A quantitative description of the extension and retraction of surface protrusions in spreading 3T3 mouse fibroblasts.

Abstract

A method of quantitating the motile actions of surface protrusions in spreading animal cells in culture is suggested. Its basis is the determination of the percentage of freshly plated cells which produce particle-free areas around them on a gold particle-coated glass cover slip within 50 min. The presence of Na+, K+, Cl- and Mg2+ or Ca2+ in a neutral or slightly alkaline phosphate or bicarbonate buffered solution is sufficient to support the optimal particle removal by the 3T3 cells [neoplastic] for at least 50 min. Two metabolic inhibitors, 2,4-dinitrophenol and Na-azide, inhibit the particle removal. If D-glucose is added along with the inhibitors, particle removal can be restored, whereas the addition of 3 glucose analoges which are generally believed to be nonmetabolizable cannot restore the activity. Serum is not required for the mechanism(s) of the motile actions of surface protrusions in spreading 3T3 cells. However, it contains components which can neutralize the inhibitory actions of bovine serum albumin and several amino acids, particularly L-cystine or L-cystein and L-methionine. Serum codetermines which of the major surface extensions, filopodia, lamellipodia or lobopodia, is predominantly active. There are 3 distinct classes of extracellular conditions under which the active surface projections are predominantly either lamellipodia, (sheetlike projections), lobopodia (blebs), or filopodia (microspikes). The quantitated dependencies on temperature, pH and the inhibition by cytochalasin B of the particle removal are very similar in all 3 cases. Preventing the cells from anchoring themselves for 15-20 min before plating in serum-free medium seems to stimulate particle removal 3-fold.