Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, the data have been interpreted by assuming free Brownian diffusion of cell surface receptors in two dimensions, an interpretation that requires that a subset of the diffusing species remains immobile. The origin of this so-called immobile fraction remains a mystery. In FPR, the motions of thousands of particles are inherently averaged, inevitably masking the details of individual motions. Recently, tracking of individual cell surface receptors has identified several distinct types of motion (Gross and Webb, 1988; Ghosh and Webb, 1988, 1990, 1994; Kusumi et al. 1993; Qian et al. 1991; Slattery, 1995), thereby calling into question the classical interpretation of FPR data as free Brownian motion of a limited mobile fraction. We have measured the motion of fluorescently labeled immunoglobulin E complexed to high affinity receptors (Fc epsilon RI) on rat basophilic leukemia cells using both single particle tracking and FPR. As in previous studies, our tracking results show that individual receptors may diffuse freely, or may exhibit restricted, time-dependent (anomalous) diffusion. Accordingly, we have analyzed FPR data by a new model to take this varied motion into account, and we show that the immobile fraction may be due to particles moving with the anomalous subdiffusion associated with restricted lateral mobility. Anomalous subdiffusion denotes random molecular motion in which the mean square displacements grow as a power law in time with a fractional positive exponent less than one. These findings call for a new model of cell membrane structure.
A procedure for the preparation of tomato chromosome suspensions suitable for flow cytometric analysis is described. Rapidly growing cell suspension cultures of Lycopersicon esculentum cv VFNT cherry and L. pennellii LA716 were treated with colchicine to enrich for metaphase chromosomes. Metaphase indices between 20 and 35% were routinely obtained when cultures were exposed to 0.1% colchicine for 15-18 h after 2 days of subculture. Mitotic cells were isolated by brief treatment with cell wall digesting enzymes in a medium with low osmolarity (∼325 mOsm/kg of H52O). The low osmolarity medium was needed to avoid the chromosome clumping and decondensation seen in standard media. Suspensions of intact chromosomes were prepared by lysing swollen protoplasts in various buffers (MgSO4, polyamines, hexylene glycol, or KCl-propidium iodide) similar in contents to the buffers used to isolate mammalian chromosomes. For univariate flow cytometric analysis, chromosome suspensions were stained with a fluorescent DNA-binding stain (propidium iodide, Hoechst 33258, mithramycin, or chromomycin A3) and analyzed using an EPICS flow cytometer (Profile Analyzer or 753). Peaks for the chromosomes, chromatids, clumps of chromosomes, nuclei, and fluorescent debris were seen on a histogram of log of fluorescence intensity, and were confirmed by microscopic examination of the objects collected by flow-sorting. Chromosome suspensions prepared in MgSO4 buffer have the highest frequency of intact chromosomes and the least fluorescent cellular debris. Peaks similar to theoretical univariate flow karyotypes of tomato chromosomes were seen on the observed univariate flow karyotypes, but were not as well resolved. Bivariate flow analysis of tomato chromosome suspension using double-stain combination, Hoechst 33258 and chromomycin A3, and two laser beams showed better resolution of some chromosomes.
The segmental flexibility of mouse immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells was compared to that of IgE in solution by measuring the steady-state anisotropy as a function of temperature and viscosity. A monoclonal IgE was used to bind the fluorescent probe N-[5-(dimethylamino)naphthalene-1-sulfonyl]-L-lysine (DNS-Lys) rigidly and specifically in the antigen combining site at the tip of the Fab region. The average rotational correlation time, phi, of 74-89 ns for the receptor-bound IgE is only slightly longer than that for IgE in solution where phi = 54 ns. Another mouse monoclonal IgE was covalently labeled in the Fab region with N-(1-pyrenyl)maleimide. Anisotropy measurements with this derivative yielded results that are very similar to those found with anti-DNS IgE and DNS-Lys. These findings are strikingly different from that expected for a rigid IgE bound to its receptor since in this case phi is likely to be very much larger. Evidently, the segmental flexibility of IgE is not greatly altered upon binding to its receptor.
The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with Cl-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units.
We have investigated the functional advantage of surface-attached ligands for TCR-mediated cell activation with flow cytometric measurements of cytoplasmic Ca2+ changes in T cells after aggregation of TCR by soluble and bead-attached mAb. Conjugation of HPB-ALL human leukemia cells with cell-sized beads coated with anti-TCR mAb causes a stronger, more sustained Ca2+ response than that produced by the soluble form of the same mAb. Addition of a large excess of the soluble mAb subsequent to stimulation with the beads causes a marked reduction in the response of the bead-conjugated cells, but only limited disruption of the conjugates. Free (nonconjugated) cells, sampled simultaneously in this mixture, respond to the soluble mAb with a transient Ca2+ increase that declines with the same kinetics as the bead-conjugated cells after addition of the soluble mAb. Fab fragments of the anti-TCR mAb cause a similar reduction in the response of the bead-conjugated cells, and they do not stimulate free cells. Following the Fab-mediated decline in cytoplasmic Ca2+ of conjugated cells to near-baseline concentrations, the addition of a second, noncompetitive, anti-TCR mab causes a Ca2+ response that is substantially reduced in magnitude compared with that for the free cells. The results indicate that soluble and surface-attached ligands cause TCR-specific desensitization of the Ca2+ response. Surface-attached ligands are more effective than soluble ligands in sustaining signaling in T cells at least in part because they facilitate steady association and/or reassociation of TCR into the bound state in the surface contact area.
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