The family of transmembrane integrin heterodimer receptors has been shown to transduce force from the extracellular matrix through the cell membrane to the cytoskeleton and nucleus. The specific role, however, of individual integrin subunits is not completely understood. To determine the role of the 1 integrin, chicken 1 integrin and ,B1 cytoplasmic deletion mutants were transfected into Swiss mouse embryo (3T3) fibroblasts. Those transfectants with a high level of 1 or mutant 1 integrin expressed on the cell surface were selected by fluorescent activated cell sorting (FACS) flow cytometry. A monoclonal antibody directed against the chicken 1 integrin extracellular domain (W 1 B 10) was grown in ascites from Balb/c mice and affinity purified using protein G. 20,000 cells were plated on 96-well tissue culture plates coated overnight with 5 mg/ml fibronectin, and were allowed to spread for a minimum of 2 hours. 4.5 mm ferromagnetic microbeads coated with WlB10 were allowed to bind to either transfected or untransfected cells in 1% BSA/DMEM for 30 min, and the unbound beads were washed away using 1% BSA/DMEM. Using magnetic twisting cytometry, the stress and strain applied by twisting the bead bound to the 1 integrin can be calculated, and the cytoskeletal parameters of deformation, recoil and stiffness can be determined. Magnetic twisting cytometry involves the application of a brief (lOmsec) but strong (1000 gauss) magnetic field that magnetizes the beads in the horizontal direction, and then applying a longer ( 1 min) and weaker (30 gauss) vertical magnetic field 90 degrees to the original orientation: the result is that the bead twists in place. With removal of the vertical magnetic field, the bead """"""""untwists"""""""" toward its original direction. The results are as follows: Cells transfected with 1 integrin exhibited slightly less deformation (p< 0.02), slightly more recoil (p< 0.02), but no difference in stiffness compared to cells transfected with the 1 cytoplasmic deletion mutants, or untransfected 3T3 cells. Beads coated with RGD peptide sequence ( ligand for integrin receptor) were bound to all cell types and twisted. All cell types exhibited approximately 4 times the stiffness when compared to WlB 10. These results indicate that the 1 integrin may only play a role in cytoskeletal rearrangement when its active site is bound. Antibody binding to the non active site may not be sufficient to cause changes in the cytoskeleton.
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