Results Hypertonic Media Inhibit Receptor-mediated Endocytosis of LDL and Bulk Uptake of HRP

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 potassium depletion appears to inhibit the assembly of coated pits, causing the cell to have a plasma membrane virtually devoid of endocytic sites (24, 26, 34), acidification appears not to affect assembly but to arrest the pinching off of coated pits to form coated vesicles (11, 41). Whether or not hypertonic treatment exerts its effects by interfering with one or another of these aspects of coated pit function has not been determined. Daukas and Zigmond (10) first showed that exposure of polymorphonuclear leukocytes to media containing 0.43 M sucrose caused the rapid and reversible inhibition of receptor-mediated internalization of chemotactic peptides, suggesting that coated pit function was inhibited by hypertonicity. We further analyze the effects of hypertonic media on coated pit function and architecture in cultured fibroblasts, taking advantage of the opportunity to obtain distinct en face images of coated pits via the quick-freeze/deep-etch method of preparing cells for electron microscopy (15, 17). This shows that hypertonic treatment rapidly and reversibly inhibits the internalization of low density lipoprotein (LDL) t and causes the receptors for this ligand to become randomly 1. Abbreviations used in this paper: HRP, horseradish peroxidase; LDL, low density lipoprotein. © The Rockefeller University Press, 0021-9525/89/02/389/12 $2.00 The Journal of Cell Biology, Volume 108, February 1989 389-400 389 on July 8, 2017 jcb.rress. org D ow nladed fom dispersed on the plasma membrane. Coordinately, coated pits become reduced in size and number, and many small "microcages" of clathrin appear on the inner membrane surface. These unusual polymers, sharply curved and devoid of membrane, become so abundant that they apparently deplete the cytoplasm of clathrin needed for normal coated pit assembly. In this sense, hypertonicity and potassium depletion appear to inhibit endocytosis in the same way. It remains to be seen, however, whether both treatments stimulate clathrin microcage formation by the same mechanism. Materials and Methods