Abstract
Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42- kdalton component that comigrates with rabbit muscle actin and a 18.5- kdalton minor component that comigrates with calmodulin as well as 110- , 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.
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- Ainsworth S. K., Karnovsky M. J. An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections. J Histochem Cytochem. 1972 Mar;20(3):225–229. doi: 10.1177/20.3.225. [DOI] [PubMed] [Google Scholar]
- Albertini D. F., Berlin R. D., Oliver J. M. The mechanism of concanavalin A cap formation in leukocytes. J Cell Sci. 1977 Aug;26:57–75. doi: 10.1242/jcs.26.1.57. [DOI] [PubMed] [Google Scholar]
- Albertini D. F., Clark J. I. Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4976–4980. doi: 10.1073/pnas.72.12.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Allison A. C., Davies P., De Petris S. Role of contractile microfilaments in macrophage movement and endocytosis. Nat New Biol. 1971 Aug 4;232(31):153–155. doi: 10.1038/newbio232153a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bourguignon L. Y., Tokuyasu K. T., Singer S. J. The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections. J Cell Physiol. 1978 Jun;95(3):239–257. doi: 10.1002/jcp.1040950302. [DOI] [PubMed] [Google Scholar]
- Brenner S. L., Korn E. D. Substoichiometric concentrations of cytochalasin D inhibit actin polymerization. Additional evidence for an F-actin treadmill. J Biol Chem. 1979 Oct 25;254(20):9982–9985. [PubMed] [Google Scholar]
- Brown S. S., Spudich J. A. Cytochalasin inhibits the rate of elongation of actin filament fragments. J Cell Biol. 1979 Dec;83(3):657–662. doi: 10.1083/jcb.83.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
- Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flanagan J., Koch G. L. Cross-linked surface Ig attaches to actin. Nature. 1978 May 25;273(5660):278–281. doi: 10.1038/273278a0. [DOI] [PubMed] [Google Scholar]
- Fox C. F., Das M. Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis. J Supramol Struct. 1979;10(2):199–214. doi: 10.1002/jss.400100210. [DOI] [PubMed] [Google Scholar]
- Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
- Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5025–5029. doi: 10.1073/pnas.75.10.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Helenius A., Kartenbeck J., Simons K., Fries E. On the entry of Semliki forest virus into BHK-21 cells. J Cell Biol. 1980 Feb;84(2):404–420. doi: 10.1083/jcb.84.2.404. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hidaka H., Yamaki T., Totsuka T., Asano M. Selective inhibitors of Ca2+-binding modulator of phosphodiesterase produce vascular relaxation and inhibit actin-myosin interaction. Mol Pharmacol. 1979 Jan;15(1):49–59. [PubMed] [Google Scholar]
- Ishikawa H., Bischoff R., Holtzer H. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J Cell Biol. 1969 Nov;43(2):312–328. [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Kennel S. J., Lerner R. A. Isolation and characterization of plasma membrane associated immunoglobulin from cultured human diploid lymphocytes. J Mol Biol. 1973 Jun 5;76(4):485–502. doi: 10.1016/0022-2836(73)90487-7. [DOI] [PubMed] [Google Scholar]
- LOWEY S., COHEN C. Studies on the structure of myosin. J Mol Biol. 1962 Apr;4:293–308. doi: 10.1016/s0022-2836(62)80007-2. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Levin R. M., Weiss B. Mechanism by which psychotropic drugs inhibit adenosine cyclic 3',5'-monophosphate phosphodiesterase of brain. Mol Pharmacol. 1976 Jul;12(4):581–589. [PubMed] [Google Scholar]
- Levy J. A., Virolainen M., Defendi V. Human lymphoblastoid lines from lymph node and spleen. Cancer. 1968 Sep;22(3):517–524. doi: 10.1002/1097-0142(196809)22:3<517::aid-cncr2820220305>3.0.co;2-a. [DOI] [PubMed] [Google Scholar]
- Lin D. C., Tobin K. D., Grumet M., Lin S. Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation. J Cell Biol. 1980 Feb;84(2):455–460. doi: 10.1083/jcb.84.2.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin S., Lin D. C., Flanagan M. D. Specificity of the effects of cytochalasin B on transport and motile processes. Proc Natl Acad Sci U S A. 1978 Jan;75(1):329–333. doi: 10.1073/pnas.75.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin S., Snyder C. E., Jr High affinity cytochalasin B binding to red cell membrane proteins which are unrelated to sugar transport. J Biol Chem. 1977 Aug 10;252(15):5464–5471. [PubMed] [Google Scholar]
- Maxfield F. R., Schlessinger J., Shechter Y., Pastan I., Willingham M. C. Collection of insulin, EGF and alpha2-macroglobulin in the same patches on the surface of cultured fibroblasts and common internalization. Cell. 1978 Aug;14(4):805–810. doi: 10.1016/0092-8674(78)90336-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Poste G., Papahadjopoulos D., Nicolson G. L. Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4430–4434. doi: 10.1073/pnas.72.11.4430. [DOI] [PMC free article] [PubMed] [Google Scholar]
- ROTH T. F., PORTER K. R. YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L. J Cell Biol. 1964 Feb;20:313–332. doi: 10.1083/jcb.20.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roth T. F., Cutting J. A., Atlas S. B. Protein transport: a selective membrane mechanism. J Supramol Struct. 1976;4(4):527–548. doi: 10.1002/jss.400040413. [DOI] [PubMed] [Google Scholar]
- Singer S. J., Ash J. F., Bourguignon L. Y., Heggeness M. H., Louvard D. Transmembrane interactions and the mechanisms of transport of proteins across membranes. J Supramol Struct. 1978;9(3):373–389. doi: 10.1002/jss.400090308. [DOI] [PubMed] [Google Scholar]
- Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
- Wagner R., Rosenberg M., Estensen R. Endocytosis in Chang liver cells. Quantitation by sucrose- 3 H uptake and inhibition by cytochalasin B. J Cell Biol. 1971 Sep;50(3):804–817. doi: 10.1083/jcb.50.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss B., Levin R. M. Mechanism for selectively inhibiting the activation of cyclic nucleotide phosphodiesterase and adenylate cyclase by antipsychotic agents. Adv Cyclic Nucleotide Res. 1978;9:285–303. [PubMed] [Google Scholar]
- Wills E. J., Davies P., Allison A. C., Haswell A. D. Cytochalasin B fails to inhibit pinocytosis by macrophages. Nat New Biol. 1972 Nov 8;240(97):58–60. doi: 10.1038/newbio240058a0. [DOI] [PubMed] [Google Scholar]
- de Petris S., Raff M. C. Normal distribution, patching and capping of lymphocyte surface immunoglobulin studied by electron microscopy. Nat New Biol. 1973 Feb 28;241(113):257–259. doi: 10.1038/newbio241257a0. [DOI] [PubMed] [Google Scholar]