Characterization of anti-actin antibodies and their use in immunocytochemical studies on the localization of actin in adrenal chromaffin cells in culture

Antibodies were produced in rabbits against purified chicken gizzard actin and were characterized. The anti-actin antibody and gizzard actin formed a single precipitin line in Ouchterlony double immunodiffusion tests but actin purified from other sources did not form precipitating complexes. To better characterize the antibody, competitive assays were used. Actins purified from various sources were used to compete with [¹²⁵I]-labelled gizzard actin for antibody binding sites. The inhibition curves produced indicated that gizzard actin had the highest affinity for the antibody while rabbit skeletal muscle actin and chromaffin cell actin bound poorly to the antibodies. No difference in binding was detected between the latter two actins. This showed that the antibodies had different immunological preferences for the various actins in spite of the actins' highly conserved structure. When the antibody was used to investigate the localization of actin in cultured bovine adrenal chromaffin cells by indirect immunofluorescence, it was found that actin is widely distributed in the cells, being associated with many cellular structures. The antibodies against actin produced a strong membrane fluorescence and a weak cytosol fluorescence in one-day-old cells in culture. Membrane patching and capping patterns were also seen. By day 7 the cultured cells exhibited a much weaker membrane fluorescence with filament and fine granular fluorescence in the cytosol of the cell body, neurites and terminal cones. Smooth muscle antibodies obtained from patients suffering from chronic active hepatitis were also used to stain the cultured cells and these antibodies produced a different fluorescence pattern which consisted of an intense and dense granular fluorescence. This speckled fluorescence did not arise from filopodia present on the cell surface because scanning electron microscopy revealed that the chromaffin cell-surface was relatively smooth and exhibited few filopodia. Most of these structures were found at the edges of the neurite terminal cones that made contact with other cells. Also seen were pits and many spherical bodies of about 200 nm in diameter that appeared to be pressing against the cytoplasmic side of the plasma membrane. These bodies may be indicative of the presence of chromaffin granules underneath the membrane.The present results, showing different immunofluorescence patterns of actin distribution in chromaffin cells, suggest that actin might be involved in several cellular functions. An important function of the chromaffin cells is secretion, a process which has many features in common with the process of muscle contraction. These similarities would suggest that contractile proteins might be involved in the secretory process. Therefore, in view of the present results, the possible functions of actin in chromaffin cells are discussed.