Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.

Abstract

Abstract. Two seemingly unrelated experimental treatments inhibit receptor mediated endocytosis: (a) depletion of intracellular K ÷ (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson. 1983. Cell. 33:273-285); and (b) treatment with hypertonic media (Daukas, G., and S. H. Zigmond. 1985. J. Cell Biol. 101:1673-1679. Since the former inhibits the formation of clathrin-coated pits (Larkin, J. M., W. D. Donzell, and R. G. W. Anderson. 1986. J. Cell Biol. 103:2619-2627, we were interested in determining whether hypertonic treatment has the same effect, and if so, why. Fibroblasts (human or chicken) were incubated in normal saline made hypertonic with 0.45 M sucrose, then broken open by sonication and freezeetched to generate replicas of their inner membrane surfaces. Whereas untreated cells display typical geodesic lattices of clathrin under each coated pit, hypertonic cells display in addition a number of empty clathrin "microcages'. At first, these appear around the edges of normal coated pit lattices. With further time in hypertonic medium, however, normal lattices largely disappear and are replaced by accumulations of microcages. Concomitantly, low density lipoprotein (LDL) receptors lose their normal clustered distribution and become dispersed all over the cell surface, as seen by fluorescence microscopy and freeze-etch electron microscopy of LDL attached to the cell surface. Upon return to normal medium at 37°C, these changes promptly reverse. Within 2 min, small clusters of LDL reappear on the surfaces of cells and normal clathrin lattices begin to reappear inside; the size and number of these receptor/clathrin complexes returns to normal over the next 10 min. Thus, in spite of their seeming unrelatedness, both K ÷ depletion and hypertonic treatment cause coated pits to disappear, and both induce abnormal clathrin polymerization into empty microcages. This suggests that in both cases, an abnormal formation of microcages inhibits endocytosis by rendering clathrin unavailable for assembly into normal coated pits. C LATHRIN coated pits are the primary plasma membrane specialization involved in the uptake of a wide variety of molecules by receptor-mediated endocytosis (4, 38). Two broad functions have been attributed to these regions of membrane: (a) molecular determinants associated with the clathrin lattice may cause receptors to become clustered; and (b) the clathrin lattice may in some way control the invagination of the membrane to form endocytic vesicles. To understand the molecular mechanisms underlying these two aspects of coated pit function, one approach is to search for treatments that inhibit endocytosis and then to characterize the effects of these treatments on coated pit structure and receptor clustering.Currently, three experimental treatments are known to inhibit receptor-mediated endocytosis: (a) depletion of intracellular potassium (22, 24-26, 29, 30, 34, 40), (b) exposure of cells to hypertonic media (10), and (c) acidification of the cytoplasm (11,16, 21,41). Whereas potassi...

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“…The relevant levels of cADPR were 3.13_+0.45 and 1.63_+0.24 pmol/mg protein in the perchloric acid extracts from NAD+-treated cells (2 h) and control cells, respectively, following extensive washing of the relevant cell populations. The presence of cycloheximide (100 ~tM) or hyperosmolar sucrose (0.45 M), an inhibitor of clathrin-dependent endocytosis [29], during incubation of the cells with NAD + did not exert any significant effect on the changes in the ratio of total to ectocellular cyclase activity (Table 1).…”

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“…9 We first verified that incubation of ΣCFTE29oϪ cells in hypertonic medium indeed modifies receptor-mediated (clathrin-dependent) endocytosis and we used fluorescein-coupled transferrin (Molecular Probes, Eugene, OR, USA) as a marker of receptor-mediated endocytosis. Incubation of cells in hypertonic medium containing 0.45 M sucrose resulted in a 90% decrease in fluoresceinated transferrin uptake, as compared with the uptake observed in control medium (P Ͻ 0.01) (Figure 2a).…”