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Journal of Neuroscience, Vol 3, 631-643, Copyright © 1983 by Society for Neuroscience

ARTICLE

Electron microscopic radioautographic localization of iodinated nerve growth factor bound to and internalized by PC12 cells

P Bernd and LA Greene

Nerve growth factor (NGF) has many effects on sympathetic and sensory neurons, but the mechanisms by which NGF exerts its actions are unknown. We have determined the localization of bound and internalized 125I-NGF by light and electron microscopic radioautography on a cell line derived from a rat pheochromocytoma (PC12). In response to NGF, PC12 cells cease mitosis and develop neuron-like processes. We have localized 125I-NGF (5 ng/ml) in cells without previous exposure to NGF (naive) after a continuous incubation with 125I-NGF for 2 min, 1 hr, 6 hr, 1 day and 1 week, as well as in cells that were grown with NGF (50 ng/ml) for 1 week (primed) and then exposed to 125I-NGF for 15 min, 1 hr, 6 hr, and 1 day. Examination of whole mount radioautographs revealed that all cells were labeled and that the distribution of grains was homogeneous. Primed cells were labeled on neurites and growth cones, as well as on cell bodies, and also had a greater density of labeling than naive cells. These patterns were identical for all time points studied, with the exception that "hot spots" of radioactivity appeared along neurites following a long incubation with 126I-NGF (longer than 6 hr). Electron microscopic radioautography revealed that these "hot spots" correspond to accumulation of grains in aggregates of lysosomes. The binding of 125I-NGF was specific, since control cultures (excess nonradioactive NGF) exhibited only background levels of grains. Quantification of electron microscopic radioautographs revealed that 70 to 95% of the grains were internalized after 15 min. The only cellular components labeled above random distribution levels were the plasma membrane, lysosomes, and the nuclear membrane. Each of these structures exhibited "down-regulation" of specific binding in naive cells after 6 hr of incubation. The cytosol, polyribosomes, dense cored granules, heterochromatin, and euchromatin showed labeling at or below the levels expected from random distribution of grains within the cell. The distribution of grains about the nuclear membrane also suggested an association with 125I-NGF. These data indicate that a large proportion of NGF "bound" to target cells is in fact internalized, and are consistent with some, but not other models for NGF's mechanism of action.

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