Our objective was to generate an experimental system to visualize the dynamic reorganization of the actin cytoskeleton of chondrocytes in a native 3D culture system. To this end, we chose lentiviral introduction of a GFP-actin fusion protein. This system efficiently infects chondrocytes and provides long-term stable protein production. Transduced chondrocytes were cultured in 3D conditions for at least four weeks with continuous production of the GFP-actin, permitting experiments that require the synthesis and assembly of extracellular matrix.
Chimeric GFP-β-actin retains the functions of endogenous β-actin in a majority of non-chondrocyte systems in which actin functions have been studied. GFP-actin incorporated into all cellular actin structures including lamellipodia, filopodia, focal contacts and stress fibers (28
), as well as more specialized structures such as podosomes in osteoclasts (29
). In most cases, GFP-actin did not interfere with cell growth (28
), although it slightly impairment cytokinesis in quickly growing Dictyostelium discoideum
). GFP-actin dynamically incorporates into rapidly remodeled actin (28
), and GFP-actin responded to actin destabilizing cytochalasin-B treatment similarly to normal actin (30
). In vitro
assays showed that low amounts of GFP-actin had little effect on either filament polymerization or movement along a surface of heavy meromyosin (32
). However, Westphal, Feng and others observed that GFP-actin function is somewhat compromised, causing filament instability (32
) and altering the initial dynamics of integrin-based cell spreading (34
). Despite this, mice expressing GFP-actin from a profilin promoter are viable, implying that there are no drastic effects of GFP-actin on overall development (33
While virtually all phalloidin-stained actin was also positive for GFP-actin, we observed additional GFP-actin in areas that were phalloidin-negative. Phalloidin only binds with F-actin. Actin binding proteins including cofilin, actin depolymerizing factor (ADF) and others, can alter the configuration of actin filaments to prevent phalloidin binding (35
). Also, actin sequestering proteins bind monomeric actin to prevent filament formation while maintaining an available pool of G-actin. These proteins include the twinfilins, beta-thymosins, ADFs/cofilins, Srv2/CAPs, profilins, and others (37
). We expect that the phalloidin-negative GFP-actin was either bound by actin-binding proteins that prevent phalloidin binding, or present as monomers ready for incorporation into filaments.
Since the actin cytoskeleton of 3D cultured chondrocytes differs substantially from the cytoskeleton in other cell types, we felt it necessary to characterize the effect of GFP-actin transduction on the chondrocyte phenotype with regard to several parameters important in chondrocyte biology. We verified that neither the viral infection, the production of a recombinant protein, or the production of GFP-actin altered the nature of the chondrocytes or disrupted the actin cytoskeleton. GFP-actin was incorporated into all actin structures visible after phalloidin staining of formaldehyde-fixed cells. GFP-actin caused no substantial differences in endogenous actin levels, actin cytoskeletal structures, cytosolic stiffness, extracellular matrix production, or cellular responses to anabolic and catabolic stimuli.
Having established that these important parameters of chondrocytes are not adversely affected by the GFP-actin expression, we used live-cell imaging to evaluate how the actin cytoskeleton responds to anabolic and catabolic growth factors and cytokines. We found that catabolic IL-1β treatment causes a cellular contraction of approximately 15% within 6 hours. Contractile forces generated through the actin cytoskeleton generally involve Rho GTPase activation, Rho Kinase, and stress fibers in other cell types, and it is likely but unproven that a similar pathway is activated by IL-1 β treatment in the chondrocytes. We also found that TGF- β treatment caused a decrease in filopodia and an increase in lamellipodia on the periphery of the chondrocytes within 4.5 hours. In other cell types, filopodia and lamellipodia are indicative of Cdc42 and Rac GTPase activity, and again it is likely but unproven that similar pathways are activated in the chondrocytes. The 3D equivalent of stress fibers, filopodia and lamellipodia have not yet been described in detail for any cell type. The molecular tools and techniques presented in this manuscript will enable us to identify the 3D equivalents of these actin structures.
In summary, we demonstrate that lentivirally transduced GFP-actin has no significant impact on several important aspects of chondrocyte biology. The ability to visualize the dynamic reorganization of the actin cytoskeleton in living chondrocytes in a three-dimensional culture without altering the nature of the chondrocyte, and without disrupting the organization and function of the cytoskeleton, is an advancement in the field of chondrocyte cell biology. This provides a powerful tool for the study of cell biomechanics and for studies in actin-dependent signaling pathways present in the chondrocyte, including responses to growth factors, cytokines, and mechanotransduction pathways.