Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Feb 1;108(2):401–411. doi: 10.1083/jcb.108.2.401

Effects of cytoplasmic acidification on clathrin lattice morphology

PMCID: PMC2115436  PMID: 2563729

Abstract

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

Full Text

The Full Text of this article is available as a PDF (5.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aggeler J., Werb Z. Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin. J Cell Biol. 1982 Sep;94(3):613–623. doi: 10.1083/jcb.94.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. G., Goldstein J. L., Brown M. S. Fluorescence visualization of receptor-bound low density lipoprotein in human fibroblasts. J Recept Res. 1980;1(1):17–39. doi: 10.3109/10799898009039253. [DOI] [PubMed] [Google Scholar]
  3. Anderson R. G., Vasile E., Mello R. J., Brown M. S., Goldstein J. L. Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution. Cell. 1978 Nov;15(3):919–933. doi: 10.1016/0092-8674(78)90276-3. [DOI] [PubMed] [Google Scholar]
  4. Aubert L., Motais R. Molecular features of organic anion permeablity in ox red blood cell. J Physiol. 1975 Mar;246(1):159–179. doi: 10.1113/jphysiol.1975.sp010884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boron W. F., De Weer P. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol. 1976 Jan;67(1):91–112. doi: 10.1085/jgp.67.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boron W. F. Intracellular pH transients in giant barnacle muscle fibers. Am J Physiol. 1977 Sep;233(3):C61–C73. doi: 10.1152/ajpcell.1977.233.3.C61. [DOI] [PubMed] [Google Scholar]
  7. Brodsky F. M. Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin. J Cell Biol. 1985 Dec;101(6):2055–2062. doi: 10.1083/jcb.101.6.2055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke B. L., Weigel P. H. Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis. J Biol Chem. 1985 Jan 10;260(1):128–133. [PubMed] [Google Scholar]
  9. Crowther R. A., Finch J. T., Pearse B. M. On the structure of coated vesicles. J Mol Biol. 1976 Jun 5;103(4):785–798. doi: 10.1016/0022-2836(76)90209-6. [DOI] [PubMed] [Google Scholar]
  10. Daiss J. L., Roth T. F. Isolation of coated vesicles: comparative studies. Methods Enzymol. 1983;98:337–349. doi: 10.1016/0076-6879(83)98162-4. [DOI] [PubMed] [Google Scholar]
  11. Daukas G., Zigmond S. H. Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes. J Cell Biol. 1985 Nov;101(5 Pt 1):1673–1679. doi: 10.1083/jcb.101.5.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davoust J., Gruenberg J., Howell K. E. Two threshold values of low pH block endocytosis at different stages. EMBO J. 1987 Dec 1;6(12):3601–3609. doi: 10.1002/j.1460-2075.1987.tb02691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foreman J. C., Mongar J. L., Gomperts B. D. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature. 1973 Oct 5;245(5423):249–251. doi: 10.1038/245249a0. [DOI] [PubMed] [Google Scholar]
  14. Goldstein J. L., Basu S. K., Brown M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. doi: 10.1016/0076-6879(83)98152-1. [DOI] [PubMed] [Google Scholar]
  15. Goodenough U., Heuser J. Structural comparison of purified dynein proteins with in situ dynein arms. J Mol Biol. 1984 Dec 25;180(4):1083–1118. doi: 10.1016/0022-2836(84)90272-9. [DOI] [PubMed] [Google Scholar]
  16. Goud B., Huet C., Louvard D. Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay. J Cell Biol. 1985 Feb;100(2):521–527. doi: 10.1083/jcb.100.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harrison S. C., Kirchhausen T. Clathrin, cages, and coated vesicles. Cell. 1983 Jul;33(3):650–652. doi: 10.1016/0092-8674(83)90007-7. [DOI] [PubMed] [Google Scholar]
  18. Heuser J. E., Anderson R. G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989 Feb;108(2):389–400. doi: 10.1083/jcb.108.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heuser J. E., Keen J. H., Amende L. M., Lippoldt R. E., Prasad K. Deep-etch visualization of 27S clathrin: a tetrahedral tetramer. J Cell Biol. 1987 Nov;105(5):1999–2009. doi: 10.1083/jcb.105.5.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heuser J. E., Kirschner M. W. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J Cell Biol. 1980 Jul;86(1):212–234. doi: 10.1083/jcb.86.1.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heuser J. E., Reese T. S., Dennis M. J., Jan Y., Jan L., Evans L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 1979 May;81(2):275–300. doi: 10.1083/jcb.81.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Heuser J. E., Reese T. S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J Cell Biol. 1973 May;57(2):315–344. doi: 10.1083/jcb.57.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Heuser J., Kirchhausen T. Deep-etch views of clathrin assemblies. J Ultrastruct Res. 1985 Jul-Aug;92(1-2):1–27. doi: 10.1016/0889-1605(85)90123-5. [DOI] [PubMed] [Google Scholar]
  24. Heuser J. Three-dimensional visualization of coated vesicle formation in fibroblasts. J Cell Biol. 1980 Mar;84(3):560–583. doi: 10.1083/jcb.84.3.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ilondo M. M., Courtoy P. J., Geiger D., Carpentier J. L., Rousseau G. G., De Meyts P. Intracellular potassium depletion in IM-9 lymphocytes suppresses the slowly dissociating component of human growth hormone binding and the down-regulation of its receptors but does not affect insulin receptors. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6460–6464. doi: 10.1073/pnas.83.17.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ishizaka T., Hirata F., Ishizaka K., Axelrod J. Stimulation of phospholipid methylation, Ca2+ influx, and histamine release by bridging of IgE receptors on rat mast cells. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1903–1906. doi: 10.1073/pnas.77.4.1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kanaseki T., Kadota K. The "vesicle in a basket". A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements. J Cell Biol. 1969 Jul;42(1):202–220. doi: 10.1083/jcb.42.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Keen J. H., Willingham M. C., Pastan I. H. Clathrin-coated vesicles: isolation, dissociation and factor-dependent reassociation of clathrin baskets. Cell. 1979 Feb;16(2):303–312. doi: 10.1016/0092-8674(79)90007-2. [DOI] [PubMed] [Google Scholar]
  29. Kirchhausen T., Harrison S. C., Heuser J. Configuration of clathrin trimers: evidence from electron microscopy. J Ultrastruct Mol Struct Res. 1986 Mar;94(3):199–208. doi: 10.1016/0889-1605(86)90067-4. [DOI] [PubMed] [Google Scholar]
  30. Kirchhausen T., Harrison S. C. Protein organization in clathrin trimers. Cell. 1981 Mar;23(3):755–761. doi: 10.1016/0092-8674(81)90439-6. [DOI] [PubMed] [Google Scholar]
  31. L'Allemain G., Paris S., Pouysségur J. Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport. J Biol Chem. 1984 May 10;259(9):5809–5815. [PubMed] [Google Scholar]
  32. Larkin J. M., Brown M. S., Goldstein J. L., Anderson R. G. Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell. 1983 May;33(1):273–285. doi: 10.1016/0092-8674(83)90356-2. [DOI] [PubMed] [Google Scholar]
  33. Larkin J. M., Donzell W. C., Anderson R. G. Modulation of intracellular potassium and ATP: effects on coated pit function in fibroblasts and hepatocytes. J Cell Physiol. 1985 Sep;124(3):372–378. doi: 10.1002/jcp.1041240303. [DOI] [PubMed] [Google Scholar]
  34. Larkin J. M., Donzell W. C., Anderson R. G. Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices. J Cell Biol. 1986 Dec;103(6 Pt 2):2619–2627. doi: 10.1083/jcb.103.6.2619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Louvard D., Morris C., Warren G., Stanley K., Winkler F., Reggio H. A monoclonal antibody to the heavy chain of clathrin. EMBO J. 1983;2(10):1655–1664. doi: 10.1002/j.1460-2075.1983.tb01640.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Madshus I. H., Sandvig K., Olsnes S., van Deurs B. Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses. J Cell Physiol. 1987 Apr;131(1):14–22. doi: 10.1002/jcp.1041310104. [DOI] [PubMed] [Google Scholar]
  37. Madshus I. H., Tønnessen T. I., Olsnes S., Sandvig K. Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport. J Cell Physiol. 1987 Apr;131(1):6–13. doi: 10.1002/jcp.1041310103. [DOI] [PubMed] [Google Scholar]
  38. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  39. Maupin P., Pollard T. D. Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J Cell Biol. 1983 Jan;96(1):51–62. doi: 10.1083/jcb.96.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Merisko E. M., Fletcher M., Palade G. E. The reorganization of the Golgi complex in anoxic pancreatic acinar cells. Pancreas. 1986;1(2):95–109. doi: 10.1097/00006676-198603000-00001. [DOI] [PubMed] [Google Scholar]
  41. Moore M. S., Mahaffey D. T., Brodsky F. M., Anderson R. G. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. doi: 10.1126/science.2883727. [DOI] [PubMed] [Google Scholar]
  42. Moya M., Dautry-Varsat A., Goud B., Louvard D., Boquet P. Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin. J Cell Biol. 1985 Aug;101(2):548–559. doi: 10.1083/jcb.101.2.548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nicol A., Nermut M. V. A new type of substratum adhesion structure in NRK cells revealed by correlated interference reflection and electron microscopy. Eur J Cell Biol. 1987 Jun;43(3):348–357. [PubMed] [Google Scholar]
  44. Oliver J. M., Seagrave J. C., Pfeiffer J. R., Feibig M. L., Deanin G. G. Surface functions during mitosis in rat basophilic leukemia cells. J Cell Biol. 1985 Dec;101(6):2156–2166. doi: 10.1083/jcb.101.6.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Patzer E. J., Schlossman D. M., Rothman J. E. Release of clathrin from coated vesicles dependent upon a nucleoside triphosphate and a cytosol fraction. J Cell Biol. 1982 Apr;93(1):230–236. doi: 10.1083/jcb.93.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pearse B. M. Clathrin and coated vesicles. EMBO J. 1987 Sep;6(9):2507–2512. doi: 10.1002/j.1460-2075.1987.tb02536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pearse B. M. Coated vesicles from pig brain: purification and biochemical characterization. J Mol Biol. 1975 Sep 5;97(1):93–98. doi: 10.1016/s0022-2836(75)80024-6. [DOI] [PubMed] [Google Scholar]
  48. Pearse B. M., Crowther R. A. Structure and assembly of coated vesicles. Annu Rev Biophys Biophys Chem. 1987;16:49–68. doi: 10.1146/annurev.bb.16.060187.000405. [DOI] [PubMed] [Google Scholar]
  49. Pressman B. C., Harris E. J., Jagger W. S., Johnson J. H. Antibiotic-mediated transport of alkali ions across lipid barriers. Proc Natl Acad Sci U S A. 1967 Nov;58(5):1949–1956. doi: 10.1073/pnas.58.5.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Pypaert M., Lucocq J. M., Warren G. Coated pits in interphase and mitotic A431 cells. Eur J Cell Biol. 1987 Dec;45(1):23–29. [PubMed] [Google Scholar]
  51. Rogers J., Hesketh T. R., Smith G. A., Beaven M. A., Metcalfe J. C., Johnson P., Garland P. B. Intracellular pH and free calcium changes in single cells using quene 1 and quin 2 probes and fluorescence microscopy. FEBS Lett. 1983 Sep 5;161(1):21–27. doi: 10.1016/0014-5793(83)80722-4. [DOI] [PubMed] [Google Scholar]
  52. Rogers J., Hesketh T. R., Smith G. A., Metcalfe J. C. Intracellular pH of stimulated thymocytes measured with a new fluorescent indicator. J Biol Chem. 1983 May 25;258(10):5994–5997. [PubMed] [Google Scholar]
  53. Roos A., Boron W. F. Intracellular pH. Physiol Rev. 1981 Apr;61(2):296–434. doi: 10.1152/physrev.1981.61.2.296. [DOI] [PubMed] [Google Scholar]
  54. Sager P. R., Brown P. A., Berlin R. D. Analysis of transferrin recycling in mitotic and interphase HeLa cells by quantitative fluorescence microscopy. Cell. 1984 Dec;39(2 Pt 1):275–282. doi: 10.1016/0092-8674(84)90005-9. [DOI] [PubMed] [Google Scholar]
  55. Sandvig K., Olsnes S., Petersen O. W., van Deurs B. Acidification of the cytosol inhibits endocytosis from coated pits. J Cell Biol. 1987 Aug;105(2):679–689. doi: 10.1083/jcb.105.2.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sandvig K., Sundan A., Olsnes S. Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin. J Cell Physiol. 1985 Jul;124(1):54–60. doi: 10.1002/jcp.1041240110. [DOI] [PubMed] [Google Scholar]
  57. Schlossman D. M., Schmid S. L., Braell W. A., Rothman J. E. An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol. 1984 Aug;99(2):723–733. doi: 10.1083/jcb.99.2.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Schook W., Puszkin S., Bloom W., Ores C., Kochwa S. Mechanochemical properties of brain clathrin: interactions with actin and alpha-actinin and polymerization into basketlike structures or filaments. Proc Natl Acad Sci U S A. 1979 Jan;76(1):116–120. doi: 10.1073/pnas.76.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sullivan P. C., Ferris A. L., Storrie B. Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles. J Cell Physiol. 1987 Apr;131(1):58–63. doi: 10.1002/jcp.1041310110. [DOI] [PubMed] [Google Scholar]
  60. Thomas J. A., Buchsbaum R. N., Zimniak A., Racker E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 1979 May 29;18(11):2210–2218. doi: 10.1021/bi00578a012. [DOI] [PubMed] [Google Scholar]
  61. Van Jaarsveld P. P., Nandi P. K., Lippoldt R. E., Saroff H., Edelhoch H. Polymerization of clathrin protomers into basket structures. Biochemistry. 1981 Jul 7;20(14):4129–4135. doi: 10.1021/bi00517a028. [DOI] [PubMed] [Google Scholar]
  62. Vigers G. P., Crowther R. A., Pearse B. M. Location of the 100 kd-50 kd accessory proteins in clathrin coats. EMBO J. 1986 Sep;5(9):2079–2085. doi: 10.1002/j.1460-2075.1986.tb04469.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vigers G. P., Crowther R. A., Pearse B. M. Three-dimensional structure of clathrin cages in ice. EMBO J. 1986 Mar;5(3):529–534. doi: 10.1002/j.1460-2075.1986.tb04242.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Warren G., Davoust J., Cockcroft A. Recycling of transferrin receptors in A431 cells is inhibited during mitosis. EMBO J. 1984 Oct;3(10):2217–2225. doi: 10.1002/j.1460-2075.1984.tb02119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Woodward M. P., Roth T. F. Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly. J Supramol Struct. 1979;11(2):237–250. doi: 10.1002/jss.400110213. [DOI] [PubMed] [Google Scholar]
  66. Young J. D., Unkeless J. C., Kaback H. R., Cohn Z. A. Macrophage membrane potential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1357–1361. doi: 10.1073/pnas.80.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Young J. D., Unkeless J. C., Kaback H. R., Cohn Z. A. Mouse macrophage Fc receptor for IgG gamma 2b/gamma 1 in artificial and plasma membrane vesicles functions as a ligand-dependent ionophore. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1636–1640. doi: 10.1073/pnas.80.6.1636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zaremba S., Keen J. H. Assembly polypeptides from coated vesicles mediate reassembly of unique clathrin coats. J Cell Biol. 1983 Nov;97(5 Pt 1):1339–1347. doi: 10.1083/jcb.97.5.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES