A user's guide for characterizing plasma membrane subdomains in living cells by spot variation fluorescence correlation spectroscopy.

Due to the intrinsic molecular Brownian agitation within plasma membrane and the vast diversity of membrane components, it is expected that the plasma membrane organization is highly heterogeneous with the formation of local complex multicomponent assemblies of lipids and proteins on different time scales. Still, deciphering this lateral organization on living cells and on the appropriate length and temporal scales has been challenging but is crucial to advance our knowledge on the biological function of the plasma membrane. Among the methodological developments based on biophotonics, the spot variation FCS (svFCS), a fluorescent correlation spectroscopy (FCS)-based method, has allowed the significant progress in the characterization of cell membrane lateral organization at the suboptical level, including to providing compelling evidence for the in vivo existence of lipid-dependent nanodomains. The aim of this chapter is to serve as a guide for setting and applying the svFCS methodology to study the plasma membrane of both adherent and nonadherent cell types.

H-Ras transfers from B to T cells via tunneling nanotubes.

Lymphocytes form cell-cell connections by various mechanisms, including intercellular networks through actin-supported long-range plasma membrane (PM) extensions, termed tunneling nanotubes (TNTs). In this study, we tested in vitro whether TNTs form between human antigen-presenting B cells and T cells following cell contact and whether they enable the transfer of PM-associated proteins, such as green fluorescent protein (GFP)-tagged H-Ras (GFP-H-Ras). To address this question, we employed advanced techniques, including cell trapping by optical tweezers and live-cell imaging by 4D spinning-disk confocal microscopy. First, we showed that TNTs can form after optically trapped conjugated B and T cells are being pulled apart. Next, we determined by measuring fluorescence recovery after photobleaching that GFP-H-Ras diffuses freely in the membrane of TNTs that form spontaneously between B and T cells during coculturing. Importantly, by 4D time-lapse imaging, we showed that GFP-H-Ras-enriched PM patches accumulate at the junction between TNTs and the T-cell body and subsequently transfer to the T-cell surface. Furthermore, the PM patches adopted by T cells were enriched for another B-cell-derived transmembrane receptor, CD86. As predicted, the capacity of GFP-H-Ras to transfer between B and T cells, during coculturing, was dependent on its normal post-transcriptional lipidation and consequent PM anchorage. In summary, our data indicate that TNTs connecting B and T cells provide a hitherto undescribed route for the transfer of PM patches containing, for example, H-Ras from B to T cells.

Palmitoylation of human FasL modulates its cell death-inducing function.

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas-FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.

The extracellular glycosphingolipid-binding motif of Fas defines its internalization route, mode and outcome of signals upon activation by ligand.

Selective compartmentalization and internalization have been shown as a means for regulating specific signals of cell surface receptors to correspond to cellular requirements and conditions. Here, we present a conserved extracellular glycosphingolipid-binding motif of Fas as one of the regulatory elements in the selection of its internalization route and consequently the signals transmitted upon ligand binding. This motif is required for clathrin-mediated internalization of Fas, which allows the transduction of its cell death signal. The loss of function of the motif drives the activated receptor to an alternative internalization route that is independent of clathrin and cholesterol-dependent rafts but dependent on ezrin, and thereby extinguishing its cell death signal while promoting its non-death functions. Through biochemical, biophysical, and genetic approaches, we present a protein/lipid-based mechanism as a key to the versatility of the signal transduction by the multifunctional Fas receptor-ligand system.

Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter.

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine.

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.

Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC ), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26(-/-) mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26(-/-), mice. This inhibitor also improved glucose tolerance in GLP-1 receptor(-/-) mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

Lateral diffusion of GFP-tagged H2Ld molecules and of GFP-TAP1 reports on the assembly and retention of these molecules in the endoplasmic reticulum.

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.

Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses.

T cell receptors (TCR) and major histocompatibility complex (MHC) molecules are integral membrane proteins that have central roles in cell-mediated immune recognition. Therefore, soluble analogs of these molecules would be useful for analyzing and possibly modulating antigen-specific immune responses. However, due to the intrinsic low-affinity and inherent solubility problems, it has been difficult to produce soluble high-affinity analogs of TCR and class II MHC molecules. This report describes a general approach which solves this intrinsic low-affinity by constructing soluble divalent analogs using IgG as a molecular scaffold. The divalent nature of the complexes increases the avidity of the chimeric molecules for cognate ligands. The generality of this approach was studied by making soluble divalent analogs of two different classes of proteins, a TCR (2C TCR2Ig) and a class II MHC (MCCI-Ek2Ig) molecule. Direct flow cytometry assays demonstrate that the divalent 2C TCR2Ig chimera retained the specificity of the native 2C TCR, while displaying increased avidity for cognate peptide/MHC ligands, resulting in a high-affinity probe capable of detecting interactions that heretofore have only been detected using surface plasmon resonance. TCR2IgG was also used in immunofluorescence studies to show ER localization of intracellular peptide-MHC complexes after peptide feeding. MCCI-Ek2Ig chimeras were able to both stain and activate an MCC-specific T cell hybridoma. Construction and expression of these two diverse heterodimers demonstrate the generality of this approach. Furthermore, the increased avidity of these soluble divalent proteins makes these chimeric molecules potentially useful in clinical settings for probing and modulating in vivo cellular responses.

Thymocytes in Thy-1-/- mice show augmented TCR signaling and impaired differentiation.

Thy-1, a single variable-like immunoglobulin superfamily domain anchored in the plasma membrane by a glycosyl phosphaditylinositol tail [1], is a major surface glycoprotein in adult mammalian neurons and rodent thymocytes [2]; the function of Thy-1 has remained enigmatic since its discovery [3]. Studies in vitro have implicated Thy-1 in homotypic and heterotypic cell-cell interactions [2,4]. Ligation of Thy-1 initiates transmembrane signaling pathways that lead to diverse physiological outcomes in different cells [2,5-7]. In rodents, Thy-1 is highly expressed on the surface of CD4+CD8+ double-positive immature thymocytes and downregulated in mature T cells. Here, we report that thymocytes from Thy-1-/- mice [8] had altered cell-cell contacts, and hyperresponsiveness to T-cell receptor (TCR) triggering as demonstrated by the heightened activation of p56lck, phosphorylation of TCR subunits, Ca2+ fluxes and cell proliferation. Thy-1-/- thymocytes exhibited impaired maturation from the double positive to single positive stage of thymocyte development, possibly due to inappropriate negative selection, and were prone to T lymphomas in aged mice. These observations indicate that Thy-1 negatively regulates TCR-mediated signaling and controls activation thresholds during thymocyte differentiation.

CD10 plays a specific role in early thymic development.

Development of T lymphocyte is a complex process that depends on both thymocytestromal cell interactions and the production of soluble factors such as cytokines, peptides, and hormones. In many tissues, the concentration of active biological peptides is regulated locally by a specialized family of enzymes: the ectopeptidases. We show here that treatment of fetal thymic organ cultures (FTOC) with the specific CD10 (endopeptidase 24.11) inhibitors SCH 32615: (N-[L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine), RB25: (N-(3-[(hydroaxyamino)carbonyl]-2-benzylidene-1-oxopropyl]-N-glyci ne), and thymopentin (TP5) results in the inhibition of thymocyte differentiation. Each agent induces a significant decrease in the number of double positive (CD4+CD8+) cells in favor of the TN (TcR alpha beta-CD4-CD8-) population. RB25 also blocks T lymphocyte differentiation in FTOC when preinjected into pregnant mice. Finally, RB25 and TP5 were also shown to reduce the number of CD44+CD25- and CD44-CD25- thymocytes both in vitro and after preinjection in vivo in day 2 FTOC. Thus, agents that affect endopeptidase 24.11 activity impair T cell development both in vitro and in vivo. Our results show that the CD10 molecule plays a specific role in promoting early T cell development.

Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases.

NK cells express cell surface receptors for MHC class I proteins (KIR). Engagement of these receptors inhibits NK cell cytotoxic programs. KIR can be expressed on T cells, and their engagement also results in inhibition of effector functions initiated by the CD3/TCR complex. While human KIR genes belong to the Ig gene superfamily, mouse KIR belong to a family of dimeric lectins. Despite these distinct evolutionary origins, we show here that both HLA-Cw3-specific human p58.183 receptors and H-2D d/k-specific mouse Ly49A receptors recruit the same protein tyrosine phosphatases, PTP1C and PTP1D, upon phosphorylation of critical intracytoplasmic tyrosine residues. These results document a common pathway by which diverse KIR can down-regulate NK and T cell activation programs, and further define the sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM), initially described in FcgammaRIIB1, and expressed in both human and mouse KIR.

Reduced cell surface expression of a mutated dipeptidyl peptidase IV (DPP IV/CD26) correlates with the generation of a beta strand in its C-terminal domain.

Dipeptidyl peptidase IV (DPP IV/CD26) belongs to a non-classical subfamily of serine-proteases. Sequence comparisons have identified Asp599, Ser624, Asp657, Asp702, and His734 as highly conserved residues of mouse DPP IV. We previously reported the identification of Ser624, Asp702 and His734 as the catalytic triad of mouse DPP IV (David, F., Bernard, A. M., Pierres, M., and Marguet, D. (1993) J Biol. Chem. 268, 17247-17252). Using site-directed mutagenesis, we have shown here that substitution of Asp599 for Ala (D599A) specifically decreases the cell-surface expression of DPP IV in stably transfected mouse fibroblasts. The D599A mutant remained as a high mannose immature glycoprotein and was rapidly degraded. This retention/degradation process correlates with the generation of a beta strand in the C-terminal region of DPP IV as shown by three dimensional computer modeling. Our results suggest that conserved residue Asp599 is important for the proper folding, glycosylation and transport of mouse DPP IV.

Structure of the mouse dipeptidyl peptidase IV (CD26) gene.

Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is an ectopeptidase whose expression is modulated during thymocyte differentiation and T cell activation. We describe here the organization of the mouse DPP IV gene. This gene, which encompasses more than 90 kb, is composed of 26 exons separated by introns, the lengths of which vary from 100 bp to more than 20 kb. Reverse PCR performed on RNA from different tissues indicated that DPP IV transcripts do not contain alternatively spliced CDS sequences and, therefore, are supposed to yield a single polypeptide. However, two types of specific mRNA have been detected that differ in their 3'UTR sequences. They derive from alternative polyadenylation of the DPP IV primary transcript, since the different 3'UTR sequences are contiguous in the mouse DPP IV gene. Sequence analysis of the gene 5'-flanking region revealed several structural features found in the TATAA-box-less promoters, including a G+C-rich segment, a high frequency of dinucleotide CpG, and an imperfect symmetrical dyad. The DPP IV gene was assigned by in situ hybridization to the mouse [2C2-2D] region, which is syntenic with human chromosome 2. These data indicate that the human Dpp4 locus is located within this synteny region (i.e., 2q14-q37). The genomic organization of the mouse DPP IV gene is compared to that of classical serine proteases and serine hydrolases. As structural and mechanistic conservation in the absence of sequence similarity is the most remarkable feature among alpha/beta hydrolases [Ollis, D. L., et al. (1992) Protein Eng. 5, 197-211], we report the possible evolutionary link between the DPP IV related family and alpha/beta hydrolases.

Factors involved in entry of the human immunodeficiency virus type 1 into permissive cells: lack of evidence of a role for CD26.

It has been proposed recently that the cell surface peptidase CD26 acts in concert with CD4, the human immunodeficiency virus (HIV) primary receptor molecule, to mediate HIV entry into permissive cells. We have failed to detect significant levels of CD26 cell surface expression and enzymatic activity in a number of commonly propagated human CD4+ cell lines, although CD26 mRNA was present at very low levels, as detected by reverse transcription PCR. No relationship existed between the expression of CD26 and the ability of these cells to be infected with HIV or to fuse to form syncytia. We have tested two inhibitors of CD26 enzymatic activity and several anti-CD26 monoclonal antibodies and found that they inhibit neither HIV infection nor HIV-induced syncytium formation. NIH 3T3 cells stably transfected with the cDNAs for human CD4 and CD26 expressed these molecules at the cell surface and had CD26 enzymatic activity. Inoculation of the double transfectants with HIV did not result in virus entry above the background level, as verified by PCR amplification of viral DNA. We were unable to recover infectious virus from the HIV-inoculated NIH 3T3 double transfectants either by transfer of supernatants or by cocultivation with human CD4+ indicator cells. Moreover, the transfectants did not fuse with HIV-infected cells to form syncytia, nor were syncytia observed in HIV-inoculated cultures. These results are inconsistent with the CD26 molecule being a cofactor for entry of HIV in CD4+ cells.

Identification of serine 624, aspartic acid 702, and histidine 734 as the catalytic triad residues of mouse dipeptidyl-peptidase IV (CD26). A member of a novel family of nonclassical serine hydrolases.

Dipeptidyl-peptidase IV (DPP IV, CD26, EC 3.4.14.5), a multifunctional ectoenzyme, is involved not only in the proteolytic cleavage of X-Pro from the NH2 terminus of a variety of biologically active peptides, but also in activation signal transduction and cell matrix adherence processes. We recently characterized mouse DPP IV cDNA and identified the serine protease Gly-X-Ser-X-Gly consensus motif in its extracellular domain. Mouse DPP IV does not exhibit sequence similarity with any of the classical members of this enzyme family (e.g. chymotrypsin and subtilisin) but shares a conserved structural domain of approximately 200 amino acids with several nonclassical serine hydrolases. In this study, analysis of the similarity of secondary structures and amino acid sequences between these enzymes led us to identify several conserved residues likely to be involved in the catalytic site of these DPP IV-related enzymes. These amino acids (Ser624, Asp702, and His734) were found to be arranged in a novel sequential order as compared with that of archetypal serine proteases (e.g. nucleophile (Ser)-acid-His versus His-acid-nucleophile (Ser), respectively). To directly explore the involvement of these residues in the catalytic function of these enzymes, we performed in vitro site-directed mutagenesis on mouse DPP IV cDNA. Our results indicate that although conservative or non-conservative permutations at these positions do not significantly alter the surface expression and biochemical properties of the mutant molecules, they completely impair their DPP IV enzymatic function. In contrast, mutagenesis of two other aspartic residues (Asp599 and Asp657), also conserved between these DPP IV-related enzymes, did not affect the enzymatic properties of the mouse enzyme. These data provide evidence that DPP IV and its related enzymes belong to a novel family that displays a catalytic triad distinct from that of the classical serine proteases.