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S J Singer

Publications and source records attributed to S J Singer.

At least 55 records · Page 3Linked to original sources
PubMed1989

The specific interaction of helper T cells and antigen-presenting B cells. IV. Membrane and cytoskeletal reorganizations in the bound T cell as a function of antigen dose.

We have used double-immunofluorescence labeling to determine the surface distributions of LFA-1 and CD4, and the intracellular distributions of the cytoskeletal protein talin and of the microtubule organizing center (MTOC) of cloned Th cells in 1:1 cell couples with antigen (Ag)-specific APC of the B cell type (B-APC). The Th cell was directed to a peptide fragment of the Ag OVA in the context of IAd. The B-APC was the transfected A20 B hybridoma cell A20-HL, bearing on its surface a surface Ig specific for the hapten TNP, and pulsed with different concentrations of DNP-OVA. At sufficiently high doses of DNP-OVA (greater than 100 ng/ml), in essentially all couples, LFA-1, CD4, and talin were each concentrated at the Th cell membrane where it was in contact with the B-APC, and the MTOC inside the Th cell was reoriented to face the contact region. At lower doses of DNP-OVA (between 50 and 10 ng/ml), in all couples, LFA-1 and talin were concentrated at the Th/B-APC contact region, but the extent of CD4 clustering, MTOC reorientation, and Th cell proliferation all decreased with decreasing Ag dose. With no Ag, none of these effects was observed. These and other data indicate that two distinct signals are received by the Th cell that is specifically bound to its B-APC. The first signal, at low Ag doses, stimulates a linkage of LFA-1 and talin in the Th cell, and a specific LFA-1-mediated intercellular adhesion; the second signal, at higher Ag doses, is required to induce Th cell proliferation, with which the Th-MTOC reorientation and CD4 clustering are correlated.

Animals↗
PubMed1989

Identification of a developmentally regulated protein-tyrosine kinase by using anti-phosphotyrosine antibodies to screen a cDNA expression library.

To identify the protein-tyrosine kinases that are expressed during chicken embryonic development, a 10-day chicken embryo cDNA expression library was screened with anti-phosphotyrosine antibodies. Of the positive clones isolated, many encoded the same protein-tyrosine kinase, which we designate Cek1 (chicken embryo kinase 1). Its amino acid sequence suggests that the Cek1 protein is a transmembrane tyrosine kinase and presumably the receptor for an unknown ligand. Antibodies prepared to the cloned Cek1 kinase recognize, in immunoblotting experiments, two protein bands with apparent molecular weights of 100,000 and 110,000. The Cek1 protein was detected in many chicken embryonic tissues, but not in the corresponding adult tissues (with the exception of brain). The Cek1 kinase appears to be very closely related to two protein-tyrosine kinases whose partial sequences have been recently determined, human Flg and mouse Bek. Cloning using anti-phosphotyrosine antibodies has allowed us to detect, in addition to Cek1, several other protein-tyrosine kinases that are expressed during chicken embryonic development, some of which have not been previously identified.

Amino Acid Sequence↗
PubMed1989

Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

Animals↗
PubMed1988

Comparative study of the tyrosine phosphorylation of proteins in Swiss 3T3 fibroblasts stimulated by a variety of mitogenic agents.

We have carried out a comparative study of the protein tyrosine phosphorylation induced by a wide range of mitogenic stimuli on a single cell type, Swiss 3T3 mouse fibroblasts. For this purpose we have used high-affinity antibodies directed to phosphotyrosine residues on proteins (Wang: Mol. Cell. Biol. 5:3640-3643, 1985) in immunoblotting and immunofluorescence microscopy experiments. Immunoblotting experiments showed that all of the mitogens tested, including epidermal growth factor, platelet-derived growth factor, basic fibroblast growth factor, insulin, fetal calf serum, trypsin, and 12-O-tetradecanoylphorbol-13-acetate, increased the phosphorylation on tyrosine of a number of proteins. Most of the increase in tyrosine phosphorylation induced by each factor involved a small set of proteins with apparent molecular weights (Mr) above 50,000. Following stimulation with epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor, increased phosphotyrosine modification of proteins with molecular weights corresponding to those of the respective receptors was observed. A protein band of apparent Mr 160,000 contained substantially increased levels of phosphotyrosine following insulin treatment, but tyrosine phosphorylation of the insulin receptor was apparently below the level of detectability. The phosphotyrosine content of proteins with apparent Mr of 220,000, 120,000, and 70,000 was increased by all the agents tested. Phosphorylation on tyrosine of most of the proteins increased within a few minutes of the mitogenic stimulation, reached a peak, and returned more slowly to basal levels. Immunofluorescence labeling with the antibodies specific for phosphotyrosine showed a substantial increase in the amount of phosphotyrosine containing proteins only in the presence of platelet-derived growth factor and fetal calf serum. This finding suggests that most of the proteins phosphorylated on tyrosine in Swiss 3T3 fibroblasts are not concentrated in specific subcellular structures, but rather are diffusely distributed throughout the cell and are therefore not detectable by immunofluorescence microscopy.

Animals↗
PubMed1988

Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.

Members of the family of transmembrane integral membrane proteins called integrins have been implicated in forming attachments to actin microfilaments of the cytoskeleton. These attachments are thought to involve one or more intervening peripheral membrane proteins linked to integrin. To detect such possible linkages in vivo, the integrin molecules on the surfaces of intact chicken peripheral blood lymphocytes were collected into caps by cross-linking with specific antibodies, and the capped cells were examined by double immunofluorescence to determine whether particular cytoskeletal proteins were co-collected with the integrin. With resting lymphocytes, the capping of integrin did not result in any detectable redistribution of either talin, vinculin, or alpha-actinin inside the cells. However, if the capping was carried out upon the addition of phorbol 12-myristate 13-acetate (PMA) to the cells, then talin, but not vinculin or alpha-actinin, was found associated with the integrin caps. PMA is known to activate protein kinase C. These results suggest that after, but not before, PMA stimulation of intact cells, talin becomes linked either directly or indirectly with integrin, reflecting the formation of a membrane-cytoskeletal association that is metabolically regulated.

Animals↗
PubMed1988

Molecular dynamics in the membranes of helper T cells.

We provide evidence that redistributions and interactions of integral proteins in the fluid membranes of helper T (Th) cells may play important roles in Th-cell activation. A particular monoclonal antibody, 3D3, directed to a clonotypic determinant on the T-cell receptor (TCR) of the cloned Th-cell line D10, had previously been shown to be distinctively capable of directly activating D10 cells at low concentrations. We demonstrate here by immunofluorescence experiments that it is also distinctively able itself to produce a clustering (capping) of the TCRs on the D10 cell surface. Simultaneously, by means of double-immunofluorescence experiments, we find that the 3D3-induced clustering of the TCRs distinctively produces a co-clustering of the accessory molecule CD4 with the TCR clusters, although the CD4 and TCR molecules are normally independent of one another in the D10 cell membrane. These results, and related ones previously obtained from studies of the interactions of D10 Th cells with antigen-presenting cells, are analyzed to suggest that the membrane clustering of TCRs and the induced TCR-CD4 interactions are critical to the signaling events in Th-cell activation.

Antibodies, Monoclonal↗
PubMed1988

Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos.

We have used high affinity polyclonal antibodies specific for phosphotyrosine (PTyr) residues to examine the localization in various chick embryonic tissues in situ of PTyr-modified proteins by immunocytochemical methods. During the period from 9 to 21 d of development, most tissues exhibit elevated levels of PTyr-modified proteins as determined by immunoblotting experiments of tissue extracts with the anti-PTyr antibodies (Maher, P. A., and E. B. Pasquale. 1988. J. Cell Biol. 106:1747-1755). By immunofluorescence labeling of semithin frozen sections, the highest concentrations of PTyr immunolabeling in all of the embryonic tissues examined were localized to the membranes of the epithelial and endothelial cells with other cells showing no detectable labeling. These results were confirmed by immunoelectron microscopic labeling, which showed particularly high concentrations of PTyr-modified proteins close to the membranes at the apical junctions. The corresponding adult tissues showed no labeling. It is proposed that these results reflect the molecular basis for the functional plasticity of epithelial and endothelial cell junctions during embryonic development.

Animals↗
PubMed1988

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain.

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.

Actins↗
PubMed1988

Forceful pulsatile local infusion of enzyme accelerates thrombolysis: in vivo evaluation of a new delivery system.

Forceful local pulsatile infusion of fibrinolytic enzyme disrupts thrombi, increases clot surface area, and thereby hastens enzyme action compared with conventional constant infusion methods, which are time consuming and therefore expensive. Prolonged thrombolytic therapy is associated with increased patient morbidity. A prototype for a clinically applicable pulsatile jet infusion system for accelerating thrombolysis was designed. The system is adaptable to standard angiographic catheters and techniques. The core of the system is a reciprocating syringe pump that delivers small volumes of thrombolytic enzyme in short, rapid, frequent pulses at high exit-jet velocity through any side-hole catheter (the smallest used was a 3-F catheter). Comparison of this system with a constant infusion system was made in vivo in a 48-hour-old thrombus model in rabbit inferior vena cava (IVC). One hour of lysis by streptokinase was conducted with each of the methods. In the first experiment, the IVC thrombi were left intact before chemical lysis. Pulsatile infusion lysed 61% of the thrombus by weight in an hour, whereas constant infusion lysed only 15% (P less than .001). In the second experiment, IVC thrombi were subjected initially to standardized mechanical perturbation by a guide wire before chemical lysis. In the latter experiment, pulsatile infusion lysed 54% of the thrombus by weight, and constant infusion lysed only 26% (P less than .005). The difference in percentage of lysis by weight between pulsatile infusion groups in the two experiments (61% vs 54%) was not significant (P greater than .1). The same was true of the difference between the two constant infusion groups (26% vs 15%, P greater than .05). The effect of initial perturbation of the thrombus by a guide wire appears to be less important than the thrombus disruption and accelerated thrombolysis caused by the pulsatile delivery system. No angiographic or macroscopically visible damage was seen in any IVC. Accelerated thrombolysis may reduce the expense, duration, and morbidity associated with conventional constant infusion methods.

Animals↗
PubMed1988

Immunocytochemical studies of endothelial cells in vivo. II. Chicken aortic and capillary endothelial cells exhibit different cell surface distributions of the integrin complex.

Frozen sections of chicken tissues containing aortic and capillary endothelial cells were immunolabeled with two mouse monoclonal antibodies directed to different epitopes of the chicken integrin beta-chain. Integrin is an integral membrane protein complex that is believed to mediate a transmembrane linkage between the extracellular matrix and the actin cytoskeleton. In immunofluorescence experiments with semi-thin frozen sections, the aortic endothelial cells were labeled for integrin all around their surfaces, whereas capillary endothelial cells of heart and kidney were labeled only on their basal surfaces. At the immunofluorescence level of resolution, the distribution of integrin appeared to be correlated with that of F-actin in double-labeling experiments with NBD-phallacidin. These different distributions of integrin on the two types of endothelial cells were definitively confirmed by immunoelectron microscopic labeling with the monoclonal antibodies on ultra-thin frozen sections. These results therefore indicate that the luminal surfaces, as well as the underlying cytoskeleton of capillary endothelial cells, are significantly different in structure from those of aortic endothelial cells. These differences may reflect the vastly different hemodynamic stress to which the two types of endothelial cells are subjected, and in addition may mediate different adhesion properties of the luminal surfaces of the two cell types.

Animals↗
PubMed1988

Localization of high concentrations of phosphotyrosine-modified proteins in mouse megakaryocytes.

Phosphorylation of tyrosine residues of cellular proteins is a rare event and is considered to be related to the regulation of cellular growth, differentiation, and some forms of neoplastic transformation. Using high-affinity antibodies specific to phosphotyrosine (P-Tyr), we have shown the presence at high concentrations of P-Tyr-modified proteins in mouse bone-marrow megakaryocytes. Immunofluorescence microscopy of semithin frozen sections revealed that P-Tyr labeling was localized in a punctate pattern in the majority of the cytoplasm. The thin outer rim of the cytoplasm and the cell membrane was devoid of the label. Immunogold electron microscopy of ultrathin frozen sections showed that P-Tyr labeling was concentrated mostly on the membranes of the vesicles in the cytoplasm. The membrane demarcation system characteristic of megakaryocytes was not labeled. The intensity of P-Tyr labeling varied from one megakaryocyte to another. These results suggest that tyrosine phosphorylation of specific proteins might be correlated with the developmental stage of megakaryocytes, possibly related to the formation and deposition of the granules.

Animals↗
PubMed1987

The specific direct interaction of helper T cells and antigen-presenting B cells. II. Reorientation of the microtubule organizing center and reorganization of the membrane-associated cytoskeleton inside the bound helper T cells.

We have produced and investigated cell couples formed between cloned Th cells or T hybridoma cells, and either Ag-presenting B hybridoma or B lymphoma cells. The specific direct interaction between a Th and B-APC is here demonstrated by two rearrangements occurring inside the bound Th cell; the MTOC (and presumably the GA) is oriented to face the cell contact region with the B cell, and a membrane-associated cytoskeletal protein, talin, becomes concentrated under the contacting Th membrane. In the absence of the specific Ag or the correct Ia determinant, nonspecific T-B cell couples form that are morphologically indistinguishable from specific cell couples in the light microscope, but neither the MTOC nor the talin rearrangement occurs inside the bound T cell of such nonspecific couples. Furthermore, Ag processing by the B cell is required to produce the MTOC and talin rearrangements within the T cell in specific T-B couples. In the case of allogeneic Th-B cell couples, similar specific MTOC and talin rearrangements are observed inside the Th. Extracellular Ca2+ is required for the MTOC orientation to occur inside the specifically bound Th cell, but not for the talin rearrangement. It is proposed that the MTOC (and GA) reorientation and the talin rearrangement are involved in the directed secretion of GA-derived lymphokines from the Th cell to the bound B cell.

Animals↗
PubMed1987

A critical role for the polarization of membrane recycling in cell motility.

This paper is concerned with the proposition that the insertion of membrane mass into the leading edge of a motile cell plays a critical role in directed cell migration. We show by immunofluorescence, with cells transfected with a cloned cDNA encoding the G-protein of a temperature-sensitive mutant of vesicular stomatitis virus, that the first cell surface appearance of the G-protein is indeed at the leading edge of the motile cell. Two drugs capable of inhibiting directed cell migration, cytochalasin D and monensin, appear to function independently, the former by affecting the actin cytoskeleton without affecting the polarized insertion of membrane mass into the cell surface and the latter by abrogating membrane mass insertion without affecting the actin cytoskeleton.

Animals↗
PubMed1987

Coclustering of CD4 (L3T4) molecule with the T-cell receptor is induced by specific direct interaction of helper T cells and antigen-presenting cells.

Blocking studies with monoclonal antibodies have suggested that helper T cell recognition and triggering involve the CD4 (L3T4) accessory molecule as well as the T-cell receptor (TCR) that is linked to the T3 complex. We have investigated the surface distribution of L3T4 and TCR during the direct interaction of a cloned murine helper T-cell line with an antigen-presenting B-cell line. Using immunofluorescence microscopy, we show that in 1:1 cell couples formed between the two cells, in which a specific interaction can be demonstrated, the L3T4 and the TCR become redistributed on the T-cell surface so that they are concentrated in the cell-cell contact region. This coclustering of L3T4 with TCR occurs only when the relevant antigen and appropriate major histocompatibility class II molecules are presented to the T cell, and it therefore requires the specific interaction of the TCR with its complex ligand on the antigen-presenting cell.

Animals↗
PubMed1987

On the translocation of proteins across membranes.

Many proteins of intracellular organelles are first synthesized in the cytoplasm and are then specifically transferred across the membranes of the organelles. On the assumption that these transfers all occur by the same basic mechanism, we enumerate the rather stringent requirements that the mechanism must satisfy. A unitary molecular mechanism is then proposed that meets these requirements.

Amino Acid Sequence↗