Partitioning of Proteins into Plasma Membrane Microdomains
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29538
ISSN1083-351X
AutoresElla Fire, Claire M. Brown, Michael G. Roth, Yoav I. Henis, Nils O. Petersen,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoInternalization of membrane proteins involves their recruitment into plasma membrane clathrin-coated pits, with which they are thought to interact by binding to AP-2 adaptor protein complexes. To investigate the interactions of membrane proteins with coated pits at the cell surface, we applied image correlation spectroscopy to measure directly and quantitatively the clustering of influenza hemagglutinin (HA) protein mutants carrying specific cytoplasmic internalization signals. The HA system enables direct comparison between isolated internalization signals, because HA itself is excluded from coated pits. The studies presented here provide, for the first time, a direct quantitative measure for the degree of clustering of membrane proteins in coated pits at the cell surface. The degree of clustering depended on the strength of the internalization signal and on the integrity of the clathrin lattices and correlated with the internalization rates of the mutants. The clustering of the HA mutants fully correlated with their ability to co-precipitate α-adaptin from whole cells, the first such demonstration for a membrane protein that is not a member of the epidermal growth factor receptor family. Furthermore, both the clustering in coated pits and the co-precipitation with α-adaptin were dramatically reduced in the cold, suggesting that low temperature can interfere with the sorting of proteins into coated pits. In addition to the specific results reported here, the general applicability of the image correlation spectroscopy approach to study any process involving the clustering or oligomerization of membrane receptors at the cell surface is discussed. Internalization of membrane proteins involves their recruitment into plasma membrane clathrin-coated pits, with which they are thought to interact by binding to AP-2 adaptor protein complexes. To investigate the interactions of membrane proteins with coated pits at the cell surface, we applied image correlation spectroscopy to measure directly and quantitatively the clustering of influenza hemagglutinin (HA) protein mutants carrying specific cytoplasmic internalization signals. The HA system enables direct comparison between isolated internalization signals, because HA itself is excluded from coated pits. The studies presented here provide, for the first time, a direct quantitative measure for the degree of clustering of membrane proteins in coated pits at the cell surface. The degree of clustering depended on the strength of the internalization signal and on the integrity of the clathrin lattices and correlated with the internalization rates of the mutants. The clustering of the HA mutants fully correlated with their ability to co-precipitate α-adaptin from whole cells, the first such demonstration for a membrane protein that is not a member of the epidermal growth factor receptor family. Furthermore, both the clustering in coated pits and the co-precipitation with α-adaptin were dramatically reduced in the cold, suggesting that low temperature can interfere with the sorting of proteins into coated pits. In addition to the specific results reported here, the general applicability of the image correlation spectroscopy approach to study any process involving the clustering or oligomerization of membrane receptors at the cell surface is discussed. Receptor-mediated endocytosis constitutes a crucial part in the life cycle of cell surface proteins and provides a major mechanism for receptor down-regulation and signal termination (1Goldstein J.L. Brown M.S. Anderson R.G.W. Russel D.W. Schneider W.J. Annu. Rev. Cell Biol. 1985; 1: 1-39Crossref PubMed Scopus (1119) Google Scholar, 2Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar, 3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (128) Google Scholar, 4Hopkins C.R. Trends Biochem. Sci. 1992; 17: 27-32Abstract Full Text PDF PubMed Scopus (48) Google Scholar). The clathrin-coated pits, which are the major port of entry into the endocytic pathway in many cell types, represent the best studied example of a sorting domain where a coat of proteins at the cytosolic face of the membrane selectively binds membrane proteins containing specific motifs that serve as internalization signals (5Kirchhausen T. Curr. Opin. Struct. Biol. 1993; 3: 182-188Crossref Scopus (95) Google Scholar, 6Schmid S.L. Curr. Opin. Cell Biol. 1993; 5: 621-627Crossref PubMed Scopus (22) Google Scholar, 7Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (306) Google Scholar). Two classes of clathrin-associated assembly proteins (APs) 1The abbreviations used are: AP, assembly protein; EGF, epidermal growth factor; HA, influenza virus hemagglutinin; ICS, image correlation spectroscopy; HBSS, Hanks' balanced salt solution; Sulfo-NHS-LC-biotin, sulfosuccinimidyl-6-(biotinamido)hexanoate; FITC-GAR Fab′, fluorescein-coupled affinity purified Fab′ of goat IgG directed against rabbit F(ab′)2; wt, wild type; BSA, bovine serum albumin. have been identified, one specific for plasma membranes (AP-2) and one for the trans-Golgi network (AP-1) (5Kirchhausen T. Curr. Opin. Struct. Biol. 1993; 3: 182-188Crossref Scopus (95) Google Scholar, 6Schmid S.L. Curr. Opin. Cell Biol. 1993; 5: 621-627Crossref PubMed Scopus (22) Google Scholar, 7Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (306) Google Scholar, 8Pearse B.M. Crowther R.A. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 49-68Crossref PubMed Scopus (143) Google Scholar, 9Brodsky F.M. Science. 1988; 242: 1396-1402Crossref PubMed Scopus (205) Google Scholar, 10Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar). AP-2 is known to function both in assembly of the clathrin lattice (10Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (536) Google Scholar, 11Rodriguez M.C. Xie Y.B. Wang H. Collison K. Segaloff D.L. Mol. Endocrinol. 1992; 6: 327-336PubMed Google Scholar, 12Zaremba S. Keen J.H. J. Cell Biol. 1983; 97: 1339-1347Crossref PubMed Scopus (153) Google Scholar, 13Pearse B.M. Robinson M.S. EMBO J. 1984; 3: 1951-1957Crossref PubMed Scopus (149) Google Scholar) and in binding to membrane proteins carrying internalization signals, which are concentrated in clathrin-coated pits for endocytosis (5Kirchhausen T. Curr. Opin. Struct. Biol. 1993; 3: 182-188Crossref Scopus (95) Google Scholar, 14Nesterov A. Kurten R.C. Gill G.N. J. Biol. Chem. 1995; 270: 6320-6327Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 15Sorkin A. Carpenter G. Science. 1993; 261: 612-615Crossref PubMed Scopus (213) Google Scholar, 16Pearse B.M.F. EMBO J. 1988; 7: 3331-3336Crossref PubMed Scopus (233) Google Scholar, 17Glickman J.N. Conibear E. Pearse B.M.F. EMBO J. 1989; 8: 1041-1047Crossref PubMed Scopus (207) Google Scholar, 18Beltzer J.P. Spiess M. EMBO J. 1991; 10: 3735-3742Crossref PubMed Scopus (81) Google Scholar, 19Sosa M.A. Schmidt B. von Figura K. Hille-Rehfeld A. J. Biol. Chem. 1993; 268: 12537-12543Abstract Full Text PDF PubMed Google Scholar, 20Gilboa L. Ben-Levy R. Yarden Y. Henis Y.I. J. Biol. Chem. 1995; 270: 7061-7067Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Boll W. Gallusser A. Kirchhausen T. Curr. Biol. 1995; 5: 1168-1178Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Ohno H. Stewart J. Fournier M.C. Bosshart H. Rhee I. Miyatake S. Saito T. Gallusser A. Kirchhausen T. Bonifacino J.S. Science. 1995; 269: 1872-1875Crossref PubMed Scopus (830) Google Scholar). Recent evidence identifies several distinct classes of cytoplasmic internalization signals (reviewed in Refs. 3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (128) Google Scholar, 4Hopkins C.R. Trends Biochem. Sci. 1992; 17: 27-32Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 23Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar, and 24Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar). The best characterized are those containing an essential aromatic residue, typically a tyrosine. They conform mostly to one of two major subclasses: NPXY, where Tyr is preceded by an asparagine-proline dipeptide and a random amino acid, or YXXZ, where Z is an amino acid with a hydrophobic side chain (3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (128) Google Scholar, 23Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar, 24Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 25Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar). It was suggested that a type 1 tight turn conformation might constitute a general feature of tyrosine-based internalization signals (3Trowbridge I.S. Curr. Opin. Cell Biol. 1991; 3: 634-641Crossref PubMed Scopus (128) Google Scholar, 23Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (704) Google Scholar, 26Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S.Q. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (394) Google Scholar, 27Ktistakis N.T. Thomas D. Roth M.G. J. Cell Biol. 1990; 111: 1393-1407Crossref PubMed Scopus (132) Google Scholar, 28Bansal A. Gierasch L.M. Cell. 1991; 67: 1195-1201Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 29Eberle W. Sander C. Klaus W. Schmidt B. von Figura K. Peters C. Cell. 1991; 67: 1203-1209Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 30Backer J.M. Shoelson S.E. Weiss M.A. Hua Q.X. Cheatham R.B. Haring E. Cahill D.C. White M.F. J. Cell Biol. 1992; 118: 831-839Crossref PubMed Scopus (87) Google Scholar). However, a recent study employing combinatorial selection methods on the binding of YXXZ-containing peptides to the μ2 chain of AP-2 indicated no requirement for a prefolded structure around the tetrapeptide signal (31Boll H.O. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (236) Google Scholar). A different class of internalization signals contains a di-leucine motif (LL or LI) (24Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 32Letourneur F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 33Johnson K.F. Kornfeld S. J. Biol. Chem. 1992; 267: 17110-17115Abstract Full Text PDF PubMed Google Scholar, 34Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2967Crossref PubMed Scopus (220) Google Scholar). Although much has been learned about clathrin-mediated endocytosis in recent years, currently it is not well understood how membrane proteins bind to coated pits at the surface of the intact cell. In vivo endocytosis studies generally measured the sequestration of proteins into vesicles or deeply invaginated pits and did not allow direct measurement of many events that occur earlier during coated vesicle formation (e.g. the selection of certain proteins for inclusion in coated pits and the exclusion of others). Furthermore, experimental evidence for interactions of the internalization signals of membrane receptors with AP-2 subunits has been limited mostly toin vitro assays employing solubilized and immobilized proteins (16Pearse B.M.F. EMBO J. 1988; 7: 3331-3336Crossref PubMed Scopus (233) Google Scholar, 17Glickman J.N. Conibear E. Pearse B.M.F. EMBO J. 1989; 8: 1041-1047Crossref PubMed Scopus (207) Google Scholar, 18Beltzer J.P. Spiess M. EMBO J. 1991; 10: 3735-3742Crossref PubMed Scopus (81) Google Scholar, 19Sosa M.A. Schmidt B. von Figura K. Hille-Rehfeld A. J. Biol. Chem. 1993; 268: 12537-12543Abstract Full Text PDF PubMed Google Scholar, 21Boll W. Gallusser A. Kirchhausen T. Curr. Biol. 1995; 5: 1168-1178Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 31Boll H.O. Ohno H. Songyang Z. Rapoport I. Cantley L.C. Bonifacino J.S. Kirchhausen T. EMBO J. 1996; 15: 5789-5795Crossref PubMed Scopus (236) Google Scholar, 35Heilker R. Manning Krieg U. Zuber J.F. Spiess M. EMBO J. 1996; 15: 2893-2899Crossref PubMed Scopus (156) Google Scholar). These assays have detected only a subset of the sequences known or suspected to function as internalization signals in vivo (36Thomas D.C. Roth M.G. J. Biol. Chem. 1994; 269: 15732-15739Abstract Full Text PDF PubMed Google Scholar, 37Naim H.Y. Roth M.G. J. Biol. Chem. 1994; 269: 3928-3933Abstract Full Text PDF PubMed Google Scholar). In particular, association of AP-2 with receptors by co-immunoprecipitation from cells has thus far been demonstrated only for receptors from the epidermal growth factor (EGF) family (14Nesterov A. Kurten R.C. Gill G.N. J. Biol. Chem. 1995; 270: 6320-6327Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 15Sorkin A. Carpenter G. Science. 1993; 261: 612-615Crossref PubMed Scopus (213) Google Scholar, 20Gilboa L. Ben-Levy R. Yarden Y. Henis Y.I. J. Biol. Chem. 1995; 270: 7061-7067Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Boll W. Gallusser A. Kirchhausen T. Curr. Biol. 1995; 5: 1168-1178Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 38Sorkin A. McKinsey T. Shih W. Kirchhausen T. Carpenter G. J. Biol. Chem. 1995; 270: 619-625Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Even for these receptors, it is not clear whether this in vitro association is directly related to the efficiency of their internalization via coated pits (21Boll W. Gallusser A. Kirchhausen T. Curr. Biol. 1995; 5: 1168-1178Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 39Nesterov A. Wiley H.S. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8719-8723Crossref PubMed Scopus (75) Google Scholar, 40Sorkin A. Mazzotti M. Sorkina T. Scotto L. Beguinot L. J. Biol. Chem. 1996; 271: 13377-13384Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). It is therefore important to explore the relationships between binding to AP-2, clustering in coated pits, and the efficiency of the internalization process as a function of the internalization signal; this manuscript presents measurements to that end. Our studies were performed on a series of influenza hemagglutinin (HA) mutants carrying specific cytoplasmic internalization signals (37Naim H.Y. Roth M.G. J. Biol. Chem. 1994; 269: 3928-3933Abstract Full Text PDF PubMed Google Scholar, 41Lazarovits J. Roth M.G. Cell. 1988; 53: 743-752Abstract Full Text PDF PubMed Scopus (141) Google Scholar,42Zwart D.E. Brewer C.B. Lazarovits J. Henis Y.I. Roth M.G. J. Biol. Chem. 1996; 271: 907-917Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This system is advantageous, because wild-type HA (HA wt) lacks internalization signals and serves as a natural control, enabling investigation of specific internalization sequences introduced into its cytoplasmic tail (41Lazarovits J. Roth M.G. Cell. 1988; 53: 743-752Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 43Fire E. Zwart D.E. Roth M.G. Henis Y.I. J. Cell Biol. 1991; 115: 1585-1594Crossref PubMed Scopus (44) Google Scholar, 44Fire E. Gutman O. Roth M.G. Henis Y.I. J. Biol. Chem. 1995; 270: 21075-21081Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Using this system, we employed comparative studies of the lateral mobility of these mutants to characterize the mode of their interactions with coated pits at the surface of intact cells (43Fire E. Zwart D.E. Roth M.G. Henis Y.I. J. Cell Biol. 1991; 115: 1585-1594Crossref PubMed Scopus (44) Google Scholar, 44Fire E. Gutman O. Roth M.G. Henis Y.I. J. Biol. Chem. 1995; 270: 21075-21081Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). However, in these studies, measurement of the binding of internalization-competent HA mutants to immobile structures presumed to represent coated pits was indirect and was inferred from the reduction in the lateral diffusion rate or mobile fraction of the mutants relative to HA wt. Because numerous factors (and not only interactions with coated pits) may inhibit the lateral mobility of membrane proteins, it was important to measure the association of the HA mutants with coated pits at the cell surface in a direct and independent manner. The current studies, which are potentially applicable for studying any type of clustering or oligomerization at the cell surface, provide a direct demonstration and measure for internalization signal-dependent clustering of HA mutants in cell surface coated pits. Together with the findings on signal-dependent co-precipitation of AP-2 with the various HAs in strict correlation with the ICS data, our results suggest that the degree of clustering in coated pits for a given protein depends on the strength of the association of its internalization signal with the clathrin-associated adaptor complexes. In turn, this clustering plays an important role in determining the endocytosis rate and restricts the lateral mobility of the internalization-competent proteins at the cell surface. Restriction enzymes and T4-DNA ligase were from New England Biolabs (Beverly, MA). Trypsin, amiloride hydrochloride, protein A-Sepharose 4B, BSA, and n-propylgallate were from Sigma. Hanks' balanced salt solution (HBSS) was from Sigma or Life Technologies, Inc. Protein A-peroxidase was from Jackson ImmunoResearch Laboratories (West Grove, PA). Sulfosuccinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin) and streptavidin-peroxidase were from Pierce, and ECL reagent (Renaissance) was from NEN Life Science Products. Airvol 205 was from Air Products and Chemicals (Allentown, PA). Polyclonal rabbit antiserum that recognized all the mutants of the Japan HA (A/Japan/305/57 strain) was employed throughout (43Fire E. Zwart D.E. Roth M.G. Henis Y.I. J. Cell Biol. 1991; 115: 1585-1594Crossref PubMed Scopus (44) Google Scholar, 44Fire E. Gutman O. Roth M.G. Henis Y.I. J. Biol. Chem. 1995; 270: 21075-21081Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Fluorescein-labeled affinity purified F(ab′)2 of goat IgG directed against rabbit F(ab′)2 (FITC-GAR F(ab′)2) and normal goat IgG were purchased from Jackson ImmunoResearch. Monovalent Fab′ fragments were prepared from the IgG fraction of the anti-HA antibodies and from normal goat IgG as described (45Henis Y.I. Gutman O. Loyter A. Exp. Cell Res. 1985; 160: 514-526Crossref PubMed Scopus (23) Google Scholar). The FITC-GAR F(ab′)2were converted to monovalent Fab′ fragments (FITC-GAR Fab′) by reduction with mercaptoethanol followed by alkylation with iodoacetamide (45Henis Y.I. Gutman O. Loyter A. Exp. Cell Res. 1985; 160: 514-526Crossref PubMed Scopus (23) Google Scholar). To eliminate any possible IgG traces, all Fab′ preparations were treated with protein A-Sepharose. The resulting Fab′ were free of contamination by IgG or F(ab′)2 as judged by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. AC1-M11 mouse monoclonal antibodies specific for the αa and αc chains of AP-2 (46Robinson M.S. J. Cell Biol. 1987; 104: 887-895Crossref PubMed Scopus (155) Google Scholar) were donated by Dr. Margaret S. Robinson (University of Cambridge, UK). The cDNA encoding the HA wt protein from the A/Japan/305/57 strain or several HA mutants were introduced into the SV40-based vector pkSVE (47Roth M.G. Doyle C. Sambrook J. Gething M.-J. J. Cell Biol. 1986; 102: 1271-1283Crossref PubMed Scopus (70) Google Scholar). The mutants are depicted in Table I. The derivation of the mutants HA+8 (containing a carboxyl-terminal extension of eight amino acids, which generates a strong internalization signal based on the motif YKSF) and HA+4 (which is HA+8 minus the last four residues, with the internalization motif truncated after YK) is detailed elsewhere (42Zwart D.E. Brewer C.B. Lazarovits J. Henis Y.I. Roth M.G. J. Biol. Chem. 1996; 271: 907-917Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). HA-Y543, which is a point mutant of HA wt (cysteine 543 replaced by tyrosine), was described previously (41Lazarovits J. Roth M.G. Cell. 1988; 53: 743-752Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Recombinant SV40 virus stocks were prepared in CV-1 monkey fibroblasts (American Type Culture Collection, Rockville, MD) using the appropriate pkSVE vector together with the dl1055 helper virus as described (48Naim H.Y. Roth M.G. J. Virol. 1993; 67: 4831-4841Crossref PubMed Google Scholar). The CV-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (from Biological Industries, Beth Haemek, Israel, or from Life Technologies, Inc.), 100 units/ml penicillin, and 100 μg/ml streptomycin (Biological Industries or Life Technologies, Inc.). For ICS experiments on the degree of aggregation and for co-immunoprecipitation studies, subconfluent CV-1 cells were infected in suspension with second or third passage recombinant virus stocks as described (37Naim H.Y. Roth M.G. J. Biol. Chem. 1994; 269: 3928-3933Abstract Full Text PDF PubMed Google Scholar, 48Naim H.Y. Roth M.G. J. Virol. 1993; 67: 4831-4841Crossref PubMed Google Scholar). The cells were plated either on glass coverslips (for immunofluorescent labeling and ICS studies) or in 150-mm dishes (for co-immunoprecipitation studies). Experiments were performed 36–38 h post infection (for HA wt, HA+4, and HA-Y543) or after 44–46 h (for HA+8). The longer post infection time for HA+8 was selected to achieve cell surface density closer to that of the other mutants, because the percentage of HA+8 at the cell surface is significantly lower (42Zwart D.E. Brewer C.B. Lazarovits J. Henis Y.I. Roth M.G. J. Biol. Chem. 1996; 271: 907-917Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar).Table ICytoplasmic domain sequences of mutant HA proteinsJapan HA mutant in pkSVE vectorSequenceReferenceHA wtNGSLQCRICI*47Roth M.G. Doyle C. Sambrook J. Gething M.-J. J. Cell Biol. 1986; 102: 1271-1283Crossref PubMed Scopus (70) Google ScholarHA-Y543.....Y....*41Lazarovits J. Roth M.G. Cell. 1988; 53: 743-752Abstract Full Text PDF PubMed Scopus (141) Google ScholarHA+8NGSLQCRICIYDYKSFYN*42Zwart D.E. Brewer C.B. Lazarovits J. Henis Y.I. Roth M.G. J. Biol. Chem. 1996; 271: 907-917Abstract Full Text Full Text PDF PubMed Scopus (18) Google ScholarHA+4..........Y.Y.*42Zwart D.E. Brewer C.B. Lazarovits J. Henis Y.I. Roth M.G. J. Biol. Chem. 1996; 271: 907-917Abstract Full Text Full Text PDF PubMed Scopus (18) Google ScholarThe sequences of the short cytoplasmic domains of each HA mutant are shown. The dots in HA-Y543 and HA+4 stand for amino acids similar to those in wt HA or HA+8, respectively. An asterisk is a stop codon. Open table in a new tab The sequences of the short cytoplasmic domains of each HA mutant are shown. The dots in HA-Y543 and HA+4 stand for amino acids similar to those in wt HA or HA+8, respectively. An asterisk is a stop codon. CV-1 cells infected and grown as above were washed twice with cold HBSS containing 20 mm HEPES and 2% BSA (HBSS/HEPES/BSA, pH 7.2) and labeled successively in this buffer at 4 °C (washing three times after each incubation) with the following antibodies: (a) anti-HA Fab′ (100 μg/ml, 30 min); (b) Fab′ of normal goat IgG (to block nonspecific staining; 200 μg/ml, 20 min); and (c) FITC-GAR Fab′ (30 μg/ml, 30 min). The labeling was done in the cold to allow only surface labeling and to avoid endocytosis. In cases where the cells were preincubated in specific buffers to alter coated pit structure (see below), the specific buffers employed for each treatment were used throughout all antibody incubation and subsequent steps. In most cases (except where indicated), the labeled cells were warmed to 22 °C for 10 min prior to fixation in methanol (−20 °C, 5 min) and acetone (−20 °C, 2 min) to enable stronger interactions with coated pits while avoiding significant internalization of some mutants at 37 °C (43Fire E. Zwart D.E. Roth M.G. Henis Y.I. J. Cell Biol. 1991; 115: 1585-1594Crossref PubMed Scopus (44) Google Scholar, 44Fire E. Gutman O. Roth M.G. Henis Y.I. J. Biol. Chem. 1995; 270: 21075-21081Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The fixed cells were mounted in Slowfade solution (Molecular Probes, Eugene, OR) or in Airvol 205 containing n-propylgallate and taken for the ICS studies. ICS (described in detail in Refs. 49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar and 50.Wiseman, P. W. (1995) Image Correlation Spectroscopy: Development and Application to Studies of PDGF Receptor Distribution. Ph.D. Thesis, University of Western Ontario, London, Ontario.Google Scholar) is an adaptation of the fluorescence correlation spectroscopy method (51Ehrenberg M. Rigler R. Q. Rev. Biophys. 1976; 29: 69-81Crossref Scopus (73) Google Scholar, 52Elson E.L. Magde D. Biopolymers. 1974; 13: 1-27Crossref Scopus (1323) Google Scholar, 53Eigen M. Rigler R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5740-5747Crossref PubMed Scopus (908) Google Scholar) used to analyze fluorescence images collected on a confocal laser scanning microscope. It is sensitive to and capable of quantifying differences in the aggregation state and distribution of fluorescently labeled components at the cell surface. In fluorescence correlation spectroscopy, one examines the volume or area illuminated by a laser beam, usually in a microscope. In ICS, instead of observing fluorescent particles as they diffuse in and out of a fixed laser beam, one generates an image of the distribution of the fluorescence intensity on the cell surface by scanning a fluorescently labeled cell with a laser beam, recording fluctuations in fluorescence intensity as a function of position rather than of time (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 53Eigen M. Rigler R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5740-5747Crossref PubMed Scopus (908) Google Scholar). The signal analysis is based on calculating the correlation function of the fluorescence intensity fluctuations. When a single fluorescently labeled component is analyzed (as in the current measurements), the autocorrelation function, g(ξ,η), is derived and fitted to a two-dimensional gaussian function determined by the intensity profile of the laser beam (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar), g(ξ,η)=g(0,0)e−(ξ2+η2)/ω2+g0Equation 1 where ξ and η are the position coordinates (for thex and y axes, respectively) of the autocorrelation function, ω is the gaussian radius of the laser beam, and g(0,0) is the value of the autocorrelation function upon extrapolation of ξ and η to zero; g 0 is an offset introduced to account for the finite sample size, which may result in a decay of g(ξ,η) to a nonzero value.g(0,0) and ω are extracted from the fitting procedure (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar). Importantly, 1/g(0,0) is equal toN p, the average number of independent fluorescently labeled particles in the area illuminated by the beam (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar, 54Petersen N.O. Biophys. J. 1986; 49: 809-815Abstract Full Text PDF PubMed Scopus (136) Google Scholar). The term “particles” refers to any fluorescently labeled species, i.e. a fluorescent monomer is one particle, and an aggregate containing many monomers is also one particle. Therefore, aggregation leads to a reduction in N p (larger and fewer particles), which is measured by the 1/g(0,0) value (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar). After labeling and fixation as described under “immunofluorescent labeling”, fluorescent images were obtained using a Bio-Rad MRC-600 confocal microscope, illuminating at 488 nm with a 25 mW argon ion laser attenuated to 1%. Images were collected in the photon-counting mode (accumulating 25 scans) to ensure linear scaling. Zoom-10 images of flat 16.3 × 16.3-μm2 areas on the cell surface (avoiding regions around the nuclei, which are not in the same focal plane) were submitted for autocorrelation analysis (49Petersen N.O. Hoddelius P.L. Wiseman P.W. Seger O. Magnusson K.-E. Biophys. J. 1993; 65: 1135-1146Abstract Full Text PDF PubMed Scopus (315) Google Scholar) on a massively parallel computer (MP-2, MasPar Computer Corporation, Sunnyvale, CA). S.E. values were calculated at the 99% confidence level using the formula S.E. = t(S.D./n 0.5), where S.D. is the sample standard deviation, n is the number of measurements (cells imaged), and t is the tstatistic for n − 1 degrees of freedom (equal to 2.576 at the 99% confidence level in view of the high n in the experiments). p values were determined based on at test for two independent samples with different variance (55Crow E.L. Davis F.A. Maxfield M.W. Statistics Manual. Dover Publications Inc., Mineola, NY1960: 60-62Google Scholar), and the null hypothesis was rejected for
Referência(s)