Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP, and dynamin but not clathrin.

Abstract

Rapid endocytosis (RE) occurs immediately after an exocytotic burst in adrenal chromaffin cells. Capacitance measurements of endocytosis reveal that recovery of membrane is a biphasic process that is complete within 20 sec. The ultimate extent of membrane retrieval is precisely controlled and capacitance invariably returns to its prestimulation value. The mechanism of RE specifically requires intracellular The physiological and molecular basis of secretion has been studied in some detail (1), but the nature of the endocytotic processes that are coupled to exocytosis is poorly understood. Endocytosis serves the dual function of maintaining cell surface area constant and retrieving vesicular components for recycling. In neurons, synaptic vesicle recycling is rapid and it is thought that endocytosed vesicles are recovered virtually intact and quickly returned to the releasable pool (2). This is a critical feature of neuronal communication as, without recycling, neural firing would rapidly deplete the pool of transmitter-containing vesicles ready for release. Whether a similar phenomenon occurs in other types of secretory cell is unknown, but as these cells may also need to secrete repetitively under various conditions, it seems likely that rapid recycling must be present (3).Recently, studies on several preparations have suggested that rapid endocytotic mechanisms can be assessed by measurement of cell capacitance. By this criterion, adrenal chromaffin cells (4), pituitary melanotrophs (5), and nerve terminals (6) rapidly recapture membrane after secretion. Little is known about the kinetics and regulation of this type of endocytosis, termed rapid endocytosis (RE) to distinguish it from other better-studied phenomena such as receptormediated endocytosis (mediated by clathrin-coated vesicles) or various kinds of pinocytosis (7). We have conducted a detailed analysis of RE in bovine adrenal chromaffin cells subjected to physiological stimulation. We show that it is a kinetically complex and highly regulated process that reproducibly follows each round of secretion. The final amount of membrane recovered seems to be precisely controlled, as most cells return to their prestimulation capacitance value. RE requires intracellular Ca2+ and GTP hydrolysis and we provide direct evidence that the guanine nucleotide binding protein (G protein) dynamin is intimately involved in the process. In contrast, our results suggest that RE does not involve a clathrin-coated vesicle-based mechanism. MATERIALS AND METHODSPreparation of Cells and Patch-Clamp. Chromaffin cells were isolated from calf adrenal medullae, cultured, and patchclamped as described (8). Capacitance was measured by a computer program using a phase-tracking technique (9). Secretion was elicited by a train of 10 depolarizations, from a holding potential of -90 mV to + 10 mV, each lasting 50 msec; 500 msec separated each depolarization. Each depolarization was preceded by a 50-msec prepulse to +120 mV to recruit facilitation Ca2+ channels (8). All experiments...

Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells

“…Alternatively, the resealing process may require the recruitment of a dedicated fission machinery either before or after exocytosis. The endocytic protein dynamin has been proposed to serve this function (10,14,39). Two-color evanescent-field microscopy seems well suited to test this idea and to test for the transient recruitment of other proteins to spatially defined sites in the plasma membrane (22).…”

Properties of fast endocytosis at hippocampal synapses

“…20^30 s (Ryan et al 1996;Wu & Betz 1996). This is at least half of the total recycling timeöfar slower than endocytosis in other secretory systems (Thomas et al 1994;Artalejo et al 1995;Smith & Neher 1997;Engisch & Nowycky 1998). It seems paradoxical that the neurosecretory terminals that might bene¢t the most from rapid endocytosis do not appear to take advantage of such a mechanism.…”

The SH3 Domain of Amphiphysin Binds the Proline-rich Domain of Dynamin at a Single Site That Defines a New SH3 Binding Consensus Sequence

“…Ultrastructural studies have shown that dynamin I forms rings at the neck of clathrin-coated pits, and it has been hypothesized that a conformational change of the ring that correlates with GTP hydrolysis represents a key step leading to vesicle fission from the plasmalemma (6,7). In addition, dynamin I has also been implicated in rapid endocytosis, a form of Ca 2ϩ -triggered endocytosis detectable by capacitance measurement in neuroendocrine cells (8). Dynamin isoforms (dynamin II and dynamin III) are expressed in non-neuronal cells (9 -11) where they are thought to play a general role in clathrin-mediated endocytosis (12,13).…”