Observation of cell shortening and dynamic changes of actin filaments during cell detachment from thermoresponsive-gelatin-coated substrate

We observed cell shortening and dynamic changes of actin filaments during detachment from the substrate by using a thermoresponsive-gelatin. The thermoresponsive-gelatin, a mixture of the poly(N-isopropylacrylamide) grafted gelatin (PNIPAAm-grafted-gelatin) and PNIPAAm, was coated on the glass bottom culture dishes. Rat aortic smooth muscle cells (SMC) expressed GFP-actin were cultured on the thermoresponsive-gelatin-coated dishes filled with serum free Dulbecco's Modified Eagle's medium (DMEM) or Ca2+-Mg2+-free Hank's balanced salt solution (HBSS(-)). They adhered normally at 37 degrees C, and became shortened and detached from the dishes when the ambient temperature was dropped below 34 degrees C due to melting of the gelatin substrate. The shortening of SMCs was larger in DMEM (51.0 +/- 3.0%, mean +/- SEM, n = 14) than in HBSS(-) (35.8 +/- 3.6%, n = 14). Actin filaments remained straight during detachment in HBSS(-), while in DMEM, they locally concentrated and disappeared at cell periphery. The shortening of SMCs upon detachment from ordinary plastic culture dishes by trypsinization was more than for both media. No significant difference was observed between the two, indicating detrimental effects of the trypsin. The present method was useful to study the cell contraction and the actin filament behavior during cell detachment for this causes minimal damage to cells.