scholarly journals Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function.

1984 ◽  
Vol 99 (5) ◽  
pp. 1624-1633 ◽  
Author(s):  
K Jacobson ◽  
D O'Dell ◽  
J T August
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Lateral Diffusion ◽  
Leading Edge ◽  
Fluorescence Recovery ◽  
Lateral Mobility ◽  
Cell Surface Glycoprotein ◽  
Murine Fibroblasts ◽  
Interphase Cells ◽  
Cell Fragments

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell-cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.

scholarly journals Lateral diffusion of wild-type and mutant Ld antigens in L cells.

1984 ◽  
Vol 99 (6) ◽  
pp. 2333-2335 ◽  
Author(s):  
M Edidin ◽  
M Zuniga
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Histocompatibility Antigen ◽  
Wild Type ◽  
Major Histocompatibility Antigen ◽  
Cytoplasmic Side

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

scholarly journals Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

1986 ◽  
Vol 103 (2) ◽  
pp. 327-331 ◽  
Author(s):  
E Livneh ◽  
M Benveniste ◽  
R Prywes ◽  
S Felder ◽  
Z Kam ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Diffusion Coefficients ◽  
Egf Receptor ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Protein Kinase Activity ◽  
Lateral Mobility ◽  
Cos Cells ◽  
Epidermal Growth

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

scholarly journals Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids.

1993 ◽  
Vol 120 (1) ◽  
pp. 25-35 ◽  
Author(s):  
G M Lee ◽  
F Zhang ◽  
A Ishihara ◽  
C L McNeil ◽  
K A Jacobson
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Colloidal Gold ◽  
Plasma Membranes ◽  
Membrane Surface ◽  
Mean Square Displacement ◽  
Lateral Diffusion ◽  
Pericellular Matrix ◽  
Cultured Fibroblasts ◽  
Average Diffusion

Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.

scholarly journals Components of the plasma membrane of growing axons. II. Diffusion of membrane protein complexes.

1984 ◽  
Vol 98 (4) ◽  
pp. 1434-1443 ◽  
Author(s):  
R K Small ◽  
M Blank ◽  
R Ghez ◽  
K H Pfenninger
Keyword(s):  
Plasma Membrane ◽  
Diffusion Coefficients ◽  
Moving Boundary ◽  
Protein Complexes ◽  
Lateral Diffusion ◽  
Membrane System ◽  
Density Gradients ◽  
Axon Elongation ◽  
Stages Of Growth ◽  
Membrane Protein Complexes

Intramembrane particles (IMPs) of the plasmalemma of mature, synapsing neurons are evenly distributed along the axon shaft. In contrast, IMPs of growing olfactory axons form density gradients: IMP density decreases with increasing distance from the perikarya, with a slope that depends upon IMP size (Small, R., and K. H. Pfenninger, 1984, J. Cell Biol., 98: 1422-1433). These IMP density gradients resemble Gaussian tails, but they are much more accurately described by the equations formulated for diffusion in a system with a moving boundary (a Stefan Problem), using constants that are dependent upon IMP size. The resulting model predicts a shallow, nearly linear IMP density profile at early stages of growth. Later, this profile becomes gradually transformed into a steep nonlinear gradient as axon elongation proceeds. This prediction is borne out by the experimental evidence. The diffusion coefficients calculated from this model range from 0.5 to 1.8 X 10(-7) cm2/s for IMPs between 14.8 and 3.6 nm, respectively. These diffusion coefficients are linearly dependent upon the inverse IMP diameter in accordance with the Stokes-Einstein relationship. The measured viscosity is approximately 7 centipoise. Our findings indicate (a) that most IMPs in growing axons reach distal locations by lateral diffusion in the plasma membrane, (b) that IMPs--or complexes of integral membrane proteins--can diffuse at considerably higher rates than previously reported for iso-concentration systems, and (c) that the laws of diffusion determined for macroscopic systems are applicable to the submicroscopic membrane system.

scholarly journals Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

1986 ◽  
Vol 102 (4) ◽  
pp. 1350-1357 ◽  
Author(s):  
T N Metcalf ◽  
M A Villanueva ◽  
M Schindler ◽  
J L Wang
Keyword(s):  
Monoclonal Antibody ◽  
Plasma Membrane ◽  
Cytochalasin B ◽  
Previous Analysis ◽  
Lateral Diffusion ◽  
Soybean Agglutinin ◽  
Modulation Effect ◽  
Lateral Mobility ◽  
Fold Reduction ◽  
Membrane Bound

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

scholarly journals Evidence from lateral mobility studies for dynamic interactions of a mutant influenza hemagglutinin with coated pits.

1991 ◽  
Vol 115 (6) ◽  
pp. 1585-1594 ◽  
Author(s):  
E Fire ◽  
D E Zwart ◽  
M G Roth ◽  
Y I Henis
Keyword(s):  
Plasma Membrane ◽  
Lattice Structure ◽  
Lateral Diffusion ◽  
Live Cells ◽  
Lateral Mobility ◽  
Coated Pits ◽  
Dynamic Interactions ◽  
Intact Cells ◽  
Hypertonic Medium ◽  
Mobility Measurement

Replacement of cysteine at position 543 by tyrosine in the influenza virus hemagglutinin (HA) protein enables the endocytosis of the mutant protein (Tyr 543) through coated pits (Lazarovits, J., and M. G. Roth. 1988. Cell. 53:743-752). To investigate the interactions between Tyr 543 and the clathrin coats in the plasma membrane of live cells, we performed fluorescence photobleaching recovery measurements comparing the lateral mobilities of Tyr 543 (which enters coated pits) and wild-type HA (HA wt, which is excluded from coated pits), following their expression in CV-1 cells by SV-40 vectors. While both proteins exhibited the same high mobile fractions, the lateral diffusion rate of Tyr 543 was significantly slower than that of HA wt. Incubation of the cells in a sucrose-containing hypertonic medium, a treatment that disperses the membrane-associated coated pits, resulted in similar lateral mobilities for Tyr 543 and HA wt. These findings indicate that the lateral motion of Tyr 543 (but not of HA wt) is inhibited by transient interactions with coated pits (which are essentially immobile on the time scale of the lateral mobility measurements). Acidification of the cytoplasm by prepulsing the cells with NH4Cl (a treatment that arrests the pinching-off of coated vesicles from the plasma membrane and alters the clathrin lattice morphology) led to immobilization of a significant part of the Tyr 543 molecules, presumably due to their entrapment in coated pits for the entire duration of the lateral mobility measurement. Furthermore, in both untreated and cytosol-acidified cells, the restrictions on Tyr 543 mobility were less pronounced in the cold, suggesting that the mobility-restricting interactions are temperature dependent and become weaker at low temperatures. From these studies we conclude the following. (a) Lateral mobility measurements are capable of detecting interactions of transmembrane proteins with coated pits in intact cells. (b) The interactions of Tyr 543 with coated pits are dynamic, involving multiple entries of Tyr 543 molecules into and out of coated pits. (c) Alterations in the clathrin lattice structure can modulate the above interactions.

Sign in / Sign up

Export Citation Format

Share Document