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Involvement of a Triton-insoluble Floating Fraction inDictyostelium Cell-Cell Adhesion

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m010016200

ISSN

1083-351X

Autores

Tony Harris, Donald E. Awrey, Brian Cox, Amir Ravandi, Adrian Tsang, Chi‐Hung Siu,

Tópico(s)

Cellular Mechanics and Interactions

Resumo

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) fromDictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those ofDictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals thatDictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role forDictyostelium TIFF in cell-cell adhesion during development. We have isolated and characterized a Triton-insoluble floating fraction (TIFF) fromDictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those ofDictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals thatDictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role forDictyostelium TIFF in cell-cell adhesion during development. Triton-insoluble floating fraction 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid CHAPS-insoluble floating fraction matrix-assisted laser desorption ionization time-of-flight green fluorescent protein regulatory myosin light chain glycosylphosphatidylinositol polyacrylamide gel electrophoresis expressed sequence tag monoclonal antibody 4-morpholine ethanesulfonic acid Cell membranes are thought to exist primarily in a fluid, liquid crystalline phase. However, certain membranes display elevated acyl chain order and exist in a liquid-ordered phase. These membranes exhibit Triton X-100 insolubility and can be separated from other insoluble cellular material by floatation into density gradients after isopycnic centrifugation (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2545) Google Scholar, 2Brown D.A. London E. J. Memb. Biol. 1998; 164: 103-114Crossref PubMed Scopus (833) Google Scholar, 3Hooper N.M. Mol. Membr. Biol. 1999; 16: 145-156Crossref PubMed Scopus (357) Google Scholar). We will refer to these membranes as a Triton X-100-insoluble floating fraction (TIFF).1 TIFF has distinctive structural properties and is involved in a variety of cellular functions. TIFF is typically isolated as membrane vesicles (4Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 5Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (860) Google Scholar, 6Kubler E. Dohlman H.G. Lisanti M.P. J. Biol. Chem. 1996; 271: 32975-32980Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). These membranes are enriched in lipids, such as cholesterol and those with saturated acyl chains, which are expected to pack closely within the liquid-ordered environment (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8047) Google Scholar). In addition, many cell membrane-associated structural and signaling proteins have been found in TIFF (8Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1719) Google Scholar). TIFF proteins are often anchored to the membranes through a lipid moiety (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8047) Google Scholar). TIFF was originally characterized in vertebrate cells (4Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 5Sargiacomo M. Sudol M. Tang Z. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Crossref PubMed Scopus (860) Google Scholar). However, TIFF analyses have been extended to yeast and Drosophila (6Kubler E. Dohlman H.G. Lisanti M.P. J. Biol. Chem. 1996; 271: 32975-32980Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar,9Rietveld A. Neutz S. Simons K. Eaton S. J. Biol. Chem. 1999; 274: 12049-12054Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 10Bagnat M. Keranen S. Shevchenko A. Shevchenko A. Simons K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3254-3259Crossref PubMed Scopus (502) Google Scholar), and a CHAPS-insoluble floating fraction (CHIFF) has been reported in Dictyostelium discoideum (11Xiao Z. Devreotes P.N. Mol. Biol. Cell. 1997; 8: 855-869Crossref PubMed Scopus (37) Google Scholar). The physiological importance of TIFF has been shown by perturbation of TIFF protein activities by cholesterol sequestration and through the co-localization of TIFF components at sites of cellular activity. Processes that involve TIFF include cellular trafficking (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8047) Google Scholar), T-cell signaling (12Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8: 723-732Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 13Webb Y Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 14Wulfing C. Davis M.M. Science. 1998; 282: 2266-2269Crossref PubMed Scopus (534) Google Scholar, 15Stulnig T.M. Berger M. Sigmund T. Raederstorff D. Stockinger H. Waldhausl W. J. Cell Biol. 1998; 143: 637-644Crossref PubMed Scopus (237) Google Scholar, 16Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (840) Google Scholar), integrin signaling (17Green J.M. Zhelesnyak A. Chung J. Lindberg F.P. Sarfati M. Frazier W.A. Brown E.J. J. Cell Biol. 1999; 146: 673-682Crossref PubMed Scopus (154) Google Scholar, 18Krauss K. Altevogt P. J. Biol. Chem. 1999; 274: 36921-36927Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and bacterial interactions with macrophages (19Gatfield J. Pieters J. Science. 2000; 288: 1647-1650Crossref PubMed Scopus (472) Google Scholar) and mast cells (20Shin J.-S. Gao Z. Abraham S.N. Science. 2000; 289: 785-788Crossref PubMed Scopus (272) Google Scholar). Dictyostelium is a favorable model organism for the study of plasma membrane structure and function. Dictyostelium is amenable to both biochemical and molecular genetic analyses of its cellular and developmental processes. During Dictyosteliumdevelopment, unicellular amoeboid cells aggregate through chemotaxis toward cAMP and embark on a multicellular developmental program (21Loomis W.F. Dictyostelium discoideum: A Developmental System. Academic Press, Inc., New York1975Google Scholar). Three components of the cAMP signaling pathway (the cAMP receptor cAR1, adenylate cyclase, and the cell surface phosphodiesterase) are found in CHIFF, suggesting that they are components of specialized microdomains on the plasma membrane (11Xiao Z. Devreotes P.N. Mol. Biol. Cell. 1997; 8: 855-869Crossref PubMed Scopus (37) Google Scholar). Multicellularity in Dictyostelium is maintained by several cell adhesion molecules, including DdCAD-1/gp24 (22Knecht D.A. Fuller D.L. Loomis W.F. Dev. Biol. 1987; 121: 277-283Crossref PubMed Scopus (70) Google Scholar, 23Brar S.K. Siu C.-H. J. Biol. Chem. 1993; 268: 24902-24909Abstract Full Text PDF PubMed Google Scholar, 24Wong E.F.S. Brar S.K. Sesaki H. Yang C. Siu C.-H. J. Biol. Chem. 1996; 271: 16399-16408Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), gp150/LagC (25Geltosky J.E. Weseman J. Bakke A. Lerner R.A. Cell. 1979; 18: 391-398Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 26Gao E.N. Shier P. Siu C.-H. J. Biol. Chem. 1992; 267: 9409-9415Abstract Full Text PDF PubMed Google Scholar, 27Wang J. Hou L. Awrey D. Loomis W.F. Firtel R.A. Siu C.-H. Dev. Biol. 2000; 227: 734-745Crossref PubMed Scopus (47) Google Scholar), and gp80 (28Muller K. Gerisch G. Nature. 1978; 274: 445-449Crossref PubMed Scopus (200) Google Scholar, 29Siu C.-H. Lam T.Y. Choi A.H.C. J. Biol. Chem. 1985; 260: 16030-16036Abstract Full Text PDF PubMed Google Scholar). gp80 is expressed during the aggregation stage of development (30Noegel A. Gerisch G. Stadler J. Westphal M. EMBO J. 1986; 5: 1473-1476Crossref PubMed Google Scholar, 31Wong L.M. Siu C.-H. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4248-4252Crossref PubMed Scopus (32) Google Scholar, 32Desbarats L. Brar S.K. Siu C.-H. J. Cell Sci. 1994; 107: 1705-1712PubMed Google Scholar) and mediates the so-called contact sites A by a Ca2+/Mg2+-independent homophilic binding mechanism (33Siu C.-H. Cho A. Choi A.H.C. J. Cell Biol. 1987; 105: 2523-2533Crossref PubMed Scopus (42) Google Scholar, 34Kamboj R.K. Wong L.M. Lam T.Y. Siu C.-H. J. Cell Biol. 1988; 107: 1835-1843Crossref PubMed Scopus (28) Google Scholar, 35Kamboj R.K. Gariepy J. Siu C.-H. Cell. 1989; 59: 615-625Abstract Full Text PDF PubMed Scopus (42) Google Scholar). gp80 is both necessary for strong adhesion during development (36Harloff C. Gerisch G. Noegel A.A. Genes Dev. 1989; 3: 2011-2019Crossref PubMed Scopus (77) Google Scholar, 37Siu C.-H. Kamboj R.K. Dev. Genet. 1990; 11: 377-387Crossref PubMed Scopus (37) Google Scholar, 38Ponte E. Bracco E. Faix J. Bozzaro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9360-9365Crossref PubMed Scopus (55) Google Scholar) and sufficient for the aggregation of otherwise nonadhesive vegetative cells (39Faix J. Gerisch G. Noegel A.A. EMBO J. 1990; 9: 2709-2716Crossref PubMed Scopus (32) Google Scholar, 40Kamboj R.K. Lam T.Y. Siu C.-H. Cell Regul. 1990; 1: 715-729Crossref PubMed Scopus (31) Google Scholar). It is apparent that gp80-gp80 interactions mediate cell-cell adhesion among Dictyostelium cells. However, structural details of gp80 adhesion complexes are largely unknown. Intriguingly, gp80 is enriched in Triton X-100-insoluble, cytoskeleton-associated contact regions (41Ingalls H.M. Goodloe-Holland C.M. Luna E.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4779-4783Crossref PubMed Scopus (42) Google Scholar). This subcellular fraction contains stacked membranes with dimensions of intact cell-cell contacts and can only be isolated after the cell aggregation stage of development. Considering that gp80 is phospholipid-anchored (42Sadeghi H. da Silva A.M. Klein C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5512-5515Crossref PubMed Scopus (28) Google Scholar, 43Stadler J. Keenan T.W. Bauer G. Gerisch G. EMBO J. 1989; 8: 371-377Crossref PubMed Scopus (87) Google Scholar), we hypothesized that these Triton X-100-insoluble contact regions may be a form of TIFF and that gp80 adhesion complexes may be organized as distinct membrane domains within the plasma membrane. In the present study, we have isolated Dictyostelium TIFF, characterized its morphology, identified its protein constituents, and analyzed its lipid composition. We demonstrate that TIFF membranes share many common physical and biochemical properties with the Triton X-100-insoluble contact regions. Furthermore, a role for TIFF components during gp80-mediated adhesion is established through colocalization, co-capping, and adhesion perturbation studies. Dictyostelium cells, including the wild-type axenic strain AX2 and the gp80-null strain GT10 (36Harloff C. Gerisch G. Noegel A.A. Genes Dev. 1989; 3: 2011-2019Crossref PubMed Scopus (77) Google Scholar), were cultured either in association with Klebsiella aerogenes or axenically in HL5 liquid medium (44Sussman M. Methods Cell Biol. 1987; 28: 9-29Crossref PubMed Scopus (533) Google Scholar). For development, cells at the late exponential growth phase were collected, washed, and resuspended at 1.5 × 107 cells/ml in MCM buffer (2 mm MgCl2, 0.2 mmCaCl2, 20 mm MES, pH 6.8) (45Aizawa H. Sutoh K. Yahara I. J. Cell Biol. 1996; 132: 335-344Crossref PubMed Scopus (129) Google Scholar) and then shaken at 180 rpm. To stimulate gp80 expression, cells were pulsed with cAMP at a final concentration of 2 × 10−8m every 7 min. Cells were also developed in MCM buffer on coverslips. A GFP-comitin construct was produced by end-filling an EcoRI fragment containing the comitin cDNA (46Greenwood M.T. Tsang A. Biochem. Cell Biol. 1992; 70: 1047-1054Crossref PubMed Scopus (2) Google Scholar), followed by subcloning into the end-filledHindIII site of the Dictyostelium expression vector pBS18-74E (obtained from Dr. R. Firtel, University of California at San Diego, La Jolla, CA). Expression of comitin was under the control of the actin 15 promoter. The BamHI/BglII fragment of pBS-GFPII (obtained from Dr. H. MacWilliams, Ludwig Maximillian University, Munich, Germany), containing the entire coding region of GFP, was then subcloned into the BglII site of comitin in the resulting plasmid. Hence, GFP was inserted in-frame between Arg15 and Ser16 of comitin. Plasmid DNA was introduced into AX2 cells by the calcium phosphate co-precipitation method (47Early A.E. Williams J.G. Gene ( Amst. ). 1987; 59: 99-106Crossref PubMed Scopus (71) Google Scholar). Stable transformants were selected and maintained in HL5 medium containing 20 μg/ml of G418. TIFF was isolated from cell aggregates that were collected at 12 h of development and gently resuspended at 5 × 107 cells/ml in cold buffer 1 (40 mm sodium pyrophosphate, 0.4 mm dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, 2 mm EDTA, 1 mm EGTA, 3 mm sodium azide, 10 mmTris-HCl, pH 7.6). Triton X-100 was added to a final concentration of 0.2% (v/v), and the suspension was shaken at 180 rpm for 1 min at 4 °C. The insoluble material was centrifuged at 14,500 ×g, and the pellet was washed with buffer 1, centrifuged at 14,500 × g, washed with buffer 2 (1 mmEGTA, 5 mm Tris-HCl, pH 7.6), and centrifuged at 14,500 × g. The resulting pellet was resuspended in buffer 2 and mixed at a 1:2 ratio with 60% (w/w) sucrose, placed at the bottom of a centrifuge tube, and then overlaid with an 11-ml continuous gradient of 10–40% (w/w) sucrose. The gradients were centrifuged at 120,000 × g for 15–17 h at 2 °C using a Beckman SW40 rotor. The TIFF material banded at ∼34% sucrose was collected. In subsequent experiments, TIFF was isolated in discontinuous gradients from the interface between 28 and 38% sucrose. TIFF collected from the sucrose gradients was washed with 20 mm sodium phosphate buffer, pH 7.6, and then pelleted by centrifugation at 39,000 × g for 20 min at 2 °C. Plasma membranes were isolated using the aqueous two-phase polymer system (48Siu C.-H. Lerner R.A. Loomis W.F. J. Mol. Biol. 1977; 116: 469-488Crossref PubMed Scopus (48) Google Scholar). Triton X-100-insoluble contact regions were isolated according to Ingalls et al. (41Ingalls H.M. Goodloe-Holland C.M. Luna E.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4779-4783Crossref PubMed Scopus (42) Google Scholar). Cells were treated with 0.2% Triton X-100 at 4 °C and then centrifuged at 4000 × g. The pellet was washed and resuspended in buffer 2 and then layered on the top of a discontinuous gradient of 5.5 ml of 50% and 5.5 ml of 60% (w/w) sucrose in 20 mmphosphate buffer, pH 6.8, for centrifugation at 120,000 ×g for 3 h at 2 °C. The material banded at the interface was collected, washed, and dialyzed in a cytoskeleton depolymerizing solution containing 0.1 mm EDTA, 0.2 mm sodium phosphate buffer, pH 7.6. The dialyzed material was pelleted, resuspended in 1.5 ml of buffer 2, and layered on top of an 11-ml continuous gradient of 26–51% (w/w) sucrose. The gradient was centrifuged at 120,000 × g for 3 h at 2 °C. Membranes banded at 32–34% sucrose were collected. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce). For SDS-PAGE, proteins were solubilized and reduced by boiling for 5 min in 3% (w/v) SDS, 3 m urea and 5% (v/v) β-mercaptoethanol and then separated in a slab gel (49Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206597) Google Scholar). For immunoblot analysis, bound antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and the enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Immunoblots were quantified using the Bio-Rad Fluor-S Max multiimager system. For protein identification by mass spectrometry, silver-stained gel bands were excised, macerated, reduced, alkylated, and then digested with trypsin. The tryptic peptides were extracted using the protocol of Shevchenko et al. (50Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 58: 850-858Crossref Scopus (7783) Google Scholar). The peptides were purified using Sephasil C18 resin, applied to MALDI plates, and mixed with a matrix solution of α-cyano-4-hydroxy-trans-cinnamic acid (Sigma) at 20 mg/ml in 50% (v/v) acetone and 50% (v/v) isopropyl alcohol. The samples were dried and then subjected to mass spectrometry. Peptide masses were determined using a PerSeptive Biosystems Voyager Elite MALDI-TOF mass spectrometer (PerSeptive Biosystems, Inc.) in the linear mode, with 92% grid voltage, 0.15% guide wire voltage, laser intensities between 1600 and 2100, and delayed extraction of 200 ns. Trypsin autolysis products and matrix molecules were used for calibration. The remaining masses were submitted to the ProFound search engine and the Protein Prospector search engine (both available on the World Wide Web) for matches. Search parameters were held constant, including tolerance for peptide mass error of ±1 Da, tolerance for protein mass error of ±10 kDa from apparent molecular masses determined by SDS-PAGE, and a maximum of one missed cut per peptide. Cells expressing GFP-comitin were collected at 10 h of development for TIFF isolation. TIFF membranes were incubated overnight with anti-gp80 mAb 80L5C4 (29Siu C.-H. Lam T.Y. Choi A.H.C. J. Biol. Chem. 1985; 260: 16030-16036Abstract Full Text PDF PubMed Google Scholar) or anti-GFP rabbit antibody (Molecular Probes, Inc., Eugene, OR) in TBS (20 mm Tris, pH 7.6, 137 mm NaCl) plus 0.1% (w/v) bovine serum albumin, with rotation at 4 °C. After washing, samples were incubated with either goat anti-mouse or goat anti-rabbit antibodies conjugated to 10-nm gold (Sigma) at 1:10 dilution in TBS plus 0.1% (w/v) bovine serum albumin. Samples were rotated overnight at 4 °C. After four washes, pellets were fixed overnight at 4 °C with 1% (v/v) glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.2. After several washes, the pellets were incubated in 1% (w/v) OsO4 in phosphate buffer for 30 min at room temperature. Samples were dehydrated with an ethanol series followed by propylene oxide and then embedded in Spurr's standard resin. Ultrathin sections were cut with a diamond knife and then stained with uranyl acetate and lead citrate prior to examination under a Hitachi 8600 analytical transmission electron microscope. To perform Triton X-100 extraction of living cells, cell aggregates on coverslips were chilled to 4 °C by replacing the MCM buffer with cold buffer 1 and placing the coverslips on an ice water bath for 15 min, and Triton X-100 was added to a final concentration of 0.2% (v/v). After 5 min, the Triton X-100 solution was aspirated, and the coverslips were fixed with cold 10% (v/v) formaldehyde for 10 min on ice, followed by 3.7% (v/v) formaldehyde at 22 °C for 10 min. Cells were stained and prepared for confocal microscopy. Cells were developed at 2 × 107 cells/ml for 2 h in 17 mmphosphate buffer, pH 6.1, containing streptomycin (0.5 mg/ml). Cells were collected and resuspended in phosphate buffer containing 0.1 mCi/ml of [9,10-3H]palmitic acid (PerkinElmer Life Sciences) and developed for another 6 h with cAMP pulsing. Cell aggregates were collected, and proteins in different subcellular fractions were analyzed by SDS-PAGE. Protein blots were coated with EN3HANCE spray (PerkinElmer Life Sciences) and exposed to Biomax MR film (Eastman Kodak Co.) at −70 °C for 3 weeks. Samples were analyzed by gas-liquid chromatography as described previously (51Ravandi A. Kuksis A. Shaikh N.A. J. Biol. Chem. 1999; 274: 16494-16500Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, sample lipids were extracted with chloroform/methanol (2:1, v/v) and digested with phospholipase C (Clostridium welchii). The mixture was extracted with chloroform/methanol (2:1, v/v) containing 100 μg of tridecanoylglycerol as an internal standard. The samples were then incubated in SYLON BFT plus 1 part dry pyridine for 30 min at 20 °C. All extracted lipids are converted into neutral species by this procedure, and they were quantified after separation on a nonpolar capillary column. Cells were developed for 12 h on coverslips and then fixed, stained, and mounted as previously described (52Sesaki H. Siu C.-H. Dev. Biol. 1996; 177: 504-516Crossref PubMed Scopus (28) Google Scholar, 53Sesaki H. Wong E.F.S. Siu C.-H. J. Cell Biol. 1997; 138: 939-951Crossref PubMed Scopus (56) Google Scholar). Samples were first incubated with primary antibodies against gp80 or porin, followed by Alexa 568/488-conjugated secondary antibodies (Molecular Probes) at 1:300 dilution. Filamentous actin (F-actin) was stained with fluorescein-phalloidin (Molecular Probes) at 1:10 dilution. Laser-scanning confocal microscopy was performed using a Zeiss Axiovert 135 inverted microscope equipped with a × 63 Neofluor objective and an LSM 410 confocal attachment. Detection was maintained within the range of the gray scale to prevent signal saturation. For filipin staining, cells were grown axenically, developed on coverslips, and then fixed in 3.7% (v/v) formaldehyde in MCM buffer for 15 min at room temperature. Cells were incubated with 0.025% (w/v) filipin in MCM buffer for 15 min at room temperature, washed, and then mounted for epifluorescence microscopy. Cells were developed in suspension for 6–8 h with cAMP pulsing. Cell aggregates were dispersed and deposited on coverslips in MCM buffer (3 × 105cells/coverslip). After 10 min, anti-gp80 mAb or polyclonal antibodies were added to cells for 15 min at room temperature. The coverslips were washed gently with two changes of 10 ml of MCM buffer for 3 min. Alexa 568-conjugated secondary antibodies were added at 1:50 dilution for 15 min, followed by two washes. The cells were fixed at 40 min after the initial addition of the primary antibody. To identify co-capped molecules, fixed cells were stained with appropriate antibodies. Cell cohesion was assayed as described previously (54Lam T.Y. Pickering G. Geltosky J. Siu C.-H. Differentiation. 1981; 20: 22-28Crossref PubMed Scopus (58) Google Scholar). Alternatively, the effects of various reagents on cell cohesion were assayed by measuring aggregate dissociation under high shear force. Cells were cultured on bacteria plates and then collected for development in liquid culture at 2 × 107 cells/ml. At different time points, cell aliquots were taken and diluted in 17 mm phosphate buffer, pH 6.1, plus the reagent under test. Stock solutions of filipin and digitonin were prepared fresh in Me2SO prior to each experiment. Equivalent amounts of Me2SO were added to control samples. Cell aggregates were incubated in various reagents for 10 min at room temperature with gentle shaking. Shear force was then applied by continuous vortexing for 30 s using a Vortex Genie 2 at setting 8. Cell dissociation was quantified by counting cells with a hemacytometer. Singlets, doublets, and triplets were scored as dissociated cells, and the percentage of cell dissociation was calculated relative to the number of cells obtained at 0 h, which was ∼2 × 106 cells/ml. TIFF was isolated fromDictyostelium cells after 10–12 h of development in liquid medium. After fractionation of detergent-insoluble material in a continuous sucrose density gradient (Fig.1 A), TIFF formed a sharp band at ∼34% sucrose (Fig. 1 A, lane 9), whereas the cytoskeleton pelleted (Fig. 1 A, lane 13). The highly resolved banding pattern suggested that TIFF had a relatively homogeneous composition. This isolation protocol routinely yielded 0.8–0.9 mg of TIFF protein from 1010cells. Silver staining of TIFF proteins revealed a highly reproducible profile (Fig. 1 B). The 10 most strongly stained bands were designated with "t" followed by their apparent molecular mass in kDa. Protein identification was attempted using MALDI-TOF mass spectrometry and data base search engines. Protein bands designated t103, t88, t43, t28, t23, and t20 were identified to be theDictyostelium expressed sequence tag (EST) C84888, gp80, actin, porin, comitin, and regulatory myosin light chain (RMLC), respectively (Fig. 1 C). These proteins produced the strongest mass spectra of the 10 analyzed. As an example, the mass spectrum of t103 is shown (Fig. 1 D). Their identifications displayed similar ranks and scores, based on two search engines. They each had similar sequence coverage with their matches and displayed the expected electrophoretic mobility. Since the identification of t103 was limited to one search engine, a postsource decay analysis was performed (Fig. 1, D andE). Every postsource decay product of the 2498-Da peptide corresponded to the predicted sequence of the EST, thus confirming the match (Fig. 1 E). To annotate the EST, the position-specific iterated basic local alignment search tool was used to search for similar proteins. The search results showed that the C termini of members of the CD36 family had the highest scores, and t103 was therefore designated DdCD36. The identities of gp80, actin, porin, and comitin were confirmed by Western blot analysis (Fig.1 F). Since proteins anchored to the plasma membrane via a lipid moiety are known to be associated with TIFF (7Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8047) Google Scholar), we labeled cells metabolically with [3H]palmitic acid and determined whether labeled proteins were enriched in the TIFF fraction. Labeled cells were fractionated into three parts: the Triton X-100-soluble fraction, the Triton X-100-insoluble pelleted fraction after floatation centrifugation, and TIFF. Equal amounts of protein from each fraction were separated by SDS-PAGE and compared after fluorography (Fig.2). TIFF was enriched with many palmitoylated proteins. Several intensely labeled species displayed gel mobilities corresponding to gp80, t46, actin, RMLC, and t18, suggesting that these proteins or some co-migrating species were heavily palmitoylated. Since membrane insolubility in cold Triton X-100 is a characteristic of liquid-ordered membrane structure (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2545) Google Scholar, 2Brown D.A. London E. J. Memb. Biol. 1998; 164: 103-114Crossref PubMed Scopus (833) Google Scholar, 3Hooper N.M. Mol. Membr. Biol. 1999; 16: 145-156Crossref PubMed Scopus (357) Google Scholar), we expected Dictyostelium TIFF to be enriched in lipids conforming to this structure. Total lipids in plasma membranes and TIFF were analyzed and compared (TableI). TIFF contained a 15-fold higher sterol level and a 1.5-fold higher phospholipid level than the plasma membrane. The elevated lipid/protein ratios in TIFF probably accounted for its low density (1.13–1.14 g/ml). The sterol/phospholipid ratio in TIFF was ∼10-fold higher than that of plasma membranes. The sterol species in TIFF were identified to be stigmasterol (78%), campesterol (14%), and sitosterol (8%) based on column retention times. The stigmasterol species was probably Δ22-stigmasten-3β-ol because it accounts for 88% of the sterols in Dictyosteliumcells (55Murray B.A. Loomis W. The Development of Dictyostelium discoideum. Academic Press, Inc., New York1982: 71-104Crossref Google Scholar). Both TIFF and plasma membranes had similar sterol compositions.Table ILipid compositions of Dictyostelium plasma membranes and TIFFSterolsPhospholipidsSterols/Phospholipidsmg/mg proteinmg/mg proteinmg/mgmol/molPlasma membrane0.236 ± 0.029 (1.0)0.350 ± 0.055 (1.0)0.678 ± 0.038 (1.0)1.26 ± 0.071 (1.0)TIFF3.620 ± 0.593 (15.3)0.541 ± 0.072 (1.55)6.750 ± 1.15 (9.96)12.5 ± 2.13 (9.92)The values represent the means ± S.D. (for plasma membrane,n = 3; for TIFF, n = 5). Relative amounts are indicated in parentheses. Open table in a new tab The values represent the means ± S.D. (for plasma membrane,n = 3; for TIFF, n = 5). Rel

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