Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

Abstract

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.