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Proper assembly of MHC class I (MHCI) molecules is dependent on the dedicated ER-resident chaperone tapasin (tpn). Tpn binds both MHCI and TAP through different domains. We created three fluorescently tagged constructs: wild type tpn, soluble tpn, which no longer interacts with TAP, and N300 tpn, which no longer interacts with MHCI. Despite previous biochemical data, using fluorescence microscopy we find that soluble tpn is mostly located within the ER, similarly to wild type tpn. FRAP analysis shows that soluble tpn is more mobile than both wild type and N300, possibly because the latter constructs interact with TAP. Transfecting tpn negative cells with soluble tpn does not increase MHCI surface expression to the same level as wild type. When wild type is transfected in combination with N300, MHCI surface levels are reduced to those found with soluble, suggesting that N300 blocks wild type tpn access to TAP. Although resulting MHCI surface levels differ between wild type, soluble, and N300 tpn constructs, MHCI surface lifetimes are the same. Our data suggests that tpn functions to bring together the MHCI subunits, a process which is aided by proximity to TAP, but does not act to increase stability of the resulting MHCI complex.

Polyunsaturated fatty acids (PUFAs) can be used as adjuvant immunosuppressants to alleviate symptoms of inflammation associated with obesity, heart disease, autoimmune disorders, and the metabolic syndrome. However, the targets and molecular mechanisms by which PUFAs affect pathways not directly linked to inflammatory processes are poorly understood. In the present study, we tested the effects of the ω-6 PUFA arachidonic acid (AA) and the ω-3 PUFA docosahexaenoic acid (DHA) on antigen presentation through the MHC class I pathway. Treatment of human B lymphoblasts (APCs) with AA and DHA lowered their susceptibility to alloreactive CD8+ T cell lysis. Similar effects were observed for mouse APCs. Peptide specific lysis of PUFA-modified B lymphoblasts by CD8+ T cells transgenic for the 2C TCR was lower than lysis of controls. We found that extracellular release of lipids by PUFA-modified APCs was responsible for the reduction in lysis by cognate T cells. Our data suggest that T cell uptake of molecules from lipid modified APCs may be a mechanism by which T cell responses are regulated in response to dietary intake of fatty acids. In addition, elimination of pathogen-derived peptides could be compromised by using PUFAs as immunosuppressants.

The cell surface mediates the flow of information and metabolites between a cell and its environment. We are interested in understanding the lateral organization of the surface, as part of our program in understanding its function. The model of lateral organization of cell surface membranes is evolving from one which emphasizes mobility and autonomy of membrane constituent molecules, to another which emphasizes the lateral concentration of membrane proteins and lipids into patches and membrane microdomains. Indirect evidence suggests that diameters of these patches and microdomains are often ≤ a wavelength of visible light, and so cannot be readily resolved by conventional light microscopy.We have developed two complementary techniques for detecting patches and microdomains on the nm scale: image fluorescence resonance energy transfer, FRET, and near-field scanning optical microscopy, NSOM. The first of these techniques detects proximity of membrane proteins and lipids on a scale of a few nm in terms of the interactions of donor and acceptor fluorophores.

PD-1, a member of the CD28/CTLA-4/ICOS costimulatory receptor family, delivers negative signals that have profound effects on T and B cell immunity. The 2.0 A crystal structure of the extracellular domain of murine PD-1 reveals an Ig V-type topology with overall similarity to the CTLA-4 monomer; however, there are notable differences in regions relevant to function. Our structural and biophysical data show that PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Furthermore, our structure-based mutagenesis studies identify the ligand binding surface of PD-1, which displays significant differences compared to those present in the other members of the family.

We describe a near-field scanning optical microscope capable of imaging biological samples in liquid. The microscope uses a straight optical fiber near-field probe and optical shear-force feedback for tip-sample distance regulation. Physical aspects of the design are discussed, and phenomena related to operation in liquid are revealed. Careful calibration of the instrument in air and in liquid is shown, and for the first time to our knowledge, near-field fluorescence images of a biological cell in liquid are presented.

TerC, a cell line derived from a strain 129 teratocarcinoma 402AX, was used to sensitize syngeneic 129 (H-2bc) splenic lymphocytes in vitro. The effector cells generated inhibited in vitro growth of TerC as measured by an 125I-IUDR post-labeling technique. It was also shown, with a modified Winn assay, that the sensitized cells were effective in preventing TerC growth in vivo. The effector lymphocyte was nonadherent to nylon wool, was sensitive to anti-Thy-1·2 + C, and was phenotypically Ly 1-2+. The anti-TerC effector T lymphocytes were not functional in a 51Cr-release assay. However, this failure to lyse appears not to be due to some intrinsic membrane resistance since both BCG and ConA-activated killers were able to lyse TerC. The TerC-sensitized lymphocytes displayed no H-2 restriction and were able to growth inhibit in vitro a wide range of tumorigenic cell lines, e.g., P815 (H-2d), EL-4 (H-2b),Sal (H-2a), and BALB/c (H-2d) 3T12. Mouse blastocyst cell lines were also inhibited. BALB/c 3T3 and mouse fibroblast cell strains were not growth inhibited. Thus, it appears that oncofetal antigens expressed on TerC are capable of initiating a cell-mediated response and that these antigenic specificities are shared by many transformed cell lines.

In mouse-human heterokaryons, the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membrane is enhanced by treatment of parent cells with ouabain. Ouabain treatment is ineffective if the medium lacks calcium ion, or if Verapamil, a blocker of calcium channels, is present. The divalent ionophore A23187 also enhances lateral diffusion of MHC antigens, to the same extent as ouabain, A23187 is effective only if calcium is present in the medium. Thus it appears that increased levels of cell calcium release constraints to lateral diffusion of MHC antigens.

The rate of collection of embryo fibroblast single cells by an embryo fibroblast monlayer was realted to the H-2 haplotype of the fibroblast monolayer. The rate was highest for the H-2s strains and lowest for the H-2k strains with all other strains examined being intermediate. As opposed to monolayers prepared from the A and C3H background animals, monolayers from B10 background mice only demonstrated an H-2 haplotype dependent rate differential after treatment with fetal calf serum or neuraminidase. The relationship that was seen between monolayer H-2 haplotype and rate of adhesion with embryonic monolayers was not observed with either congenic 3T3 cell lines or fibroblasts derived from adult tissues. It was further shown that the rate of single cell pick-up could be substantially reduced by incubating the monolayers with the appropriate polyspecific anti-H-2 antisera. The inhibition observed appeared to be directly related to anti-H-2 antibody binding and was not merely a function of ligand binding to the cell surface, as antisera directed against other fibroblast cell surface antigens did not significantly inhibit the adhesive rate. These results indicate a role for the H-2 gene complex in modulating fibroblast-fibroblast intercellular adhesion.

We review experiments on the presence of strong transplantation antigens, H-2 antigens, in mouse gametes and early embryos. Parallels are drawn between antigenic structure of embryos and tumour cells, and experiments with a model tumour system, a teratoma, are presented indicating the presence in early embryos of surface antigens which are shared with the tumour.

We have used fluorescence photobleaching recovery to measure the diffusion of lipid probes (1,2-acyl-2-(N-4-nitro-benzo-2-oxo-1,3-diazole)aminocaproylphosphatidylcholine (NBD-PC)) and the 3,3′-diacylindocarbocyanine iodides (CndiI) with even acyl chain lengths (n = 10 to 18) in the plasma membranes of the sea urchin egg Strongylocentrotus purpuratus before and after fertilization. At 15°C measured diffusion coefficients, D, ranged from (1 to 5) × 10−9 cm2/sec, diffusing fractions from 36 to 77% for the CndI's. D shows a relative minimum at n = 12 for unfertilized eggs, a relative maximum at n = 14 for fertilized eggs. Thus the effect of fertilization on diffusion coefficient is dependent on probe structure. The fraction of molecules free to diffuse also depends upon acyl chain length; this fraction was smaller in fertilized eggs than in unfertilized eggs for all chain lengths. Our results indicate that different lipid analogs probe different microenvironments and suggest the existence of domains of lipids differing in composition or physical state from the average for the egg plasma membrane.

Four cell lines derived from four-day-old SWR/J♀×SJL/J♂ mouse blastocysts have been assayed for their expression of H-2 specificities with pauci- and monospecific H-2 typing sera. Direct microcytotoxicity and indirect absorption studies reveal many deviations from expected expression of particular H-2 specificities based on the cell lines' genotypes and onH-2 typing of adult F1 lymphocytes. No pattern of selective expression of public or private specificities ofD-end orK- end specificities or of inclusion groups was noted. At least one public or private specificity of eachD q ,K q ,D s , andK s region is present, indicating that part of each product is expressed. The partial expression of H-2 specificities is discussed structurally, in terms of how incomplete H-2 molecules may be present on the cell surface, and developmentally, in terms of how the variant H-2 specificities may be involved in cell positioning during ontogenesis.

A variety of genetic and biochemical evidence points to an association between major histocompatibility complex (MHC) haplotype and several types of cell surface receptors including epidermal growth factor and insulin receptors. We report evidence for such associations between human class I MHC antigens, HLA antigens, and specific insulin binding sites on human B lymphoblasts. We have measured insulin binding to cells of an HLA-heterozygous, Epstein-Barr virus-transformed B-cell line, LCL 721, and to derivative mutants from which all or part of the HLA complex had been deleted. The affinity, Ka, of insulin binding sites is approximately 10(8) M-1 in mutants expressing antigen HLA-B5 together with other HLA antigens and in mutants expressing only HLA-C. HLA-A1; HLA-A1,B8; HLA-A2,C; and HLA null mutants (not expressing any HLA antigens) bind insulin to sites with an affinity of approximately 10(9) M-1.

Single mouse intestinal epithelial cells (IEC) may be isolated by the use of a combination of methods used for the isolation of IEC from other species. Isolated cells remain viable for several hours. The membrane integral enzymes alkaline phosphatase and leucine aminopeptidase of isolated IEC are localized to the brush borders of IEC in tissue and in most newly isolated IEC. With time, both enzymes are found distributed over the entire cell surface. Redistribution appears to occur by diffusion in the plane of the membrane. It is slowed, but not blocked, if cells are maintained at 0 degrees C instead of at 37 degrees C, and it is not blocked by fixation in 0.5-3% paraformaldehyde. Drugs that alter cell membrane potential or that affect cell levels of ATP enhance the rate of redistribution of the enzymes.

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.

Site-directed mutagenesis in vitro was used to introduce stop codons in the genomic DNA of the alpha and beta chains of the murine class II major histocompatibility complex antigen, I-Ak. Mutated DNA was transfected into B lymphoma cells that were then selected by neomycin resistance and for their ability to express I-Ak molecules on their plasma membrane. The translational diffusion coefficient (Dlat) of I-Ak molecules composed of a wild-type beta chain paired with an alpha chain missing either 6 or 12 amino acids from the cytoplasmic domain is on the average threefold higher than the Dlat of wild-type I-Ak molecules as measured by fluorescence photobleaching and recovery. The removal of 12 amino acids from the cytoplasmic domain of the beta chain did not change the Dlat value from that of wild-type I-Ak if the truncated beta chain was paired with a wild-type alpha chain. Removing all amino acids of the cytoplasmic domains of both the alpha and beta chains resulted in a 10-fold increase in the Dlat, the highest value for any of the truncated I-Ak molecules tested. These data indicate that the carboxy-terminal six amino acids of the cytoplasmic domain of the alpha chain and the six plasma membrane-proximal amino acids of the beta chain are important in constraining the translational diffusion of I-Ak molecules in the plasma membrane.

Specifically absorbed rabbit antisera to a mouse testicular teratoma were used to study the expression of three heteroantigens on cell surfaces of early mouse embryos. This antiserum is known to define three antigens on the surface of murine tumor cells, though it does not bind to cells of normal adult tissue. Antigen I, physically associated with H-2 antigens in L-cell plasma membranes and found on many transplantable mouse tumors (7), is expressed by ova and morulae. Antigen I persists on cells of the inner-cell mass beyond implantation, but is not detected on trophoblast. In contrast, antigen II, found on teratoma and hepatoma cells, is absent from cleavage embryos and is first expressed on differentiation of the trophoblast prior to implantation. Antigen III, which is apparently teratoma specific, is absent from all embryonic material studied.

Steady-state level of liver 3′,5′-cyclic monophosphate, cAMP, has been shown to be under genetic control linked to the mouseH-2 complex. Liver cAMP levels are associated withH-2 haplotype in fully segregating crosses of strains C3H and C57BL/10. In crosses involving strain A, other loci have an effect that swamps that ofH-2. Results withH-2 recombinants indicate that liver cAMP levels are affected by more than oneH-2-linked locus.

Rabbit antisera to a mouse testicular teratoma, absorbed with normal mouse tissues, react by immunofluorescence with plasma membrane antigens of a variety of transplantable mouse tumor cells and transformed fibroblast cell lines including Clone 1D, SV-40-3T3, and 3T12. Trypsin treatment of cells of "normal" lines, 3T3 and FR-SV-3T3, uncovers reactivity on these as well. Early passage mouse embryo fibroblast cell cultures do not react even after trypsinization. By cross-absorbtion studies, the anti-teratoma serum appears to react with an antigen common to most tumor cells investigated thus far. When this antigen on Clone 1D cells is "capped," H-2 antigens collect with the teratoma antigens in the cap indicating a physical association between the molecules. Molecules specified by both the H-2D and H-2K regions are bound to the teratoma antigens in the Clone 1D plasma membrane. This antigen is also found in soluble tumor cell fractions where it is believed to be free of H-2. A second cell surface antigen defined by anti-teratoma serum is expressed only by hepatoma and teratoma itself. This second antigen is apparently a secretory product of teratoma cells. A third surface antigen defined by anti-teratoma serum appears to be specific for the teratoma.

Since murine teratocarcinomas and early embryos are known to share cell surface antigens, we investigated the possibility of maternal immune responses to normal pregnancy using teratocarcinoma-derived cell lines as targets. We found that an adherent cell population from both the spleen and peritoneum of syngeneically mated 129/SvSl pregnant females stimulated the uptake of [125I]iododeoxyuridine ( [125I]IUdR) by a teratocarcinoma-derived cell line, TerCs in vitro. Adherent cells from multiparous females did not stimulate the growth of other tumor cell lines. However, levels of natural anti-tumor activity detected in peritoneal cell populations of 129/SvSl virgin females were greatly reduced during pregnancy. Peritoneal cells from multiparous females with growth-stimulating activity were retained on nylon-wool columns and not eliminated by treatment with anti-theta antiserum and complement. Peritoneal cells from virgin females, treated with anti-theta antiserum and complement to eliminate cytotoxic lymphocytes, gained the ability to stimulate the uptake of [125I]IUdR by TerCs cells. [125I]IUdR uptake by cultured normal mouse blastocysts was significantly enhanced by peritoneal cells from multiparous females, while cells from age-matched virgin females had no effect. These results suggest that changes in immunocyte populations occur during pregnancy in the mouse; these changes could promote the growth of the embryo in utero.

Rabbit and mouse antisera prepared against teratocarcinoma cells precipitate both glycoproteins and glycolipids from detergent extracts of radiolabeled cells. Extracts of immunoprecipitates with chloroform/methanol, 2:1 (vol/vol) have been resolved on thin-layer gels into multiple peaks. There are more species seen in extracts of teratocarcinoma cells than in extracts of the crossreacting cultured cell line, cl 1d. The teratocarcinoma antigens may be extracted out of chloroform/methanol into buffered saline. Incubation in these secondary extracts converts unreactive cells (lymphocytes to cells reactive with antisera against teratocarcinoma. Furthermore, the coated cells absorb at least 80% of the activity of antisera against teratocarcinoma targets.

This review records evidence for diffusion of membrane components within membranes themselves. It is restricted to evaluation of the magnitude of these movements and to some consideration of the ways in which they may be speeded or hindered. However, it is appropriate to ant ic ipate w hat we may learn about their place in cell physiology. In the words of Robert Boyle: "There may be more accounts than we have yet thought of, upon which local motion may perform considerable things, either w ithout being much heeded, or wit hout seeming other than faint, at least in relation to t he considerableness of the effects produced by them" (1). In the following pages we shall attempt to documen t motions in the plane of the membrane, discussing numerical estimates of d iffusion in mem branes as well as less direct evidence for such diffusion.

We describe a method for the assay of adenyl cyclase in whole tissue homogenates. Adenosine 3′:5′-cyclic monophosphate (cAMP) formed from α-32P-, 14C- or 3H-labeled adenosine 5′-triphosphate (ATP) substrate is isolated from all known ATP metabolites and an unknown metabolite by electrophoresis in 1% sodium borate for 40 min, followed by overnight descending chromatography in 95% ethanol:1 m ammonium acetate (70:30). The purity of the cAMP isolated is established by chromatographic techniques as well as by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here also makes possible the measurement of phosphodiesterase activity in homogenates. It is rapid enough to allow routine assay of 180 samples per day, although the number of samples processed depends on the number of electrophoretic and chromatographic units available.

The mouse teratocarcinoma cell line 402AX is known to express surface antigens[hereafter called teratoma-defined antigen(s)] shared with early mouse embryos and some other murine cell lines, as detected by an absorbed rabbit antiserum. As gauged by immunofluorescence, human teratocarcinoma cells (tera 2) following neuraminidase treatment display antigen(s) cross-reactive with mouse teratoma-defined antigens. On the human tumor cells the teratoma-defined antigen(s) is density dependent, being present on subconfluent cultures, but absent on confluent cultures. Neuraminidase-treated control subconfluent human fibroblasts are nonreactive with the antiserum. Sera from some human patients presenting with teratocarcinoma, absorbed on normal mouse tissue to remove the nonspecific hetero-anti-species antibodies, show anti-mouse teratoma reactivity by immunofluorescence and microcytotoxicity. Sera from age- and sex-matched individuals with nonteratoma tumors are nonreactive with the mouse teratoma cells. The antigenic determinant(s) detected by the human teratoma sera co-caps with H-2 antigens on the mouse mastocytoma line P815, as do the rabbit antiserum-defined teratoma antigens. These data suggest that human and mouse teratomas share common antigens, and that these antigens may be integrally related to the major histocompatibility antigens in both species.

Cells from established tissue culture lines of mouse (cIID) and human (VA-2) origin were fused together with Sendai virus, producing heterokaryons bearing both mouse and human surface antigens which were then followed by the indirect fluorescent antibody method. Within 40 mm following fusion, total mixing of both parental antigens occurred in over 90% of the heterokaryons. Mouse H-2 (histocompatibility) and human surface antigens were visualized by successive treatment of the heterokaryons with a mixture of mouse alloantiserum and rabbit anti-VA-2 antiserum, followed by a mixture of fluorescein-labelled goat anti-mouse IgG and tetramethyl-rhodamine-labelled goat anti-rabbit IgG(Fc). The cIIDxVA-2 fusions were carried Out in suspension and maintained at 37°C in a shaking water bath; aliquots were removed at various intervals and stained with the above reagents. The heterokaryon population was observed to change from an initial one (5-min post-fusion) of non-mosaics (unmixed cell surfaces of red and green fluorescence) to one of over 90% mosaics (total intermixing of the 2 fluorochromes) by 40 min after fusion. Mouse-human hybrid lines, derived from similar fusions, gave fluorescence patterns identical to those of the mosaic heterokaryons. Four possible mechanisms would yield such results: (i) a very rapid metabolic turnover of the antigens; (ii) integration of units into the membrane from a cytoplasmic precursor pool; (iii) movement, or ‘diffusion’of antigen in the plane of the membrane; or (iv) movement of existing antigen from one membrane site into the cytoplasm and its emergence at a new position on the membrane. In an effort to distinguish among these possibilities, the following inhibitor treatments were carried out: (1) both short- and long-term (6-h pre-treatment) inhibition of protein synthesis by puromycin, cycloheximide, and chloramphenicol; (2) short-term inhibition of ATP formation by dinitrophenol (DNP) and NaF; (3) short- and long-term inhibition of glutamine dependent pathways with the glutamine analogue 6-diazo-5-oxonorleucine; and (4) general metabolic suppression by lowered temperature. The only treatment found effective in preventing the mosaicism was lowered temperature, from which resulted a sigmoidal curve for per cent mosaics versus incubation temperature. These results would be consistent with mechanisms iii and/or iv but appear to rule out i and ii. From the speed with which the antigen markers can be seen to propagate across the cell membrane, and from the fact that the treatment of parent cells with a variety of metabolic inhibitors does not inhibit antigen spreading, it appears that the cell surface of heterokaryons is not a rigid structure, but is ‘fluid’ enough to allow free ‘diffusion’ of surface antigens resulting in their intermingling within minutes after the initiation of fusion.

Treatment with chelating agents binding divalent cations has been found to effect the dissociation of a variety of tissues of both embryo and adult animals (reviewed in Steinberg, 1958). In the course of dissociation it appears that materials are released from cell surfaces which play a part in their specific adhesion, and which may be shown experimentally to promote selectively the re-aggregation of dissociated cells (Humphreys, 1963; Moscona, 1963). The extracted materials appear to be glycoprotein complexes (Humphreys, 1965), made up of fairly small subunits, estimated to be of 13000-20000 molecular weight (Margoliash et al. 1965). Units of about the same size appear to be the antigenic sites involved in the blocking of sponge cell aggregation by rabbit anti-sponge serum, specific for a given sponge species (MacLennan, 1963). I shall here present evidence that materials of similar molecular weight bearing immunological specificities of the H-2 alloantigen system are released from the tissues of certain mouse embryos during the course of their dissociation by the chelating agent Versene (ethylenediaminetetraacetic acid). Antigens of the H-2 system function as transplantation antigens determining the fate of allogeneic homografts, as immunogens stimulating the production of circulating antibody, and as antigens of erythrocytes and leucocytes reacting with antibody (Stimpfling & Snell, 1961; Snell, Hoecker, Amos & Stimpfling, 1964). The antigens extracted by Versene are most conveniently detected by their ability to inhibit the cytotoxic action of antibody produced by tissue allografts made between mouse strains differing at the H-2 locus. The potency of the antigens extracted is thus expressed in terms of their inhibition of a standard cytotoxic alloantiserum. This technique allows determination of very small amounts of material and it avoids the problems of immunization and quantitative scoring of skin graft survival, which are encountered when H-2

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23 degrees -25 degrees C than at 0 degrees -4 degrees C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1-3 x 10(-9) cm(2) s(-1) can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.

Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited.

Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.

We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and alpha-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in alpha-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for alpha-spectrin-deficient and normal MEL cells, differing by a factor of approximately 2, on three different timescales: tens of seconds, 1-10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the alpha-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.

Cooling populations of newly formed mouse human heterokaryons has effects on the intermixing of mouse and human surface antigens which indicate the occurrence of phase separations in membrane lipids. Antigen mixing, previously shown to be due to diffusion in the plane of the membrane, is retarded when cells are cooled from 37 degrees to 21 degrees C, but is then speeded by further cooling to 15 degrees C. This result is in accord with observations on phase separations of lipids in artificial and bacterial membranes.

To consider the effect of surface microvilli on measurements of lateral diffusion by fluorescence photobleaching recovery, we have measured the diffusion of the lipid probe 3,3'-dihexadecyl indocarbocyanine iodide on the villated main body and unvillated budding polar body of unfertilized mouse eggs. On the main body we found D = (6.41 +/- 0.62) x 10(-9) cm(2)/s with (77.0 +/- 2.1)% recovery, and on the budding polar body we found D = (7.05 +/- 0.75) x 10(-9) cm(2)/s with (84.7 +/- 1.3)% recovery. We thus find only slight differences in diffusion in the two regions.

We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.

The binding of epidermal growth factor (EGF) by A-431 human epidermoid carcinoma cells was reduced after exposure of the cells to low concentrations (0.01-1 mM) of ATP and other nucleoside 5'-triphosphates at 37 degrees C, but not at 0 degree C. This was due to loss of high-affinity EGF binding sites. The modulation was associated with transient increases in inositol phosphate synthesis and intracellular Ca2+ and with phosphorylation of the EGF receptor on serine and threonine. There was no evidence for entry of labeled ATP into the cells. ATP appeared to bind to specific cell surface receptors. Such binding was demonstrated directly with the nonmetabolizable ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate.

Derivatives of mouse L cells, cl-ld and LM, are capable of self- or cis-glycosylation. This reaction appears to require interaction of transferase and acceptor by collision in the course of diffusion in the plane of the membrane. The effect of temperature on glycosylation indicates this may well be the case since anomalies in the rate of the reaction, unexpectedly high rates, are found at temperatures below 20 °C. The anomalies are similar to those previously found for lateral diffusion of surface antigens in these cells. Such anomalies are not found for the transfer of galactose by the cells' enzymes to a water-soluble acceptor.

The murine teratocarcinoma-derived cell line TerC, previously thought to lack products of the major histocompatibility locus H-2, was shown to express low levels of K- and D-end H-2 specificities with the use of a sensitive cytotoxic assay. The assay, based on the ability of antibody and complement to inhibit uptake of the thymidine analogue [125I]5-iodo-2'-deoxyuridine, detected appropriate public and private specificities, as demonstrated with the use of oligospecific antisera and by absorption analyses. A series of clone of TerC varied only slightly in H-2 expression, and there was no tendency for expression to increase with time in culture; thus the low levels of H-2 were not the result of a differentiating subpopulation.

Lateral diffusion of receptors binding fluorescein labeled concanavalin A and its succinylated derivative has been measured by bleaching portions of the labeled surface and following return of fluorescence to the bleached spot. Binding of either concanavalin A or its succinylated derivative causes restriction of mobility of the surface receptors for this lectin. The degree of restriction is a function of time after binding the lectin.