Mitochondrial (Delta)(psi) and cytoplasmic environment · 60-180 minutes, 1.5 µM colcemid...

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INTRODUCTION The use of potential-sensitive mitochondrial probes, namely of the lipophilic cation 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl- benzimidazol-carbocyanine iodide (JC-1), has revealed the presence of mitochondria with differing membrane potential (m∆ψ) within the same cell. m∆ψ is generally correlated with mitochondrial location. In particular, m∆ψ has been found to be higher in peripheral mitochondria of spread cells (Bereiter-Hahn et al., 1983; Smiley et al., 1991; Chen and Smiley, 1993; Bereiter-Hahn and Vöth, 1994; Kirischuk et al., 1995). This fact, which has not yet been investigated, should not be linked to a closer association to the plasma membrane, since the distance between mitochondria and plasma membrane is nearly equivalent in the cell center as in the cell periphery owing to the almost uniform thickness of spread cells. Rather, the peripheral location of high-polarized mitochondria should refer to their closeness to the cell edge, topologically defined as the region of transition between the substrate-adhering surface and the free surface of the cell. This view is supported by the observation that confluent cells possess an m∆ψ consistently lower than non- confluent cells (Chen and Smiley, 1993). In fact the edge of confluent cells is hidden by cell-cell contacts whereas that of non-confluent cells is exposed to the extracellular environment. The relationship between m∆ψ and pathological conditions such as anoxia, hypoxia and apoptosis has been extensively investigated (Skowronek et al., 1992; Ankarcrona et al., 1995; DiLisa et al., 1995; Castedo et al., 1996). However, to date, no information is available on subcellular m∆ψ changes in these conditions. Results based on ‘one cell – one value’ data, such as those obtainable from flow cytometry, may be misleading since averaged or integrated m∆ψ estimates may derive from very different spatial and intensity distributions. With particular concern for apoptosis, different conclusions have been drawn regarding the hypotheses that m∆ψ is lost early as a consequence of permeability transition pore opening (Marchetti et al., 1996; Zamzami et al., 1996) or is maintained longer for mitochondrial ATP requirements by cell undergoing apoptosis (Ankarcrona et al., 1995). More recent findings indicate that apoptosis does not require mitochondrial ATP synthesis but a functional electron transport chain (Jia et al., 1997a) and that the release of cytochrome c from the mitochondrial intermembrane space, a key step of apoptosis, is not accompanied by m∆ψ changes (Kluck et al., 1997). To verify whether m∆ψ is locally influenced by the intracellular and extracellular environments and to what extent m∆ψ varies within and between cells in different physiological and pathological conditions, we investigated the pattern of mitochondrial polarization in confluent and non-confluent cultures of four cell types (human astrocytes, HEp-2, MDCK and Vero cells) and the relevant changes induced by hypoxia and apoptosis. 1077 Journal of Cell Science 112, 1077-1084 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS9897 The subcellular heterogeneity of mitochondrial membrane potential (m∆ψ) was investigated in confluent and sub-confluent cultures of four cell types (human astrocytes, HEp-2, MDCK and Vero cells) in normal growth conditions, hypoxia and apoptosis. The distribution of high-polarized mitochondria, detected by the potential-sensitive probe JC-1, was found to depend on: (1) the proximity to the cell edge; (2) the local absence of cell-cell contacts; and (3) the local absence of acidic vesicles. Both hypoxia and apoptosis produced a general m∆ψ increase with different redistributions of high- polarized mitochondria. Hypoxic cells maintained high- polarized mitochondria for over 24 hours, until cells underwent necrosis. On the other hand, apoptotic cells showed an unexpected convergence of high-polarized mitochondria into an extremely packed mass at one side of the nucleus, in a stage preceding nuclear condensation, but correlated to the retraction of cell-cell contacts. Key words: Mitochondrion, Membrane potential, Acidic vesicle, Cell edge, JC-1 SUMMARY Subcellular heterogeneity of mitochondrial membrane potential: relationship with organelle distribution and intercellular contacts in normal, hypoxic and apoptotic cells Giacomo Diaz 1, *, Maria Dolores Setzu 1 , Andrea Zucca 1 , Raffaella Isola 1 , Andrea Diana 1 , Roberto Murru 2 , Valeria Sogos 1 and Fulvia Gremo 1 Departments of Cytomorphology 1 and Medical Sciences 2 , University of Cagliari, 09124 Cagliari, Italy *Author for correspondence (e-mail: [email protected]) Accepted 18 January; published on WWW 10 March 1999

Transcript of Mitochondrial (Delta)(psi) and cytoplasmic environment · 60-180 minutes, 1.5 µM colcemid...

Page 1: Mitochondrial (Delta)(psi) and cytoplasmic environment · 60-180 minutes, 1.5 µM colcemid (demecolcine) for 120 minutes, 10 µM colchicine for 30 minutes, 0.1 µM vinblastine for

INTRODUCTION

The use of potential-sensitive mitochondrial probes, namely ofthe lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), has revealed thepresence of mitochondria with differing membrane potential(m∆ψ) within the same cell. m∆ψ is generally correlated withmitochondrial location. In particular, m∆ψ has been found to behigher in peripheral mitochondria of spread cells (Bereiter-Hahnet al., 1983; Smiley et al., 1991; Chen and Smiley, 1993;Bereiter-Hahn and Vöth, 1994; Kirischuk et al., 1995). This fact,which has not yet been investigated, should not be linked to acloser association to the plasma membrane, since the distancebetween mitochondria and plasma membrane is nearlyequivalent in the cell center as in the cell periphery owing to thealmost uniform thickness of spread cells. Rather, the peripherallocation of high-polarized mitochondria should refer to theircloseness to the cell edge, topologically defined as the region oftransition between the substrate-adhering surface and the freesurface of the cell. This view is supported by the observation thatconfluent cells possess an m∆ψ consistently lower than non-confluent cells (Chen and Smiley, 1993). In fact the edge ofconfluent cells is hidden by cell-cell contacts whereas that ofnon-confluent cells is exposed to the extracellular environment.

The relationship between m∆ψ and pathological conditionssuch as anoxia, hypoxia and apoptosis has been extensively

investigated (Skowronek et al., 1992; Ankarcrona et al., 1995;DiLisa et al., 1995; Castedo et al., 1996). However, to date, noinformation is available on subcellular m∆ψ changes in theseconditions. Results based on ‘one cell – one value’ data, suchas those obtainable from flow cytometry, may be misleadingsince averaged or integrated m∆ψ estimates may derive fromvery different spatial and intensity distributions. Withparticular concern for apoptosis, different conclusions havebeen drawn regarding the hypotheses that m∆ψ is lost early asa consequence of permeability transition pore opening(Marchetti et al., 1996; Zamzami et al., 1996) or is maintainedlonger for mitochondrial ATP requirements by cell undergoingapoptosis (Ankarcrona et al., 1995). More recent findingsindicate that apoptosis does not require mitochondrial ATPsynthesis but a functional electron transport chain (Jia et al.,1997a) and that the release of cytochrome c from themitochondrial intermembrane space, a key step of apoptosis, isnot accompanied by m∆ψ changes (Kluck et al., 1997).

To verify whether m∆ψ is locally influenced by theintracellular and extracellular environments and to what extentm∆ψ varies within and between cells in different physiologicaland pathological conditions, we investigated the pattern ofmitochondrial polarization in confluent and non-confluentcultures of four cell types (human astrocytes, HEp-2, MDCKand Vero cells) and the relevant changes induced by hypoxiaand apoptosis.

1077Journal of Cell Science 112, 1077-1084 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS9897

The subcellular heterogeneity of mitochondrialmembrane potential (m∆ψ) was investigated in confluentand sub-confluent cultures of four cell types (humanastrocytes, HEp-2, MDCK and Vero cells) in normalgrowth conditions, hypoxia and apoptosis. Thedistribution of high-polarized mitochondria, detected bythe potential-sensitive probe JC-1, was found to dependon: (1) the proximity to the cell edge; (2) the local absenceof cell-cell contacts; and (3) the local absence of acidicvesicles. Both hypoxia and apoptosis produced a generalm∆ψ increase with different redistributions of high-

polarized mitochondria. Hypoxic cells maintained high-polarized mitochondria for over 24 hours, until cellsunderwent necrosis. On the other hand, apoptotic cellsshowed an unexpected convergence of high-polarizedmitochondria into an extremely packed mass at one sideof the nucleus, in a stage preceding nuclear condensation,but correlated to the retraction of cell-cell contacts.

Key words: Mitochondrion, Membrane potential, Acidic vesicle, Celledge, JC-1

SUMMARY

Subcellular heterogeneity of mitochondrial membrane potential: relationship

with organelle distribution and intercellular contacts in normal, hypoxic and

apoptotic cells

Giacomo Diaz 1,*, Maria Dolores Setzu 1, Andrea Zucca 1, Raffaella Isola 1, Andrea Diana 1, Roberto Murru 2,Valeria Sogos 1 and Fulvia Gremo 1

Departments of Cytomorphology1 and Medical Sciences2, University of Cagliari, 09124 Cagliari, Italy*Author for correspondence (e-mail: [email protected])

Accepted 18 January; published on WWW 10 March 1999

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MATERIALS AND METHODS

HEp-2, MDCK and Vero cell lines (Flow Laboratories, Inc., McLean,VG) and primary cultures of human fetal astrocytes were grown inNaH2PO4 and NaHCO3 buffered Dulbecco’s modified Eagle medium(Life Technologies, Inc., Gaithersburg, MD). Cells were allowed toadhere to uncoated glass coverslips at least three days prior toexperiments. Treatments consisted of 10 nM bafilomycin A1 for 60minutes, 16 µM camptothecin for 90-180 minutes, 2 mM glutamate for60-180 minutes, 1.5 µM colcemid (demecolcine) for 120 minutes, 10µM colchicine for 30 minutes, 0.1 µM vinblastine for 30 minutes and 1M NaCl for a few seconds (all from Sigma Chemical Co., St Louis, MO).

Cells were supravitally stained with 0.5 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1), 0.1µM pyronin-Y (PY), 0.1 µM Acridine Orange (AO), 1 µM ethidiumbromide (EB) and 5 µM Calcium Green-1 acetoxylmethyl ester(CaGreen-1). JC-1, AO and CaGreen-1 AM were obtained fromMolecular Probes (Eugene, OR); EB from Sigma (St Louis, MO); PYfrom ICN Biomedicals (Aurora, OH). All stains were dissolved in theculture medium: JC-1 from a 200 µM DMSO stock solution, added 20minutes before observation; PY and AO from aqueous stock solutions,added 10 minutes before observation; EB from an aqueous stocksolution, added few seconds before observation. The presence of thedyes in the medium did not interfere with observations. JC-1 is a dyesensitive to the mitochondrial transmembrane potential (m∆ψ) butinsensitive to transmembrane acidic gradient (m∆pH) (Chen andSmiley, 1993). It stains mitochondria with an m∆ψ (negative inside)between neutrality and 140 mV in green-orange. At m∆ψ>140 mV,JC-1 forms characteristic J-aggregates which emit in red (Chen andSmiley, 1993; Cossarizza et al., 1996). The terms high-polarized andlow-polarized mitochondria conventionally refer to this m∆ψ step. AOstains acidic vesicles in green-yellow or orange-red, depending on theirpH (Zelenin, 1993). However, at a concentration as low as 0.1 µM, AOstains acidic vesicles only in green. PY is a potential-insensitivemitochondrial stain (Darzynkiewicz et al., 1986; Diaz et al., 1997)useful in the observation of the mitochondrial morphology(arrangement, shape, and thickness/swelling) independently from themetabolic status. PY is specifically and rapidly sequestered bymitochondria. As compared to other mitochondrial stains, PY shows avery low inhibition of mitochondrial F1-ATPase (Bullough et al., 1989)and sharp mitochondrial profiles. EB is a nuclear dye largely used totest cell viability: both the low permeability of the intact plasmamembrane and the low accessibility of chromatin-structured DNAexclude EB from nuclei of healthy cells. Membrane and chromatinalterations result in a progressive nuclear staining which is graduallyhigher in apoptotic cells and strongly intense in dead (post-apoptoticas well as accidentally necrotic) cells (Ferlini et al., 1996). In parallelwith EB staining, apoptosis was assessed by plasma membrane affinityto annexin V and sensitivity of DNA in situ to denaturation by HCl/AOassay (Darzynkiewicz et al., 1997) (data not shown).

CaGreen-1 AM is a nonfluorescent cell-permeant compound. Onceinside the cell, CaGreen-1 AM is cleaved by cytosolic esterases intothe calcium-sensitive green-fluorescent, membrane-impermeantCaGreen-1 probe (Kd = 190 nM).

Observations were made using a Zeiss Axioskop microscope (Zeiss,Oberkochen, Germany) equipped with a differential interferencecontrast (DIC) Plan Achro ×40/0.75 NA water-immersion objective.For fluorescence and white light imaging, optical DIC components(polarizer, prism and condenser) were excluded. JC-1 (both green andred fluorescence) and CaGreen-1 were observed with a standardfluorescein filter set (excitation BP 450-490, emission LP 520) (Figs1A, 5B). JC-1-red and AO-green fluorescences were observed with anupper emission threshold (LP 550) to exclude the green fluorescenceof JC-1 (Fig. 1D,F). JC-1-red fluorescence, PY and EB were observedwith a standard rhodamine filter set (excitation BP 534-558, emissionLP 590) (Figs 1B,C,E,G, 3B,D, 4A-F,H,I, 5A,C-E,G). 8-bit,1024×1024 grayscale images, embracing a field of 140 µm2 (nominal

resolution: 0.13 µm/pixel), were acquired by a Kodak Megaplus 1.4slow scan camera (Eastman Kodak Co., San Diego, CA) with variableexposure (usually in the range of 200 to 800 milliseconds) and gain (0to +18 db) controlled by an IBAS 2000 image analyzer (KontronElektronik, GmbH, München, Germany). Post-processing wasperformed using ImagePro Plus software (Media Cybernetics, SilverSpring, MD). Noise due to high gain levels and long exposure timeswas removed by a conditional median filter applied only to pixels witha 5×5 neighbour variance exceeding a preset threshold value.

Preliminary experiments using a closed slide chamber revealedspontaneous m∆ψ changes due to hypoxia. Therefore all sessionswere carried out in open dishes under normal air atmosphere with theobjective directly immersed into the medium. The medium pH did notvary appreciably during experiments. Only to induce hypoxia, cellswere kept in the slide chamber completely filled with 300 µl ofmedium for at least 30 minutes. In this case, images were acquiredthrough the culture coverslip used as chamber window, with the sameoptical settings used for dishes. Cells were maintained at 37°C by aircurtain through the microscope stage hole.

RESULTS

Cell type variabilityIn normal cultural conditions, only astrocytes and HEp-2 cellsshow high-polarized mitochondria. MDCK and Vero cellmitochondria do not present any trace of JC-1-redfluorescence. This fact reflects differences already observedbetween some cell lines and correlated to the basal m∆ψspecific of each cell type (Chen and Smiley, 1993).

Influences of the intracellular environmentHigh-polarized mitochondria are almost exclusively found inthe peripheral cytoplasm (Fig. 1B,C). They represent the outerfringes of a complex network of low-polarized mitochondriaparticularly concentrated around the nucleus (Fig. 1A). DualJC-1/AO staining reveals that high-polarized mitochondria arealways external to the territory occupied by acidic vesicles.Very frequently, high-polarized and acidic vesicles areseparated by a precise virtual boundary (Figs 1D,E and 2).One-hour treatment with 10 nM bafilomycin A1, a specificblocker of vesicular protonic pump V-ATPase, results in asignificant depletion of acidic vesicles accompanied by theappearance of newly high-polarized mitochondria in the cell(Fig. 1F,G).

Influences of the extracellular environmentIsolated cells show high-polarized mitochondria along theirentire periphery. On the other hand, contacting cells showhigh-polarized mitochondria only in correspondence of thefree tracts of the cell edge. Clusters of confluent cells show aunique ring of high-polarized mitochondria along the freeedge regions of outer cells, so that the whole cluster resemblesa single giant cell (Fig. 3A,B). However, even in proximity ofthe cell edge, high-polarized mitochondria are missingwhenever the cloud of acidic vesicles is found close to the celledge (Fig. 3C,D). This predominant effect of acidic vesicleson the high-polarization of edge mitochondria is also presentin single cells (Fig. 1D,E).

Changes induced by hypoxiaPhysical hypoxia induces the elongation of pre-existing high-

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polarized traces in the cell periphery and the appearance of newhigh-polarized mitochondria in the cell center (Fig. 4A, to becompared with Fig. 1B and 1C). High-polarized traces assumea characteristic rectilinear shape and their elongation increaseswith the duration of the treatment. A 24 hour-hypoxia resultsin 10 to 20 µm long, straight high-polarized mitochondrialtraces within still apparently integer cells, as judged by theabsence of nuclear EB staining. The effect of 24-hours physicalhypoxia is reversed by medium renewal.

Changes induced by glutamate and camptothecin(apoptosis)An increase in the number of high-polarized mitochondria isobserved after glutamate treatment. This initial change isfollowed by the migration of high-polarized mitochondriatowards the cell center (Fig. 4B) and their eventualcondensation into an extremely packed mass at one side ofthe nucleus (Fig. 4C to H). Mitochondrial phenomena arefollowed by the typical apoptotic events such as the reductionof cell volume and the condensation of the nucleus (Fig.4D,E). However mitochondrial clustering is earlier, so that itappears to be an active process, not a consequence of the

apoptotic cell shrinkage. All these changes are more or lesssynchronized in isolated cells. On the contrary, in clusters ofconfluent cells, mitochondrial and nuclear changes inducedby glutamate are delayed, depending on the cell positionwithin the cluster. Outer cells undergo apoptosis firstfollowed by inner cells (Fig. 4F). These undergo apoptosisonly after the retraction of contacts with outer cells. Thissuggests that initiation of apoptotic changes (including earlymitochondrial clustering) requires that the cell edge isexposed to the extracellular environment. Analogousmitochondrial changes are visible in cells treated with theapoptotic inducer camptothecin (data not shown). EB assayshows a weak nuclear staining throughout the process ofapoptosis, even at the stage of the highest nuclearcondensation (Fig. 4E). On the other hand, a very intensenuclear staining, indicative of plasma membrane damage andchromatin alteration, accompanied by an m∆ψ drop,characterizes accidental necrosis (Figs 4I, 5A). Cell necrosis,as judged by the size of stained nuclei, may occur at any stageof apoptosis. Since EB binds mitochondrial DNA (Coppey-Moisan et al., 1996) and, if chronically administered, inducesmitochondrial depletion (King and Attardi, 1989), EB was

Fig. 1. (A-B) Image pair of the same cell (HEp-2)which shows the whole mitochondrial network(left, JC-1-green emission) and the fraction ofhigh-polarized mitochondria (right, JC-1-redemission). These appear as short fluorescenttraces distributed along the cell edge. (C) Thesame pattern is found in astrocytes. However, inlarger astrocytes (D-E), high-polarizedmitochondria may spread over a wider region ofperipheral cytoplasm comprised between the celledge and the mass of acidic vesicles. Broken linesshow the virtual boundary between high-polarizedmitochondria and acidic vesicles. High-polarizedmitochondria are excluded from the territoryoccupied by acidic vesicles. For this reason, high-polarized mitochondria are not found in thoseregions of the cell edge which are invaded by themass of acidic vesicles (D,E: see diagonal bordersof cells). For the same reason, high-polarizedmitochondria are completely lacking from smallcells whose cytoplasm is full of acidic vesicles(F,G: left cell, positive to AO but negative to JC-1). If acidic vesicles are successfully depleted bythe V-ATPase blocker bafilomycin A1,mitochondria become high-polarized (F,G: rightcell, negative to AO – except the nucleolous – butpositive to JC-1). Cell types: A-B: HEp-2 cells;C-G: human astrocytes. Bars, 10 µm.

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added to the medium only at the end of sessions andimmediately observed. In this way, any toxic or staininginterference of EB on cells and mitochondria can beexcluded. Indeed, the mitochondrial pattern observed using

both EB and JC-1 (Fig. 4E) is fully identical to that observedwith JC-1 only (Fig. 4G,H).

In cells preloaded with CaGreen-1, glutamate induces theappearance of intensely fluorescent hotspots proximal to thecell edge. Gradually, calcium hotspots propagate into the innercytoplasm at the speed of 20-25 µm/minute so that they appearin the perinuclear cytoplasm within few minutes from theinduction (Fig. 5B).

Changes induced by high NaClHigh (>1 M) NaCl induces a third pattern of mitochondrialpolarization, different from those described above, andconsisting in the appearance of a myriad of very small, high-fluorescent spots in the central cytoplasm (Fig. 5C). Thephenomenon is not due to increased osmotic or ionic strengthnor to membrane depolarization, since it is not replicated byequimolar sucrose or KCl solutions. It is interesting to observethat JC-1 microspots induced by high NaCl represents thesmallest areas of high-polarization ever observed in ourexperiments as well as in the whole repertoire of JC-1 imagespublished in literature.

Analogous changes in cells with low basal m ∆ΨMDCK and Vero cells, which normally show only low-polarized mitochondria, react to hypoxia, apoptosis and high

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JC-1-red fluorescence intensity(high-polarized mitochondria)

AO

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Fig. 2. Bivariate analysis of JC-1-red and AO-green fluorescenceintensities detected in small cytoplasmic areas (sampling radius: 2µm) of a normal human astrocyte. The mutual exclusion of high-polarized mitochondria and acidic vesicles from their relativeterritories is evident from the orthogonality of distributions.

Fig. 3. (A-B) High-polarizedmitochondria are distributed alongthe cell edge but are excluded fromthose edge regions which contactadjacent cells. As a result, clustersof confluent cells exhibit a uniqueperipheral ring of high-polarizedmitochondria which reproduces thepattern of a single cell. (C-D). However, even when the celledge is free from cell-cell contacts,mitochondrial high-polarization islocally prevented by the presenceof acidic vesicles in the same cellregion. In the picture, the virtualboundary between acidic vesicles(visible in the PhC image) andhigh-polarized mitochondria(visible in the fluorescent image) isapproximated by the curve. Starslabel nuclei. Cell types: humanastrocytes. Bars, 10 µm.

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NaCl with an m∆ψ increase basically similar to that found inastrocytes and HEp-2 cells (Fig. 5F,G).

Mitochondrial polarization is unrelated to the lengthof mitochondrial segmentsPeripheral high-polarized mitochondria are generally shorterthan mitochondria present in the cell center. This fact suggestedthe hypothesis, consistent with the propagation of electricalsignals in mitochondria (Amchenkova et al., 1988; Ichas et al.,1997), that the higher polarization could be due to thedisconnection of peripheral mitochondria from themitochondrial network. To verify this, we treated cells withmicrotubule-disrupting agents known to induce fragmentationof the mitochondrial network (Johnson et al., 1980). Indeed,colchicine, colcemid and vinblastine induce a generaldisarrangement and fragmentation of the mitochondrialnetwork (Fig. 5D,E) but the phenomenon has no effect on themitochondrial polarization (data not shown).

DISCUSSION

Though the molecular basis of mitochondrial polarization iswell explained by the chemiosmotic theory (Mitchell, 1979), nohypotheses have been formulated concerning the heterogeneityof the mitochondrial potential (m∆ψ) within the same cell andthe same mitochondrion (Bereiter-Hahn et al., 1983; Smiley etal., 1991; Chen and Smiley, 1993; Bereiter-Hahn and Vöth,1994). This paper represents the first attempt to investigate thesubcellular m∆ψ heterogeneity and its relationship with othernon-mitochondrial cell compartments. In particular, thepresence of high-polarized mitochondria appears to becorrelated to three environmental conditions: (1) proximity tothe cell edge, (2) local absence of intercellular contacts and (3)local absence of acidic vesicles. These conditions, in turn,depend largely on the size, shape and degree of confluency ofcells. For instance, the average distance of peripheralmitochondria from central acidic vesicles depends on the cell

Fig. 4. (A) Hypoxia results in a straightening andelongation of high-polarized mitochondria(compare with Fig. 1B and C). The extension ofhigh-polarized traces increases with duration ofthe treatment. (B-E) Glutamate induces a generalm∆ψ increase accompanied by the migration ofhigh-polarized mitochondria (arrows) toward thecenter of the cell. The phenomenon is followedby the condensation of high-polarizedmitochondria into an extremely packed mass atone side of the nucleus and, later, by typicalapoptotic events such as the contraction of thecell volume and nuclear condensation. Nuclei areonly weakly stained by EB (compare withnecrotic nuclei in Figs 4I and 5A). (F) The timingof mitochondrial changes in confluent cells iscorrelated to the cell position. Clustering andcondensation of high-polarized mitochondria(arrows) is visibly advanced in outer cells. (G-H) Mitochondrial events (arrows) in HEp-2 cellsundergoing apoptosis are identical to thoseobserved in astrocytes. (I) Accidental necrosis,recognizable by the parallel disappearance of JC-1-red-stained mitochondria and the appearance ofintensely EB-stained nuclei, affects cells at anystage of apoptosis, as judged by the variable sizeof positive nuclei. Cell types: A-F, I: humanastrocytes; G-H: HEp-2 cells. Bars, 10 µm.

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size. Likewise, the proximity of mitochondria to edge regionsfree from cell-cell contacts depends on the degree of confluencyof cells. These morphological determinants may be at the originof the developmental m∆ψ switch observed inoligodendrocytes, where high-polarized mitochondria areinitially found only at the tips of short processes of immaturecells. When oligodendrocytes mature, in parallel with the cellbody enlargement, high-polarized mitochondria spread into thewhole dendritic tree (Kirischuck et al., 1995).

Several experimental evidences indicate that the cell edgeis a specific site of the plasma membrane provided withspecific calcium-sensitive components (Mittal and Bereiter-Hahn, 1985) and implicated in the initiation of calcium waves(Cornell-Bell et al., 1992; Empson and Galione, 1997). Ourfindings show that calcium hotspots, commonly interpretedas sites of Ca2+ induced Ca2+ release, progress from the celledge toward the cell center. In addition, in clusters ofconfluent cells, apoptosis due to calcium overloadingpropagates from outer cells to inner cells. Both phenomena

could not be easily explained if the extracellular calcium hadaccess from the whole cell surface. The hypothesis of calciumchannels located on the cell edge is also consistent with thehigher polarization of mitochondria present in that cellregion, since an important role of peripheral mitochondriaconsists in buffering extracellular calcium influx (Babcock etal., 1997), the force to drive calcium inside mitochiondriabeing provided by the m∆ψ. On the other hand, when theexposition of the cell edge to the extracellular environment iscovered by cell-cell contacts, peripheral mitochondriabecome low-polarized. With this respect, our data are inperfect agreement with the finding that high-polarizedmitochondria, present in isolated CV-1 cells, are lacking inconfluent cells of the same cell line (Chen and Smiley, 1993).The relationship between mitochondria and cell edge isstrictly local since, in the same cell, high- and low-polarizedmitochondria alternate in correspondence of tracts of the celledge free from or bearing contacts with adjacent cells. Inaddition, low-polarized mitochondria of confluent cells

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Fig. 5. (A) Cells undergoing necrosisshow the progressive decrease of JC-1 mitochondrial staining(arrowheads) and the parallelincrease of EB nuclear staining(stars). (B) 1 mM glutamate inducesthe appearance of calcium hotspots,revealed by CaGreen-1, propagatingfrom the cell edge to the center of thecell at the speed of 20-25 µm/minute.(C) High (>1 M) NaCl gives rise tothe simultaneous appearance of amyriad of high-polarizedmitochondria throughout thecytoplasm. The effect is not due tothe higher ionic strength nor tomembrane depolarization, sinceequivalent sucrose or KCl solutionsare unable to replicate the effect.(D E) PY staining showing the intactmitochondrial network in normalconditions (left) and itsfragmentation after 0.1 M vinblastinetreatment (right). (F-G) MDCK andVero cell mitochondria, which arebasally low-polarized, become high-polarized in apoptosis. Two MDCKcells undergoing apoptosis(encircled) are recognizable in theDIC image by the convex cell shape,the poor contrast of cytoplasmicgranulations and thin radialprocesses due to the cell retraction(arrows). Five normal cells visible inthe same field (stars) are flattenedand rich in well contrastedcytoplasmic granulations. Thefluorescent image shows thepresence of high-polarizedmitochondria only in the pair ofapoptotic cells. Cell types: A-E:human astrocytes; F-G: MDCK cells.Bars, 10 µm.

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become high-polarized again as soon as contacts withadjacent cells are retracted in cells undergoing apoptosis.

A second aspect of m∆ψ heterogeneity concerns theexclusion of high-polarized mitochondria from regionsoccupied by acidic vesicles. A suggestive hypothesis is that them∆ψ may be influenced by the sequestration of protons byneighbouring acidic vesicles. In support of this view there isthe fact that depleting acidic vesicles by the V-ATPase blockerbafilomycin A1 results in a significant m∆ψ increase. Thepresence of a Ca2+/nH+ antiport in acidic vesicles (Calvert andSanders, 1995; Bode et al., 1996) suggests also the possibilitythat a local release of calcium by endoplasmic reticulum,besides stimulating mitochondrial respiration, could elicitprotons release by acidic vesicles. In this way, the proton-motive force decrease due to calcium entry into themitochondrial matrix (m∆ψ down) could be compensated atleast in part by local acidification (m∆pH up). An involvementof acidic vesicles in the mitochondrial metabolism is alsoconsistent with the ubiquitary presence of these organelles,independent from the cell (auto)phagocytic activity, since alarge number of acidic vesicles is found even in cells withminimal amounts of histochemically detectable lysosomalhydrolases (data not shown). Acidic vesicles are so commonthat their staining by AO is suggested as standard assay of cellviability (Darzynkiewicz et al., 1997; Zelenin, 1993).

Hypoxia produces an increase of the number and length ofhigh-polarized tracts which appear as straight segments, mostlyin the periphery of the cell. Whereas the higher polarizationmay be considered as a result of the lower oxygen availability(Broekemeier et al., 1998), the nature of the rod-likemorphology of high-polarized mitochondria is less clear.However, in view of their conspicuous extension (up to 20 µmlong), an active role of the cytoskeleton is likely to be involved(Hollenbeck, 1996). The hypothesis is attractive, since apreferential dragging of energized mitochondria by motilecytoskeletal components could deliver ATP to cytoplasmicsites with low ATP/ADP ratio in a more efficient way than anaspecific mitochondrial transport, independent of the metabolicstatus. This hypothesis is at present under investigation.

At variance with hypoxia, camptothecin, glutamate and highNaCl produce an increase in the number of small, high-polarized mitochondrial tracts in the center of the cell. It is notan easy task to reveal which mechanisms are responsible forthe different mitochondrial changes. An element ofdiscrimination may be the fact that hypoxia induces anaerobicmetabolism and cytoplasmic acidosis which protect againstmitochondrial depolarization and permeability transition(Lemasters et al., 1997), as well as against cytochome c-mediated apoptosis (Jia et al., 1997b). Actually we observedthat after 24 hours hypoxia cells apparently retained theirnormal morphological features and negativity to EB. On theother hand, a common denominator of glutamate,camptothecin and high NaCl may be the elevation of thecytosolic calcium via metabotrophic glutamate receptors(Lodge and Collingridge, 1991), calcium mobilizationassociated to apoptosis (Nicotera et al., 1994) or the activationof the Na+/Ca2+ mitochondrial exchanger (Babcock et al.,1997; Uceda et al., 1995). With reference to the last point,hyperosmotic NaCl (but not KCl) has been found to induce aspecific decrease in state 3 respiratory flux (Devin et al., 1998).So, diffuse high-polarization of centrocellular mitochondria

could arise from a slowing down of oxygen consumption, notparalleled by a decreased activity of the electron transportchain proton-pumping activity.

The relationship between m∆ψ and apoptosis has beenalready evaluated in a number of studies, but no univocalconclusions have been drawn as to whether apoptosis involvesan early m∆ψ drop associated to permeability transition poreopening (Marchetti et al., 1996; Zamzami et al., 1996) or them∆ψ loss is only a conclusive event of apoptosis (Ferlini et al.,1996) which requires functional mitochondria for its energy-dependent processes (Ankarcrona et al., 1995). Otherinvestigations suggest that apoptosis is in fact independent ofmitochondrial ATP synthesis but requires the integrity of theelectron transport chain (Jia et al., 1997a) and that the key eventof apoptosis, represented by the cytochrome c release from themitochondrial intermembrane space, is not accompanied bym∆ψ changes (Kluck et al., 1997). Consistent with the latterfinding, our data show that m∆ψ is sustained in cellsundergoing apoptosis. However, we wish to emphasize the factthat high-polarized mitochondria of late apoptotic cellsrepresent a residual fraction of the original mitochondrialpopulation, the major part of which degenerate in the courseof apoptosis. This dual aspect may be the cause of the diversityof views reported in previous papers.

In all our experiments, the highest level of mitochondrialpolarization was seen in cells subjected to prolonged hypoxia.We do not know whether the m∆ψ increase dependsexclusively on the limited oxygen availability (Broekemeier etal., 1998) or is generated by reverse ATP synthase activitysupported by glycolytic ATP (Skowronek et al., 1992; DiLisaet al., 1995). What seems evident is that mitochondrial high-polarization is not synonymous of enhanced mitochondrialactivity. Indeed, an inverse relationship may be assumedbetween the average level of mitochondrial polarization and therate of ATP synthesis, at least in intact cells where complexhomeostatic mechanisms are established betweenmitochondria and other cytoplasmic compartments.Mitochondria actively involved in ATP synthesis undergo acontinuous m∆ψ dissipation which should be reflected by anaverage low-polarized condition. This is expected to occur incentrocellular mitochondria which interact with theendoplasmic reticulum with bi-directional calcium flows(Babcock et al., 1997; Montero et al., 1997) but also with acidicvesicles which may play an important role in the regulation ofthe proton gradient across the mitochondrial membrane.

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