Lateral diffusion of H-2 antigens on mouse fibroblasts

We have used fluorescence photobleaching and recovery (FPR) to measure the lateral diffusion of mouse H-2 antigens, labeled with fluorescent Fab fragments, in the membrane of cl 1d fibroblasts. Diffusion coefficients, D, vary more than 20-fold from cell to cell, though they vary no more than twofold when measured at different points on a single cell. The fraction of H-2 antigens mobile, R, also varies from cell to cell, and no lateral diffusion of H-2 antigens can be detected in approximately 20% of the cells examined. Treatment of cells with NaCN + NaF, reducing their levels of ATP reduces the proportion of cells in which no lateral diffusion can be detected. The maximum values of D seen in poisoned cells are less than those in controls. Treatment of cells with the divalent inophore, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, greatly increases the proportion of cells in which diffusion of H-2 is rapid, D greater than 2 x 10(-9) cm2 s-1. The data obtained on diffusion by FPR can be replotted in the form of an experiment in which lateral diffusion of H- 2 antigens is measured in a population of heterokaryons. There is good agreement between this transformation and actual data on heterokaryons. Thus the two methods appear to measure the same transport process.     

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 is regulated by actin filaments and 4.1N in neuronal dendrites

Inositol 1,4,5-trisphosphate receptor type1 (IP3R1) plays an important role in neuronal functions; however, the lateral diffusion of IP3R1 on the endoplasmic reticulum membrane and its regulation in the living neurons remain unknown. We expressed green fluorescent protein-tagged IP3R1 in cultured rat hippocampal neurons and observed the lateral diffusion by the fluorescence recovery after photobleaching technique. IP3R1 showed lateral diffusion with an effective diffusion constant of approximately 0.3 microm2/s. Depletion of actin filaments increased the diffusion constant of IP3R1, suggesting that the diffusion of IP3R1 is regulated negatively through actin filaments. We also found that protein 4.1N, which binds to IP3R1 and contains an actin-spectrin-binding region, was responsible for this actin regulation of the IP3R1 diffusion constant. Overexpression of dominant-negative 4.1N and blockade of 4.1N binding to IP3R1 increased the IP3R1 diffusion constant. The diffusion of IP3R type 3 (IP3R3), one of the isoforms of IP3Rs lacking the binding ability to 4.1N, was not dependent on actin filaments but became dependent on actin filaments after the addition of a 4.1N-binding sequence. These data suggest that 4.1N serves as a linker protein between IP3R1 and actin filaments. This actin filament-dependent regulation of IP3R1 diffusion may be important for the spatiotemporal regulation of intracellular Ca2+ signaling.     

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

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.     

用荧光漂白恢复技术研究竹红菌乙素在小鼠肝癌细胞内的扩散过程

利用竹红菌乙素自身的荧光特征,在ＦＰＲ装置上直接测定了乙素在ＡＨ细胞内的侧向扩散系数和荧光漂白的恢复率。实验结果表明乙素在ＡＨ细胞内的扩散系数Ｄ＝３．２×１０＾－９ｃｍ＾２／ｓ＾－１荧光漂白恢复比率Ｒ＝９７．８％,上述实验说明乙素在细胞膜内与生物大分子之间没有形成共价键形式的结合状态。     

A milling crowd model for local and long-range obstructed lateral diffusion. Mobility of excimeric probes in the membrane of intact erythrocytes.

A new model for lateral diffusion, the milling crowd model (MC), is proposed and is used to derive the dependence of the monomeric and excimeric fluorescence yields of excimeric membrane probes on their concentration. According to the MC model, probes migrate by performing spatial exchanges with a randomly chosen nearest neighbor (lipid or probe). Only nearest neighbor probes, one of which is in the excited state, may form an excimer. The exchange frequency, and hence the local lateral diffusion coefficient, may then be determined from experiment with the aid of computer simulation of the excimer formation kinetics. The same model is also used to study the long-range lateral diffusion coefficient of probes in the presence of obstacles (e.g., membrane proteins). The dependence of the monomeric and excimeric fluorescence yields of 1-pyrene-dodecanoic acid probes on their concentration in the membranes of intact erythrocytes was measured and compared with the prediction of the MC model. The analysis yields an excimer formation rate for nearest neighbor molecules of approximately 1 X 10(7) s-1 and an exchange frequency of approximately greater than 2 X 10(7) s-1, corresponding to a local diffusion coefficient of greater than 3 X 10(-8) cm2 s-1. This value is several times larger than the long-range diffusion coefficient for a similar system measured in fluorescence photobleaching recovery experiments. The difference is explained by the fact that long-range diffusion is obstructed by dispersed membrane proteins and is therefore greatly reduced when compared to free diffusion. The dependence of the diffusion coefficient on the fractional area covered by obstacles and on their size is derived from MC simulations and is compared to those of other theories lateral diffusibility.     

Restricted mobility of membrane constituents in cell-substrate focal contacts of chicken fibroblasts

We studied the lateral mobility of membrane components in cell- substrate focal contacts using the fluorescence photobleaching recovery method. The measurements were performed on isolated substrate-attached membranes of chicken gizzard fibroblasts. The diffusion coefficients of a fluorescent lipid probe and rhodamine-conjugated surface proteins within contact regions (identified by interference-reflection microscopy) were significantly lower than those measured in nonattached areas along the ventral membrane. Complete recovery of fluorescence after photobleaching of the lipid probe was measured both in focal contacts and in nonattached areas with lateral diffusion coefficient (D) of approximately 10(-8) cm2/s. This indicated that the lipid probe is free to diffuse from and into the contact regions. Rhodamine-labeled surface components (mostly proteins) exhibited almost complete recovery after bleaching (approximately 90%) in unattached regions of the ventral membrane with D congruent to 10(-9 cm2/s. The rhodamine-labeled proteins in focal contacts showed only partial recovery (approximately 50%), suggesting that large proportion of the membrane proteins in cell- substrate contacts are immobile (within the time scale of the experiments, D less than or equal to 5 x 10(-12) cm2/s. The implications of these findings on the molecular dynamics of cell contacts are discussed.     

Lateral mobility in membranes as detected by fluorescence recovery after photobleaching.

The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.     

Dynamics of the Ins(1,4,5)P3 receptor during polarization of MDCK cells

BACKGROUND INFORMATION: The uneven distribution of the Ins(1,4,5)P3R [Ins(1,4,5)P3 receptor] within the ER (endoplasmic reticulum) membrane generates spatially complex Ca2+ signals. The ER is a dynamic network, which allows the rapid diffusion of membrane proteins from one part of the cell to another. However, little is known about the localization and the dynamics of the Ins(1,4,5)P3R in the ER of living cells. We have used a MDCK (Madin-Darby canine kidney) clone stably expressing the Ins(1,4,5)P3R1-GFP (where GFP stands for green fluorescent protein) to investigate the effect of cell polarity on the lateral mobility of the Ins(1,4,5)P3R. RESULTS: In non-confluent MDCK cells, the chimaera is homogeneously distributed throughout the ER and the nuclear envelope. FRAP (fluorescence recovery after photobleaching) experiments showed that the receptor can move freely in the ER with a diffusion constant (D=0.01 microm2/s) approx. ten times lower than other ER membrane proteins. In confluent polarized cells, two populations of receptor can be defined: one population is distributed in the cytoplasm and is mobile but with a slower diffusion constant (D=0.004 microm2/s) compared with non-confluent cells, whereas the other population is concentrated at the periphery of the cells and is apparently immobile. CONCLUSIONS: The observed differences in the mobility of the Ins(1,4,5)P3R are most probably due to its interactions with stable protein complexes that form at the periphery of the polarized cells.     

Lateral mobility of membrane-binding proteins in living cells measured by total internal reflection fluorescence correlation spectroscopy

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows us to measure diffusion constants and the number of fluorescent molecules in a small area of an evanescent field generated on the objective of a microscope. The application of TIR-FCS makes possible the characterization of reversible association and dissociation rates between fluorescent ligands and their receptors in supported phospholipid bilayers. Here, for the first time, we extend TIR-FCS to a cellular application for measuring the lateral diffusion of a membrane-binding fluorescent protein, farnesylated EGFP, on the plasma membranes of cultured HeLa and COS7 cells. We detected two kinds of diffusional motion-fast three-dimensional diffusion (D(1)) and much slower two-dimensional diffusion (D(2)), simultaneously. Conventional FCS and single-molecule tracking confirmed that D(1) was free diffusion of farnesylated EGFP close to the plasma membrane in cytosol and D(2) was lateral diffusion in the plasma membrane. These results suggest that TIR-FCS is a powerful technique to monitor movement of membrane-localized molecules and membrane dynamics in living cells.     

Microinjection in combination with microfluorimetry to study proton diffusion along phospholipid membranes

Proton diffusion along the surface of a planar bilayer lipid membrane was measured by means of acid/base injection with a micropipette and recording of the kinetics of fluorescence changes of fluorescein-labelled lipid on the surface. The dimensionality of the process was assayed by fitting the kinetic curves with two-dimensional (2D) or three-dimensional (3D) diffusion equations. In agreement with Serowy et al. (Biophys J 84:1031-1037, 2003), lateral proton diffusion proceeded via bulk phase by means of buffer molecules as proton carriers (D = 600 microm2/s) under the conditions of 1 mM buffer in the solution. Introduction of proton binding sites on the membrane surface led to the appearance of a considerable contribution of two-dimensional proton diffusion on the membrane surface with D = 1,100 mum(2)/s. The system described can be used to study the dependence of the proton diffusion rate on the phospholipid and protein composition of the membrane.     

Small intestinal differentiation in human colon carcinoma HT29 cells has distinct effects on the lateral diffusion of lipids (ganglioside GM1) and proteins (HLA class 1, HLA class 2, and neoplastic epithelial antigens) in the apical cell membrane

We have studied the effect of maturation to small intestinal-like epithelial cells of the human colonic carcinoma cell line HT29 on the lateral mobility of different representative membrane components (lipid, proteins), as assessed with fluorescence recovery after photobleaching (FRAP). Maturation was induced in vitro in the HT29 cells by replacing glucose (Glu) with galactose (Gal) in the growth medium (DMEM) during a 21-day period. Scanning electron microscopy revealed an increased number of microvilli in the apical cell membrane, and enzyme analyses (alkaline phosphatase, aminopeptidase) in combination with aqueous countercurrent distribution, indicated that maturation was induced with DMEM-Gal. In comparison to control cells grown in DMEM-Glu medium, the more small intestinal-like cells grown in DMEM-Gal displayed no alteration of the lateral mobility of either cholera toxin (B subunit)-labelled ganglioside GM1 (diffusion coefficient, D [x 10(8)] = 0.8-0.9 cm2s-1; mobile fraction, R = 50-60%) or antibody-stained Class 2 histocompatibility (HLA-DR) antigen (D [x 10(9)] = 2 cm2s-1; R = 60-70%). However, antibody-labelled beta 2-microglobulin of HLA Class 1 antigen displayed increased mobility in HT29-Gal cells; D was x 1.4 and R x 1.8 larger in the HT29-Gal cells. By contrast, the mobility of a neoplastic antigen was reduced; D and R were x0.60 and x0.69 of the values seen in HT29-Glu cells. It is thus concluded that DMEM-Gal-induced differentiation in confluent HT29 cells is accompanied by specific rather than general effects on the lateral mobility of different membrane components.     

Translational mobility of the type 3 inositol 1,4,5-trisphosphate receptor Ca2+ release channel in endoplasmic reticulum membrane

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an integral membrane protein in the endoplasmic reticulum (ER) which functions as a ligand-gated Ca2+ release channel. InsP3-mediated Ca2+ release modulates the cytoplasmic free Ca2+ concentration ([Ca2+]i), providing a ubiquitous intracellular signal with high temporal and spatial specificity. Precise localization of the InsP3R is believed to be important for providing local [Ca2+] regulation and for ensuring efficient functional coupling between Ca2+ release sites by enabling graded recruitment of channels with increasing stimulus strength in the face of the intrinsically unstable regenerative process of Ca2+-induced Ca2+ release. Highly localized Ca2+ release has been attributed to the ability of the InsP3R channels to cluster and to be localized to discrete areas, suggesting that mechanisms may exist to restrict their movement. Here, we examined the lateral mobility of the type 3 isoform of the InsP3R (InsP3R3) in the ER membrane by performing confocal fluorescence recovery after photobleaching of an InsP3R3 with green fluorescent protein fused to its N terminus. In Chinese hamster ovary and COS-7 cells, the diffusion coefficient D was approximately 4 x 10(-10) cm2/s at room temperature, a value similar to that determined for other ER-localized integral membrane proteins, with a high fraction (approximately 75%) of channels mobile. D was modestly increased at 37 degrees C, and it as well as the mobile fraction were reversibly reduced by ATP depletion. Although disruption of the actin cytoskeleton (latrunculin) was without effect, disruption of microtubules (nocodazole) reduced D by half without affecting the mobile fraction. We conclude that the entire ER is continuous in these cells, with the large majority of InsP3R3 channels free to diffuse throughout it, at rates that are comparable with those measured for other polytopic ER integral membrane proteins. The observed InsP3R3 mobility may be higher than its intrinsic diffusional mobility because of additional ATP- and microtubule-facilitated motility of the channel.     

Regulation of the lateral diffusion of WGA-labeled glycoconjugates in human leukocytes. Comparison between adult granulocytes and differentiating promyelocytic HL60 cells

The regulation of the membrane mobility of glycoconjugates in human polymorphonuclear leukocytes (PMNL) was studied by comparing adult PMNL with promyelocytic HL60 cells before and after stimulation of differentiation in HL60 cells with phorbol-myristate acetate (PMA) with respect to lateral diffusion of wheat germ agglutinin (WGA)-labeled glycoconjugates. For this purpose we developed a novel variant of microscope equipment for the study of fluorescence recovery after photobleaching (FRAP) and continuous fluorescence microphotolysis (CFM) using a mini-computer for handling of shutters, data acquisition, and calculations. This equipment is presented in the report. We found that PMA-induced differentiation in HL60 cells reduced the lateral diffusion coefficient (D) of WGA-labeled membrane entities from about 1.5 to 1.0 x 10(-10) cm2/s, which was close to that found for adult blood PMNL, i.e., 1-1.2 x 10(-10) cm2/s. The lateral mobility (D x 10(10)) of succinylated WGA (S-WGA) was 2.3 and 1.7 cm2/s in undifferentiated and PMA-differentiated HL60 cells, respectively, indicating that WGA might have cross-linked membrane receptors, resulting in the slower diffusion. The results are discussed in relation to the effect of phagocyte maturation on the mobility of membrane components.     

Lateral mobility and organization of phospholipids and proteins in rat myocyte membranes. Effects of aging and manipulation of lipid composition

The effects of aging and of liposome treatment on the lateral mobility of phospholipids and proteins in the plasma membrane of cultured rat heart myocytes were studied by fluorescence photobleaching recovery. Both the mobile fraction (R) and the lateral diffusion coefficient (D) of the fluorescent phospholipid N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine were found to depend on the culture's age. Aged myocyte cultures (15 days old) demonstrated higher R and lower D as compared with young ones (5 days old). Treatment of aged cultures with phosphatidylcholine (PC) liposomes, which increases the PC/sphingomyelin (SM) ratio and decreases the cholesterol level, reversed the D value to the level observed in young cultures and decreased R below the value encountered in young cells. Treatments with SM liposomes (which induce cholesterol depletion without altering the PC/SM ratio) and with PC/cholesterol (1:0.9) liposomes (which increase the PC/SM ratio without cholesterol depletion) have indicated that the PC-liposome effect is due to changes in both the PC/SM ratio and in the cholesterol level. Analogous experiments on the mobility of succinyl-concanavalin A receptors yielded similar effects on R, without altering the D value. The changes in the D and R values of the markers studied are most likely initiated by the observed alterations in the myocyte lipid composition under the conditions employed. The possible involvement of changes in the organization of membrane lipids in domains in the observed phenomena is discussed.     

Lateral diffusion of PDGF β-receptors in human fibroblasts

When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum or PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding normal and starved cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0×10^–10 cm²s^–1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor, and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.     

Cell-specific constraints to the lateral diffusion of a membrane glycoprotein.

We have previously shown that the lateral diffusion, D, of the class I Major Histocompatibility Complex (MHC) glycoprotein H-2Ld is constrained by its glycosylation, when expressed in mouse L-cells. Removal of one or more of the 3 N-linked oligosaccharides of H-2Ld glycoproteins results in an increase in D. In order to further examine the influence of glycosylation on D, we compared lateral diffusion of H-2Ld expressed in wild-type CHO cells with lateral diffusion of the same molecule expressed in mutant CHO cells with aberrant surface glycosylation. In addition, we compared lateral diffusion of wild-type and unglycosylated H-2Ld antigens in these cells. In contrast to the large effect of glycosylation state on lateral diffusion of H-2Ld in mouse L-cells, there was little effect of glycosylation on lateral diffusion of H-2Ld in any of the CHO cells. This, together with similar results on hamster class I antigens, indicates that the constraints to D of H-2Ld and other class I MHC molecules are different in CHO cells than in L-cells. Measurements of lateral diffusion after treatment of cells with cytochalasin D make it clear that interactions between MHC class I molecules and a cytoskeleton are important in reducing the mobile fraction of diffusing molecules, R, though they cannot be shown to directly affect the diffusion coefficient, D.     

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.     

Lateral diffusion of nerve growth factor receptor: modulation by ligand-binding and cell-associated factors.

We compared the properties in human melanoma cell line A875 and rat pheochromocytoma cell line PC12 of nerve growth factor receptor (NGFr). We also analyzed NGFr and a truncated NGFR lacking the cytoplasmic domain, which were transiently expressed in COS cells. The full-length NGFR expressed in COS cells bound nerve growth factor (NGF) with positive cooperativity, but A875 NGFr and truncated NGFr in COS cells did not display positive cooperativity. The anti-human NGFr monoclonal antibody NGFR5 was characterized and found not to compete with NGF for binding to NGFr. Fabs were prepared from NGFR5 and 192, an anti-rat NGFR monoclonal antibody that was previously shown not to compete with NGF for binding. Fluorescein-labeled Fabs were used to measure the distribution and lateral diffusion of the NGFr. NGFr expressed on COS and A875 cells are diffusely distributed, but NGFr on the surface of PC12 cells appeared, for some cells, to be patched. In A875 cells, 51% of the NGFr was free to diffuse with diffusion coefficient (D) approximately 7 X 10(-10) cm2/s. In COS cells, 43% diffused with D approximately 5 X 10(-10) cm2/s. There was no significant difference in diffusibility between the full-length NGFr and the truncated NGFr. We compared NGFr diffusion on PC12 cells in suspension or adherent to collagen-coated coverslips. For suspension cells, we obtained 32% recovery with D approximately 2.5 X 10(-9) cm2/s. On adherent cells, we obtained 17% recovery with 6 X 10(-9) cm2/s. Binding of NGF enhanced lateral diffusion of NGFr in A875 cells and in PC12 cells in suspension but did not alter lateral diffusion of NGFr in COS cells or in adherent PC12 cells. NGF had no effect on the diffusing fraction or the distribution of NGFR for any cell line.     

The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells

Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N-hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures.     

Lowering the barriers to random walks on the cell surface

We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and alpha-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in alpha-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for alpha-spectrin-deficient and normal MEL cells, differing by a factor of approximately 2, on three different timescales: tens of seconds, 1-10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the alpha-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.