 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.

Original languageEnglish (US)
Pages (from-to)135-144
Number of pages10
JournalMCB Molecular and Cellular Biomechanics
Volume6
Issue number3
StatePublished - 2009
Externally publishedYes

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ASJC Scopus subject areas

  • Cell Biology
  • Biophysics
  • Molecular Biology
  • Molecular Medicine

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