Domain formation in models of the renal brush border membrane outer leaflet.

The plasma membrane outer leaflet plays a key role in determining the existence of rafts and detergent-resistant membrane domains. Monolayers with lipid composition mimicking that of the outer leaflet of renal brush border membranes (BBM) have been deposited on mica and studied by atomic force microscopy. Sphingomyelin (SM) and palmitoyloleoyl phosphatidylcholine (POPC) mixtures, at molar ratios varying from 2:1 to 4:1, were phase-separated into liquid condensed (LC) SM-enriched phase and liquid expanded (LE) POPC-enriched phase. The LC phase accounted for 33 and 58% of the monolayers surface for 2:1 and 4:1 mixtures, respectively. Addition of 20-50 mol % cholesterol (Chl) to the SM/POPC (3:1) mixtures induced marked changes in the topology of monolayers. Whereas Chl promoted the connection between SM domains at 20 mol %, increasing Chl concentration progressively reduced the size of domains and the height differences between the phases. Lateral heterogeneity was, however, still present at 33 mol % Chl. The results indicate that the lipid composition of the outer leaflet is most likely responsible for the BBM thermotropic transition properties. They also strongly suggest that the common maneuver that consists of depleting membrane cholesterol to suppress rafts does not abolish the lateral heterogeneity of BBM membranes.

Temperature dependence of the topology of supported dimirystoyl-distearoyl phosphatidylcholine bilayers.

Topology of fluid and gel domains in the supported bilayer two-component system formed from equimolar mixtures of dimyristoylphosphatidylcholine (DMPC) and distearoylphosphatidylcholine (DSPC) was determined by AFM, at various temperatures corresponding to the gel and the gel + fluid region of the phase diagram. The data show that, in the disconnected fluid part of the DMPC/DSPC gel-liquid crystal-phase-separation region, the size of fluid domains markedly exceeds that predicted from spectroscopic experiments or from Monte Carlo simulations. They provide a direct evidence for the transition from the disconnected fluid to the disconnected gel region of the phase diagram, again with gel-phase domains much larger than expected. Finally, images of the gel phase at different temperatures suggest that structural rearrangements of the phospholipids can disrupt the continuity of the supported bilayer.

In situ imaging of detergent-resistant membranes by atomic force microscopy.

Purified detergent-resistant membranes (DRMs) are powerful tools for the biochemical study of plasma membrane domains. To what extent these isolated DRMs correspond to native membrane domains remains, however, a matter of debate. The most immediate question to be answered concerns the in situ size range of DRMs, a determination that escapes classical microscopy techniques. In this study we show that in situ three-dimensional images of a material as fragile as Triton X-100-treated cells can be obtained, in buffer, by tapping mode atomic force microscopy. These images establish that, prior to the isolation procedure, the detergent plasma membrane fragments form domains whose size frequently exceeds 15-20 microm(2). This DRMs size range is about 1 order of magnitude higher than that estimated for the larger microdomains of living cells, which strongly suggests that membrane microdomains rearrange into larger DRMs during Triton X-100 treatment. Concomitantly, the images also reveal the presence of the cytoskeleton, which is resistant to detergent extraction, and suggest that, in situ, DRMs are associated with the membrane cytoskeleton.

Tapping-mode atomic force microscopy on intact cells: optimal adjustment of tapping conditions by using the deflection signal.

Difficulties in the proper adjustment of the scanning parameters are often encountered when using tapping-mode atomic force microscopy (TMAFM) for imaging thick and soft material, and particularly living cells, in aqueous buffer. A simple procedure that drastically enhances the successful imaging of the surface of intact cells by TMAFM is described. It is based on the observation, in liquid, of a deflection signal, concomitant with the damping of the amplitude that can be followed by amplitude-distance curves. For intact cells, the evolution of the deflection signal, steeper than the amplitude damping allows a precise adjustment of the feedback value. Besides its use in finding the appropriate tapping conditions, the deflection signal provides images of living cells that essentially reveal the organization of the membrane cytoskeleton. This allows to show that changes in the membrane surface topography are associated with a reorganization of the membrane skeleton. Studies on the relationships between the cell surface topography and membrane skeleton organization in living cells open a new field of applications for the atomic force microscope.

In situ determination of intracellular membrane physical state heterogeneity in renal epithelial cells using fluorescence ratio microscopy.

6-Lauroyl-2-dimethylaminonaphtalene (laurdan) shows a spectral sensitivity to the lipid phase state with a 50 nm red shift of the emission maximum when passing from the gel to the liquid crystalline phase. This spectral sensitivity allows one to determine the membrane physical state using Generalized Polarization (GP). In the present experiments, we used fluorescence ratio imaging microscopy to determine the laurdan GP in living kidney cells. Two renal epithelial cells lines, MDCK and LLC-PK1 cells, and CV-1 cells, a fibroblast-like renal cell line were investigated. In these cells, laurdan labels both the plasma membrane and intracellular membranes. Comparison of spectrofluorimetry and fluorescence ratio imaging data obtained from liposomes and cells suspensions labeled with laurdan demonstrates that the GP can be accurately determined using common fluorescence microscopy equipment. The GP mean values determined from individual cells varied from 0.2 to 0.4 for the epithelial cells as compared to 0.0-0.1 for CV1 cells. Using living MDCK cells grown as a monolayer, the GP maps indicated that, within a single cell, the intracellular GP values varied from 0.0 to 0.6, i.e., from the equivalent of a liquid-crystalline state to a gel or a lipid-ordered state, and that there was a marked heterogeneity in the spatial distribution of the GP values. To further characterize this intracellular heterogeneity, co-localization experiments with specific organelle markers were undertaken. The results strongly suggest that in intact cells at physiological temperature, GP values decrease in the following order: plasma membranes > endosomes > mitochondria > Golgi apparatus.

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.

Atomic force microscopy of renal cells: limits and prospects.

In this brief review, we present three-dimensional images of living Madin-Darby canine kidney (MDCK) cells and CV-1 cells that illustrate the possibilities and limits in the use of atomic force microscopy (AFM) for studying the topography of the cell surfaces and of isolated biological membranes. We show that microvilli can be imaged at the surface of living epithelial cells. However, when these microvilli are abundant and close to each other, the geometry of the AFM tip only allows an access to the upper part of the structures and precludes nanometer range imaging of the cell surface. Such a nanometer range imaging was obtained with other cell types like CV-1 cells and with isolated biological membranes. It reveals that protruding particles 5 to 60 nm xy size, likely corresponding to membranes proteins, occupy most of the membrane surface. These images indicate that the AFM already gives an access to the cell surface structure at the mesoscopic scale, which constitutes a major step for the understanding of the structure-function relationships in membranes. Perspectives for a further step, the imaging at molecular resolution of membranes, are discussed.

Simultaneous imaging of the surface and the submembraneous cytoskeleton in living cells by tapping mode atomic force microscopy.

Contact and tapping mode atomic force microscopy have been used to visualize the surface of cultured CV-1 kidney cells in aqueous medium. The height images obtained from living cells were comparable when using contact and tapping modes. In contrast, the corresponding, and simultaneously acquired, deflection images differed markedly. Whereas, as expected, deflection images enhanced the surface features in the contact mode, they revealed the presence of a filamentous network when using the tapping mode. This network became disorganized upon addition of cytochalasin, which strongly suggests that it corresponded to the submembraneous cytoskeleton. Examination of fixed cells further supported this assumption. These data show that, in addition to the structural information on the cell surface, the use of the tapping mode in liquid can also provide a good visualization of the membrane cytoskeleton. Tapping mode atomic force microscopy appears to be a promising technique for studying interactions between cell surface and subsurface structures, a critical step in many biological processes.

Temperature dependence of endocytosis in renal epithelial cells in culture.

Temperature dependence of fluid-phase endocytosis was determined in two renal epithelial cell lines, MDCK cells and LLC-PK1 cells, using Lucifer Yellow or horseradish peroxidase as markers. For both cell lines, grown on solid support as a confluent monolayer, biphasic curves of marker uptake vs. temperature were obtained. The changes in slope occurred around 27 degrees C, a critical temperature at which the lipids of the plasma membrane of MDCK cells enter in the gel state. Activation energies were significantly higher above 27 degrees C (15-22 kcal/mol) than below that critical temperature (9-12 kcal/mol). These data indicate that changes in membrane physical state have marked effects on endocytic processes. They suggest that two mechanisms, with different activation energies are involved in the fluid phase endocytosis by renal epithelial cells in culture.

Imaging of the cytoplasmic leaflet of the plasma membrane by atomic force microscopy.

The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on glass coverslips was imaged by atomic force microscopy (AFM) in air and under aqueous medium, in "contact" mode. Micrometer range scans on air-dried samples revealed a heterogeneous structure with some filaments, likely corresponding to actin filaments that abut the inner leaflet of the membrane, and a few semi-organized lattice structures that might correspond to clathrin lattices. Experiments in phosphate-buffered saline confirmed the heterogeneity of the inner membrane surface with the presence of large (> 100 nm) globular structures emerging from the surface. Using sub-micrometer scan ranges, protruding particles, that occupy most of the membrane surface, were imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, high magnification images of the inner membrane surface were heterogeneous with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proximity.

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles.

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.

Benzyl alcohol differently affects fluid phase endocytosis and exocytosis in renal epithelial cells.

The effects of benzyl alcohol, a local anaesthetic commonly used for modification of membrane fluidity, on fluid phase endocytosis and on exocytosis have been investigated in MDCK cells. Fluid phase endocytosis in confluent cells monolayer grown on solid support was determined, at 37 degrees C, by the uptake of the fluorescent dye Lucifer Yellow (LY). Exocytosis was estimated from the release of LY by cells preloaded with the dye. Addition of benzyl alcohol resulted in a concentration dependent inhibition of fluid phase endocytosis. For 30 mM benzyl alcohol, the inhibition obtained (83%) compared with that produced by preincubating the cells in a solution made hypertonic with 0.25 M sucrose. The inhibitory effect of benzyl alcohol was reversed within 30 min by washing. Endocytosis inhibition by benzyl alcohol was also observed in LLC-PK1 cells and OK cells, two renal epithelial cell lines of proximal tubule origin. In contrast, benzyl alcohol had no effect on exocytosis in LLC-PK1 cells, a limited but significant (15% at 30 mM) stimulatory effect on exocytosis in MDCK cells and a marked stimulatory effect (75% at 30 mM) in OK cells. These data demonstrate that benzyl alcohol affects endocytosis and exocytosis processes in renal epithelial cells. They suggest that membrane fluidity may alter membrane trafficking in living renal epithelial cells.

Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.

Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2-benzyl-1-oxopropyl]- glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor-NEP complexes. In the present paper, we demonstrate that, for short incubation times, the fluorescent probe preferentially labeled brush border membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in videomicrofluorimetry suggesting that part of the inhibitor was bound or localized close to the basolateral plasma membrane. Confocal experiments confirmed the transcytosis of FTI/NEP complex, from the apical to the basolateral domain. Using [3H]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral membrane-bound molecules was estimated to be about 12 and 23%, respectively. The coincubation of the cells with FTI and 2B12, a monoclonal antibody raised against the rabbit enzyme, greatly modified the fluorescence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (> 1 h), in addition to the basolateral labeling, some intracellular fluorescent vesicles were observed essentially localized in the vicinity of the nucleus. The colocalization of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC-Con A) strongly suggests an endosomal/lysosomal internalization pathway when FTI was incubated in the presence of 2B12 mAb.

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1.

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC6 (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10-70 nm for MDCK cells and 20-90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.

Water permeation in Madin-Darby canine kidney cells is modulated by membrane fluidity.

Simultaneous determinations of water and antipyrine permeations in monolayers of Madin-Darby canine kidney (MDCK) cells grown on a permeant support were done to study the relationships between water transport and membrane fluidity in these epithelial cells. The changes in permeation of the lipophilic non-electrolyte antipyrine were used to probe the modifications in membrane fluidity. In controls, the apparent diffusional permeability coefficient for water (PDw) was three times higher than the antipyrine's one, PDAp (4.2.10(-5) vs. 1.4.10(-5) cm s-1). Addition of vasopressin or dibutyryl cyclic AMP to the monolayers induced a biphasic increase in antipyrine permeation with peak values at t = 2 min, 3-4-fold that of controls. Variations in water permeation were of similar amplitude and obeyed the same time course, leaving the water to antipyrine permeation ratios unchanged. Compound H7, an inhibitor of protein kinases, blunted the increase in permeation for both antipyrine and water. Finally, addition of the fluidizing agent benzyl alcohol to the monolayers resulted in a parallel increase in PDAp and PDw. These results suggest that the physical state of membrane lipids may control water permeation in MDCK cells.

ADH modulates plasma membrane lipid order of living MDCK cells via a cAMP-dependent process.

Using 1-[4-(trimethylamino)phenyl]-6-phenyl-hexa-1,3,5-triene, a fluorescent probe that specifically labels the external leaflet of the plasma membrane of living cells, we examined the effects of antidiuretic hormone (ADH) and various agents known to raise intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on the physical state of the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In polarized cells grown as a monolayer, [desamino-Cys1, DArg8]-vasopressin (V2-agonist) elicited a biphasic decrease in the lipid order as estimated from the decrease in fluorescence anisotropy (from r = 0.317 to r = 0.304, 37 degrees C) of the apical domain of the plasma membrane, equivalent at the peak response (t = 5 min) to that produced by an upward shift in temperature of 5-6 degrees C. A similar response was obtained by adding dibutyryl cAMP to the monolayers. Experiments on cell suspensions further indicated that the biphasic decrease in lipid order could also be evoked by forskolin, prostaglandin E2, and bradykinin but not by bradykinin plus indomethacin and was inhibitable by the protein kinases inhibitor compound H7. These data demonstrate that the lipid order of the plasma membrane of MDCK cells can be modulated in situ by cAMP-dependent processes probably involving protein kinase A activity, i.e., that membrane "fluidity" might act in the regulation of the cellular function of living epithelial cells. They provide a rationale for the changes in lipophilic solute permeability that accompany the increase in water permeability of target cells on ADH administration.

Temperature dependence of plasma membrane physical state in living Madin-Darby canine kidney cells.

Using 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) as a probe, we have done fluorescence anisotropy experiments to estimate, in situ, the temperature dependence of the lipid order of the plasma membrane of MDCK cells. In intact monolayers, anisotropy vs temperature biphasic curves were obtained, indicative of discontinuous change in lipid physical state starting around 27 degrees C, in the apical domain of the plasma membrane. The very high anisotropy values (r greater than 0.33) determined below this temperature strongly suggested it corresponded to a phase separation phenomenon. Biphasic curves with similar critical temperatures zone were also obtained when measuring anisotropy or fluorescence lifetime of suspensions of MDCK cells. This suggests that changes in lipid physical state might be involved in the marked dependence on temperature of tight junction formation and of electrical conductance in MDCK monolayers.

Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells.

Fluorescence anisotropy experiments have been done to estimate, in situ, the lipid order of the plasma membrane of polarized Madin-Darby Canine Kidney cells (MDCK) grown on glass cover slips and labeled by 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific marker of the plasma membrane of living cells. Fluorescence microscopy, back-exchange, and quenching experiments indicated that TMA-DPH labeled the highly ordered (r greater than or equal to 0.32, 37 degrees C) apical domain of the plasma membrane of confluent monolayers. Opening of tight junctions or addition of the probe to cell suspensions resulted in a homogeneous distribution of TMA-DPH over the cell surface and in a marked decrease in anisotropy (0.27 less than or equal to r less than or equal to 0.29) that was due neither to a direct effect of Ca2+ on the probe nor to a change in fluorescence lifetime. Our data indicate that the apical domain, likely the external leaflet, of the plasma membrane of polarized MDCK cells is much more ordered than its basolateral counterpart.