Biomedical subjects

U Kubitscheck

Publications and source records attributed to U Kubitscheck.

26 records · Page 2Linked to original sources

PubMed1995

Modulation of CD4 lateral mobility in intact cells by an intracellularly applied antibody.

This study shows that the lateral mobility of CD4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracellularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) insect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for fluorescence microphotolysis (FM) studies was constructed. By these means it was found that the commercial anti-CD4 antibody Leu3a-PE, in contrast with several other anti-CD4 antibodies, could be used as a fluorescent label of CD4 without interfering greatly with CD4 mobility. Labelled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm2/s and a mobile fraction of fm = 80 +/- 16% (room temperature). Within experimental accuracy the truncated CD4 had the same mobility as full-length CD4. Introduction of the C6 antibody into Sf9 cells by microinjection or by fusion with C6-loaded liposomes decreased the mobility of full-length CD4 (fm = 40%) but not of truncated CD4 (fm = 80%). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (fm = 50%) but not truncated CD4 (fm = 90%). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect.

Amino Acid Sequence↗

PubMed1995

Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.

Adenosine Triphosphate↗

PubMed1995

Mapping of nucleoporins to the center of the nuclear pore complex by post-embedding immunogold electron microscopy.

Ultrathin sections of Lowicryl K4M embedded cultured 3T3 cells, human keratinocytes and mouse/rat liver tissue were incubated with polyspecific primary antibodies against p62 and other nucleoporins followed by 10 nm gold labeled secondary antibodies. By quantitatively evaluating both cross sections and tangential sections of the NPC, we found that irrespective of the cell type antibodies predominantly bound within a radius of 25 nm around the central axis of the nuclear pore complex (NPC). Superposition of a current structural model of the NPC with the nucleoporin distribution observed by us showed that nucleoporins mapped predominatly to the controversely discussed 'central granule'. Our experimental approach was verified by mapping gp210, another nuclear pore protein, at or very close to the NPC in the perinuclear cisterna thus establishing a distribution pattern completely different from that of the nucleoporins.

3T3 Cells↗

PubMed1994

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope.

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.

3T3 Cells↗

PubMed1994

Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging.

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the 'Scamper'. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result, almost any desired pattern could be bleached ('written') into fluorescent samples at high definition and then imaged ('read') at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.

3T3 Cells↗

PubMed1993

Distribution of type I Fc epsilon-receptors on the surface of mast cells probed by fluorescence resonance energy transfer.

The aggregation state of type I Fc epsilon-receptors (Fc epsilon RI) on the surface of single living mast cells was investigated by resonance fluorescence energy transfer. Derivatization of Fc epsilon RI specific ligands, i.e., immunoglobulin E or Fab fragments of a Fc epsilon RI specific monoclonal antibody, with donor and acceptor fluorophores provided a means for measuring receptor clustering through energy transfer between the receptor probes. The efficiency of energy transfer between the ligands carrying distinct fluorophores was determined on single cells in a microscope by analyzing the photobleaching kinetics of the donor fluorophore in the presence and absence of receptor ligands labeled with acceptor fluorophores. To rationalize the energy transfer data, we developed a theoretical model describing the dependence of the energy transfer efficiency on the geometry of the fluorescently labeled macromolecular ligands and their aggregation state on the cell surface. To this end, the transfer process was numerically calculated first for one pair and then for an ensemble of Fc epsilon RI bound ligands on the cell surface. The model stipulates that the aggregation state of the Fc epsilon RI is governed by an attractive lipid-protein mediated interaction potential. The corresponding pair-distribution function characterizes the spatial distribution of the ensemble. Using this approach, the energy transfer efficiency of the ensemble was calculated for different degrees of receptor aggregation. Comparison of the theoretical modeling results with the experimental energy transfer data clearly suggests that the Fc epsilon RI are monovalent, randomly distributed plasma membrane proteins. The method provides a novel approach for determining the aggregation state of cell surface components.

Animals↗

PubMed1992

Peptide binding to class I molecules of the major histocompatibility complex on the surface of living target cells.

Molecules encoded by the class I major histocompatibility genes bind short (nonameric) peptides produced by intracellular proteolysis of antigens. These complexes formed intracellularly are then expressed on membranes of target cells and recognized by the antigen receptor of cytolytic T cells. No binding of externally added peptides could so far be monitored directly on the antigen presenting cells, although cytotoxicity experiments and indirect binding assays provided evidence for its existence. Here we report experiments where specific binding to class I molecules, of externally added peptides, has been monitored on living cells. N-terminal biotin-labelled Kd-restricted peptides (residues 147-155, residues 147-158, and an analogue lacking the arginine at position 156, derived from the sequence of the influenza A virus nucleoprotein) were incubated with murine H-2Kd mastocytoma cells (line P815) at 4 degrees C. The binding on surface of live, intact cells was then demonstrated fluorometrically via the interaction of a streptavidin-phycoerythrin conjugate with the biotin-labelled peptides. Thus, this binding does not involve processing, and its specificity in terms of peptide structure was established by competition with the respective unmodified peptides. The specificity of binding to class I molecules was demonstrated by blocking experiments using monoclonal antibodies specific for H-2Kd. Finally, a correlation was observed between the results of peptide binding measurements and those of cytotoxicity assays.

Amino Acid Sequence↗

PubMed1991

Fluorescence resonance energy transfer on single living cells. Application to binding of monovalent haptens to cell-bound immunoglobulin E.

We have determined the specific binding of 2,4-dinitrophenyl (DNP)-haptens to two different monoclonal immunoglobulin (IgE) molecules bound to Fc epsilon-receptors on the cell surface of single, living rat basophilic leukemia cells subclone 2H3 cells. The measurements were performed at 4 degrees, 15 degrees, and 25 degrees C using a recently developed technique that permits the quantitative determination of fluorescence resonance energy transfer between two fluorophores on single cells in a microscope from the photobleaching kinetics of the donor fluorophore. We introduce here a method for performing binding studies on individual attached cells. At 25 degrees C, the titration studies yielded equilibrium binding constants Kint of 9 x 10(8), 8 x 10(8), and 8 x 10(7) M-1 for the monovalent haptens N-2,4-DNP-epsilon-amino-n-caproic acid, N epsilon-2,4-DNP-L-lysine, and N-2,4-DNP-gamma-amino-n-butyric acid, respectively. Our data indicate that the affinity constants for the first two haptens binding to IgE on adherent cells are 4 to 11 times larger than that of the corresponding values obtained by fluorescence quenching experiments with the same haptens and IgE molecules either in solution or bound to cells in suspension.

Animals↗