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Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells
Cell Calcium 36 (2004) 499–508
Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells Aniko Varadi a,1 , Vincenzo Cirulli b , Guy A. Rutter a,∗ a
Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK b The Whittier Institute for Diabetes, Laboratory of Developmental Biology, University of California San Diego, La Jolla, CA 92037, USA Received 20 January 2004; received in revised form 4 May 2004; accepted 14 May 2004
Abstract Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+ ]c ), mitochondria ([Ca2+ ]m ) and ER ([Ca2+ ]ER ) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+ ] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+ ]c and [Ca2+ ]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cytoplasmic dynein; Calcium; Mitochondria; Motor proteins
1. Introduction Mitochondria play important roles both in controlling and in decoding calcium signals in mammalian cells [1,2]. Thus, uptake of Ca2+ by mitochondria is important for the regulaAbbreviations: [ATP]c , cytosolic free ATP concentration; [Ca2+ ]c , cytosolic free Ca2+ concentration; CPA, cyclopiazonic acid; CRAC, Ca2+ release-activated Ca2+ channels; DMEM, Dullbeco’s modified Eagle’s medium; EM, electron microscopy; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; ER.Aq, ER targeted aequorin; FCS, foetal calf serum; GFP, green fluorescent protein; His, histidine; IP3 , inositol 1,4,5-trisphosphate; KRH, Krebs–Ringer–Hepes–Bicarbonate; m, mitochondria; mit.Aq, mitochondrially-targeted aequorin; SERCA, sarco(endo)plasmic Ca2+ -ATPase; TMRE, tetramethylrhodamine ethyl ester; SOC, store-operated Ca2+ entry; YFP, yellow fluorescent protein ∗ Corresponding author. Tel.: +44 117 954 6401; fax: +44 117 928 8274. E-mail address: [email protected] (G.A. Rutter). 1 Present address: Centre for Research in Biomedicine, Bristol Genomics Research Institute, Faculty of Applied Sciences, University of West of England, Bristol BS16 1QY, UK. 0143-4160/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2004.05.003
tion of intramitochondrial oxidative metabolism [3,4] whilst the resulting changes in cytosolic [Ca2+ ] ([Ca2+ ]c ) appear to influence the generation and propagation of Ca2+ oscillations and waves [1,5]. Suggesting a role for highly localised changes in [Ca2+ ]c in determining the rate of mitochondrial Ca2+ uptake in living cells, rapid increases in mitochondrial [Ca2+ ] observed following agonist stimulation and the mobilisation of ER Ca2+ [6–8] are incompatible with the relatively low affinity of the mitochondrial Ca2+ uniporter in isolated mitochondria [9]. This discrepancy has led to the proposal [10] that domains of particularly high Ca2+ concentration may exist transiently at the mouth of inositol 1,4,5-trisphosphate (IP3 ) receptors juxtaposed to sites of mitochondrial Ca2+ uptake, hence permitting rapid mitochondrial Ca2+ sequestration. In recent years, a good deal of evidence has accumulated in support of the existence of such “Ca2+ microdomains” [5,11–13]. Furthermore, it has also been proposed that close physical contacts between mitochondria and ER may permit a direct “channelling” of Ca2+ ions from the ER into mitochondria [5,10,11,13]. Whilst
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the nature of the putative channel complex remains obscure, the existence of a direct interaction between IP3 receptors and mitochondrial Ca2+ uptake systems, possibly including the mitochondrial outer membrane voltage-dependent anion channel (VDAC) [13–15] is now considered a strong possibility. In opposition to these views, however, other workers [16] have suggested that agonist-induced uptake of Ca2+ by mitochondria can be equally efficient irrespective of the extent of association between mitochondria and ER, implying instead that the rapid accumulation of Ca2+ into mitochondria may result from the activation, by unknown diffusible factors, of the Ca2+ uniporter. Uptake of Ca2+ by mitochondria has also been suggested [17,18] to play an important role in store-operated Ca2+ influx (also called capacitative Ca2+ entry) [19] across the plasma membrane. In this case, mitochondrial Ca2+ accumulation is hypothesised to prevent the local accumulation of Ca2+ ions in the cytosol and hence feedback inhibition of calcium release-activated (CRAC) channels in the plasma membrane [20,21]. However, current evidence for this view is based largely on the observation that carbonyl cyanide-derived uncouplers of mitochondrial respiration [17] or respiratory chain inhibitors [18] suppress Ca2+ entry after the depletion of intracellular Ca2+ stores. However, these experimental approaches (a) involve changes in mitochondrial oxidative function, and hence likely local decreases in ATP concentration; (b) are complicated by the effects of protonophores on ER Ca2+ release [22] as well as the polarisation of the plasma [23] and possibly other non-mitochondrial membranes; and (c) provide no direct information on the role of different subsets of mitochondria, e.g. those beneath the plasma membrane versus those located elsewhere in the cell. In an earlier study, Lawrie et al. [12] provided evidence for the importance for close interactions between mitochondria and the ER or plasma membrane for efficient uptake of Ca2+ ions by mitochondria. Thus, mitochondrial Ca2+ accumulation in response to release from intracellular stores was found to be much more rapid in HeLa cells, which displayed extensive ER-mitochondria contacts, than in endothelial cells, which had many fewer. This position was reversed with respect to Ca2+ entry where mitochondrial Ca2+ accumulation was more efficient in endothelial cells, which possessed more mitochondria beneath the plasma membrane [12]. As an alternative to the above, correlative strategy, we sought here to explore the importance of close contacts between individual mitochondria and the ER (or the plasma membrane) by selectively altering the extent of these interactions in a single cell type by molecular means. We have recently demonstrated that mitochondrial distribution within cells can be altered, with relatively little impact on the distribution of other cellular organelles including the ER, by over-expressing the dynactin subunit, dynamitin [24]. Dynactin is a multisubunit complex through which cytoplasmic dynein, a retrograde motor protein, is able to transport specific cargoes along microtubules [25]. Dyna-
mitin over-expression in HeLa cells leads to the redistribution of mitochondria away from the plasma membrane and towards the nuclear periphery and microtubule organising centre. This appears to result, at least in part, from the loss of dynamin-related protein (Drp-1 or DLP-1) from the mitochondrial surface [24], since the dynamitin + phenotype is rescued by over-expression of Drp-1 and mimicked by overexpression of dominant negative Drp-1. However, the latter manoeuvre tends also to cause changes in the distribution of the ER [24–26] making dominant negative Drp-1 a less selective tool through which to alter mitochondrial structure. We show that whilst the mis-localization of mitochondria leads to a dramatic increase in the number of points of close contact between mitochondria and the ER, this does not significantly affect the efficiency of transfer of Ca2+ ions between the two organelles in response to the opening of IP3 receptors. On the other hand, expression of dynamitin causes a marked inhibition of Ca2+ influx likely via store-operated channels, consistent with a requirement for strategically localised mitochondria, positioned just beneath the plasma membrane, for optimal capacitative Ca2+ entry. 2. Materials and methods 2.1. Materials cDNAs encoding dynamitin and dynamitin.EGFP (Valetti et al., 1999) were kindly provided by Dr. Trina Schroer (Johns Hopkins University, Baltimore) and Dr. Vladimir Gelfand (Urbana, Illinois), respectively. cDNA encoding endoplasmic reticulum-targeted green fluorescent protein (ER.GFP) was from Dr. Rosario Rizzuto (University of Ferrara, Italy) [11]. Rabbit polyclonal pan anti-inositol 1,4,5-trisphosphate (IP3 ) receptor antibody [27,28] was a gift from Prof. Jan B. Parys (University of Leuven, Belgium). Cell culture reagents were from GibcoBRL (Life Science Research, Paisley, UK). All molecular biologicals were obtained from Roche Diagnostics Ltd. (Lewes, UK). EM grade paraformaldehyde, glutaraldehyde and sodium cacodylate trihydrate were purchased from Electron Microscopy Sciences (Fort Washington, PA). 2.2. Cell culture HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) tissue-culture medium supplemented with 10% (v/v) foetal calf serum (FCS) penicillin (100 units ml−1 ), streptomycin (0.1 mg ml−1 ) and l-glutamine (2 mM) at 37 ◦ C in an atmosphere of humidified air (95%) and CO2 (5%) as described previously [29]. 2.3. Plasmids cDNA encoding mitochondrially-targeted disodium red (mito.DsRed) construct was generated as described earlier [30].
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2.4. Immunocytochemistry and confocal microscopy Cells were co-transfected with 1 g of plasmid DNA encoding mito.DsRed and dynamitin or dynamitin.EGFP, using 10 g ml−1 lipofectamine in Optimem ITM medium (GibcoBRL, Life Science Research, Paisley, UK) for 4 h. Immunocytochemistry was performed as described earlier [29]. Images were captured on a Leica TCS-NT confocal laser-scanning microscope attached to a DM IRBETM epifluorescence microscope using an ×63 PL Apo 1.4 NA oil-immersion objective (Leica, Heidelberg, Germany) or on an UltraVIEWTM Nipkow spinning disc live cell confocal imaging system (Perkin-Elmer Life Sciences, Boston, MA). For live imaging, cells were kept in Krebs–Ringer–Hepes–Bicarbonate (KRH) buffer comprising 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2 PO4 , 0.5 mM MgSO4 , 2.0 mM NaHCO3 , 5.5 mM glucose, 10 mM Hepes (pH 7.4) and 1.0 mM CaCl2 equilibrated with O2 /CO2 (95:5, v/v) at 37 ◦ C. 2.5. Image analysis Z-series stacks (0.2 m steps) of dynamitin, mito.DsRed and ER.GFP co-expressing cells were acquired with the UltraVIEWTM confocal microscope. Subsequent image restoration and all image analysis and processing were performed using VolocityTM software (Improvision, Coventry, UK). For the analysis of the spatial relationship between ER and mitochondria, each three-dimensional image was first corrected to eliminate background. The percentage of mitochondrial surface colocalised with the ER was expressed as the number of voxels (volume pixels) occupied by both signals (ER.GFP, mito.DsRed) divided by voxels occupied by mito.DsRed. 2.6. Measurement of cytosolic free ([Ca2+ ]c ) with Fura-2
Ca2+
concentration
Changes in [Ca2+ ]c were measured at 37 ◦ C with entrapped Fura-2 [31] using a Leica DM-IRBI inverted microscope (40× objective) and a Hamamatsu C4742-995 charge-coupled device camera driven by OpenLabTM software (Improvision, Coventry, UK) [32]. Cells transfected with dynamitin.EGFP or the empty vector (pAdTrack-CMV) were loaded with 5 M Fura-2/AM and 0.1% (w/v) Pluronic F-127 (BASF, Mount Olive, NJ) for 40 min in KRH buffer containing 3 mM glucose. 2.7. Measurement of aequorin and luciferase luminescence Cells cultured on 13 mm diameter poly-l-lysine-coated coverslips were co-transfected with 0.5 g plasmid DNA encoding ER.Aq, mit.Aq, cyt.Aq or cyt.Luc and 1 g dynamitin or empty vector (pcDNA3) using Lipofectamine (Promega). For [Ca2+ ] measurements, cells were de-
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pleted of Ca2+ by incubation with 10 M ionomycin and 10 M cyclopiazonic acid (CPA) in KRH buffer containing 3 mM glucose and supplemented with 1 mM EGTA for 10 min at 4 ◦ C [32]. Aequorin was reconstituted in 0.1 mM EGTA, 5 M coelenterazine (mit.Aq) or coelenterazine n (ER.Aq) for 1–2 h at 4 ◦ C. Cell perifusion and signal calibration were performed as described previously [7,33]. For monitoring changes in mitochondrial ATP synthesis, cells expressing mitochondrially-targeted firefly luciferase cells were constantly perifused in KRH buffer supplemented with 20 M luciferin, 0.1 mM pyruvate and 1 mM lactate [34] then challenged with 100 M histamine as shown. 2.8. Electron microscopy Cells were fixed in 0.1 M sodium cacodylate trihydrate buffer (pH 7.4, at 37 ◦ C) containing 2% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 3 M CaCl2 for 4 h at 22 ◦ C. Samples were then post-fixed with osmium tetraoxide (1% (w/v) in H2 O) and counterstained with uranyl acetate (2% (w/v) in H2 O). Following gradual dehydration in ethanol, samples were embedded in Durcupan resin (Sigma Immunochemicals) and polymerized overnight at 60 ◦ C and −20 mmHg, as described [35]. Ultrathin sections (80 nm) were cut using a 35◦ angle Diatome diamond knife, and mounted on 300 mesh gold grids (Electron Microscopy Sciences, Fort Washington, PA). Following counterstaining with uranyl acetate (1% (w/v) in H2 O) and Sato lead (1% (w/v) in H2 O) sections were imaged at 80 keV using an electron microscope (1200FX JEOL Ltd., Akashima, Japan). 2.9. Statistical analysis Data are presented as the mean ± S.E.M. for the number of observations given, and statistical significance calculated using Student’s t-test under Microsoft ExcelTM .
3. Results 3.1. Dynamitin over-expression relocalizes mitochondria away from the plasma membrane and increases the number of close contact sites between mitochondria and the ER As previously described in detail [24], over-expression of dynamitin in HeLa cells leads to a dramatic relocalisation of mitochondria towards the nuclear periphery, assessed either with mitochondrially-targeted DsRed [30] (Fig. 1D versus A), or the mitochondrial stain, MitoTrackerRedTM (not shown). The effect of dynamitin expression on mitochondrial distribution was highly reproducible and was unaffected by the fusion of EGFP at the carboxy-terminus
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Fig. 1. Over-expression of dynamitin induces retreat of mitochondria from the cell membrane towards the nucleus. HeLa cells were co-transfected with 0.5 g mito.DsRed and 1 g dynamitin.EGFP. Twelve to forty-eight hours after transfection cells were imaged on Leica TCS-NT confocal microscope. Dynamatin-expressing cells were identified by exciting EGFP at 488 nm and DsRed fluorescence was visualised in the same cells by exciting at 568 nm. (A, D) Typical DsRed confocal images of mitochondria in control or dynamitin-expressing cells. (B, E) Intrinsic EGFP fluorescence. (C, F) Composite images. The broken lines (A–F) indicate the position of the plasma membrane obtained as an overlay from the transmitted image of the cell. Bars, 10 m. Note the re-localisation of mitochondria close to the nucleus in dynamitin-expressing cells (D vs. A).
of dynamitin (not shown). Thus, in dynamitin-expressing cells, the density of mitochondria was substantially greater around the nucleus compared to control cells, a region where the density of ER (Fig. 2A) [16] and IP3 receptors (Fig. 2D) [36] is high. By contrast, mitochondria were rarely if ever found within 2–3 m the plasma membrane after dynamitin expression (Fig. 1D). To determine whether dynamitin over-expression led to a significant increase in the number of close overlaps between mitochondria and ER, the latter compartment was labelled by expressing cDNA encoding an IgG chain-GFP chimera [11], and mitochondria were tagged with mitochondrial DsRed [30] (Fig. 2A). Cells were imaged with a Nipkow spinning disc confocal microscope (see Section 2). This approach allowed rapid (0.1–0.25 s/image) data acquisition and thus largely avoided artefacts arising from the movements of organelles during data collection, whilst providing a spatial resolution of close to 200 nm [37]. The number of close associations between the two organelle types, estimated in living cells by determining the extent of overlap of mitochondrial and ER images with VolocityTM software [16] (see Section 2), was increased >2.5-fold by dynamitin over-expression (Fig. 2B). Correspondingly, close associations between ER and mitochondria were also frequently apparent by EM in
dynamitin-expressing cells (Fig. 2C) but were rare in control cells [24]. 3.2. Effects of dynamitin over-expression on Ca2+ release from the ER, and uptake by mitochondria We next determined whether the observed changes in mitochondria/ER overlap might affect either (i) the kinetics of Ca2+ release from the ER; or (ii) Ca2+ uptake by mitochondria. These experiments were performed in the absence of external Ca2+ (1 mM EGTA) in order to eliminate any contribution of Ca2+ influx across the plasma membrane to the observed changes in cytosolic or organellar [Ca2+ ]. Stimulation of control or dynamitin-over-expressing cells with the phospholipase C␥-linked agonist, histamine, led to identical, sharp increases in [Ca2+ ]c , assessed using Fura-2 (Fig. 3A) or cytosolically-targeted aequorin (Cyt.Aq, Fig. 3B). Similarly, there were no differences in the apparent rate or extent of release of Ca2+ from the ER in response to histamine (Fig. 3C) nor in apparent Ca2+ uptake by the ER [38] after cellular Ca2+ depletion with the sarco(endo)plasmic Ca2+ -ATPase (SERCA) inhibitor, thapsigargin [39] (not shown). Likewise, we were unable to detect any differences in the apparent rate of Ca2+ uptake into
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Fig. 2. Dynamitin over-expression alters the proximity of mitochondria to the ER. (A) HeLa cells were co-transfected with 0.25 g mito.DsRed, 0.5 g ER.GFP and 1 g pcDNA3 (empty vector/control; a, c) or dynamitin.pcDNA3 (b, d) and 12 or 24 h later Z-series stacks (0.2 m steps) of cells were acquired with an UltraVIEWTM confocal microscope. Subsequent image restoration was achieved with the VolicityTM software. The regions bounded with white boxes in (a) and (b) are shown on expanded scales in (c) and (d), respectively. Bars represent 10 m (a, b) and 1 m (c, d). The arrows (c, d) denote where ER and mitochondria co-localise within a resolution of ∼150 nm. (B) Total and co-localised surface areas of mitochondria (see Section 2). (C) Transmission electron micrographs of dynamitin-expressing HeLa cell cross-sections at high magnification show unusual branched mitochondria (star). Close association between ER and mitochondria are indicated with arrows (m: mitochondria, ER: endoplasmic reticulum). (D) Distribution of IP3 receptors in HeLa cells visualised using a rabbit polyclonal pan-IP3 receptor antibody (see Section 2 for further details).
mitochondria, estimated by monitoring increases in [Ca2+ ]m using a matrix-targeted aequorin (Fig. 4) [6,8]. Moreover, apparent mitochondrial Ca2+ uptake was identical in control and dynamitin-expressing cells even at very low histamine concentrations (Fig. 4A–C). These changes could not be ascribed to any loss of mitochondrial oxidative capacity since increases in mitochondrial ATP synthesis in response to 100 M histamine in the presence of oxidative substrates (lactate plus pyruvate; see Section 2) was not significantly different in dynamatin-expressing ([ATP]c peak value 115% of basal, mean of two independent experiments) and control cells ([ATP]c peak value 113% of basal, n = 2).
We would stress, however, that these measurements leave open the possibility that changes in free ATP concentration in subcellular microdomains, for example, immediately beneath the plasma membrane, may be altered by changes in mitochondrial distribution. Moreover, mitochondrial membrane potential (ψmit ) was also unaffected by dynamitin over-expression [24]. 3.3. Effects of dynamitin over-expression on Ca2+ entry We next explored the impact of dynamitin over-expression and mitochondrial relocalisation on Ca2+ entry. In these
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Fig. 3. Increased association of mitochondria with the ER does not affect Ca2+ sequestration by mitochondria following release of ER Ca2+ . HeLa cells were transfected with 1 g dynamitin.EGFP or pAdTrack-CMV (empty vector/control) then loaded with 5 M Fura-2/AM (A). Alternatively, cells were co-transfected with 1 g dynamitin.pcDNA3 with 0.5 g Cyt.Aq (B); 0.5 g ER.Aq (C). Histamine was then applied during perifusion in medium containing 1 mM EGTA. In (A), measurements of Fura-2 fluorescence ratio were performed on 78 single cells in five independent experiments, and data are shown as mean ± S.E.M. Aequorin bioluminescence (B, C) was calibrated to give mean free Ca2+ concentrations as described in Section 2, using data obtained in three independent experiments. Bars, 10 s.
experiments, Ca2+ entry was activated by depleting intracellular Ca2+ stores with the SERCA inhibitor, cyclopiazonic acid, in the presence of EGTA, and then reintroducing extracellular CaCl2 (Fig. 5A–E). Under these conditions, the activation of Ca2+ entry provoked very similar increases in [Ca2+ ]c and in [Ca2+ ]m (Fig. 5B and D, open points), indicating that the reported increases in mitochondrial [Ca2+ ] likely reflect changes in mitochondria located throughout the cytoplasm, and not just those located beneath the plasma membrane where an elevated [Ca2+ ]c microdomain is likely to pertain [40,41]. In contrast to effects on ER Ca2+ release (Figs. 3 and 4), CRAC-dependent Ca2+ entry was markedly diminished in dynamitin over-expressing cells, which displayed a smaller increase in both [Ca2+ ]c (Fig. 5A and B)
Fig. 4. Impact of dynamitin over-expression on ER Ca2+ mobilisation stimulated by varying histamine concentrations. Cells were co-transfected with 1 g dynamtin-expressing or empty vector, and 0.5 g mit.Aq and then the aequorin luminescence was measured by photon-counting 48 h later (see Section 2). Ca2+ release from the ER was triggered by perifusing with 0.1–100 M histamine (Hist) in Ca2+ free medium containing 1 mM EGTA. Bars, 10 s. See the legend to Fig. 3 and Section 2 for other details.
and [Ca2+ ]m (Fig. 5D). In order to eliminate the possibility that the observed differences in Ca2+ entry were due to altered membrane potential, we clamped the latter at close to 0 mV by using 140 mM KCl in the buffer and performed the same experiment shown in Fig. 5A. Under these condition the Ca2+ entry was markedly reduced in dynamitin over-expressing cells (Fig. 5E). Furthermore, the extent of store depletion induced by CPA was not significantly different in dynamitin-expressing and control cells (Fig. 5C).
4. Discussion 4.1. Effect of mitochondrial redistribution on mitochondrial Ca2+ uptake from the ER Contrary to our expectations, over-expression of dynamitin and a substantial increase in the number of close contact sites between mitochondria and ER (Fig. 2) had no significant effect on basal or stimulated Ca2+ concentrations in the cell cytosol, mitochondria, or ER (Fig. 3). Importantly, we detected no differences in the apparent rate
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Fig. 5. Effect of mitochondrial redistribution on store-operated Ca2+ entry. HeLa cells were transfected with 1 g dynamitin.EGFP or pAdTrack-CMV (empty vector/control) then loaded with 5 M Fura-2/AM (A, E). Alternatively, cells were co-transfected with 1 g dynamitin.pcDNA3 with 0.5 g Cyt.Aq (B); 0.5 g ER.Aq (C); or 0.5 g mit.Aq (D) and then aequorin luminescence was measured by photon-counting (see Section 2). The ER/Golgi Ca2+ pool was depleted by pre-treatment of cells with 1 M thapsigargin in 1 mM EGTA for 10 min before perifusion of cells with 10 M CPA in 1 mM EGTA. Ca2+ entry was then initiated by substituting the EGTA in the extracellular medium with 1.5 mM CaCl2 . The membrane potential was clamped at close to 0 mV by using 140 mM KCl and 10 mM NaCl in the perifusion buffer (E). Bars, 10 s.
of Ca2+ accumulation by mitochondria in control and dynamitin over-expressing cells even during stimulation with a very low agonist dose (Fig. 4A) where ER Ca2+ release is slow and the effects of increasing the proximity between ER and mitochondria might be expected to be greatest. The latter finding thus eliminates concerns that rapid saturation of mitochondrial aequorin during fast Ca2+ release from the ER might explain the apparent lack of effect of dynamitin over-expression. Instead, our observations are most compatible with two extreme models: (a) that local domains of high Ca2+ concentration have no role whatsoever to play in the transfer of Ca2+ from ER to the mitochondria, i.e. that mitochondria respond to changes in bulk cytosolic Ca2+ concentration; or (b) that Ca2+ microdomains are
minutely localised to specific and defined “contact” points between the ER and mitochondrial surface such that they are unaffected by a gross relocalisation of mitochondria (see scheme, Fig. 6). In essence, possibility (b) is equivalent to the existence of physical “tunnels”, i.e. stable entities perhaps formed between adjacent pairs (or groups of) IP3 receptors and VDAC channels, and through which Ca2+ ions might flow with little if any exchange with the bulk cytosol (see Section 1). Although the present studies cannot discriminate definitively between these two possibilities, it should be stressed that substantial evidence exists which would argue against model (a) [42]. Moreover, and also in line with model (b), a high density of IP3 receptors has been reported in domains of the ER that face towards
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Fig. 6. Scheme: role of sub-plasma membrane mitochondria in the regulation of store-operated calcium entry. (A) In control cells, phospholipase C-linked agonists cause Ca2+ release from the ER (1); and uptake by mitochondria via VDAC channels on the outer mitochondria membrane [12], potentially via points of intimate contact between the two organelles (2). ER Ca2+ depletion (3); and the activation of calcium influx via, for example, store-operated Ca2+ channels or other Ca2+ entry channels also leads to the uptake of Ca2+ by mitochondria (4); and possibly the release of undefined mitochondrial regulators (“X”, see Section 4) which ensure sustained flux of Ca2+ ions into the cell [17,18]. (B) Expression of dynamitin and relocalization of mitochondria from the cell surface and cell cytosol towards the nuclear periphery has no detectable effect on mitochondrial Ca2+ accumulation in response to IP3 production, but inhibits calcium entry since microdomains of high [Ca2+ ] accumulate at the mouth of Ca2+ entry channels.
mitochondria [43–45]. Furthermore, selective clustering of VDAC channels was recently reported in HeLa cells [13] whilst VDAC and ryanodine receptors are both clustered at sarcoplasmic reticulum/mitochondria contact points in skeletal muscle [15]. Finally, Pozzan and coworkers have proposed [46] that multiple populations of mitochondria may exist even within relatively simple mammalian cells, with “high-responding” mitochondria being stably located close to sites of ER Ca2+ release. The present findings provide further support for this view, and suggest that the number of these selective contact points is unchanged after gross alterations in mitochondrial distribution and an apparent increase in mitochondria-ER proximity. 4.2. Effect of mitochondrial redistribution on Ca2+ entry The present findings support previous results which suggested that functional mitochondria play an important role in ensuring efficient Ca2+ entry via store-operated channels [17,18,47,48]. However, these earlier findings largely involved exploring the effects of global inhibition of mitochondrial function, usually achieved with mitochondrial respiratory poisons, and provided no information on the respective roles of mitochondria at different subcellular
locations. By using molecular tools to manipulate, with reasonable selectivity [24], the spatial distribution of mitochondria in cells, the current data show that Ca2+ uptake by mitochondria located strategically beneath the plasma membrane, rather than global Ca2+ clearance by mitochondria located throughout the cell, is likely to be the key regulator of store-operated Ca2+ influx. Although no attempt was made here to measure [Ca2+ ]m changes in specific subsets of mitochondria, given the very limited spatial resolution which can be obtained by aequorin imaging [8], these data are also consistent with the observation that in pancreatic acinar cells, store-operated Ca2+ entry caused specific uptake of Ca2+ into mitochondria situated very close to the basal membrane [49]. Moreover, the present results also support findings, obtained by measuring the activity of large conductance Ca2+ -activated potassium channels [50], that sub-plasma membrane Ca2+ concentrations are lowest in regions adjacent to mitochondria, and that decreased mitochondrial Ca2+ buffering caused by inhibition of mitochondrial Na+ /Ca2+ exchange [51] diminishes store-dependent Ca2+ influx in endothelial cells. As shown in the scheme (Fig. 6) we propose that Ca2+ uptake by both the ER and by strategically-positioned mitochondria reduces [Ca2+ ]c near Ca2+ entry sites at the plasma membrane and therefore diminishes the negative feedback effect of locally high sub-plasma membrane Ca2+ concentrations [41] on the open state probability of CRAC channels [20]. As a refinement of this model, it should be mentioned that Glitsch et al. [18] have proposed that Ca2+ uptake by mitochondria, and hence lowering of local Ca2+ concentrations, is not sufficient for the activation of CRAC currents, but rather triggers the stimulation of intramitochondrial oxidative metabolism [3]. This in turn may prompt the release from the mitochondrial matrix of other factors (ATP? amino acids?) which serve as channel activators. Future studies, involving permeabilised or patch-clamped cells, will be necessary to explore the relative contribution of local changes in [Ca2+ ]c , and mitochondria-derived metabolic factors, more fully. The present results are distinct from the recently-described effects of mitochondrial reorganisation achieved by vinblastine treatment of neurones [52]. By destabilising microtubules, this agent led to mitochondrial rounding, aggregation and to amplified increases in cytosolic Ca2+ concentration in response to the activation of glutamate receptors. These changes were ascribed to a decrease in mitochondrial Ca2+ accumulation, and hence a decrease in cytosolic Ca2+ buffering. However, in the neuronal setting [52], Ca2+ influx was achieved by the activation of ligandand voltage-gated, rather than store-operated, channels, leading to much more rapid increases in cytosolic [Ca2+ ] (half-time to peak ∼5 s for neuron depolarisation versus ∼20 s and CaCl2 reintroduction to HeLa cells). Under these conditions of more rapid [Ca2+ ]c increase it is likely that the most important role of sub-plasma membrane mitochondria is to buffer relatively large local [Ca2+ ]c changes, rather
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than to ensure the sustained opening of voltage-dependent channels in the face of smaller local [Ca2+ ]c increases. However, it might also be mentioned that microtubule disruption, as achieved with vinblastine, may elicit more widespread effects on the distribution of other organelles and Ca2+ stores, including the ER. Such changes are in contrast to the effects of dynamitin which had no gross impact on the structures of secretory pathway organelles in the current study [24], and caused only a small increase in the average length of mitochondria, as opposed to the rounding up seen in response to vinblastine [52]. 4.3. Conclusions Controlled changes in mitochondrial distribution in the context of a single cell type, as achieved here, provide a useful tool to explore the role of mitochondrial localisation in regulating Ca2+ influx and release from intracellular stores. The application of this approach to other, more specialised cells types [53], may now allow a detailed assessment of the role of mitochondrial organisation in orchestrating more complex intracellular Ca2+ signals.
Acknowledgements G.A.R. was supported by Wellcome Trust Programme Grant 067081/Z/02/Z, Human Science Frontiers Program grant RGP 0347/2001-M, and by project grants from the Wellcome Trust, Biotechnology and Biological Research Council, UK and Diabetes UK. V.C. was supported by NIH grants DK55183 and DK63443, JDRFI Research Grant 197009, and a Network Grant from The Larry L. Hillblom Foundation. Electron microscopy and imaging analysis was performed at The National Center for Microscopy and Imaging Research (UCSD), supported by NIH grant RR04050 to Dr. Mark H. Ellisman. We thank Dr. Mark Jepson and Alan Leard of the Bristol MRC Imaging Facility for technical assistance, and Professor Peter Cullen for the use of the UltraVIEWTM microscope. G.A.R. is a Wellcome Trust Research Leave Fellow.
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Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells Aniko Varadi a,1 , Vincenzo Cirulli b , Guy A. Rutter a,∗ a
Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK b The Whittier Institute for Diabetes, Laboratory of Developmental Biology, University of California San Diego, La Jolla, CA 92037, USA Received 20 January 2004; received in revised form 4 May 2004; accepted 14 May 2004
Abstract Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+ ]c ), mitochondria ([Ca2+ ]m ) and ER ([Ca2+ ]ER ) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+ ] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+ ]c and [Ca2+ ]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles. © 2004 Elsevier Ltd. All rights reserved. Keywords: Cytoplasmic dynein; Calcium; Mitochondria; Motor proteins
1. Introduction Mitochondria play important roles both in controlling and in decoding calcium signals in mammalian cells [1,2]. Thus, uptake of Ca2+ by mitochondria is important for the regulaAbbreviations: [ATP]c , cytosolic free ATP concentration; [Ca2+ ]c , cytosolic free Ca2+ concentration; CPA, cyclopiazonic acid; CRAC, Ca2+ release-activated Ca2+ channels; DMEM, Dullbeco’s modified Eagle’s medium; EM, electron microscopy; ER, endoplasmic reticulum; EGFP, enhanced green fluorescent protein; ER.Aq, ER targeted aequorin; FCS, foetal calf serum; GFP, green fluorescent protein; His, histidine; IP3 , inositol 1,4,5-trisphosphate; KRH, Krebs–Ringer–Hepes–Bicarbonate; m, mitochondria; mit.Aq, mitochondrially-targeted aequorin; SERCA, sarco(endo)plasmic Ca2+ -ATPase; TMRE, tetramethylrhodamine ethyl ester; SOC, store-operated Ca2+ entry; YFP, yellow fluorescent protein ∗ Corresponding author. Tel.: +44 117 954 6401; fax: +44 117 928 8274. E-mail address: [email protected] (G.A. Rutter). 1 Present address: Centre for Research in Biomedicine, Bristol Genomics Research Institute, Faculty of Applied Sciences, University of West of England, Bristol BS16 1QY, UK. 0143-4160/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2004.05.003
tion of intramitochondrial oxidative metabolism [3,4] whilst the resulting changes in cytosolic [Ca2+ ] ([Ca2+ ]c ) appear to influence the generation and propagation of Ca2+ oscillations and waves [1,5]. Suggesting a role for highly localised changes in [Ca2+ ]c in determining the rate of mitochondrial Ca2+ uptake in living cells, rapid increases in mitochondrial [Ca2+ ] observed following agonist stimulation and the mobilisation of ER Ca2+ [6–8] are incompatible with the relatively low affinity of the mitochondrial Ca2+ uniporter in isolated mitochondria [9]. This discrepancy has led to the proposal [10] that domains of particularly high Ca2+ concentration may exist transiently at the mouth of inositol 1,4,5-trisphosphate (IP3 ) receptors juxtaposed to sites of mitochondrial Ca2+ uptake, hence permitting rapid mitochondrial Ca2+ sequestration. In recent years, a good deal of evidence has accumulated in support of the existence of such “Ca2+ microdomains” [5,11–13]. Furthermore, it has also been proposed that close physical contacts between mitochondria and ER may permit a direct “channelling” of Ca2+ ions from the ER into mitochondria [5,10,11,13]. Whilst
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the nature of the putative channel complex remains obscure, the existence of a direct interaction between IP3 receptors and mitochondrial Ca2+ uptake systems, possibly including the mitochondrial outer membrane voltage-dependent anion channel (VDAC) [13–15] is now considered a strong possibility. In opposition to these views, however, other workers [16] have suggested that agonist-induced uptake of Ca2+ by mitochondria can be equally efficient irrespective of the extent of association between mitochondria and ER, implying instead that the rapid accumulation of Ca2+ into mitochondria may result from the activation, by unknown diffusible factors, of the Ca2+ uniporter. Uptake of Ca2+ by mitochondria has also been suggested [17,18] to play an important role in store-operated Ca2+ influx (also called capacitative Ca2+ entry) [19] across the plasma membrane. In this case, mitochondrial Ca2+ accumulation is hypothesised to prevent the local accumulation of Ca2+ ions in the cytosol and hence feedback inhibition of calcium release-activated (CRAC) channels in the plasma membrane [20,21]. However, current evidence for this view is based largely on the observation that carbonyl cyanide-derived uncouplers of mitochondrial respiration [17] or respiratory chain inhibitors [18] suppress Ca2+ entry after the depletion of intracellular Ca2+ stores. However, these experimental approaches (a) involve changes in mitochondrial oxidative function, and hence likely local decreases in ATP concentration; (b) are complicated by the effects of protonophores on ER Ca2+ release [22] as well as the polarisation of the plasma [23] and possibly other non-mitochondrial membranes; and (c) provide no direct information on the role of different subsets of mitochondria, e.g. those beneath the plasma membrane versus those located elsewhere in the cell. In an earlier study, Lawrie et al. [12] provided evidence for the importance for close interactions between mitochondria and the ER or plasma membrane for efficient uptake of Ca2+ ions by mitochondria. Thus, mitochondrial Ca2+ accumulation in response to release from intracellular stores was found to be much more rapid in HeLa cells, which displayed extensive ER-mitochondria contacts, than in endothelial cells, which had many fewer. This position was reversed with respect to Ca2+ entry where mitochondrial Ca2+ accumulation was more efficient in endothelial cells, which possessed more mitochondria beneath the plasma membrane [12]. As an alternative to the above, correlative strategy, we sought here to explore the importance of close contacts between individual mitochondria and the ER (or the plasma membrane) by selectively altering the extent of these interactions in a single cell type by molecular means. We have recently demonstrated that mitochondrial distribution within cells can be altered, with relatively little impact on the distribution of other cellular organelles including the ER, by over-expressing the dynactin subunit, dynamitin [24]. Dynactin is a multisubunit complex through which cytoplasmic dynein, a retrograde motor protein, is able to transport specific cargoes along microtubules [25]. Dyna-
mitin over-expression in HeLa cells leads to the redistribution of mitochondria away from the plasma membrane and towards the nuclear periphery and microtubule organising centre. This appears to result, at least in part, from the loss of dynamin-related protein (Drp-1 or DLP-1) from the mitochondrial surface [24], since the dynamitin + phenotype is rescued by over-expression of Drp-1 and mimicked by overexpression of dominant negative Drp-1. However, the latter manoeuvre tends also to cause changes in the distribution of the ER [24–26] making dominant negative Drp-1 a less selective tool through which to alter mitochondrial structure. We show that whilst the mis-localization of mitochondria leads to a dramatic increase in the number of points of close contact between mitochondria and the ER, this does not significantly affect the efficiency of transfer of Ca2+ ions between the two organelles in response to the opening of IP3 receptors. On the other hand, expression of dynamitin causes a marked inhibition of Ca2+ influx likely via store-operated channels, consistent with a requirement for strategically localised mitochondria, positioned just beneath the plasma membrane, for optimal capacitative Ca2+ entry. 2. Materials and methods 2.1. Materials cDNAs encoding dynamitin and dynamitin.EGFP (Valetti et al., 1999) were kindly provided by Dr. Trina Schroer (Johns Hopkins University, Baltimore) and Dr. Vladimir Gelfand (Urbana, Illinois), respectively. cDNA encoding endoplasmic reticulum-targeted green fluorescent protein (ER.GFP) was from Dr. Rosario Rizzuto (University of Ferrara, Italy) [11]. Rabbit polyclonal pan anti-inositol 1,4,5-trisphosphate (IP3 ) receptor antibody [27,28] was a gift from Prof. Jan B. Parys (University of Leuven, Belgium). Cell culture reagents were from GibcoBRL (Life Science Research, Paisley, UK). All molecular biologicals were obtained from Roche Diagnostics Ltd. (Lewes, UK). EM grade paraformaldehyde, glutaraldehyde and sodium cacodylate trihydrate were purchased from Electron Microscopy Sciences (Fort Washington, PA). 2.2. Cell culture HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) tissue-culture medium supplemented with 10% (v/v) foetal calf serum (FCS) penicillin (100 units ml−1 ), streptomycin (0.1 mg ml−1 ) and l-glutamine (2 mM) at 37 ◦ C in an atmosphere of humidified air (95%) and CO2 (5%) as described previously [29]. 2.3. Plasmids cDNA encoding mitochondrially-targeted disodium red (mito.DsRed) construct was generated as described earlier [30].
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2.4. Immunocytochemistry and confocal microscopy Cells were co-transfected with 1 g of plasmid DNA encoding mito.DsRed and dynamitin or dynamitin.EGFP, using 10 g ml−1 lipofectamine in Optimem ITM medium (GibcoBRL, Life Science Research, Paisley, UK) for 4 h. Immunocytochemistry was performed as described earlier [29]. Images were captured on a Leica TCS-NT confocal laser-scanning microscope attached to a DM IRBETM epifluorescence microscope using an ×63 PL Apo 1.4 NA oil-immersion objective (Leica, Heidelberg, Germany) or on an UltraVIEWTM Nipkow spinning disc live cell confocal imaging system (Perkin-Elmer Life Sciences, Boston, MA). For live imaging, cells were kept in Krebs–Ringer–Hepes–Bicarbonate (KRH) buffer comprising 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2 PO4 , 0.5 mM MgSO4 , 2.0 mM NaHCO3 , 5.5 mM glucose, 10 mM Hepes (pH 7.4) and 1.0 mM CaCl2 equilibrated with O2 /CO2 (95:5, v/v) at 37 ◦ C. 2.5. Image analysis Z-series stacks (0.2 m steps) of dynamitin, mito.DsRed and ER.GFP co-expressing cells were acquired with the UltraVIEWTM confocal microscope. Subsequent image restoration and all image analysis and processing were performed using VolocityTM software (Improvision, Coventry, UK). For the analysis of the spatial relationship between ER and mitochondria, each three-dimensional image was first corrected to eliminate background. The percentage of mitochondrial surface colocalised with the ER was expressed as the number of voxels (volume pixels) occupied by both signals (ER.GFP, mito.DsRed) divided by voxels occupied by mito.DsRed. 2.6. Measurement of cytosolic free ([Ca2+ ]c ) with Fura-2
Ca2+
concentration
Changes in [Ca2+ ]c were measured at 37 ◦ C with entrapped Fura-2 [31] using a Leica DM-IRBI inverted microscope (40× objective) and a Hamamatsu C4742-995 charge-coupled device camera driven by OpenLabTM software (Improvision, Coventry, UK) [32]. Cells transfected with dynamitin.EGFP or the empty vector (pAdTrack-CMV) were loaded with 5 M Fura-2/AM and 0.1% (w/v) Pluronic F-127 (BASF, Mount Olive, NJ) for 40 min in KRH buffer containing 3 mM glucose. 2.7. Measurement of aequorin and luciferase luminescence Cells cultured on 13 mm diameter poly-l-lysine-coated coverslips were co-transfected with 0.5 g plasmid DNA encoding ER.Aq, mit.Aq, cyt.Aq or cyt.Luc and 1 g dynamitin or empty vector (pcDNA3) using Lipofectamine (Promega). For [Ca2+ ] measurements, cells were de-
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pleted of Ca2+ by incubation with 10 M ionomycin and 10 M cyclopiazonic acid (CPA) in KRH buffer containing 3 mM glucose and supplemented with 1 mM EGTA for 10 min at 4 ◦ C [32]. Aequorin was reconstituted in 0.1 mM EGTA, 5 M coelenterazine (mit.Aq) or coelenterazine n (ER.Aq) for 1–2 h at 4 ◦ C. Cell perifusion and signal calibration were performed as described previously [7,33]. For monitoring changes in mitochondrial ATP synthesis, cells expressing mitochondrially-targeted firefly luciferase cells were constantly perifused in KRH buffer supplemented with 20 M luciferin, 0.1 mM pyruvate and 1 mM lactate [34] then challenged with 100 M histamine as shown. 2.8. Electron microscopy Cells were fixed in 0.1 M sodium cacodylate trihydrate buffer (pH 7.4, at 37 ◦ C) containing 2% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde, 3 M CaCl2 for 4 h at 22 ◦ C. Samples were then post-fixed with osmium tetraoxide (1% (w/v) in H2 O) and counterstained with uranyl acetate (2% (w/v) in H2 O). Following gradual dehydration in ethanol, samples were embedded in Durcupan resin (Sigma Immunochemicals) and polymerized overnight at 60 ◦ C and −20 mmHg, as described [35]. Ultrathin sections (80 nm) were cut using a 35◦ angle Diatome diamond knife, and mounted on 300 mesh gold grids (Electron Microscopy Sciences, Fort Washington, PA). Following counterstaining with uranyl acetate (1% (w/v) in H2 O) and Sato lead (1% (w/v) in H2 O) sections were imaged at 80 keV using an electron microscope (1200FX JEOL Ltd., Akashima, Japan). 2.9. Statistical analysis Data are presented as the mean ± S.E.M. for the number of observations given, and statistical significance calculated using Student’s t-test under Microsoft ExcelTM .
3. Results 3.1. Dynamitin over-expression relocalizes mitochondria away from the plasma membrane and increases the number of close contact sites between mitochondria and the ER As previously described in detail [24], over-expression of dynamitin in HeLa cells leads to a dramatic relocalisation of mitochondria towards the nuclear periphery, assessed either with mitochondrially-targeted DsRed [30] (Fig. 1D versus A), or the mitochondrial stain, MitoTrackerRedTM (not shown). The effect of dynamitin expression on mitochondrial distribution was highly reproducible and was unaffected by the fusion of EGFP at the carboxy-terminus
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Fig. 1. Over-expression of dynamitin induces retreat of mitochondria from the cell membrane towards the nucleus. HeLa cells were co-transfected with 0.5 g mito.DsRed and 1 g dynamitin.EGFP. Twelve to forty-eight hours after transfection cells were imaged on Leica TCS-NT confocal microscope. Dynamatin-expressing cells were identified by exciting EGFP at 488 nm and DsRed fluorescence was visualised in the same cells by exciting at 568 nm. (A, D) Typical DsRed confocal images of mitochondria in control or dynamitin-expressing cells. (B, E) Intrinsic EGFP fluorescence. (C, F) Composite images. The broken lines (A–F) indicate the position of the plasma membrane obtained as an overlay from the transmitted image of the cell. Bars, 10 m. Note the re-localisation of mitochondria close to the nucleus in dynamitin-expressing cells (D vs. A).
of dynamitin (not shown). Thus, in dynamitin-expressing cells, the density of mitochondria was substantially greater around the nucleus compared to control cells, a region where the density of ER (Fig. 2A) [16] and IP3 receptors (Fig. 2D) [36] is high. By contrast, mitochondria were rarely if ever found within 2–3 m the plasma membrane after dynamitin expression (Fig. 1D). To determine whether dynamitin over-expression led to a significant increase in the number of close overlaps between mitochondria and ER, the latter compartment was labelled by expressing cDNA encoding an IgG chain-GFP chimera [11], and mitochondria were tagged with mitochondrial DsRed [30] (Fig. 2A). Cells were imaged with a Nipkow spinning disc confocal microscope (see Section 2). This approach allowed rapid (0.1–0.25 s/image) data acquisition and thus largely avoided artefacts arising from the movements of organelles during data collection, whilst providing a spatial resolution of close to 200 nm [37]. The number of close associations between the two organelle types, estimated in living cells by determining the extent of overlap of mitochondrial and ER images with VolocityTM software [16] (see Section 2), was increased >2.5-fold by dynamitin over-expression (Fig. 2B). Correspondingly, close associations between ER and mitochondria were also frequently apparent by EM in
dynamitin-expressing cells (Fig. 2C) but were rare in control cells [24]. 3.2. Effects of dynamitin over-expression on Ca2+ release from the ER, and uptake by mitochondria We next determined whether the observed changes in mitochondria/ER overlap might affect either (i) the kinetics of Ca2+ release from the ER; or (ii) Ca2+ uptake by mitochondria. These experiments were performed in the absence of external Ca2+ (1 mM EGTA) in order to eliminate any contribution of Ca2+ influx across the plasma membrane to the observed changes in cytosolic or organellar [Ca2+ ]. Stimulation of control or dynamitin-over-expressing cells with the phospholipase C␥-linked agonist, histamine, led to identical, sharp increases in [Ca2+ ]c , assessed using Fura-2 (Fig. 3A) or cytosolically-targeted aequorin (Cyt.Aq, Fig. 3B). Similarly, there were no differences in the apparent rate or extent of release of Ca2+ from the ER in response to histamine (Fig. 3C) nor in apparent Ca2+ uptake by the ER [38] after cellular Ca2+ depletion with the sarco(endo)plasmic Ca2+ -ATPase (SERCA) inhibitor, thapsigargin [39] (not shown). Likewise, we were unable to detect any differences in the apparent rate of Ca2+ uptake into
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Fig. 2. Dynamitin over-expression alters the proximity of mitochondria to the ER. (A) HeLa cells were co-transfected with 0.25 g mito.DsRed, 0.5 g ER.GFP and 1 g pcDNA3 (empty vector/control; a, c) or dynamitin.pcDNA3 (b, d) and 12 or 24 h later Z-series stacks (0.2 m steps) of cells were acquired with an UltraVIEWTM confocal microscope. Subsequent image restoration was achieved with the VolicityTM software. The regions bounded with white boxes in (a) and (b) are shown on expanded scales in (c) and (d), respectively. Bars represent 10 m (a, b) and 1 m (c, d). The arrows (c, d) denote where ER and mitochondria co-localise within a resolution of ∼150 nm. (B) Total and co-localised surface areas of mitochondria (see Section 2). (C) Transmission electron micrographs of dynamitin-expressing HeLa cell cross-sections at high magnification show unusual branched mitochondria (star). Close association between ER and mitochondria are indicated with arrows (m: mitochondria, ER: endoplasmic reticulum). (D) Distribution of IP3 receptors in HeLa cells visualised using a rabbit polyclonal pan-IP3 receptor antibody (see Section 2 for further details).
mitochondria, estimated by monitoring increases in [Ca2+ ]m using a matrix-targeted aequorin (Fig. 4) [6,8]. Moreover, apparent mitochondrial Ca2+ uptake was identical in control and dynamitin-expressing cells even at very low histamine concentrations (Fig. 4A–C). These changes could not be ascribed to any loss of mitochondrial oxidative capacity since increases in mitochondrial ATP synthesis in response to 100 M histamine in the presence of oxidative substrates (lactate plus pyruvate; see Section 2) was not significantly different in dynamatin-expressing ([ATP]c peak value 115% of basal, mean of two independent experiments) and control cells ([ATP]c peak value 113% of basal, n = 2).
We would stress, however, that these measurements leave open the possibility that changes in free ATP concentration in subcellular microdomains, for example, immediately beneath the plasma membrane, may be altered by changes in mitochondrial distribution. Moreover, mitochondrial membrane potential (ψmit ) was also unaffected by dynamitin over-expression [24]. 3.3. Effects of dynamitin over-expression on Ca2+ entry We next explored the impact of dynamitin over-expression and mitochondrial relocalisation on Ca2+ entry. In these
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Fig. 3. Increased association of mitochondria with the ER does not affect Ca2+ sequestration by mitochondria following release of ER Ca2+ . HeLa cells were transfected with 1 g dynamitin.EGFP or pAdTrack-CMV (empty vector/control) then loaded with 5 M Fura-2/AM (A). Alternatively, cells were co-transfected with 1 g dynamitin.pcDNA3 with 0.5 g Cyt.Aq (B); 0.5 g ER.Aq (C). Histamine was then applied during perifusion in medium containing 1 mM EGTA. In (A), measurements of Fura-2 fluorescence ratio were performed on 78 single cells in five independent experiments, and data are shown as mean ± S.E.M. Aequorin bioluminescence (B, C) was calibrated to give mean free Ca2+ concentrations as described in Section 2, using data obtained in three independent experiments. Bars, 10 s.
experiments, Ca2+ entry was activated by depleting intracellular Ca2+ stores with the SERCA inhibitor, cyclopiazonic acid, in the presence of EGTA, and then reintroducing extracellular CaCl2 (Fig. 5A–E). Under these conditions, the activation of Ca2+ entry provoked very similar increases in [Ca2+ ]c and in [Ca2+ ]m (Fig. 5B and D, open points), indicating that the reported increases in mitochondrial [Ca2+ ] likely reflect changes in mitochondria located throughout the cytoplasm, and not just those located beneath the plasma membrane where an elevated [Ca2+ ]c microdomain is likely to pertain [40,41]. In contrast to effects on ER Ca2+ release (Figs. 3 and 4), CRAC-dependent Ca2+ entry was markedly diminished in dynamitin over-expressing cells, which displayed a smaller increase in both [Ca2+ ]c (Fig. 5A and B)
Fig. 4. Impact of dynamitin over-expression on ER Ca2+ mobilisation stimulated by varying histamine concentrations. Cells were co-transfected with 1 g dynamtin-expressing or empty vector, and 0.5 g mit.Aq and then the aequorin luminescence was measured by photon-counting 48 h later (see Section 2). Ca2+ release from the ER was triggered by perifusing with 0.1–100 M histamine (Hist) in Ca2+ free medium containing 1 mM EGTA. Bars, 10 s. See the legend to Fig. 3 and Section 2 for other details.
and [Ca2+ ]m (Fig. 5D). In order to eliminate the possibility that the observed differences in Ca2+ entry were due to altered membrane potential, we clamped the latter at close to 0 mV by using 140 mM KCl in the buffer and performed the same experiment shown in Fig. 5A. Under these condition the Ca2+ entry was markedly reduced in dynamitin over-expressing cells (Fig. 5E). Furthermore, the extent of store depletion induced by CPA was not significantly different in dynamitin-expressing and control cells (Fig. 5C).
4. Discussion 4.1. Effect of mitochondrial redistribution on mitochondrial Ca2+ uptake from the ER Contrary to our expectations, over-expression of dynamitin and a substantial increase in the number of close contact sites between mitochondria and ER (Fig. 2) had no significant effect on basal or stimulated Ca2+ concentrations in the cell cytosol, mitochondria, or ER (Fig. 3). Importantly, we detected no differences in the apparent rate
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Fig. 5. Effect of mitochondrial redistribution on store-operated Ca2+ entry. HeLa cells were transfected with 1 g dynamitin.EGFP or pAdTrack-CMV (empty vector/control) then loaded with 5 M Fura-2/AM (A, E). Alternatively, cells were co-transfected with 1 g dynamitin.pcDNA3 with 0.5 g Cyt.Aq (B); 0.5 g ER.Aq (C); or 0.5 g mit.Aq (D) and then aequorin luminescence was measured by photon-counting (see Section 2). The ER/Golgi Ca2+ pool was depleted by pre-treatment of cells with 1 M thapsigargin in 1 mM EGTA for 10 min before perifusion of cells with 10 M CPA in 1 mM EGTA. Ca2+ entry was then initiated by substituting the EGTA in the extracellular medium with 1.5 mM CaCl2 . The membrane potential was clamped at close to 0 mV by using 140 mM KCl and 10 mM NaCl in the perifusion buffer (E). Bars, 10 s.
of Ca2+ accumulation by mitochondria in control and dynamitin over-expressing cells even during stimulation with a very low agonist dose (Fig. 4A) where ER Ca2+ release is slow and the effects of increasing the proximity between ER and mitochondria might be expected to be greatest. The latter finding thus eliminates concerns that rapid saturation of mitochondrial aequorin during fast Ca2+ release from the ER might explain the apparent lack of effect of dynamitin over-expression. Instead, our observations are most compatible with two extreme models: (a) that local domains of high Ca2+ concentration have no role whatsoever to play in the transfer of Ca2+ from ER to the mitochondria, i.e. that mitochondria respond to changes in bulk cytosolic Ca2+ concentration; or (b) that Ca2+ microdomains are
minutely localised to specific and defined “contact” points between the ER and mitochondrial surface such that they are unaffected by a gross relocalisation of mitochondria (see scheme, Fig. 6). In essence, possibility (b) is equivalent to the existence of physical “tunnels”, i.e. stable entities perhaps formed between adjacent pairs (or groups of) IP3 receptors and VDAC channels, and through which Ca2+ ions might flow with little if any exchange with the bulk cytosol (see Section 1). Although the present studies cannot discriminate definitively between these two possibilities, it should be stressed that substantial evidence exists which would argue against model (a) [42]. Moreover, and also in line with model (b), a high density of IP3 receptors has been reported in domains of the ER that face towards
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Fig. 6. Scheme: role of sub-plasma membrane mitochondria in the regulation of store-operated calcium entry. (A) In control cells, phospholipase C-linked agonists cause Ca2+ release from the ER (1); and uptake by mitochondria via VDAC channels on the outer mitochondria membrane [12], potentially via points of intimate contact between the two organelles (2). ER Ca2+ depletion (3); and the activation of calcium influx via, for example, store-operated Ca2+ channels or other Ca2+ entry channels also leads to the uptake of Ca2+ by mitochondria (4); and possibly the release of undefined mitochondrial regulators (“X”, see Section 4) which ensure sustained flux of Ca2+ ions into the cell [17,18]. (B) Expression of dynamitin and relocalization of mitochondria from the cell surface and cell cytosol towards the nuclear periphery has no detectable effect on mitochondrial Ca2+ accumulation in response to IP3 production, but inhibits calcium entry since microdomains of high [Ca2+ ] accumulate at the mouth of Ca2+ entry channels.
mitochondria [43–45]. Furthermore, selective clustering of VDAC channels was recently reported in HeLa cells [13] whilst VDAC and ryanodine receptors are both clustered at sarcoplasmic reticulum/mitochondria contact points in skeletal muscle [15]. Finally, Pozzan and coworkers have proposed [46] that multiple populations of mitochondria may exist even within relatively simple mammalian cells, with “high-responding” mitochondria being stably located close to sites of ER Ca2+ release. The present findings provide further support for this view, and suggest that the number of these selective contact points is unchanged after gross alterations in mitochondrial distribution and an apparent increase in mitochondria-ER proximity. 4.2. Effect of mitochondrial redistribution on Ca2+ entry The present findings support previous results which suggested that functional mitochondria play an important role in ensuring efficient Ca2+ entry via store-operated channels [17,18,47,48]. However, these earlier findings largely involved exploring the effects of global inhibition of mitochondrial function, usually achieved with mitochondrial respiratory poisons, and provided no information on the respective roles of mitochondria at different subcellular
locations. By using molecular tools to manipulate, with reasonable selectivity [24], the spatial distribution of mitochondria in cells, the current data show that Ca2+ uptake by mitochondria located strategically beneath the plasma membrane, rather than global Ca2+ clearance by mitochondria located throughout the cell, is likely to be the key regulator of store-operated Ca2+ influx. Although no attempt was made here to measure [Ca2+ ]m changes in specific subsets of mitochondria, given the very limited spatial resolution which can be obtained by aequorin imaging [8], these data are also consistent with the observation that in pancreatic acinar cells, store-operated Ca2+ entry caused specific uptake of Ca2+ into mitochondria situated very close to the basal membrane [49]. Moreover, the present results also support findings, obtained by measuring the activity of large conductance Ca2+ -activated potassium channels [50], that sub-plasma membrane Ca2+ concentrations are lowest in regions adjacent to mitochondria, and that decreased mitochondrial Ca2+ buffering caused by inhibition of mitochondrial Na+ /Ca2+ exchange [51] diminishes store-dependent Ca2+ influx in endothelial cells. As shown in the scheme (Fig. 6) we propose that Ca2+ uptake by both the ER and by strategically-positioned mitochondria reduces [Ca2+ ]c near Ca2+ entry sites at the plasma membrane and therefore diminishes the negative feedback effect of locally high sub-plasma membrane Ca2+ concentrations [41] on the open state probability of CRAC channels [20]. As a refinement of this model, it should be mentioned that Glitsch et al. [18] have proposed that Ca2+ uptake by mitochondria, and hence lowering of local Ca2+ concentrations, is not sufficient for the activation of CRAC currents, but rather triggers the stimulation of intramitochondrial oxidative metabolism [3]. This in turn may prompt the release from the mitochondrial matrix of other factors (ATP? amino acids?) which serve as channel activators. Future studies, involving permeabilised or patch-clamped cells, will be necessary to explore the relative contribution of local changes in [Ca2+ ]c , and mitochondria-derived metabolic factors, more fully. The present results are distinct from the recently-described effects of mitochondrial reorganisation achieved by vinblastine treatment of neurones [52]. By destabilising microtubules, this agent led to mitochondrial rounding, aggregation and to amplified increases in cytosolic Ca2+ concentration in response to the activation of glutamate receptors. These changes were ascribed to a decrease in mitochondrial Ca2+ accumulation, and hence a decrease in cytosolic Ca2+ buffering. However, in the neuronal setting [52], Ca2+ influx was achieved by the activation of ligandand voltage-gated, rather than store-operated, channels, leading to much more rapid increases in cytosolic [Ca2+ ] (half-time to peak ∼5 s for neuron depolarisation versus ∼20 s and CaCl2 reintroduction to HeLa cells). Under these conditions of more rapid [Ca2+ ]c increase it is likely that the most important role of sub-plasma membrane mitochondria is to buffer relatively large local [Ca2+ ]c changes, rather
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than to ensure the sustained opening of voltage-dependent channels in the face of smaller local [Ca2+ ]c increases. However, it might also be mentioned that microtubule disruption, as achieved with vinblastine, may elicit more widespread effects on the distribution of other organelles and Ca2+ stores, including the ER. Such changes are in contrast to the effects of dynamitin which had no gross impact on the structures of secretory pathway organelles in the current study [24], and caused only a small increase in the average length of mitochondria, as opposed to the rounding up seen in response to vinblastine [52]. 4.3. Conclusions Controlled changes in mitochondrial distribution in the context of a single cell type, as achieved here, provide a useful tool to explore the role of mitochondrial localisation in regulating Ca2+ influx and release from intracellular stores. The application of this approach to other, more specialised cells types [53], may now allow a detailed assessment of the role of mitochondrial organisation in orchestrating more complex intracellular Ca2+ signals.
Acknowledgements G.A.R. was supported by Wellcome Trust Programme Grant 067081/Z/02/Z, Human Science Frontiers Program grant RGP 0347/2001-M, and by project grants from the Wellcome Trust, Biotechnology and Biological Research Council, UK and Diabetes UK. V.C. was supported by NIH grants DK55183 and DK63443, JDRFI Research Grant 197009, and a Network Grant from The Larry L. Hillblom Foundation. Electron microscopy and imaging analysis was performed at The National Center for Microscopy and Imaging Research (UCSD), supported by NIH grant RR04050 to Dr. Mark H. Ellisman. We thank Dr. Mark Jepson and Alan Leard of the Bristol MRC Imaging Facility for technical assistance, and Professor Peter Cullen for the use of the UltraVIEWTM microscope. G.A.R. is a Wellcome Trust Research Leave Fellow.
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