Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

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Abstract
The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the [rat adrenal] PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 days of exposure to NGF, these cells acquire many properties of normal sympathetic neurons. By phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells within 1 min, become prominent by 3 min and almost disappear by 7 min. Microvilli disappear as the dorsal ruffles become prominent. Ruffles are seen at the periphery of the cell at 3 min, are prominent on most cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli appear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with .alpha.-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earliest NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor''s action.