Plasma membrane sterol distribution resembles the surface topography of living cells

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8-2.5 microm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1--a putative raft marker--did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Summary Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothyocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.This study was supported by a grant from the Cancer Society of Saint Joseph County, Indiana and from the Phi Beta Psi Sorority     

Surface morphology and functions of pharyngeal structures in the larval lamprey Petromyzon marinus

To gain insight into the multiple functions of a complex biological structure, the morphology of the pharynx of the larva (ammocoete) of the lamprey Petromyzon marinus was investigated with scanning electron microscopy and histochemistry (PAS and Alcian blue). Features studied include the gills, the parabranchial chambers external to the gills, intrapharyngeal ciliary tracts, the ridged pharyngeal roof, the floor, and the intrapharyngeal taste buds. Significant findings are: (1) All (nonciliated) cells lining these structures are covered with microvilli or microridges. The pattern and packing density of these membrane features vary among different pharyngeal structures. The lumenar membranes of pharyngeal lining cells overlie a mucous prosecretion in the apical cytoplasm, suggesting that the microvilli/ridges on these membranes function to anchor mucus. (2) Patterns of microvilli/ridges on the gill respiratory lamellae differ among ammocoetes of different species. (3) Pharyngeal osmo-regulatory cells (“chloride cells”) could not be identified on the basis of the microvillus/ridge pattern. (4) Two types of ciliary tracts are present within the pharynx. One has tall (x = 13 μm) and densely packed cilia, whereas the other has shorter (x = 7 μm) and less densely packed ones. Because mucus covers both types of tracts their function appears to involve the transport of mucus. (5) Food particles were found on the lateral surfaces of the gill filaments and on the surfaces of the parabranchial chambers. It appears that goblet cells in the epithelia of these regions secrete mucus in which the particles are trapped.     

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.     

Morphometric analysis of epithelial cells of frog urinary bladder, II. Effect of ADH, calcium ionophore (A23187) and verapamil on isolated dissociated cells

Isolated frog urinary bladder epithelial cells, upon dissociation lose their polarity and develop microridges and occasional microvilli in a global fashion. These cells, when exposed only to isotonic Ringer's solution manifest a membrane conformation with smooth discontinuous microridges, a cytoplasm with numerous free ribosomes, rough ER, thin Golgi cisternae, mitochondria, small vacuoles, electron-dense granules, few microtubules, and numerous microfilaments and intermediate filaments with an apparent random distribution, the dissociated cells, when treated with ADH or calcium ionophore (A23187), have the appearance of numerous elongated microvilli over the entire cell surface. The cytoplasm, under these conditions, is occupied by large vacuoles with a distribution of long profiles of aggrephores and associated vesicles. The peripheral cytoplasm as well as the cavities of the elongated microvilli of these cells contain large concentrations of microfilaments often showing a strong axial orientation to the long axis of the microvilli. Many of these filamentous elements appear in contact with the apical membrane of these microvilli with an alignment with the external glycocalyx. There is an indication that these morphocytological changes as revealed by SEM and TEM studies, correlated with a redistribution and realignment of microfilaments and possibly microtubules as detected by fluorescent microscopy using immunofluorescent antibody labeling for actin and tubulin. Cells treated with verapamil, a calcium antagonist, presented dwarf and stout microvilli with little detectable alterations in the cytoplasmic compositions from that of non-hormonal treated cells. Verapamil prevented ADH induction of microvilli, with the membrane, under these conditions, appearing as compact microridges. The results indicate that calcium ionophore, like ADH, produces intense formation of microvilli in dissociated cells, mobilization and realignment of microfilaments, microtubules, increase in the density of vesicles, aggrephores and possibly secretory granules, whereas the calcium antagonist, verapamil, opposes these actions. The results suggests a prominent role of calcium in the morphological changes induced by ADH.     

Diffusion, patching, and capping of stearoylated dextrans on 3T3 cell plasma membranes

Fluorescence-labeled trinitrophenylated stearoylated dextrans have been used as controllable analogues of cell membrane proteins on model membranes and on a variety of natural cell membranes. This paper reports their behavior on 3T3 mouse fibroblast plasma membranes. Spatial distribution on the membrane was studied by fluorescence microscopy, and molecular mobility was measured by fluorescence photobleaching recovery. At concentrations from 10(2) to 3 X 10(3) molecules/micron2 essentially homogeneous fluorescence was observed after treatment with these stearoyldextrans in culture. Diffusion coefficients and fractional recovery of fluorescence after photobleaching were cvoncentration independent. For 3 X 10(3) molecules/micron2 we found at 23 degrees C D = (3.0 +/- 1.8) X 10(-10) cm2/s with 65 +/- 17% recovery and at 37 degrees C D = (7.0 +/- 5.0) X 10(-10) cm2/s without a change of the fractional recovery. Cross-linking with antibodies stopped diffusion on a macroscopic scale and sometimes induced patching, mottling (defined as the development of gaps in the fluorescence layer), and capping (defined as the confinement of the fluorescence to less than 50% of the cell). Capping required approximately 3 h at 37 degrees C and was inhibited by metabolic poisons and cytochalasin B. These drugs did not affect stearoyldextran diffusion or fractional recovery. Colchicine, which did not dramatically affect capping, slowed diffusion two- to threefold but did not affect fractional recovery. The antibody inhibition of the diffusion of stearoyldextrans precedent to capping did not affect the diffusion of a lipid probe or fluorescein isothiocyanate labeled membrane proteins. When the trinitrophenylated stearoyldextran was cleared from most of the surface by capping and the surface subsequently relabeled with stearoyldextran, the diffusion coefficient and fractional recovery of the second label were identical with those of the first label prior to capping. Thus, capping does not clear an immobilizing factor from the membrane.     

Changes of the lingual epithelium in Ambystoma mexicanum

Changes in the lingual epithelium during ontogenesis and after induced metamorphosis in Ambystoma mexicanum are described as observed by light microscopy and scanning electron microscopy. The epithelium of the tongue is always multilayered in the larva as well as in the adult. It consists of a stratum germinativum with little differentiated basal cells and a stratum superficiale (superficial layer) with specialized superficial cells and goblet cells. Usually, there are more than two layers because of a stratum intermedium consisting of replacement cells. The apical cell membrane of the superficial cells is perforated by fine pores. Its most typical feature are microridges. Maturing superficial cells possess microvilli. Goblet cells occur in early larvae primarily in the centre of the tongue. They spread throughout the dorsal face of the tongue as their numbers increase during ontogenesis. The small apices of the goblet cells are intercalated in the wedges between the superficial cells. Leydig cells are not found on the larval tongue but on that of adults. Due to metamorphosis, the epithelium of the tongue changes. It is furrowed in its anterior part. The furrows house the openings of the lingual glands. The surface is further modulated by ridges which are densely coated by microvilli and which bear the taste buds. The villi of the tongue which lack extrusion pores show cilia and microvilli but lack microridges. The Leydig cells disappear during metamorphosis. In addition to the two types of goblet cells found in different regions of the glandular tubules, goblet cells occur in the caudal part. They secrete directly into the cavity of the mouth. The posterior part is characterised by a dense coat of cilia.     

Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.     

Lactadherin detects early phosphatidylserine exposure on immortalized leukemia cells undergoing programmed cell death.

BACKGROUND: Phosphatidylserine (PS) appears on the outer membrane leaflet of cells undergoing programmed cell death and marks those cells for clearance by macrophages. Macrophages secrete lactadherin, a PS-binding protein, which tethers apoptotic cells to macrophage integrins. METHODS: We utilized fluorescein-labeled lactadherin together with the benchmark PS Probe, annexin V, to detect PS exposure by flow cytometry and confocal microscopy. Immortalized leukemia cells were treated with etoposide, and the kinetics and topology of PS exposure were followed over the course of apoptosis. RESULTS: Costaining etoposide-treated leukemoid cells with lactadherin and annexin V indicated progressive PS exposure with dim, intermediate, and bright staining. Confocal microscopy revealed localized plasma membrane staining, then diffuse dim staining by lactadherin prior to bright generalized staining with both proteins. Annexin V was primarily localized to internal cell bodies at early stages but stained the plasma membrane at the late stage. Calibration studies suggested a PS content less, less than or approximately equal to 2.5%-8% for the membrane domains that stained with lactadherin but not annexin V. CONCLUSIONS: Macrophages may utilize lactadherin to detect PS exposure prior to exposure of sufficient PS to bind annexin V. The methodology enables detection of PS exposure at earlier stages than established methodology.     

Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine-like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine-labeled polyclonal anti-human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 +/- 2.0 (mean value +/- SD; n = 29) and 7.1 +/- 2.3 (n = 30) x 10(-10) cm2s-1 for Ig-and Fab-labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 +/- 13% and 71 +/- 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope-based impression that the basolateral surface primarily harboured the SC receptor.     

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.     

Ras diffusion is sensitive to plasma membrane viscosity

The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

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.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

Summary The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Distribution pattern of acid phosphatase activity in human peripheral blood leukocytes: a cytochemical scanning electron microscopy study

The distribution patterns of acid phosphatase hydrolytic activity were studied in human peripheral blood cells with enzymocytochemical techniques together with light and scanning electron microscopy in the secondary and backscattered electron imaging modes. The acid phosphatase reaction product was seen in three different patterns of distribution: focal, granular and diffuse. These patterns were correlated with similar findings obtained with light microscopy. Acid phosphatase distribution patterns seen with SEM in the BEI mode were also correlated with the surface morphology of peripheral blood cells seen in the SEI mode. Cells exhibiting the focal pattern were smooth-surfaced with few microvilli; cells showing a granular pattern presented microvilli and microridges; ruffles were characteristic of cells with a diffuse pattern of activity. No reaction product was seen in cells bearing microvilli or ridges. Our findings demonstrate the correlation between acid phosphatase activity patterns and surface features in different subpopulations of peripheral blood cells.     

Intrinsically fluorescent luteinizing hormone receptor demonstrates hormone-driven aggregation

The possibility that LH receptors exist as isolated molecules when unbound and aggregate upon binding gonadotropins has previously been untestable in viable cells for want of a suitable nonhormone probe. We have now expressed in CHO cells an intrinsically-fluorescent LH receptor involving enhanced green fluorescent protein (GFP) fused to the C-terminus of the rat LH receptor (rLHR-GFP). More than half of these receptors (54 +/- 4%) are located on the plasma membrane and are functional: cAMP levels increase 3-5 fold in response to 10 nM LH or hCG. In fluorescence photobleaching recovery studies at 37 degrees C, 54 +/- 13% of unoccupied rLHR-GFP were laterally mobile with a diffusion coefficient D of 16 +/- 3.5 x 10(-10)cm2sec-1. Introduction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 +/- 1.3 x 10(-10)cm2sec-1 and reduced fluorescence recovery after photobleaching to 27 +/- 1%. Following treatment with 1 nM hCG, rLHR-GFP were laterally immobile and were distributed into small fluorescent patches over the cell surface. Thus, unoccupied rLHR-GFP receptors apparently exist as dispersed plasma membrane proteins with comparatively fast lateral diffusion. Interaction of receptors with LH or hCG caused clustering of rLHR-GFP receptors, significantly restricting lateral diffusion.     

Expression and localization of four uroplakins in urothelial preneoplastic lesions

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.     

Creating biological membranes on the micron scale: forming patterned lipid bilayers using a polymer lift-off technique

We present a new method for creating patches of fluid lipid bilayers with conjugated biotin and other compounds down to 1 microm resolution using a photolithographically patterned polymer lift-off technique. The patterns are realized as the polymer is mechanically peeled away in one contiguous piece in solution. The functionality of these surfaces is verified with binding of antibodies and avidin on these uniform micron-scale platforms. The biomaterial patches, measuring 1 micro m-76 microm on edge, provide a synthetic biological substrate for biochemical analysis that is approximately 100x smaller in width than commercial printing technologies. 100 nm unilamellar lipid vesicles spread to form a supported fluid lipid bilayer on oxidized silicon surface as confirmed by fluorescence photobleaching recovery. Fluorescence photobleaching recovery measurements of DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiIC(18)(3))) stained bilayer patches yielded an average diffusion coefficient of 7.54 +/- 1.25 microm(2) s(-1), equal to or slightly faster than typically found in DiI stained cells. This diffusion rate is approximately 3x faster than previous values for bilayers on glass. This method provides a new means to form functionalized fluid lipid bilayers as micron-scale platforms to immobilize biomaterials, capture antibodies and biotinylated reagents from solution, and form antigenic stimuli for cell stimulation.