atp depletion: a novel method to study junctional ... · johns hopkins university school of...

INTRODUCTION

Many of the morphological features that characterize epithe-lial tissues result from the specialized arrangement of both theactin cytoskeleton and intercellular junctions connecting thecells. In the case of MDCK cells, some steps involved in theformation of the cytoskeleton have been elucidated (R.Bacallao et al., unpublished data; Mills and Lubin, 1986). Asearly as 6 hours in culture, the actin fibers form a ring that cir-cumscribes the plasma membrane and defines their apical pole.This apical ring appears to precede and to form independentlyof the intercellular junctions such as tight junctions and zonulaadherens. However, when intercellular junctions form, theapical ring and the junctions are located at the same level inthe cells. The establishment of the actin cytoskeleton ofMDCK cells is completed with the formation of stress fiberslocated at the surface that attaches to the substratum, followedby the formation of a cortical network extending along theentire apico-basal axis of the cell. Thus, in confluent MDCKepithelia, the actin cytoskeleton exhibits three distinct arrange-ments: an apical ring that includes and circumscribes the

microvilli, a cortical network and the stress fibers at the baseof the cells.

Several studies in a number of epithelia have suggested thatthe actin cytoskeleton interacts specifically with the tightjunction and the zonula adherens (Hirokawa and Tilney, 1982;Madara et al., 1986; Madara and Dharmsathaphorn, 1985;Madara, 1989; Meza et al., 1980, 1982). The junctions and theapical ring are located at the same level. Drugs, such ascytochalasin B and D, that depolymerize fibrillar actin (F-actin) into globular actin (G-actin) increase the diffusion ofions and small molecules through the tight junction (Rassat etal., 1982; Madara, 1988; Madara et al., 1986, Kellerman et al.,1990). Prior work has shown that ATP depletion can be usedto separate the gate and fence functions of the tight junctions(Mandel et al., 1993). Ultrastructural analysis showing theclose apposition between the tight junction strands and theactin cytoskeleton further support the idea that the actincytoskeleton modulates tight junction permeability (Madara,1987). This study explores the modification of the actincytoskeleton after ATP depletion and attempts to correlatethese changes to both the gate and fence function of the tight

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The effect of cellular injury caused by depletion of intra-cellular ATP stores was studied in the Madin-Darby caninekidney (MDCK) and JTC cell lines. In prior studies, it wasshown that ATP depletion uncouples the gate and fencefunctions of the tight junction. This paper extends theseobservations by studying the changes in the actin cytoskele-ton and tight junction using electron microscopy andconfocal fluorescence microscopy in combination withcomputer-aided three-dimensional reconstruction. Markedregional differences in the sensitivity to the effects of ATPdepletion were observed in the actin cytoskeleton. Actindepolymerization appears to first affect the cortical actinnetwork running along the apical basal axis of the cell. Thenext actin network that is disrupted is the stress fibers

found at the basal surface of the cell. Finally, the actin ringat the level of the zonulae occludens and adherens is com-promised. The breakup of the actin ring correlates withultrastructural changes in tight junction strands and theloss of the tight junction’s role as a molecular fence. Duringthe process of actin network dissolution, polymerized actinaggregates form in the cytoplasm. The changes in the junc-tional complexes and the potential to reverse the ATPdepletion suggest that this may be a useful method to studyjunctional complex formation and its relationship to theactin cytoskeletal network.

Key words: actin cytoskeleton, ATP depletion, tight junction

SUMMARY

ATP depletion: a novel method to study junctional properties in epithelial

tissues

I. Rearrangement of the actin cytoskeleton

Robert Bacallao1,*, Alan Garfinkel2, Steven Monke2, Guido Zampighi3 and Lazaro J. Mandel4

1Division of Nephrology and Hypertension, Department of Medicine S-208, Northwestern University, 303 E Chicago Avenue,Chicago, IL 60611, USA2Department of Physiological Science and 3Department of Anatomy and Cell Biology, University of California, Los Angeles, CA,USA4Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA

*Author for correspondence

Journal of Cell Science 107, 3301-3313 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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junction. We show that the actin cytoskeleton of MDCKepithelia became altered after 20-30 minutes of ATP depletion.The three actin arrangements (the cortical actin network, theapical ring and stress fibers) show varying degrees of sensitiv-ity to the effects of ATP depletion. The loss of the molecularfence function correlates with disruption of the actin ring after60 minutes of ATP depletion.

MATERIALS AND METHODS

Cell culture MDCK-clone II (Fuller and Simons, 1986) and JTC cells (Finemanet al., 1992) were grown in DMEM media supplemented with 5%newborn calf serum (Gemini Bioproducts, CA), 2 mM glutamine(Sigma, MO) and penicillin/streptomycin (Sigma, MO). The cellswere incubated at 37°C in an air/5% CO2 atmosphere and werepassaged twice a week. The cells were plated on Nunc filter supportsat a low density (50,000 cells/cm2) and were allowed to grow to con-fluence over a 5 day period. Experiments were performed on confluentcells grown on these filter supports, as previously described (Bacallaoet al., 1989).

ATP depletionSince cultured cells display a combination of oxidative and glycolyticenergy metabolism, ATP depletion required inhibition of bothpathways (Mandel, 1986). Inhibition of glycolysis was accomplishedby initially washing the filters in a glucose-free modified Ringer’sbuffer supplemented with 2 mM glutamine as the sole metabolicsubstrate followed by a 3 hour incubation in this buffer at 37°C todeplete the tissue of endogenous metabolic substrates. The composi-tion of this modified Ringer’s buffer was (in mM): NaCl (115),NaHCO3 (25), K2HPO4 (5), MgSO4 (2), CaCl2 (1), and glutamine (2).This solution was bubbled with air/5%CO2 to obtain a pH of 7.4.After the initial incubation, rapid ATP depletion was achieved bytransferring the filters to modified Ringer’s buffer containing themitochondria inhibitor antimycin A (10

µM) and the glycolyticinhibitor 2-deoxyglucose (10 mM). At various times following theaddition of the metabolic inhibitors, the cells on the filters were pre-cipitated with ice-cold 3% perchloric acid. After obtaining a neu-tralized extract, the samples were analyzed for ATP, ADP, AMP andhypoxanthine (HX) contents by high pressure liquid chromatogra-phy, as previously described (Mandel et al., 1988), and normalizedto their protein content. The latter was measured by the Bradford(1976) method.

Samples designated as controls were obtained from culturessubjected to either the initial 3 hour incubation in the modifiedRinger’s buffer, or incubated for a fourth hour in this buffer. No dif-ferences were noted in any of the measured properties between thesetwo groups, nor was there a measurable deterioration due to the 3 hourinitial incubation.

Measurement of the transepithelial resistance (TER)After the 3 hour preincubation in modified Ringer’s buffer, the filterswere removed from the ring support and mounted in a speciallydesigned Ussing chamber maintained at 37°C by a water jacket. Cellmonolayers grown on filter supports were initially equilibrated inmodified Ringer’s buffer to obtain the initial TER values. Thesolutions were subsequently changed to the same buffer containing10 mM 2-deoxyglucose and 10 µM antimycin A to achieve ATPdepletion, as before. After 20 minutes, 5 mM EGTA was added toboth solutions to eliminate any remaining tight junctional resistance.The TER was measured by passing a constant current pulse (5 µA)through the tissue once per minute and measuring the resulting changein transepithelial voltage using an automatic voltage clamp (WorldPrecision Instruments, CN). The initial TER was calculated as the dif-

ference between the measured TERs at the beginning (prior toinhibitor treatment) and end of each experiment (after EGTAaddition). The MDCK cells used for these experiments are a low resis-tance clone (Fuller and Simons, 1986). The initial TER of these cellsgrown on the Nunc filters was consistently lower than reported byother investigators using this clone (Stevenson et al., 1988). Never-theless as previously described these cells have well-developed tightjunctions and normal gate and fence functions (Fuller and Simons,1986; Mandel et al., 1993).

Depolymerization of the microtubule networkIn some experiments, the effects of microtubule depolymerizationwere studied. For these studies, the cells were washed in themodified Ringer’s buffer and incubated at 37°C for 2.5 hours. Thecells were then transferred to the modified Ringer’s buffer contain-ing 30 µM nocodazole at 4°C for 30 minutes. Preliminary experi-ments had shown that 30 minutes of such treatment completelydepolymerized the microtubules, as determined by immunofluores-cence microscopy and by western blot analysis of the pool of poly-merized and depolymerized tubulin (results not shown). Thesamples were then separated into two groups, one group subjectedto ATP depletion as described above and the second group servedas an ATP replete control.

AntibodiesAntibodies that bind ZO-1 (clone R26), α- and β-tubulin (Amersham,IL), were used for the immunofluorescence studies. R26 (anti-ZO-1)was obtained from the Developmental Studies Hybridoma Bank main-tained by the Department of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, Baltimore, MD, andthe Department of Biology, University of Iowa, Iowa City, IA, undercontract NO1-HD-2-3144 from the NICHD. The actin cytoskeletonwas labeled with Bodipy-phalloidin (Molecular Probes, OR). All flu-orescence-conjugated secondary antibodies were obtained fromJackson Immunoresearch, PA.

ImmunofluorescenceThe cells were fixed and stained as previously described (Bacallaoet al., 1989). For actin and tubulin staining, the filters were dippedin 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2 (PEMbuffer) and were fixed with 0.25% glutaraldehyde (Polysciences, PA)in PEM buffer plus 0.1% Triton X-100 (Sigma, MO). The sampleswere fixed for 10 minutes at room temperature. The reaction wasquenched with three successive incubations with 1 mg/ml NaBH4 inPBS, pH 8.0, for 10 minutes at room temperature. The samples werewashed twice in PBS and were incubated in PBS + 0.1% Triton X-100 with 0.2% fish skin gelatin (Sigma, MO). All antibodies werediluted in this buffer. Antibody incubations were performed at 37°Cfor 45 minutes. A second incubation with a fresh sample of antibodyfor an additional 45 minutes was performed in all the specimens toensure adequate labeling. The cells were washed 6 times with PBS+ 0.1% Triton X-100 for 15 minutes at room temperature followingeach antibody incubation. Following the second antibody labeling,the cells were washed as described above with the exception that thefinal wash step was replaced with three successive washes with PBSfor 5 minutes each.

Samples that were stained for ZO-1 were fixed with 2%paraformaldehyde (Sigma, MO) prepared as previously described(Bacallao and Stelzer, 1989). The fixation reaction was quenched asdescribed above.

After the immunolabeling was completed the samples were post-fixed in 4% paraformaldehyde for 30 minutes at room temperature.The reaction was quenched with 50 mM NH4Cl dissolved in PBS.The samples were stored at 4°C in PBS. Prior to examination with aconfocal fluorescence microscope the specimen was mounted in 50%glycerol/PBS with 100 mg/ml of diamino-bicyclo-[2.2.2] octane(DABCO; Sigma, MO).

R. Bacallao and others

3303ATP depletion: I. Rearrangement of the actin cytoskeleton

Data acquisition, processing and visualizationData sets were gathered using a confocal scanning laser microscope(CLSM, Leitz, USA). The optical pathway in a confocal microscopeenables these instruments to collect light from a narrow focal plane(in the order of 0.2-1 µm), thus discriminating against out-of-focusinformation (Inoué, 1990). Each ‘optical section’ is stored in digitizedform (typically 512 × 512 × 1 byte), and a data set may consist of 50slices at 0.4 µm spacing. The digitized data sets can then be visual-ized by a variety of methods.

Extended focus imagesIn an extended focus image, all focal planes are stacked together,resulting in an image in which every observed structure is in focus.Figs 4 and 5 are extended focus images encompassing the entire imagefrom the apical portion of the cell to the filter support.

Three-dimensional image reconstructionData sets were transferred to a RISC-type workstation (Kubota Pacific750), were the intermediate slices were interpolated mathematicallyto produce a solid three-dimensional volume of data. The datavolumes were visualized by rendering methods available within thevisualization software AVS (Advanced Visualization Systems).

A data volume consists of a three-dimensional array of voxels(volume elements), with each voxel having an (

x,y,z) address and anintensity value (a) ranging from 0 to 255, representing the amount offluorescence detected at the point (x,y,z). Each voxel is assigned acolor based on its intensity value. In all the figures, the color scaleruns monotonically from the blue end of the spectrum for the lowestintensities of fluorescence through green and yellow for the interme-diate values and to orange and red for the highest values. The colorassignment was chosen to enhance contrasts and make them visible.This was sometimes necessary, as the intensity values that wererecorded by the microscope sometimes had narrow ranges (on theorder of 60 values out of 256).

In addition to the color assigned to a voxel (x,y,z) on the basis ofits intensity value (a), the software enabled us to independently assigneach voxel an opacity, ranging from 0 (completely transparent) to 1(completely opaque). This feature was important, because it allowedus to melt away uninteresting regions and background noise byrendering them transparent. Without this feature, foreground struc-tures would inevitably obscure objects deeper in the volume set.

Visualization schemes most suited to confocal microscopy arebased on volume rendering methods, in which the observer can seeinto the volume, which has been made somewhat transparent. Theadvantage of confocal microscopy lies in its ability to image throughthe object and collect a three-dimensional volume of data. Creatingopaque solid structures fails to take advantage of the power of 3-Dimage collection. The most popular volume rendering methods arebased on ray tracing. A ray from the observer is cast into the object,and each voxel that it passes through contributes some of its color,depending on its opacity. The line-of-sight through the ray is assigneda color by its opacity, and summing the results. This method by itselftends to produce images that are murky and clouded. To make objectswithin the volume more visible, we used gradient-based shadingmethods to enhance boundaries between regions with significantlydifferent intensity values (Toga and Payne, 1990; Bacallao andGarfinkel, 1994). This has the net effect of enhancing the detailsobservable in the final images.

Incorporation of fluorescent phosphatydyl choline into theapical membraneFluorescent lipid incorporation in the plasma membrane was achievedby a modification of the method of Pagano and Martin (1987).Confluent MDCK cells were incubated for 1 hour at 10°C in normalRinger’s buffer on the basolateral side and the same buffer contain-ing a 1:1 mixture of 25 mM defatted bovine serum albumen andthe fluorescent lipid 1-palmitoyl-2-caproyl-sn-glycero-3-phospho-

choline-n-(lissamine rhodamine b sulfonyl) (Avanti Polar Lipids-Alabaster, AL) on the apical side. After washing 3 times in regularbuffer at 10°C, the filters were incubated at 37°C in the varioussolutions described above and subsequently viewed in the confocalmicroscope in either the x-z or the x-y mode to obtain two-dimen-sional optical sections through the tissues.

Freeze fracture and electron microscopyFreeze fracture and electron microscopy were performed as describedby Zampighi et al. (1989). The cultured cells were fixed while on thefilters by immersion for 2 hours at room temperature in 3% glu-taraldehyde, 4% paraformaldehyde in 0.2 M sodium cacodylatebuffer, pH 7.4. The cells were then infiltrated with 20% glycerol in0.2 M Na cacodylate buffer for 1 hour at room temperature, followedby scraping from the filters. Sheets of cells were deposited on Balzer’sholders with a minimum amount of solution, and frozen by immersionin liquid propane. Fracture was performed in the freeze-fractureapparatus at −100°C with a liquid nitrogen-cooled knife. Thefractured surfaces were then shadowed with platinum-carbon at 45°and carbon at 90°. The resulting replicas were released by digestingthe tissue with 4% sodium hypochloride. The replicas were depositedon single hole copper grids coated with Formvar for electronmicroscopy.

RESULTS

ATP depletion The course of ATP depletion after metabolic inhibitortreatment is shown in Fig. 1 for both MDCK and JTC cells. Inboth cell types, ATP content fell rapidly to about 50% of initialvalues within 5 minutes, to 25% after 10 minutes, and to 15%after 60 minutes. This drop in ATP was initially mirrored byconcomitant increases in ADP and AMP content, as seen inTables 1 and 2. The amount of the latter two nucleotidesincreased to the same extent, and the sum of the 3 adeninenucleotides remained constant after the first 10 minutes ofinhibitor treatment for MDCK cells (Table 1) and 20 minutesfor the JTC cells (Table 2). A continual decline in ADP content

Fig. 1. Intracellular ATP after metabolic inhibition in MDCK andJTC cells. Rapid ATP depletion was achieved with metabolicinhibitors, as described in Materials and Methods. Thesimultaneously measured contents of ADP, AMP and HX are listedin Tables 1 and 2. The contents of all these substances weremaintained at a constant level in the absence of metabolic inhibitors(control).

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was observed at the later time points, causing a significantdecrease in total adenine nucleotide content. There was only aslight accumulation of hypoxanthine (HX), a major adeninenucleotide breakdown product, suggesting that further hydrol-ysis occurred (Mandel et al., 1988).

ATP depletion caused decreased transepithelialresistance (TER)Intracellular ATP depletion caused the decrease of TER in bothMDCK and JTC cells, as shown in Fig. 2. We have previouslyshown that in MDCK cells the fall in TER was rapid and waspreceded by a dramatic decrease in the ATP/ADP ratio, whichfell by 85% in the first 2 minutes (Mandel et al., 1993).Although the declines in ATP and the ATP/ADP ratio occurredwith a similar time course in the JTC cells, the TER fell muchmore slowly in these cells, and was only inhibited by 50% afterthe 20 minute experimental protocol (Fig. 2).

Reorganization of the actin cytoskeleton after ATPdepletionThe normal organizational pattern of the F-actin cytoskeletonin MDCK cells is shown in a three-dimensional reconstructionin Fig. 3A. The similar F-actin pattern displayed by JTC cells

is seen in Fig. 4A in an extended focus image. The three char-acteristic cytoskeletal patterns described in the Introduction areseen in both cell lines. On the apical side of the cell, the actincytoskeleton forms the microvilli and in addition a ring of actinstaining is present at the level of the tight junction and thezonula adherens. Second, along the lateral membrane there arecortical actin microfilaments that run parallel to the apical-basal axis of the cells along the cytoplasmic face of the lateralmembrane. Third, at the base of the cells there are stress fibersthat stabilize the interactions with the substratum (Burridge etal., 1988).

Alterations in this pattern are first observable after 20minutes of ATP depletion, becoming more discernible after 30minutes of ATP depletion. Since all the samples were treatedand processed identically, these images are semi-quantitative.At this time, the actin cytoskeleton forms aggregates that arefound in the cytoplasm, typically around the nucleus (Fig. 3B;data not shown for JTC cells), while the cortical actin stainingat the cell borders is lost and there is an apparent thinning ofthe stress fibers in the MDCK cells and in the JTC cells (Fig.3B, data not shown for JTC cells). The total cellular F-actincontent seems to decline at this time. After 60 minutes of ATPdepletion, these structural changes become more striking inboth cell types (Figs 3C, 4E and 4F), including the disappear-ance of the actin ring and stress fiber staining. However, thetotal F-actin content in the perinuclear region appears toincrease, as compared to the 30 minute time point. The changesin the actin cytoskeleton are diagramed in the schematicpresented in Fig. 9.

ATP depletion does not affect the organization ofthe microtubule networkThe possibility of an interaction between microtubules and theactin cytoskeleton has been recently suggested (Buendia et al.,1990). Several types of experiments were performed todetermine whether such an interaction played a role in theactin alterations just described. First, we observed the micro-tubule network during ATP depletion. As seen in Fig. 5, themicrotubule network showed no evidence of depolymerizationor reorganization even with 60 minutes of ATP depletion.Second, depolymerization of the microtubule network bynocodazole, prior to the onset of ATP depletion, did not affect


Table 1. Adenine nucleotide and hypoxanthine (HX)content of MDCK cells subjected to various times of

ATP depletionTime (min) ATP ADP AMP HX ΣAXP* N

Controls0c 61±9 13±4 1.4±0.8 2.8±1.2 75±9 7

30c 70±9 13±2 2.0±0.8 1.4±1.0 85±9 660c 68±12 18±2 2.2±0.4 1.6±0.9 88±12 4

ATP depletion5 30±6† 32±5† 20±4† 6.4±1.7 88±6 7

10 15±2† 18±5 32±10† 6.6±2.0 72±10 720 14±2† 5±2† 25±5† 5.2±1.2 49±5† 430 10±2† 26±5† 4.8±1.1 41±5† 460 9±2† 26±7† 3.4±0.9 38±7† 4

All contents are in nmoles/mg protein.*ΣAXP = sum of ATP, ADP and AMP.†Significantly different from the 0c (control) time point P<0.01.

Table 2. Adenine nucleotide and hypoxanthine (HX)content of JTC cells subjected to various times of ATP

depletionTime (min) ATP ADP AMP HX ΣAXP* N

Controls0c 49±12 11±2 2.4±0.8 1.2±0.4 62±12 6

30c 52±7 12±3 2.0±0.8 1.2±0.4 67±7 660c 49±9 7±3 0.4±0.8 1.6±0.8 56±10 3

ATP Depletion5 26±7† 24±4† 21±7† 3.6±0.8† 71±7 6

10 13±2† 13±3 22±8† 4.8±2.0† 49±8 620 12±2† 10±4 40±14† 4.4±1.2† 62±14 430 9±3† 24±3† 3.2±2.0† 33±5† 360 7±4† 20±4† 2.0±0.4 27±6† 3

All contents are in nmoles/mg protein.*ΣAXP= sum of ATP, ADP and AMP.†Significantly different from the 0c (control) time point P<0.01.

Fig. 2. Fractional change in transepithelial resistance (TER) as afunction of time after inhibitor treatment. The average initial TERwas 25±4 and 20±2 ohm/cm2, respectively, for MDCK and JTCcells. Although the time course of ATP depletion was similar in thetwo cell types, the decline in TER was much slower in the JTC cells.


the aforementioned alterations of the actin cytoskeleton (datanot shown). These results suggest that the perinuclear aggre-gates of actin are not drawn in from the cell periphery by amicrotubule-dependent mechanism, but may rather be theresult of re-polymerization of G-actin at a perinuclearlocation.

Structural alterations in the tight junctions (TJ)occur only after prolonged ATP depletionPrevious results demonstrated only minimal alterations in theTJ after 10 minutes of ATP depletion (Mandel et al., 1993).Since the present results demonstrate that the time frame forthe actin alterations after ATP depletion was 20-60 minutes,

Fig. 3. Stereo pairsimages of F-actindistribution in MDCKcells after various timesof ATP depletion. Thecells were fixed,permeabilized, andstained with Bodipy-phalloidin aftermetabolic inhibitortreatment. All sampleswere treated identicallyand the three-dimensionalreconstruction of theconfocal images utilizedthe same color scalethroughout: from bluefor low intensitystaining to red for thehighest intensitystaining. (A) Controlconditions with noinhibitors. The apicalring structures aredemarcated by arrowsand the cortical actinnetwork, which runsalong the apical-basalaxis of the cell, byarrowheads. Stress fiberscan be seen at the baseof the cells (openarrows). This image is asegment of a larger fieldof cells; 16 completeMDCK cells arepresented. Thedimensions along the xand y axes areapproximately 40 µmand 35 µm, respectively.The cells are 18 µmhigh. (B) After 30minutes of ATPdepletion. The corticalactin network is nolonger present and theheight of the cells hasdecreased. Fewer stressfibers are seen comparedto the control cells. Theapical ring of actin is

intact. The total content of F-actin appears diminished. There are 14 cells visible in this section. The dimensions along the x and y axis are thesame as in A. The cells are 10-11 µm high. (C) After 60 minutes of ATP depletion. Most of the F-actin is in a perinuclear location, showinglittle structure. The apical actin ring and the stress fibers have disappeared. The total F-actin content appears larger than observed after 30minutes, but it is difficult to compare to the control condition. The dimensions of the x and y axes are the same as in A. The cells are 12-15 µmhigh.

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structural changes in the TJ and their effect on the molecularfence function were examined before and at 60 minutes of ATPdepletion.

Untreated control MDCK epithelia exhibited tight junctionscomposed of, on average, three tight junctional strands. Thesestrands fractured, leaving rows of particles on the P fracture


Fig. 4. Extended focusimage of F-actincytoskeleton in JTCcells after various timesof ATP depletion.(A,B,C) Controlconditions. (A) Anextended focus imagethat spans 5.6 µm of theapical side of the cells.The punctate stainingcorresponds tomicrovilli, while theapical ring can be seenat the periphery of thecells (arrows).(B) Extended focusimage spanning 5.6 µmin the mid-region of thecell. Punctate staining isstill observed andcorresponds tomicrovilli. Cortical actinstaining is observednear the cell border(arrowheads). (C) Basal5.6 µm extended focusimage. The linearstreaks of actin stainingare stress fibers(arrowheads) (D,E,F)After 60 minutes ofATP depletion. Theimage intensity andphotographic settingsare equivalent to thesettings used to obtainthe control images.(D) An extended focusimage that spans 5.6 µmof the apical side of thecell. Note the decreasein microvilli stainingthat is absent in somecells. The ring of actinis disrupted.(E) Extended focusimage spanning 5.6 µmin the mid-region of thecell. Aggregates ofcytoplasmic actin arefound, mostly in aperinuclear location(arrowheads). Note theabsence of the actin ringor cortical actinnetwork. (F) Basal 5.6

µm extended focus image. There is a marked decrease in the number of stress fibers as compared to control. The cortical actin network isdisrupted and some aggregates of actin are observed (arrowhead). Bar, 10 µm.


face (the protoplasmic leaflet of the membrane) and comple-mentary furrows on the E fracture face (the external leaflet ofthe membrane), as previously shown by Mandel et al. (1993).Thus, each tight junctional strand formed a continuous ringaround the entire cell perimeter. Only the P fracture face isshown in Fig. 6A.

Epithelia that were ATP depleted for 60 minutes showeddramatic alterations of this fracture pattern. The overall

appearance of the tight junction was still recognizable butthe P face strands contained extensive gaps or vacancies(Fig. 6B). The E face furrows become filled with particlesthat, in places, appeared almost as a regular P face strand.Complementary matching of the particles and pits of the Pand E faces appeared lost (data not shown). Anotherimportant difference between control and ATP-depleted cellswas detected in the P face strands. In untreated controls, the

Fig. 5. Extended focus image of microtubule network in MDCK cells. No differences were observed between control conditions (A,B) and 60minutes after ATP depletion (C,D). (A,C) Extended focus image of the apical side of the cells 4.6 µm deep. (B,D.) Extended focus image of thebasal side of the cells 6.0 µm deep. Both control and ATP-depleted samples were imaged and photographed using the same settings.Bar, 10 µm.

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particles were of uniform diameter and associated in welldefined, extensive rows. After 60 minutes of ATP depletion,the P face strands were shorter. At places, the tight junctionstrands appeared to be composed of particles of largerdiameter and irregular morphology. Careful inspection ofthese images, however, suggested that the strands broke downinto short rows formed from 5-6 particles. These short rows

curved and in some cases the strands appeared to foldtogether (Fig. 6C, arrow).

Additional information regarding the integrity of the tightjunction was obtained by examining the distribution of the tightjunction-associated protein, ZO-1 (Stevenson et al., 1986).Both cell lines showed the characteristic unbroken pattern ofZO-1 staining delineating the TJ that was observed under


Fig. 6. Freeze-fracture electron microscopy of tightjunctions in MDCK cells. (A) Control conditions. The Pfracture face shows three parallel strands of particles onaverage, and they contain a fair number of interruptions.×125,000. (B) P fracture face after 60 minutes of ATPdepletion. Three strands can still be identified, however,the number and average length of the interruptions are

greatly increased. ×125,000. (C) P fracture face after 60 minutes of ATP depletion. The higher magnification (×400,000) shows folding of thetight junction strands (arrow) and aggregation of the tight junction particles.

C


control conditions (Fig. 7A). This pattern was intact in bothcell lines until the cells were ATP depleted for 60 minutes. Atthis time, discontinuities in ZO-1 staining were observed, asillustrated in Fig. 7B. Similar results were observed in the JTCcell line (data not shown). The continuity of the molecularfence was also tested at this time through the use of apical flu-orescent phosphatydyl choline (PC). Previous work had shownthat fluorescent PC incorporated into the apical membrane ofMDCK cells did not diffuse to the basolateral side undercontrol conditions (Fig. 8A) (Dragsten et al., 1981; van Meerand Simons, 1986; Mandel et al., 1993). After 60 minutes ofATP depletion (Fig. 8B), fluorescent PC diffused to the baso-lateral side, suggesting that the TJ molecular fence had beencompromised. Therefore the time required to disrupt the fencefunction of the tight junction corresponds to the time at whichthe actin ring is compromised.

DISCUSSION

The results establish the temporal sequence of changes in tightjunctional properties and cytoskeleton organization upondepletion of intracellular stores of ATP. These experimentsexpand a previous communication, in which we reported onthe ability to separate the paracellular gate and molecular fencefunctions of the tight junctions (Mandel et al., 1993). Twodifferent renal cell lines originating from the proximal (JTC)and the distal tubule (MDCK) were used to test the generalityof the present findings. ATP depletion was achieved rapidly inboth cell types through use of simultaneous glycolytic andoxidative inhibitors. The pattern of ATP hydrolysis was similarto that found in freshly isolated renal tubules subjected toanoxia. The initial breakdown of ATP into ADP and AMP wasfollowed by continued adenine nucleotide breakdown overtime (Mandel et al., 1988). The hypoxanthine content didincrease slightly, but little accumulation of this ATPbreakdown product occurred, suggesting that further hydroly-sis took place. It should be noted that total adenine nucleotidecontent decreased with time. This information on the metabolicprofile of the cells was recently utilized to formulate a protocolthat enabled a rapid reversal of ATP depletion (Doctor et al.,

Fig. 7. The effect of ATPdepletion on ZO-1 staining inMDCK cells. Extended focusimages obtained by confocalfluorescence microscopy.(A) Control MDCK cells. ZO-1forms a linear pattern at the cellcontact sites. (B) After 60minutes of ATP depletion.Breaks in the linear pattern arereadily observed. Bar, 10 µm.

Fig. 8. Confocal optical sections of MDCK cells, showing thefluorescence of lissamine-rhodamine phosphatydylcholineincorporated into the apical membrane. (A) x-y section obtained 2mm below the apical surface under control conditions, showingmainly endocytic vesicles. No evidence of lateral membrane stainingis seen. (B) x-y section 2 mm below the apical surface delineating thelateral membranes and showing fewer endocytic vesicles.Bar, 10 µm.

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1994). Due to the decline in adenine nucleotide content by 30minutes of energy depletion, re-establishment of energy metab-olism after 30 minutes of energy depletion would not beexpected to rapidly restore ATP content, since ATP synthesisin the short term is primarily from ADP and AMP. Such aresult was previously obtained in LLC-PK1 cells followingremoval of metabolic inhibitors in which the ATP content wasonly restored to 50% of control at 6 hours (Canfield et al.,1991). More recently, ATP content was restored within 30minutes of energy depletion by inhibiting purine breakdownwith allopurinol and exogenous addition of adenosine (Doctoret al., 1994).

In both cell types, the fall in the ATP/ADP ratio precededthe decrease in TER. Both of these variables were affectedmore rapidly in the MDCK than the JTC cells. Furthermore,the TER was essentially abolished in the MDCK cells whereasit only fell by 50% during the 20 minute experimental periodin the JTC cells. The latter result is similar to that reported inLLC-PK1 cells (Canfield et al., 1991). It is presently unclearwhat could account for these differences, especially in light ofthe limited information available regarding the molecular basisof the paracellular barrier. Clearly, epithelial cells differ intheir ability to modulate paracellular permeability. It ispossible that these differences are also reflected in the reactionto ATP depletion. These early changes in TER are unlikely tobe caused by the disruption of the actin ring, since it precedesthe disruption of the actin ring by 40 minutes.

The present results with ATP depletion indicate that thefunctional alterations are quite different from those obtainedwith low Ca2+-EDTA. As described earlier, ATP depletionpermits the separation between the paracellular barrier and themolecular fence functions of the TJ, whereas Ca2+ depletionrapidly abolishes both functions (Volberg et al., 1986; vanMeer and Simons, 1986). Structural alterations in ZO-1 did notoccur until 60 minutes after ATP depletion, whereas internal-ization and breaks in both ZO-1 and cingulin structure havebeen reported after 15 minutes of low Ca2+ treatment (vanMeer and Simons, 1986; Citi, 1992). Recently it was shownthat inhibition of protein kinase C activity with H-7 preventedthe TJ disruption by low Ca2+, suggesting that protein kinaseactivity was required for this event (Citi, 1992). This result

contrasts with the conditions of ATP depletion, where the lowATP content would be expected to inhibit kinase activity dueto lack of substrate. Therefore, the expectation would be formolecular events possibly involving protein phosphatases andprotein dephosphorylation. Further experiments are needed totest the possibility that ATP depletion and low Ca2+ treatmentmay act through different pathways.

Correlation between disruption of the actin ring andthe loss of molecular fence function of the tightjunctionThe molecular fence function of the TJ was lost after 60 minutesof ATP depletion, as measured by the appearance of rhodamine-conjugated PC on the basolateral membrane. The loss of themolecular fence seemed to be accompanied by profoundrearrangements in ZO-1 and freeze fracture patterns of tightjunction strands. The normal linear ridge pattern of the TJ wastransformed to one in which the TJ particles formed large aggre-gates and left extensive gaps. The coincidence between thesestructural alterations and the loss of the fence function is con-sistent with previous models in which this function depends onthe contact between particles forming the strand within each cell(Madara, 1989). In contrast, the paracellular gate (barrier)function depends on the contact between strands located inapposing cells. These two functions of the TJ could be separatedafter 10 minutes of ATP depletion, since the gate function wasessentially abolished at this time while the fence functionappeared to be intact. No alterations in the linear structure ofthe ridges and furrows were observable after 10 minutes of ATPdepletion (Mandel et al., 1993). Our data suggests that theintegrity of the paracellular barrier function is dependent uponATP or can be altered by the phosphorylation state of TJproteins. Indirect evidence in support of this idea comes fromthe work by Citi and Stevenson that showed that cingulin andZO-1 are phosphoproteins (Stevenson et al., 1989; Nigam et al.,1991). Additional supportive evidence comes from work byOjakian, which showed that activation of protein kinase C byphorbol esters caused a drop in TER (Ojakian, 1981). The datain this paper suggest that dephosphorylation of TJ proteinscauses a rapid loss of paracellular resistance, although furtherwork is needed to prove this suggestion.


Fig. 9. Diagram of the changes in the actin cytoskeleton following ATP depletion. (A) Actin organization in control cells. The actincytoskeleton is composed of an apical ring and longitudinal fibers running along the apical basal axis of the cells. Actin structures not shownare the microvilli and the stress fibers. These were omitted to improve the clarity of the diagram. The dotted lines at the base of the cells reflectcell boundaries rather than an actin structure. (B) Actin organization after 30 minutes of ATP depletion. The longitudinal actin network hasbeen lost. The cells decrease in height but the apical ring remains intact. The dotted lines at the base of the cells delineate cell boundaries.(C) Actin organization After 60 minutes of ATP depletion. Actin has polymerized throughout the cytoplasm and the apical actin ring isdisrupted.


The effect of ATP depletion on the actincytoskeleton The results demonstrate that ATP depletion is associated witha marked reorganization of the actin cytoskeleton (Fig. 9). Theinitial step appears to be the disruption of the cortical actinnetwork followed by dissolution of the stress fibers. The lastarrangement of actin fibers that appears to be disrupted is theapical actin ring (Fig. 3). This sequence reverses the organiz-ation of actin observed during epithelial morphogenesis, sup-porting the hypothesis of Bacallao and Fine (1989) for cellinjury (R. Bacallao et al., unpublished data). The time coursefor actin disruption is roughly coincident with other events thatappear to be linked to actin microfilaments, as follows: (1) thedisappearance of cortical actin fibers within 20-30 minutes ofATP depletion coincides with the beginning of internalizationof E-cadherin and possible decrease in cell-cell contacts(Mandel et al., 1994). (2) The dissolution of stress fibers isusually accompanied by loss of adhesion to the filter supports.Whole epithelial sheets often float away from the support after60 minutes of ATP depletion (results not shown). (3) The dis-ruption of TJ structure after 60 minutes of ATP depletion cor-relates with the final disappearance of the apical actin ring. Itis tempting to speculate that the coincidence of the latter eventssuggests that the linearity of the TJ strands may be determinedby links to the actin cytoskeleton. Disruption of the apical actinring may affect these links, leading to the observed aggrega-tion of TJ particles.

The mechanisms leading to actin rearrangement after ATPdepletion are unknown. Our results are similar to thoseobtained by Bershadsky and co-workers (1980), who foundthat energy depletion caused a gradual disorganization of actin-containing microfilament bundles in fibroblasts. Other investi-gators have also noted differences in the relative susceptibilityof the actin cytoskeleton to ATP depletion. In endothelial cellsit has been shown that stress fibers are depolymerizedfollowing 20 minutes of ATP depletion whereas the cleavagefurrow formed during mitosis was not depolymerized at thistime (Sanger et al., 1983). Canfield et al. (1991) reported actinredistribution to a perinuclear location required about 2 hoursof energy depletion in LLC-PK1 cells. A similar time scale (2-3 hours) was reported by Hinshaw et al. (1991) to elicit short-ening of actin microfilaments in a murine cell line followingATP depletion. The specificity and complexity of the actindepolymerization pattern presented in this paper suggest thatthis process is not merely a reversal of actin polymerizationcaused by ATP depletion. Rather, it is more likely that thiscondition activates specific time- and location-dependent actin-severing proteins, as originally suggested by Bershadsky andGelfand (1983). Their conclusion was based on the observa-tion that ATP depletion prevented rapid depolymerization(within 5 minutes) of filamentous actin when fibroblasts weretreated with cytochalasin B and D. Since the time course ofactin depolymerization continued at the normal rate observedfor ATP depletion (20 minutes) it was unlikely that ATPdepletion itself mediated actin depolymerization (Bershadskyet al., 1980). Our own unpublished observations also show thatin MDCK cells the time course of actin depolymerization ofthe stress fibers and cortical actin network after ATP depletionis longer than the time course of cytochalasin D effects on theactin cytoskeleton.

It has been shown that actin can be phosphorylated in vitroby several proteins kinases (Machicao and Wieland, 1985;Machicao et al., 1983; Erikson et al., 1979). However, onlyrecent evidence has shown that actin monomers can be phos-phorylated in vivo (Schweiger et al., 1992; Howard et al.,1993). In addition the phosphorylation state of actin correlateswith cell shape changes in Dictyostelium (Schweiger et al.,1992; Howard et al., 1993). In vitro studies of phorphorylatedactin have demonstrated that phosphorylation of serineresidues of rabbit skeletal muscle actin decreases its affinityfor DNAse I. The phosphorylation state of actin monomers wasnot examined in this study and will require additional experi-ments.

The factors leading to the formation of polymerized actinaggregates in the cytoplasm after 60 minutes of ATP depletionare likewise unknown. However, a possible sequence of eventsmay be deduced from known in vitro properties of actin: ATPlowers the critical concentration for spontaneous polymeriza-tion of G actin, ADP-G actin monomers polymerize at a higherconcentration, and therefore ATP is not an essential cofactorfor actin polymerization (Carlier, 1990). As described above,actin filament depolymerization may be initially due tosevering of the actin filaments by actin-binding protein(s). Thekinetics of subunit addition/loss now favors depolymerizationsince the number of free ends increases and the concentrationof ATP-G actin falls due to a lack of substrate. Eventually thelevel of ADP-G actin may rise above its critical concentrationat which point polymerized actin aggregates may form. Recentwork in vitro has shown that ATP-depleted cytosol causedspontaneous actin polymerization, similarly actin polymeriza-tion has been observed following ischemia in vivo (Kurzchaliaet al., 1992; Molitoris et al., 1991).

These phenomena described in cultured cells are similar tothose observed in vivo following ischemic injury. The actincytoskeleton was rapidly rearranged in the proximal tubulewith an accompanying loss of the apical brush border (Venkat-achalam et al., 1981; Kellerman and Bogusky, 1992). In thelatter study, the actin cytoskeleton of the apical brush borderappeared to be most sensitive to the effects of ATP depletion.The changes in the cortical actin network were not determinedin these studies. Furthermore, a common observation made byMolitoris and co-workers (1989, 1991) is the rapid increase inparacellular permeability following ischemia. These resultssuggest that the present observations made in renal culturedcells may be used to understand the derangements in cytoskele-tal and junctional structure that occur in vivo.

The effects of ATP depletion on microtubulesBuendia et al. (1990) have shown that there is a dynamic inter-action between the actin cytoskeleton and microtubule networkin MDCK cells. Based on this study, we tested the possibilitythat the breakup of the actin filaments and the formation ofactin aggregates was mediated by the microtubule network.The present results demonstrate that the morphology of themicrotubule network was not affected by ATP depletion. Fur-thermore, depolymerization of the microtubule network bynocodazole prior to ATP depletion did not change the timecourse of actin depolymerization or its subsequent aggregation.These results are consistent with those obtained in fibroblasts,where it has been shown that ATP depletion stabilized themicrotubule cytoskeleton. Fibroblasts depleted of their cyto-

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plasmic ATP did not show any change in the organization ofthe microtubule cytoskeleton nor were the microtubulesdisrupted when the cells were treated with nocodazole (Ber-shadsky and Gelfand, 1983). This difference in sensitivity tothe effects of ATP depletion between the actin cytoskeletonand microtubule network may be a useful method to studymicrotubule function in the absence of a normal actincytoskeleton.

In summary, this work demonstrates the time course foractin cytoskeleton disassembly following ATP depletion andits possible relationship to epithelial tight junctional properties.There is a rapid loss of TER that does not coincide with visiblealterations in F-actin distribution. The actin cytoskeleton is firstdepolymerized along the apical-basal axis of the cell followedby the basal stress fibers. As the period of ATP depletioncontinues, the actin ring located at the apical portion of the cellbreaks up. At this point, TJ structure is also affected with coin-cident loss of the molecular fence function. This disruption ofthe actin cytoskeleton is independent of microtubule organiz-ation. These results demonstrate that ATP depletion may be auseful method to study junctional complex assembly and dis-assembly in epithelial cells.

The authors thank Dr Leon Fine for his support and encouragementfor this project, Dr Thomas Buell for his help with the TER mea-surements and Mr Mike Kreman for his technical expertise in thefreeze fracture experiments. The work was supported in part by NIHgrants DK01777 and the American Heart Association Grant in Aidno. 92016070 (to R.B.) and DK 26816 (to L.J.M.). The authors alsothank Mark Gregory of Leitz America, IL for his generous support.Steven Napier generously contributed the diagram presented in thispaper. We are grateful to Arthur Toga at UCLA, Department of Neu-roscience, for providing generous access to his computer imagingfacilities in general and allowing us to store our data sets in particu-lar.

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(Received 21 September 1993 - Accepted, in revised form,18 August 1994)

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