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ß-Actin Protein Concentration Fluctuates During Contraction of Loaded and Unloaded Fibroblast Populated Collagen Gels.  B. P. Johnson-Wint and W. Heisler.  Dept. of Biological Sciences, Northern Illinois University, DeKalb, IL.

   Fibroblasts use a cytoplasmic actin-myosin force generating machinery to contract type-I collagen gels.  ß-actin is the major actin expressed in fibroblasts and exists as monomers or microfilaments capable of force generation.  In the present study we ask if total ß-actin protein concentration changes in fibroblasts during contraction of unloaded and loaded fibroblast populated collagen gels (FPCGs).

   Fibroblasts were isolated from adult rat tail tendon and maintained in cell culture.  FPCGs were formed in wells of 48-well culture plates and maintained in 0.5 mls of culture medium.  Two glass beads weighing 19 mg were placed on top of each loaded FPCG.  FPCGs were harvested and subject to SDS-PAGE and quantitative ß-actin Western blotting.  A DNA assay was used to determine the cell number in each FPCG.  FPCGs were photographed to track gel contraction.      

    FPCGs contracted 80% in the first 24 hours.  Loaded FPCGs contracted faster than unloaded FPCGs.   Cell number in the FPCGs stayed at 3.7 X 10⁵ for 48 hours and gradually fell to 3 X 10⁵ cells at 96 hours.  The ß-actin protein content of the fibroblasts in FPCGs rose and fell twice during the 96 hour culture period.  The first elevation of ß-actin concentration occurred during the first 24 hours of contraction where it went from 100 million molecules/cell to 240 million molecules/cell.  ß-actin protein levels rose and peaked 6 hours earlier in loaded than in unloaded FPCGs.  From 24 to 58 hours ß-actin levels fell back down to 100 million molecules per cell.  This was followed by a second rise in ß-actin protein levels to 300 million molecules/cell at 74 hours and fall to 125 million molecules/cell by 96 hours.                 

   In conclusion, we have shown that ß-actin protein content of fibroblasts in contracting FPCGs rises and falls twice during a 96 hour culture period spanning a range of 100 million to 300 million ß-actin molecules/cell.  The first rise in ß-actin concentration coincides with early reduction of FPCG area and is accelerated in loaded FPCGs.  Supported by NIU and the NIU Foundation.