doi: 10.1161/01.RES.0000137875.42385.8e
© 2004 American Heart Association, Inc.
Reviews |
Examining Intracellular Organelle Function Using Fluorescent Probes
From Animalcules to Quantum Dots
From the Laboratories of Cardiovascular Sciences (D.B.Z., M.J., S.J.S.) and Clinical Investigation (E.K.), Gerontology Research Center, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, Md; and the Department of Bioenergetics (D.B.Z.), A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, Russia.
Correspondence to Steven J. Sollott, MD, or to Magdalena Juhaszova, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, Box 13, Intramural Research Program, National Institute on Aging, 5600 Nathan Shock Dr., Baltimore, MD 21224-6825. E-mail sollotts{at}grc.nia.nih.gov
This Review is part of a thematic series on Imaging of Cardiovascular Cells and Tissues, which includes the following articles:
Use of Chimeric Fluorescent Proteins and FRET to Monitor Cellular Responses
Imaging Microdomain Ca2+ in Muscle Cells
Optical Imaging of the Heart
Examining Intracellular Organelle Function Using Fluorescent Probes
Two-Photon Microscopy of Cells and Tissues
Brian O’Rourke Guest Editor
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Abstract |
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 Top Abstract IntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function.
Key Words: microscopy • calcium • redox • mitochondria • fluorescent proteins
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Introduction |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Responses of single cells undergoing physiological activation processes or pathological stresses can be quite heterogeneous in time and space. Measurements of these parameters in bulk (such as in tissue or cellular suspension), using conventional physiological and biochemical methods, can often fail to resolve discrete differences in the kinetics and magnitudes of the responses between cells, as well as complex intracellular and subcellular dynamical processes, critical to understanding how cells actually work. For example, during embryonic development or periods of environmental stress, the decision for a certain cell to undergo apoptosis may be triggered in a moment and distinct from its neighbors, the resulting intracellular processes may proceed along a more or less protracted time frame, and these events may occur heterogeneously among (and inside) other cells. Yet, bulk measures of the progression of apoptosis, such as by DNA-laddering, although undeniably valuable, would show only the overall slow progression with time. All-or-none phenomenon occurring in discrete organelles, such as the mitochondrial permeability transition (MPT), appears as a graded response when examined in bulk suspensions of cells or mitochondria. Similar arguments can be made for resolving questions of Ca2+ signaling and gene expression. Ca2+ oscillations mediate signaling in a wide variety of cell types and, unless they are synchronized (such as in the beating heart), their occurrence or nature (eg, frequency, amplitude, etc) might not be apparent from ensemble measurements. Significant information about these processes would obviously need to use a single cells studies.
Currently, one of the most useful techniques to assess the activities of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated is based on fluorescence microscopy imaging. Each of the various imaging techniques requires that the signaling molecule(s), compartment(s), or organelle(s) be labeled in a specific fashion such that they can be tracked in time and space without significantly interfering with the underlying processes. In other experiments, the combination of specialized imaging techniques together with cellular probes capable of producing photodynamic changes in the local environment enables "interactivity," because it is intentionally devised to enable dynamic perturbation of the system and to follow the responses in real time. These labels can be exogenously introduced or fortuitously may already exist as endogenous species with innate fluorescence properties that can be exploited for the purpose.
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Fluorescent Probes of Cell Signaling: Small Organic Fluorophores |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Aside from the fluorescent antibody techniques having few practical applications for intracellular imaging in living cells,1 the most significant advances came from the development of small organic fluorescent molecules by Roger Tsien’s laboratory that were responsive to ionic signals, such as Ca2+, H+, and membrane potential (
m), and could be introduced into live cells, generally without significant toxicity.2 Many of these fluorochromes are both useful for and limited to measurements in compartments to which they would naturally distribute. Perhaps one of the most useful applications of this technology is the ability to image microdomain Ca2+ dynamics.3,4 Small organic probes that have been particularly useful to assess Ca2+ flux include the ratiometric indicators, indo-1 and fura-2, which shift their excitation or emission spectra on binding Ca2+, and the nonratiometric ("single wavelength") indicators, fluo-3 and rhod-2 (and their newer derivatives), which increase fluorescence emission on Ca2+ binding.5
Although certain indicators would naturally sequester inside-specific organelles because of biophysical or biochemical properties favoring that distribution (such as m providing the accumulation of positively charged dyes, ie, tetramethylrhodamine methyl ester [TMRM] and tetramethylrhodamine ethyl ester [TMRE], and the Ca2+-indicator, rhod-2, inside mitochondria6), specific targeting for most of these dyes inside specific subcellular locations is generally not possible. Available fluorophores are multivalent organic anions that by themselves are impermeant to membranes. Because of this limitation, acetate-ester or acetoxymethyl-ester derivatives have been developed for these compounds, rendering them membrane-permeable and easily loaded into cells. Endogenous esterases regenerate the original organic anion form of the indicator, trapping it inside the cell.7 Although the distribution of dye-loading can be manipulated to favor relative localization in certain compartments by controlling the temperature and hence activity of esterases during dye-loading (for example, facilitation of the relative mitochondrial localization of the marker molecule, calcein),8 nevertheless, the highly specific localization of these ester-derivative–loaded dyes to a given compartment or organelle is frequently impractical.
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Fluorescent Protein Tags and Biosensors |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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An important advance during the 1990s enabled the introduction of genes encoding fluorescent proteins that could serve to tag expressed proteins,9 which proved to be well-suited for fluorescence microscopy and enabled in vivo tracking of proteins.10 The cloning11 and expression9 of the jellyfish green fluorescent protein (GFP) was the first of this group and has been used extensively for real-time imaging in live cells.12,13 In the intervening decade, GFP has undergone significant improvements; currently, a wide variety of GFP mutants are available that emit increased fluorescence, have favorably shifted spectral properties that emit light of different colors, and have improved photostability. Also, a range of fluorescent proteins from other organisms has been cloned and the application of the expression of the anemone red fluorescent protein, DsRed, has been demonstrated.14 Recently, a "fluorescent timer" protein was identified from the mutation of a red fluorescent protein (E5), which changes from green to red over time, and thus can be used to report the timing and regulation of gene expression.15 Mutations in a nonfluorescent chromoprotein from Anemonia sulcata has produced a "kindling" fluorescent protein that becomes fluorescent under moderate green light excitation. Light of greater intensity or longer duration causes irreversible kindling. With an organelle-targeted sequence (and irreversibly kindled), it may be used to report solitary organelle traffic within a cell.16
Another photoprotein from the jellyfish is aequorin (AEQ), which undergoes a Ca2+-induced conformational change leading to the oxidation of its coenzyme, coelenterazine, and the emission of blue light (chemiluminescence).5,17 Although AEQ has yielded valuable insights into Ca2+ signaling, several limitations have become apparent. Coelenterazine is consumed and the photoprotein is destroyed on Ca2+ binding to AEQ. Also, the inherent signal produced on Ca2+ binding is very low (<1 photon per molecule), creating problems with detection and limiting the spatial and temporal resolution.18 Fluorescent protein tags and biosensors are presented in detail in another part of this thematic series.19 However, several additional developments deserve special mention because of their importance for the present review.
Organelle-Targeted AEQs and GFPs
Organelle-targeted AEQs and GFPs can be genetically expressed in target cells; chimeric AEQs with organelle-specific targeting sequences enable cellular expression of recombinant protein to specific intracellular locations, including the cytosol,20 nucleus,20,21 sarcoplasmic reticulum,22 endoplasmic reticulum,23 mitochondria,24,25 golgi apparatus,26 secretory granules,27 and gap junctions.28 Cells can also be transfected with specific organelle-targeted GFPs,29 and combinations of the different color GFP mutants can be used together to target different organelles in the same cell.30,31 Probes are now available for compartments, including the cytosol, endoplasmic reticulum (ER), mitochondria, golgi, nucleus, and plasma membrane.10,32
GFP Emission Variants
Perhaps the most important engineered GFPs manifesting different spectral properties is the production of 2 mutants, a blue-shifted cyan fluorescent protein (ECFP) and a red-shifted yellow fluorescent protein (EYFP), because they have overlapping spectra that allows fluorescence resonance energy transfer (FRET).31,33 FRET is a quantum mechanical event describing the nonradiative energy transfer from an excited donor chromophore to an acceptor chromophore, which in turn emits light at its own wavelength.34 This only occurs when the 2 dipoles are within a range of <10 nm. Because FRET is proportional to the sixth power of the distance between dipoles, small changes in separation of the GFP pair can induce substantial changes in the signal, making this a very sensitive reporter of the biochemical event. A potential drawback of this approach is that the probe is molecularly bulky and can interfere with the underlying phenomenon or with its targeting.
GFP-Based Ion and Second Messenger Probes
An entire range of GFP-based indicators sensitive to specific ions and second messengers has been developed.35 These include sensors of Ca2+, H+, cAMP, cGMP, and protein kinase activity (PKA, PKC, tyrosine kinase), and those targetable to subcellular compartments and organelles. Recently, reduction—oxidation-sensitive GFPs (roGFPs) have been created with cysteines in appropriate positions to form disulfide bonds. These roGFPs have been targeted to both the cytosol and the mitochondria and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo.36 For the purposes of the present review, we limit the discussion to GFP-based Ca2+ sensors (designed around variations of GFP–calmodulin constructs): "cameleons," "camgaroos," and "pericams."33,37–39 In each case, the sensor relies on the change in conformation on Ca2+–calmodulin-binding to serve as the molecular switch. In the case of chameleon, this leads to changes in FRET between cyan fluorescent protein and yellow fluorescent protein (YFP); for the non-FRET–based camgaroo and pericam probes (each of which have only YFP [or a variant] interacting with calmodulin) Ca2+-binding leads to a specific change (increase) in fluorescence of YFP. Compared with cameleons, pericams show greater Ca2+ responses and improved signal-to-noise ratio.38 By contrast to low Ca2+-affinity camgaroos (Kd=7 µmol/L),37 pericams have higher Ca2+ affinity (Kd=0.7 µmol/L), favorable for sensing physiological Ca2+changes.38 It should also be noted that GFP-based probes are generally quite pH-sensitive, which can complicate their use. Cameleon probes have been targeted to the nucleus,33 endoplasmic reticulum,33 mitochondrial matrix,40 and the plasma membrane.41 Pericam probes have targeted to the nucleus,39 mitochondria,25,38 and plasma membrane.42
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Photobleaching Methods |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Photobleaching is photochemical alteration of a fluorophore resulting in depletion of fluorescence. Usually, photobleaching is performed by scanning a focused laser across defined regions of interest at high zoom and intensity. Advanced methods use an acousto-optical tunable filter, which enables rapid control of laser attenuation during scanning. Accurate measurements in defined regions of interest are achieved by rapid switching between the bleaching and normal beam.
There are different photobleaching techniques.43 Fluorescence recovery after photobleaching (FRAP) is based on selective photobleaching of fluorophores in defined regions of interest and subsequent monitoring of recovery that occurs as nonbleached molecules move into the bleached region. It provides information about kinetics of protein diffusion, binding, and trafficking. To monitor the rate of the movement out of the bleached region, inverse FRAP is performed. Fluorophores outside the region of interest are photobleached, and fluorescence loss in nonphotobleached areas is monitored. Fluorescence localization after photobleaching (FLAP) is used with proteins tagged with 2 different fluorophores. By photobleaching 1 of these fluorophores, the movement of nonphotobleached pools can be monitored over time. In fluorescence loss in photobleaching (FLIP) experiments, defined regions of interest are repeatedly photobleached, whereas the whole cell is imaged. Cell regions interconnected with the photobleached area lose fluorescence because of movement of mobile proteins into this area, but unconnected regions are not affected.
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Mitochondria |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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In addition to the long-recognized roles in energy metabolism, mitochondria play crucial roles in apoptosis, redox, and Ca2+ homeostasis. They oxidize substrates (forming a proton motive force with both
m and pH components), which also provide a major source of reactive oxygen species (ROS) production. m drives ATP synthesis, cation transport, and protein transport, and also is a signal in the cell life–death decision. In addition to ROS, mitochondria are a source of reactive nitrogen species (RNS) and a target regulated by nitric oxide (NO).44,45
Membrane Potential
All fluorescent molecules used as probes for the transmembrane potential in general (and m specifically) must meet important criteria. They should carry a delocalized charge, thus making them membrane-permeable, and should be accumulated according to the Nernst electrochemical potential: 
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where Fin and Fout are the concentrations of the (monovalent) cation inside and outside the organelle. These are hydrophobic or amphiphilic organic molecules, in which charge-delocalization is caused substantially by the electron density spreading across several coupled double bonds. If the compartment is charged negatively inside (eg, cell interior, mitochondrial matrix), lipophilic cations are used. Having a negative membrane potential of 
200 to 250 mV (the polarization of inner mitochondrial membrane with respect to the outside of the cell) results in mitochondria accumulating dye to levels 4 orders above that outside the cell (1µmol/L outside yields >1 mmol/L in the matrix, which may be toxic46). Because physical diffusion through lipid barriers is required to achieve gradient levels determined by m, these probes fall into the group of "slow" dyes,47 meaning that fluorescence changes cannot immediately follow changes in m. Because these permeant cations are extruded by P-glycoprotein (ie, multidrug-resistance transporter),48 specific precautions should be taken to avoid artifacts. Another complicating property of these dyes (which actually facilitates photodynamic therapy) is "non-Nernstian" retention inside mitochondria of some tissues, making it difficult to assign an energy dependence of their accumulation.49
One of the oldest mitochondrial fluorochromes described is Janus green (introduced >50 years ago), although it is rarely used today because of significant toxicity. Currently, the most useful mitochondrial dyes include: those from rhodamine and rosamine families (TMRM, TMRE, rhodamine 123, tetramethylrosamine); those retained in mitochondria after chemical fixation by conjugation with proteins and lipids (mitotrackers: green, red, orange); mitoFluors (green, red) and nonyl-acridine orange (staining mitochondria in live and fixed cells, but unable to be retained after fixation); carbocyanines [indo- (DiI), thia- (DiS) and oxa- (DiO) derivatives]; dual-emission dyes (JC-1, JC-9) expressing their fluorescence in a potential-sensitive mode by having green fluorescent monomers under low m and red fluorescent aggregates when m is high; styryl dyes (DASPMI and DASPEI) having large fluorescence Stokes shifts; and others.
In general, many of these dyes can measure m in the steady state if used at the low submicromolar range. It should be emphasized that sufficient time for dye equilibration must be allowed. Beyond the issue of toxicity, problems arise when dyes are used at concentrations above the point at which fluorescence becomes nonlinear as a function of concentration. In fact, at micromolar bathing concentrations, rhodamines can accumulate to levels inside mitochondria at which fluorescence self-quenching is obtained, caused by increased dissipation of excitation energy by the high probability of molecular collisions, as well as the formation of dye aggregates with altered fluorescence properties. Under these circumstances, mitochondrial depolarization (causing the redistribution of dye into the cytoplasm) can produce a superficially "paradoxical" increase of fluorescence as the intramitochondrial dye levels decrease to levels at which self-quenching is less. Although there are useful m measurements that can be made using this "dequenching" approach, it is our opinion that the lowest dye concentration possible probably provides the general all-around best balance of experimental compromises. Additionally, each of these compounds can act as photosensitizing agents. Photodynamic effects, principally resulting from the photochemical ROS production as a byproduct of dye excitation in the presence of oxygen, can cause significant and unwanted phototoxicity without proper attention to the experimental conditions. We feel that the most useful small molecule probes for dynamic measurements of m are TMRE and TMRM used at very low concentrations. Detailed examinations of practical methods for validation and calibration of fluorescence measurements of m are available.46,50
Photodynamic Depolarization, Induction of the MPT, and ROS
The photodynamic effects described in the context of toxicity can also be exploited to experimental advantage to perturb cellular systems in controlled ways, for example, to examine cellular responses in real-time by the controlled local production of free radicals in specific organelles to simulate a signaling event or pathological stress. Because probes such as TMRM localize inside mitochondria and produce ROS when photoexcited, we exploited this coincidence to track m during experimentally controlled ROS-induction of the MPT, using laser line-scanning confocal microscopy imaging.51 Repetitive laser scanning of a row of mitochondria in a cell loaded with TMRM causes additive incremental exposure of just that laser-targeted area to the photodynamic production of ROS. After a reproducible ROS exposure, the MPT occurs, which is clearly identified by the immediate dissipation of m (Figure 1A). In cardiac myocytes double-loaded with TMRM and the ROS-tracking dye, dichlorofluorescein (DCF), MPT-induction (triggered by the photodynamic production of ROS) coincided with a burst of mitochondrial ROS generation (as measured by DCF fluorescence), which we have termed mitochondrial "ROS-induced ROS release" (RIRR) (Figure 1B). Using this methodology, we examined cardioprotection signaling mechanisms. Protection involves activation of endogenous signaling, which can confer significant resistance to oxidant and other stresses associated with ischemia/reperfusion injury. We demonstrated that hypoxia/reoxygenation significantly reduces the MPT ROS threshold, that cardiac myocyte survival is steeply negatively correlated with the fraction of depolarized mitochondria, and that a wide variety of cardioprotective agents acting via distinct upstream mechanisms all promote cell survival by limiting MPT induction. We found that protection can be triggered in 2 general ways—dependent and independent of regulatory mitochondrial swelling—which converge via inhibition of GSK-3ß on the end effector, the permeability transition pore complex, preventing the MPT.52
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Because the MPT causes nonselective permeability of the inner mitochondrial membrane to solutes with molecular masses up to 1.5 kDa, its occurrence can be followed by the redistribution of marker molecules that are normally membrane-impermeable. The fluorescent molecule, calcein, has been found suitable for this purpose, and has enabled imaging of the MPT in situ. Calcein can be loaded into cells as the membrane-permeable acetoxymethyl ester forms, where it can enter essentially all organelles, and cellular esterases hydrolyze the dye to form the impermeable salt form. The success of the live cell-imaging method, however, relies on the ability to selectively load and entrap calcein initially with a sufficient gradient across the inner mitochondrial membrane to observe any quantitative redistribution later on MPT induction,7,53 or be able to selectively quench the fluorescence in the cytosol (eg, with Co2+) leaving the mitochondrial matrix-entrapped species fluorescent.54 Although each of these techniques has some significant limitations,53 careful application has permitted convincing MPT demonstration in direct cell imaging (Figure 2A).
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Aon et al examined the propagation and consequences of RIRR in cardiac myocytes loaded with TMRE and DCF using 2-photon confocal microscopy.55 They demonstrated that photo-induced mitochondrial depolarization and ROS-release in <1% of the volume of the cell can trigger self-propagated spatio-temporally synchronized, cyclosporin A–insensitive oscillations in m, mitochondrial ROS-production, and mitochondrial redox potential (as reflected in NAD(P)H autofluorescence, see later) throughout the entire volume of the cell, which had become apparently independent of exogenous photodynamic ROS. Furthermore, these mitochondrial transitions were found to be tightly coupled to activation of sarcolemmal KATP currents, causing oscillations in action potential duration, and thus might contribute to arrhythmias during ischemia–reperfusion injury.
Imaging of Mitochondrial NO
A novel fluorometric probe, 4,5-diaminofluorescein diacetate (DAF-2DA), has been developed that enables detection of NO production in living cells.56 DAF-2DA is cleaved to DAF-2, trapping it within the cell. Reaction of DAF-2 with NO to form the stable triazolofluorescein DAF-2T yields a >180-fold increase in fluorescence. In experiments from our laboratory, we found that MPT-induction produces a burst of mitochondrial ROS and also a significant increase in NO production.51 In cardiac myocytes dual-loaded with TMRM and DAF-2, a significant increase of mitochondrial DAF-2 fluorescence was observed after MPT induction, suggesting that NO production had occurred in these areas (but with relatively slower kinetics compared with the ROS burst); the specificity for NO detection in these experiments was demonstrated by ability of the NOS inhibitor, L-NAME, to abolish the increase in DAF-2 fluorescence after MPT-induction (Figure 1C).
Because oxidative and nitrosative stress can modulate the spontaneous activity of the ryanodine receptor,57,58 we examined the nature of Ca2+ spark activity in the "wake" of MPT induction.51 As shown in Figure 1D, in a representative cell dual-loaded with TMRM and the Ca2+-sensitive dye, fluo-3, there is frequently a period of significantly increased Ca2+ spark frequency at the z-lines (ie, the site of the T-tubule–sarcoplasmic reticulum junction) in the immediate vicinity of the mitochondrion soon after MPT induction. In proximity of the MPT (defined as within the sarcomere containing the involved mitochondria and within 3 seconds after MPT occurrence), the event rate approximately doubled (Figure 1D, inset). Although ordinary background Ca2+ sparks are typically single events, the MPT-associated ones frequently occurred as clusters, as seen in Figure 1D. Although it is possible that mitochondrial Ca2+ may contribute to Ca2+ spark formation,59 we were unable to exclude the possibility that such local Ca2+ release from sarcoplasmic reticulum (SR) may be driven by ROS/RNS generated in and released by these adjacent mitochondria undergoing MPT induction (a "ROS/RNS-induced Ca2+ release" mechanism). Regardless of whether Ca2+ or ROS modulate this increased spark frequency after MPT induction, this phenomenon could induce pathological disturbances in cardiac excitation and rhythm, for example, contributing to postischemic reperfusion arrhythmias.
Recently, it was noted that ER–Ca2+ stress could induce apoptosis in nonexcitable cells, such as Jurkat T-cells, in a Ca2+-dependent and NO-dependent fashion.60 Using DAF-2–loaded Jurkat cells exposed to the SERCA inhibitor, thapsigargin (TG 50 nmol/L, which results in apoptosis in approximately one-third of the population by 36 hours), together with confocal imaging, we found that mitochondria rapidly produce NO (colocalization of DAF-2 signal with TMRE added at the end of the experiment confirms mitochondrial localization; see Figure 2B) compared with control cells not TG-exposed (not shown). Specificity of NO production was provided by parallel experiments in the presence of L-NAME, which prevented the TG-related increase in mitochondrial DAF-2 fluorescence, which was restored in the presence of >10-fold L-arginine (Figure 2B).
Imaging Mitochondrial Ca2+ and pH
The nature of the Ca2+ dependence of ER–stress-mediated (TG-mediated) apoptosis was examined in Jurkat cells dual-loaded with indo-1 and rhod-2 to assess whole-cell and mitochondrial Ca2+, respectively, using confocal imaging. Figure 2C shows that TG produced a rapid, parallel increase in both whole-cell and mitochondrial Ca2+. We found that although Ca2+-dependent activation of NO production mediates apoptosis after ER stress, and that protection against apoptosis seen in Bcl-2 and Bcl-XL overexpressing cells was mediated not by any difference in TG-induced elevations in mitochondrial and global cell Ca2+, but rather by the downstream capacity to produce NO.60 Hajnóczky’s laboratory examined the transmission of cytosolic Ca2+ oscillations mediated by InsP3 into the mitochondrial matrix and found a tight, synapse-like functional coupling architecture, which would explain how periodic InsP3 receptor activation could dynamically control Ca2+-dependent mitochondrial metabolism.61 Cytosolic Ca2+ signal communication to mitochondria using confocal imaging of compartmentalized rhod-2 in H9c2 cells was further examined.62 They found that cytosolic Ca2+ sparks mediated by ryanodine receptors could elicit miniature mitochondrial matrix Ca2+ signals that they called "Ca2+-marks," which are restricted to single mitochondria. A key finding was that mitochondria also appear to have a direct effect on the properties of Ca2+ sparks, because inhibition of mitochondrial Ca2+ uptake results in an increase in the frequency and duration of Ca2+ sparks, suggesting that mitochondrial Ca2+ handling may be an important determinant of cardiac excitability through local feedback control of elementary Ca2+ signals.
The mitochondria-targeted chimeric AEQ and GFPs have been useful for understanding mitochondrial Ca2+ dynamics. In an early report using mitochondrial AEQ, together with parallel fura-2 measurements to follow cytoplasmic Ca2+, Rizzuto et al were able to observe that physiological stimulation with Ca2+-mobilizing agents induced increases of the Ca2+ level in mitochondria much larger than those reached in the bulk cytoplasm.24 Ratiometric chameleons having appropriate localization signals permitted measurement of free Ca2+ concentrations simultaneously in the nucleus and mitochondria, revealing that extramitochondrial Ca2+ transients caused rapid changes in mitochondrial Ca2+ together with the free propagation of Ca2+ across the nuclear envelope.33 In another elegant demonstration, using mitochondrial-targeted and nuclear-targeted ratiometric pericams, Pozzan’s group showed that in neonatal cardiac myocytes, mitochondrial Ca2+ oscillates synchronously with cytosolic Ca2+ and that mitochondrial Ca2+ handling rapidly adapts to inotropic and chronotropic inputs25 (Figure 3).
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Because the electrochemical potential energy formed by oxidative phosphorylation is stored in the H+ gradient (µH, which in turn is harnessed for ATP production), and hence comprises both m and pH, it is important to be able to measure both variables because perturbations can influence them independently. Whereas m-tracking probes are well-developed (see previous), methods to accurately measure mitochondrial matrix pH (and hence pH) remain problematic. Specifically, fluorescein and its derivatives, carboxy-SNARF and BCECF, are difficult to selectively localize inside mitochondria, and although certain pH-sensitive GFPs can be appropriately targeted, many have pKa that make them less sensitive to pH changes in the alkaline environment of the mitochondrial matrix.63 However, a GFP chimera targeted to the mitochondria was recently engineered that has an apparent pKa 8.5, which develops large reversible fluorescence changes, making it suitable for use as a mitochondrial alkaline pH indicator (named "mtAlpHi").64
Imaging Mitochondrial Redox State
The main source of reducing equivalents to the respiratory chain is reduced nicotinamide-adenine dinucleotide (NADH). A family of flavoproteins (which deliver reducing equivalents via flavins, FAD, or FMN) is in close redox equilibrium with the mitochondrial NAD system. The NAD redox state reflects the metabolic balance of the rate of oxidative phosphorylation by respiration and the rate of delivery of reducing equivalents to the respiratory chain. Fortuitously, NADH is fluorescent, whereas the oxidized form, NAD+, is not. In contrast, flavoprotein fluorescence increases with oxidation. Because of this, ratiometric fluorometry may be rather useful because 2 signals respond oppositely to changes in mitochondrial metabolic states. This kind of redox fluorimetry has been used for studying regulation and defects in cellular energy metabolism.65 One important application of this was from Marbán’s laboratory, which established single-cell methodologies to assay surface KATP and mitochondrial KATP channel activities by measuring membrane current and flavoprotein fluorescence simultaneously.66,67 They showed that activation of the mitochondrial KATP increases flavoprotein oxidation, and that this endogenous fluorescence could be used as a reporter of mitochondrial KATP activity (Figure 4A and 4B).
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Another redox fluorimetry application to characterize energy metabolism in single cardiomyocytes uses a perturbation–kinetics approach via rapid photobleaching of NADH, followed by "enzyme-dependent" FRAP confocal imaging as the topological measure of its resynthesis (Figure 4C and 4D).68 The mono-exponential NADH recovery curve provides a pseudo first-order reaction rate constant of regional NADH generation capacity, reflecting NADH dehydrogenase activity and NADH use (in the respiratory chain and in catabolic processes), providing information beyond conventional steady-state methods.
Imaging Mitochondrial Caspase-3 Activation
Among various apoptotic components, caspases-2, 3, 8, and 9 were recently found inside mitochondria.69,70 Uncertainty remains regarding whether "upstream" caspases and "executioner" caspase-processing takes place inside mitochondria or if they are released from mitochondria in immature form. Paradoxically, digested products of procaspase-9 have not been detected in mitochondria, whereas caspase-3 (classically "downstream" of caspase-9) was found in activated form both in cytosol and in mitochondria.71 Furthermore, there is no information about the possibility that caspases might be activated locally to serve purposes other than apoptosis (which would be below immunoblot detection limits). Cell imaging together with different permeant fluorescent caspase substrates would be useful to address these questions. Using a permeable fluorogenic caspase substrate containing the caspase-3 recognition motif, DEVD (PhiPhiLux-G1D272), together with TMRM loading, we observed increased DEVDase activity in areas corresponding to rare, spontaneously depolarized mitochondria in cardiomyocytes (Figure 5).
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The concept of programmed mitochondrial destruction was put forth as a possible function of the mitochondrial mega-channel (permeability transition pore)73 and later named "mitoptosis."74 Mitochondrial proteins have a half-life of days (depending on tissue, the slowest turnover is 17.5 days in heart and 24.4 days in brain),75 but the process of mitochondrial execution has not been characterized (except for limited EM data identifying mitochondrial degradation in lysosomes). It is unclear whether mitochondria are executed by an "apoptosis-like" program, but we speculate that the orderly disposal of mitochondria in postmitotic cells (such as in heart and brain) requires specially compartmentalized mechanisms (possibly involving caspase-3) unless premature apoptosis is accidentally triggered.
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Sarcoplasmic/Endoplasmic Reticulum |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Previously, extensive data related to the estimation of stored SR/ER Ca2+ were based on indirect measurements of the released SR/ER Ca2+ into cytosol after activation. Development of Ca2+ indicator Fluo-5N (with low Kd
400 µmol/L) facilitated direct measurements of SR Ca2+, including observing the beat-to-beat dynamics of stored SR Ca2+ in cardiac myocytes.76 Fluo-5N fluorescence was localized to z-lines and transverse tubules (confirmed by Di-8-ANNEPS staining). Combination of multiple fluorescence indicators and a method to stretch cardiac myocytes (Figure 6A and 6B) permitted study of the mechanisms of stretch-dependent modulation of Ca2+-transient amplitude, Ca2+ spark frequency, and NO production.58 Stretch increased the amplitude of the electrically stimulated Ca2+ transients assessed in cells loaded with the ratiometric indicator, indo-1 (Figure 6D and 6E). To understand the mechanisms of the stretch effect on Ca2+ transients, spontaneous Ca2+ spark frequency was compared in slack and stretched myocytes loaded with indicator Fluo-3 (Figure 6C). Spontaneous Ca2+ spark frequency (but not amplitude) increased directly with stretch and was blocked by NOS and PI(3)K inhibitors. To show that stretch directly modulated endogenous NO production, cells were loaded with DAF-2DA. Imaging experiments demonstrated that stretch induced NO production in cardiac myocytes. It was proposed that myocyte NO produced by stretch activation of the PI(3)K-Akt-eNOS axis acts as a second messenger of stretch by enhancing ryanodine receptor activity, contributing to myocardial contractile activation.58
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In live and fixed cells, the structure and dynamics of basic elements of SR/ER (the membranous sacs, tubules, polygonal reticulum, and junctions) can be revealed with a variety of lipophilic probes. Originally, Terasaki et al used the fat-soluble dicarbocyanine dye, 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], to study SR/ER morphology.77 At low concentrations, DiOC6(3) accumulates preferentially into mitochondria. High concentrations are toxic for mitochondria, which condense and depolarize, releasing DiOC6(3), which then redistributes and reveals SR/ER structure. The fixable ER-Tracker Blue-White DPX indicator is a relatively selective and photostable stain for the SR/ER in live cells. Unlike DiOC6(3), ER-Tracker does not incorporate into mitochondria, and at low concentrations appears to be nontoxic.78 Cell-permeable BODIPY FL-X- and BODIPY TR-X-conjugates of thapsigargin, ryanodine, and InsP3 proved useful to study the spatial distribution and density of SR/ER ryanodine receptors,79–81 InsP3 receptors,82 and Ca2+-ATPases.79,81,82 FRAP experiments revealed remarkable changes in the mobility of proteins in the ER lumen under conditions of cell stress,83 thus proving ER to be a very dynamic structure responding to cell stress.
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Nucleus |
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Transcriptional Factors
Regulation of transcription is a critical point in nuclear gene expression. There are important transcription factors that are translocated from cytoplasm to nucleus on activation. Fluorescent tags such as GFP allow time-lapse imaging of activation of transcription factor in live cells. Nuclear factor of activated T cells (NFAT) plays a central role in the development of cardiovascular system. Activation of NFAT requires nuclear Ca2+ elevation. Calcineurin (CaN) binds and translocates NFAT into the nucleus as a complex.84 The distribution of NFAT between nuclear and cytoplasmic compartments is dynamically regulated by CaN and nuclear kinase activities. The stimulation-dependent nuclear translocation of GFP–NFAT has been demonstrated in skeletal muscle fibers,85 in Jurkat and BHK cells,86 and in arterial smooth muscle.87 Another transcription factor, NF-
B, translocates into the nucleus after phosphorylation and degradation of an inhibitory subunit, I-kB.88 Dynamics of this translocation has been studied with a GFP–NF-B fusion protein.89 GFP–NF-B also can be used in a DNA-binding assay in vitro by applying the principle of FRET.90 FRET-based assays can be used to detect transcriptional activation inside nucleus in situ. cAMP stimulates the expression of numerous genes through the PKA-mediated phosphorylation of the cAMP-responsive element-binding protein, CREB, which recruits the coactivator CREB binding protein (CBP).91 To monitor the formation of the active transcriptional CREB/CBP complex, fusion constructs of CREB/CBP interacting domains (KID and KIX) with ECFP and EYFP were created, and interaction of KID and KIX domains was monitored by FRET.92 The important role of voltage-gated L-type Ca2+ channels in activating of CREB-dependent transcription has been shown in neurons and in recombinant systems.93,94
Nuclear Organization, RNA, and Protein Transport
Different photobleaching techniques revealed a highly dynamic nuclear organization. FRAP and FLIP experiments showed that several nuclear proteins involved in ribosomal biogenesis cycle rapidly between the nucleolus and nucleoplasm.95 These findings of the dynamic nature of nuclear structure particularly highlight the need for the application of advanced imaging techniques with sufficient spatial and temporal resolution.
Many aspects of posttranscriptional regulation of gene expression involve specific RNA–protein interactions. Selective RNA recognition by GFP sequence-specific RNA binding protein fusion constructs serves as a reliable indicator of RNA localization.96 With this technique, it is possible to visualize RNAs that are synthesized and processed by cells.97 Nuclear pore permeability of 27-kDa EGFP was examined by real-time FRAP imaging. In this elegant study, no evidence for significant nuclear pore gating or block of EGFP diffusion by depletion of perinuclear Ca2+ stores was found, as assayed by a perinuclear-targeted Ca2+ indicator.98
Nuclear Calcium
Interplay between cytoplasmic and nuclear Ca2+ regulation remains controversial. Ryanodine and InsP3 receptors are reported99 to participate in Ca2+ release from the nuclear envelope. Nucleus-targeted cDNAs encoding fluorescent protein calcium indicators (cameleons) were transfected into suprachiasmatic nucleus neurons. Circadian rhythm changes were observed for the cytosolic Ca2+ reporter, but not for the nuclear one,100 indicating that the nucleoplasmic reticulum regulates calcium signals independently of cytosol. Two-photon uncaging of InsP3 revealed spatially restricted PKC signaling for the nucleus and cytoplasm. PKC was translocated from cytoplasm to the plasma membrane by photorelease of Ca2+ into the cytoplasm. Photorelease of Ca2+ into the nucleus induced a loss of nucleoplasmic PKC, which relocated to the nuclear envelope. These findings showed that the nuclear interior contains a network of calcium stores forming functionally distinct subcomparments.101 See the online data supplement, available at http://circres.ahajournals.org, for fluorescence applications in "Other Organelles."
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Recent Advances |
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Nanoscopy
A weakness of fluorescence microscopy lies in its relatively low spatial resolution, which is limited by light diffraction to approximately one-half of the wavelength of excitation or emission light (the Rayleigh criterion). Going beyond this limit requires advanced technical measures, which theoretically can resolve 2 points in the fluorescent image separated by a distance less than the diffraction limit. There are 3 approaches to obtain this resolution enhancement: (1) specialized optical devices; (2) image processing; and (3) special fluorescent probes or physico-chemical processes for generation of the recorded signal,102,103 for example, Quantum dots (see later). Using these approaches, resolution to
28 nm can theoretically be achieved (see the online data supplement).
Quantum Dots
Existing imaging techniques use small organic fluorescent molecules, fluorescent biomolecules, and photoproteins. However, their spectral properties limit the use of >3 dyes at a time to tag and image different biological molecules simultaneously. Fluorescence of dyes also tends to fade away quickly over time. Inorganic semiconductor nanocrystals, "quantum dots" (QDs) (2 to 10 nm, on the order of average proteins), have intriguing optical properties that get around these problems and have the potential to revolutionize biological imaging. In addition to being brighter and persisting longer than organic fluorophores, QDs have a broader excitation spectrum. This means that a mixture of different QDs can be excited by a single-wavelength light source, allowing simultaneous detection and imaging in color. When the electron-hole pairs in the core of a QD are excited with a beam of light, they emit fluorescence that depends directly on the size of the dot: the larger the dot, the redder the light. Thus, QDs can be fine-tuned to emit light at a variety of wavelengths (compatible with standard fluorescent microscopes) simply by altering the core size.
Stable, soluble, and biocompatible cadmium–selenide QDs encapsulated in phospholipid micelles were developed and tested for the first time in Xenopus embryos.104 On cell division, QDs appeared to be solely distributed to the offspring of the injected parent cell and did not appear to have any detrimental effect on the frog development. The dots were stable for months in vivo, fluorescence could be followed to the tadpole stage, allowing cell-lineage tracking experiments throughout embryogenesis without significant photobleaching.
Recently Quantum Dot Corporation developed QD conjugates with streptavidin, protein A, biotin, IgGs, small molecules, and oligonucleotides. These bioconjugates are well-suited for multicolor applications because of to their narrow (typically 20 to 40 nm) and symmetric emission spectra and are a promising alternative to organic dyes for fluorescence-based applications (Figure 7). QDs linked to streptavidin and IgG were successfully used to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus.105
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QD labeling has enabled real-time assessment of protein trafficking in living cells. QDs have been used to track the motional dynamics of individual glycine receptors in neurons.106 Much larger probes would not have worked, as the receptors are only 5 nanometers wide. The ability to image QD-tagged glycine receptors by real-time fluorescence and by electron microscopy allowed both dynamic behavior and accurate localization studies to be performed with the same samples. Epidermal growth factor-conjugated QDs enabled tracking the dynamics of the interaction and activation of the EGF receptor, erbB1, in real time.107 Two-photon microscopy of QDs was used by Larson et al108 to image capillary blood flow through the skin of live mice.
These examples illustrate that the properties of QDs make them suitable for applications in many areas from tagging single proteins in cells to diagnostic imaging, with great potential for use in diverse fluorescence applications and in emerging imaging technologies.
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Conclusion |
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TopAbstractIntroductionFluorescent Probes of Cell...Fluorescent Protein Tags and...Photobleaching MethodsMitochondriaSarcoplasmic/Endoplasmic...NucleusRecent AdvancesConclusionReferences |
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Previously, anatomical and physiological studies defined the ability of organs and certain aspects of cellular function in vivo to change in time in response to perturbations, but considerable molecular mechanistic detail was lacking. Twentieth and twenty-first century advances in molecular biology and genetics identified many key molecules involved in signal transduction, and often linear and sometimes branching, network hierarchies can be deduced, but what is ultimately desired, the coordinated integration in cells in space and time, is frequently beyond the reach of these techniques alone. This review has attempted to show by example how the synergy of these disciplines and advanced microscopy imaging has provided one of the most useful techniques to assess the activities of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The future of "molecular imaging" appears bright, with such novel interdisciplinary applications as QDs from solid-state physics, super-resolution optics enabling nanoscale resolution, and developments from other disciplines, providing new opportunities in signal tracking from cells to animals. From "Animalcules" to QDs, Léeuwenhoek would be proud.
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Acknowledgments |
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This work was supported by the Intramural Research Program, National Institute on Aging, National Institutes of Health.
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Footnotes |
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Original received March 5, 2004; revision received May 11, 2004; accepted May 13, 2004.
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