Mobility of surface proteins on normal rat macrophages and on a "macrophagelike" rat tumor

Peritoneal macrophages endocytosed their histocompatibility antigens (RT1), Fc receptors (FcR), and concanavalin A (Con A) receptors after cross-linking by ligands, but did not cap these membrane proteins. The 323N cell, a "macrophage like" tumor cell, under identical conditions capped its surface proteins. Experiments measuring fluorescence recovery after photobleaching showed that the mobile fraction of RT1 was significantly greater in 323N cells than in normal peritoneal macrophages. Presumably, the membrane proteins of 323N are not as tethered to the cytoskeleton, or, if so, are in a nexus that is not the same as that which occurs between membrane proteins of normal macrophages and the cytoskeleton. The mobility of RT1 on normal lymphocytes was also different from that of macrophages. These observations suggest that the movement of membrane molecules is determined by cell type and is regulated by the cytoskeleton which varies in structure and function from cell type to cell type.     

Differential HIV-1 Endocytosis and Susceptibility to Virus Infection in Human Macrophages Correlate with Cell Activation Status

HIV-1 is an enveloped virus that enters target cells by fusion either directly at the plasma membrane or at the endosomal membrane. The latter mechanism follows a rapid engulfment of HIV-1 after its receptor engagement at the cell surface, and its scale depends on cellular endocytosis/degradation rates and virus fusion kinetics. HIV-1 has recently been shown to exploit a novel Pak1-dependent macropinocytosis mechanism as a way to productively infect macrophages. However, macrophages are highly heterogeneous cells that can adapt functionally to their changing environment, and their endosomal/lysosomal pathway is highly regulated upon cell activation. These changes might impact the ability of HIV-1 to exploit endocytosis as a way to productively infect macrophages. In this study, we compared HIV-1 endocytosis/degradation rates in nonactivated, M1-activated, and M2a-activated monocyte-derived macrophages (MDMs). We found that the rate of HIV-1 endocytosis was increased in M1-activated but decreased in M2a-activated MDMs. However, both M1 and M2a activations of MDMs led specifically to a greater clathrin-mediated endocytosis of HIV-1, which was independent of CD4 and CCR5 binding. Furthermore, clathrin-mediated endocytosis is unlikely to result in productive HIV-1 infection, given that it leads to increased viral degradation. Therefore, we suggest that viral fusion following endocytosis is restricted in activated macrophages.     

Hydrodynamics of a permeable patch in the fluid membrane.

A patch of cross-linked proteins in the fluid membrane is considered for the case in which the patch is permeable (porous) for the lipid flow in the membrane. The Bretscher flow field is studied quantitatively and the distribution of Brownian particles over the surface of the cell is given. This leads to a simple quantitative criterion for cap formation. Finally, explicit expressions for the rotational and translational diffusion coefficients of a permeable patch, as calculated from hydrodynamics, are given.     

Transmembrane interactions and the mechanisms of transport of proteins across membranes

We have made observations, by double fluorescence staining of the same cell, of the distributions of surface receptors, and of intracellular actin and myosin, on cultured normal fibroblasts and other flat cells, and on lymphocytes and other rounded cells. The binding of multivalent ligands (a lectin or specific antibodies) to a cell surface receptor on flat cells clusters the cell receptors into small patches, which line up directly over the actin- and myosin-containing stress fibers inside the cell. Similar ligands binding to rounded cells can cause their surface receptors to be collected into caps on the surface, and these caps are invariably found to be associated with concentrations of actin and myosin under the capped membrane. Although these ligand-induced surface phenomena appear to be different on flat and rounded cells, we propose that in both cases clusters of receptors become linked across the membrane to actin- and myosin-containing structures. In flat cells these structures are very long stress fibers; therefore, when clusters of receptors become linked to these fibers, the clusters are immobilized. In round cells, membrane-associated actin- and myosin-containing structures are apparently much less extensive than in flat cells; therefore, clusters of receptors linked to these structures are still mobile in the plane of the membrane. We suggest that in this case the clusters are then actively collected into a cap by an analogue of the muscle sliding filament mechanism. To explain the transmembrane linkage, we propose that actin is associated with the plasma membrane as a peripheral protein which is directly or indirectly bound to an integral protein (or proteins) X of the membrane. Individual molecules of any receptor are not bound to X, but after they are specifically clustered into patches, a patch of receptors then becomes bound to S and hence to actin/myosin. Patching and capping of specific receptors on rounded cells is often accompanied by a specific endocytosis of the ligand-receptor complexes. This represents one common transport mechanism of a protein (the ligand) across the plasma membrane. The possibility is discussed that this type of endocytosis is mediated by a transmembrane linkage of the clustered receptor to actin/myosin. Another mechanism of endocytosis involves the “coated pit” structures that are observed by electron microscopy of plasma membranes. The possible relationships between an actin/myosin and a coated pit mechanism of endocytosis are explored.     

Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytoskeleton, and protein kinases

GPI-anchored surface proteins mediate many important functions, including transport, signal transduction, adhesion, and protection against complement. They cluster into glycolipid-based membrane domains and caveolae, plasmalemmal vesicles involved in the transcytosis and endocytosis of these surface proteins. However, in lymphocytes, neither the characteristic flask shaped caveolae nor caveolin, a transmembrane protein typical of caveolae, have been observed. Here, we show that the GPI-anchored CD59 molecule on Jurkat T cells is internalized after cross-linking, a process inhibited by nystatin, a sterol chelating agent. Clustered CD59 molecules mostly accumulate in non-coated invaginations of the lymphocyte membrane before endocytosis, in marked contrast with the pattern of CD3-TCR internalization. Cytochalasin H blocked CD59 internalization in lymphocytes, but neither CD3 internalization nor transferrin uptake. Confocal microscopy analysis of F-actin distribution within lymphocytes showed that CD59 clusters were associated with patches of polymerized actin. Also, we found that internalization of CD59 was prevented by the protein kinase C inhibitor staurosporine and by the protein kinase A activator forskolin. Thus, in lymphocytes, as in other cell types, glycolipid-based domains provide sites of integration of signaling pathways involved in GPI-anchored protein endocytosis. This process, which is regulated by both protein kinase C and A activity, is tightly controlled by the dynamic organization of actin cytoskeleton, and may be critical for polarized contacts of circulating cells.     

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.     

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway.

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.     

Lateral mobility and capping of rat lymphocyte membrane proteins

Surface immunoglobulin (SIg), RT1 (rat histocompatibility proteins) and thy-1 present on rat lymphocytes cap with similar kinetics and to the same extent, the only difference being the antibody requirement. SIg requires one cross-linking antibody to induce capping, while RT1 and thy-1 require two antibodies. Fluorescence recovery after photobleaching experiments show that there is heterogeneity in the diffusion characteristics of lymphocyte membrane proteins. SIg is relatively immobile when labeled by Fab' and is immobilized by F(ab')2. RT1 diffuses 2.7 times as fast as SIg and the mobile fraction of RT1 is greater. When RT1 is cross-linked by relatively low concentrations of F(ab')2 its mobility is about the same as that of SIg. When the concentration of F(ab')2 is increased, RT1 is immobilized. Thy-1 diffuses faster than SIg and has a higher mobile fraction. When thy-1 is labeled with F(ab')2 it remains mobile and is immobilized when a second antibody layer is added. The relative order of mobility is thy-1 much greater than RT1 greater than SIg.     

Fas Death Receptor Enhances Endocytic Membrane Traffic Converging into the Golgi Region

The death receptor Fas/CD95 initiates apoptosis by engaging diverse cellular organelles including endosomes. The link between Fas signaling and membrane traffic has remained unclear, in part because it may differ in diverse cell types. After a systematic investigation of all known pathways of endocytosis, we have clarified that Fas activation opens clathrin-independent portals in mature T cells. These portals drive rapid internalization of surface proteins such as CD59 and depend upon actin-regulating Rho GTPases, especially CDC42. Fas-enhanced membrane traffic invariably produces an accumulation of endocytic membranes around the Golgi apparatus, in which recycling endosomes concentrate. This peri-Golgi polarization has been documented by colocalization analysis of various membrane markers and applies also to active caspases associated with internalized receptor complexes. Hence, T lymphocytes show a diversion in the traffic of endocytic membranes after Fas stimulation that seems to resemble the polarization of membrane traffic after their activation.     

An association of 27- and 40-kDa molecules with glycolipids that bind A-B bacterial enterotoxins to cultured cells

It is well recognized that the Shiga-like toxins (Stxs) preferentially bind to Gb3 glycolipids and the cholera toxin (CT) and heat-labile enterotoxin (LTp) bind to GM1 gangliosides. After binding to the cell surface, A-B bacterial enterotoxins have to be internalized by endocytosis. The transport of the toxin-glycolipid complex has been documented in several manners but the actual mechanisms are yet to be clarified. We applied a heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3′-dithiopropionate (SASD), to detect the membrane proteins involved in the binding and the transport of A-B bacterial enterotoxins in cultured cells. Both Stx1 and Stx2 bound to the detergent-insoluble microdomain (DIM) of Vero cells and Caco-2 cells, which were susceptible to the toxin, but neither was bound to insusceptible CHO-K1 cells. Both CT and LTp bound to the DIM of Vero cells, Caco-2 cells, and CHO-K1 cells. In a cross-linking experiment, Stx1 cross-linked only with a 27-kDa molecule, while Stx2, which was more potently toxic than Stx1, cross-linked with 27- and 40-kDa molecules of Vero cells as well as of Caco-2 cells; moreover, no molecules were cross-linked with the insusceptible CHO-K1 cells. LTp was cross-linked only to the 27-kDa molecule of these three cell types but the CT, which was more toxic than LTp, was also cross-linked with 27- and 40-kDa molecules of Vero cells, Caco-2 cells, and CHO-K1 cells. The 27- and the 40-kDa molecules might play a role in the endocytosis and retrograde transport of A-B bacterial enterotoxins.     

Interaction of a limited set of proteins with different mRNAs and protection of 5''-caps against pyrophosphatase digestion in initiation complexes.

A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.     

The 'lipid raft' microdomain proteins reggie-1 and reggie-2 (flotillins) are scaffolds for protein interaction and signalling

Reggie-1 and reggie-2 are two evolutionarily highly conserved proteins which are up-regulated in retinal ganglion cells during regeneration of lesioned axons in the goldfish optic nerve. They are located at the cytoplasmic face of the plasma membrane and are considered to be 'lipid raft' constituents due to their insolubility in Triton X-100 and presence in the 'floating fractions'; hence they were independently named flotillins. According to our current view, the reggies subserve functions as protein scaffolds which form microdomains in neurons, lymphocytes and many other cell types across species as distant as flies and humans. These microdomains are of a surprisingly constant size of less than or equal to 0.1 mm in all cell types, whereas the distance between them is variable. The microdomains co-ordinate signal transduction of specific cell-surface proteins and especially of GPI (glycosylphosphatidylinositol)-anchored proteins into the cell, as is demonstrated for PrP(c) (cellular prion protein) in T-lymphocytes. These cells possess a pre-formed reggie cap scaffold consisting of densely packed reggie microdomains. PrP(c) is targeted to the lymphocyte reggie cap when activated by antibody cross-linking, and induces a distinct Ca(2+) signal. In developing zebrafish, reggies become concentrated in neurons and axon tracts, and their absence, after morpholino antisense RNA-knockdown, results in deformed embryos with reduced brains. Likewise, defects in Drosophila eye morphogenesis occur upon reggie overexpression in mutant flies. The defects observed in the organism, as well as in single cells in culture, indicate a morphogenetic function of the reggies, with emphasis on the nervous system. This complies with their role as scaffolds for the formation of multiprotein complexes involved in signalling across the plasma membrane.     

EH proteins

Endocytosis is a protein and lipid-trafficking pathway that occurs in all eukaryotic cells. It involves the internalization of plasma membrane proteins and lipids into the cell and the subsequent degradation of proteins in the lysosome or the recycling of proteins and lipids back to the plasma membrane. Over the past decade, studies in yeast and mammalian cells have revealed endocytosis to be a very complex molecular process that depends on regulated interactions between a variety of proteins and lipids. The Eps15 homology (EH) domain is a conserved, modular protein-interaction domain found in several endocytosis proteins. EH proteins can function as key regulators of endocytosis through their ability to interact with many of the other proteins involved in this process.     

Spatially regulated recruitment of clathrin to the plasma membrane during capping and cell translocation

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.     

Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. I. Relationship to cytoplasmic myosin

A detailed kinetic analysis of the distribution of cytoplasmic myosin during the capping of various lymphocytic surface molecules revealed two distinct capping mechanisms. (a) Some cell surface molecules, including immunoglobulin, Fc receptor, and thymus leukemia antigen, all cap spontaneously in a small fraction of lymphocytes during locomotion. Cytoplasmic myosin becomes concentrated in the cytoplasm underlying these spontaneous caps. Exposure to specific antibodies causes all three of these surface molecules to cap rapidly with a concomitant redistribution of cytoplasmic myosin to the area of the cap. These antibodies also stimulate cell locomotion. (b) Other lymphocyte surface molecules, including H2 and Thy.1, do not cap spontaneously. Moreover, exposure to antibodies to these molecules causes them to cap slowly without a redistribution of cytoplasmic myosin or stimulation of cell locomotion. Exposure to concanavalin A gives a response intermediate between these two extremes. We believe that the first type of capping is active and may involve a direct link between the surface molecules and the cytoplasmic contractile apparatus. The second type of capping appears to result simply from aggregation of cross-linked molecules in the plane of the membrane.     

Basolateral Internalization of GPI-anchored Proteins Occurs via a Clathrin-independent Flotillin-dependent Pathway in Polarized Hepatic Cells

In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2–positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.