Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm

Abstract

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7-10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(-5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(-8) M) or relaxing (free Ca2+ less than 10(-8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12-18% shortenings and were complete in 1-3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(-8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(-8) M and 5 X 10(-7) M.