Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

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Abstract
Bovine adrenal chromaffin cells were maintained in culture in Dulbecco''s modified Eagle''s medium containing 20% fetal calf serum and 10 U/ml of nerve growth factor. Under these conditions, chromaffin cells developed up to 5 neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules migrated from the cell body to reach neurite endings where they were densely packed. Intense staining was observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. They showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.