using imaging methods to interrogate radiation-induced cell signaling

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Using Imaging Methods to Interrogate Radiation-Induced Cell SignalingAuthor(s): Harish Shankaran, Thomas J. Weber, Claere von Neubeck and Marianne B. SowaSource: Radiation Research, 177(4):496-507. 2012.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR2669.1URL: http://www.bioone.org/doi/full/10.1667/RR2669.1

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RADIATION RESEARCH 177, 496–507 (2012)0033-7587/12 $15.00�2012 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2669.1

Using Imaging Methods to Interrogate Radiation-Induced Cell Signaling

Harish Shankaran,a Thomas J. Weber,b Claere von Neubeckb and Marianne B. Sowab,1

a Computational Biology and Bioinformatics, b Systems Toxicology, Pacific Northwest National Laboratory, Richland, Washington 99352

Shankaran, H., Weber, T. J., von Neubeck, C. and Sowa,M. B. Using Imaging Methods to Interrogate Radiation-Induced Cell Signaling. Radiat. Res. 177, 496–507 (2012).

There is increasing emphasis on the use of systems biologyapproaches to define radiation-induced responses in cells andtissues. Such approaches frequently rely on global screeningusing various high throughput ‘omics’ platforms. Althoughthese methods are ideal for obtaining an unbiased overview ofcellular responses, they often cannot reflect the inherentheterogeneity of the system or provide detailed spatialinformation. Additionally, performing such studies withmultiple sampling time points can be prohibitively expensive.Imaging provides a complementary method with high spatialand temporal resolution capable of following the dynamics ofsignaling processes. In this review, we utilize specificexamples to illustrate how imaging approaches have fur-thered our understanding of radiation-induced cellularsignaling. Particular emphasis is placed on protein colocal-ization, and oscillatory and transient signaling dynamics.� 2012 by Radiation Research Society

INTRODUCTION

The response to stress is comprised of a chain of eventsinvolving changes in metabolite and second messengerlevels, mRNA expression, protein abundance and post-translational modifications. These distinct levels of biolog-ical information processing span a wide range of timescales. Initial events in the radiation response frequentlyinvolve the rapid assembly of DNA repair complexes,reactive oxygen species generation, lipid peroxidation, andsecond messenger generation (e.g., Ca2þ, cyclic AMP) (1).These responses all occur on the order of seconds-to-minutes and can activate and modulate several mitogenicand pro-survival signaling pathways. The subsequentchanges in gene expression and protein abundance occuron the order of hours and ultimately effect cellular decisionssuch as proliferation, differentiation, migration, and apo-ptosis.

Signaling pathways act as a nexus for integrating and

processing information from a variety of intra- and

extracellular cues (2–5). The traditional approach to

monitoring the activation of signaling pathways involves

population-based measurements at a given time point. If one

is interested in dynamics, the number of sampling time

points is increased. However, there is increasing evidence to

suggest that the spatial and temporal dynamics of signaling

molecules play a critical role in cell fate decisions (6–8),

and static population-based measurements can obscure the

true pathway dynamics.

Molecular imaging provides an important complement to

population based measurements. It provides spatial resolu-

tion at the cellular or even sub-cellular level, real-time

dynamic information of the system response, and details of

specific protein interactions. For example, live cell imaging

has been successfully used to monitor ERK, p53 and NF-jB

activation dynamics, and has revealed the presence of

sustained oscillatory behavior of these critical signaling

pathways; information that would have been missed with

static imaging methods (9–12). The field of live cell

imaging has been significantly advanced with the discovery

of the green fluorescence protein (GFP) in 1962 by

Shimomura et al. (13). The real potential of this protein

as a marker was revealed when it was functionally

expressed in a recombinant system (14). Cells expressing

fluorescently-tagged gene products can be used to directly

monitor protein movement (15–17). Now a rainbow of

fluorescent proteins are available (18), and can be used in

numerous combinations to perform multispectral imaging

(19–20). Recent advances in image analysis methods also

allow for the automated identification of various cell

phenotypes from imaging data, which greatly facilitates

investigations into the origins of cell behavior by increasing

throughput (21).

Here we examine how imaging techniques have furthered

our understanding of cell signaling by elucidating key

events in the radiation response. We do this through the

discussion of the specific examples of protein colocaliza-

tion, and both oscillatory and transient signaling dynamics.

The aim of this review is to specifically highlight how

imaging has contributed to a fuller understanding of

radiation-induced signaling. Therefore, no attempt is made

1 Address for correspondence: Pacific Northwest National Labo-ratory, P.O. Box 999, MS J4-02, Richland, WA 99352; e-mail:[email protected].

496

to provide an exhaustive review of the vast literatureregarding each biological application covered.

RADIATION AND CELL SIGNALING

DNA Damage and Associated Signaling Events

We will use the recruitment of proteins to the site of DNAdamage as an example for co-localization studies. There-fore, we provide a brief introduction regarding radiation-induced DNA damage and repair [for a recent in-depthreview, see (22)]. A critical target for radiation-induceddamage is the DNA in the cell nucleus, although non-targeted effects have received increasing attention as well(23). Fortunately, a very efficient repair system exists in thecell and the majority of DNA damage can be faithfullyrepaired without the loss of genetic information or inducedcell death. When damage occurs to only a single strand ofthe DNA, the undamaged strand functions as a template andprovides for error-free repair in most of the cases. If twosingle strand breaks (SSB) occur in close proximity onopposite strands, they lead to a double strand break (DSB).For ionizing radiation exposures, the ratio of SSB to DSB is; 20:1 (24). DSBs are considered to be the most difficult torepair since the two strand ends have to be identified, fixed,marked, and repaired. Numerous proteins are involved inthe recognition and repair of DSBs, including Mre11, CtIP,RPA, Rad51, Rad52, Rad54, BRCA2, and DNA proteinkinases (DNA-PKs) (25–27). The binding of DNA-PKleads to a cascade of phosphorylation including the histoneH2AX. Phosphorylated H2AX, c-H2AX triggers the DNAdamage response, which leads to the recruitment of MDC1and the MRN complex (Nbs1, Mre11, Rad50), therebyinducing more phosphorylation of H2AX (26). Chromatinmodification results in the recruitment of 53BP1 and theactivation of the p53 pathway (26).

Radiation-Induced Activation of Protein Kinase Pathways

We will use the imaging of ERK nuclear translocation toillustrate live cell imaging studies of the effects of radiationon signaling. Intracellular signaling pathways play a criticalrole in the radiation response, serving as both effectors andmodulators of the biological effects of radiation (see Fig. 1for a representative signaling pathway). Apart from DNAdamage and the associated signaling events (e.g., p53pathway activation), radiation can initiate and interact withother cell signaling pathways within the cell (28–29) due tothe generation of reactive oxygen species, lipid modifica-tions at the plasma membrane, and through ATP release andassociated calcium transients (30–31). Radiation at dosesabove ;0.5 Gy leads to the phosphorylation and activationof the ErbB family of receptor tyrosine kinases (32–33). TheErbB family consists of four members ErbB1 to ErbB4 (alsocalled HER1-4) that activate several key downstreamsignaling pathways involved in cell fate decisions. Of these,the PI3K pathway is predominantly activated by ErbB3 and

has been documented to play a radioprotective role due toits ability to induce anti-apoptotic effects.

Radiation can induce mitogen activated protein kinase(MAPK) activation through several direct and indirectmeans including the activation of ErbB receptors, thegeneration of ceramide at the plasma membrane, clusteringof death receptors at the plasma membrane, generation ofreactive oxygen species, the enhanced synthesis andshedding of growth factors, and calcium transients (28–29, 32, 34–35). There are three major MAPK pathways inmammals: extracellular-signal regulated kinase (ERK), JNKand p38, which are all activated to various extentsdepending upon cell type and context in response toradiation. The ERK pathway is activated in response to avariety of stimuli. The induction, regulation and conse-quences of radiation-induced ERK activation are complex,with cell type and the duration of ERK signalingdetermining the consequences of ERK activation onradiation responses. ERK activation can play either aradioprotective role or can result in radiosensitization andenhanced cell-killing. The JNK pathway (also known as thestress activated protein kinase pathway – SAPK pathway)primarily plays a proapoptotic role. The p38 pathway isactivated in response to a variety of stresses and has beenshown to promote cell death as well as enhance cell growthand survival depending upon the cell type and stimulus.Radiation-induced activation of the p38 pathway is highlyvariable, in contrast to the other two MAPK pathways,which are found to be activated over a broad range of dosesin a variety of cell types (29). It is important to note thatradiation has been documented to activate the PI3K andMAPK pathways at doses above 0.5–1 Gy. Upregulation ofthe MAPK pathway has also been implicated in propagationof the bystander effect (34).

IMAGING RADIATION EFFECTS ON CELLSIGNALING: SPECIFIC EXAMPLES

Although most studies in radiobiology include some formof molecular imaging, imaging methods are only recentlybeing exploited to understand radiation-induced signaling.In the sections that follow, we will provide illustrativeexamples of the use of imaging to understand cell-signalingdynamics and protein recruitment. We will use the examplesof radiation-induced protein colocalization, calcium tran-sients, and ERK oscillations.

Visualizing Protein Colocalization and Dynamics in theDNA Repair Pathway

One area where imaging has played a critical role in theunderstanding of radiation-induced signaling is in the areaof DNA damage and repair. The standard technique tomeasure protein recruitment to the site of DNA damage isthe visualization of fixed immunolabeled cells with confocaland epi-fluorescence microscopy (Figs. 2, 3). The visual-

IMAGING AND CELL SIGNALING 497

ization of a fluorescent focus following an ion traversal has

been demonstrated for various proteins including CDKN1A

and Mre11B (36–37) and for the DNA DSB marker c-

H2AX (38–39). Studying c-H2AX foci formation has led to

the observation that the number of foci induced after

ionizing radiation exposures is dependent on the phase of

the cell cycle (24). This is linked to the cell’s DNA content

with the number of c-H2AX foci being approximately

double in G2 cells relative to cells in G1 (40). When

interpreting foci data, care must be taken to understand and

account for changes in the endogenous levels. For instance,

S phase cells show an elevated background level of ATR-

dependent c-H2AX foci at stalled replications forks (41).

Additionally, the endogenous level of c-H2AX foci is

highly cell-type dependent. In a recent study on 25

untreated normal human fibroblast strains in G0/G1, the

FIG. 1. Feedback and crosstalk in the ERK/MAPK pathway. Signaling pathways such as the ERK/MAPKpathway can integrate information from multiple sources and can generate a diverse range of dynamic behaviorsdue to the existence of feedback interactions in the pathway structure. Here the EGFR-activated ERK/MAPKpathway is shown. Activation of the EGFR by endogenous ligands such as transforming growth factor-alpha(TGFa) induces EGFR phosphorylation leading to the recruitment of adaptor proteins such as Shc, Grb2 andSos. Adaptors couple the activation of the EGFR to the activation of Ras, which then stimulates the 3-tiered Raf! MEK! ERK cascade (depicted using bold red letters), which constitutes the core of the signaling pathway.Negative feedback (red lines) can result from the phosphorylation of either Sos or Raf by ERK, or thetranscriptional induction of negative feedback inhibitors, such as the DUSPs, through activation of transcriptionfactors, such as AP1. Positive feedback (blue lines) can result from the transcriptional up-regulation or ERK-induced shedding of ligands, such as TGFa, or the inactivation of repressors such as RKIP. G-protein coupledreceptor (GPCR) agonists induce Ca2þ transients, which can feed into the ERK pathway via the protein kinase C(PKC)-mediated inactivation of RKIP. Further, EGFR signaling by itself can activate PKC, thereby enhancingERK activation. Radiation can activate and interact with the ERK pathway through several routes: viaphosphorylation of the EGFR, shedding of EGFR ligands, and induction of calcium transients. The specificmechanism of ERK activation, and even the feedback interactions, can depend upon cell type and context. Thus,it is not surprising that considerable variability is observed in ERK-activation dynamics and in ERK-mediatedcell fate decisions.

FIG. 2. Visualization of heavy ion tracks in cell nuclei. HumanNT2 cells were exposed with 600 MeV/u silicon ions and fixed after30 min. Cells were immunostained for anti-phosphorylated H2AX atserine 139 foci, counterstained with DAPI, and the images wereacquired using confocal microscopy. The methodology used forsample preparation and imaging is similar to that described in (121).As the dose increases, the number of tracks per nuclei increases.(Image courtesy of A. Kim.)

498 SHANKARAN ET AL.

foci number ranged from 0.2 to 2.6 foci/cell on average

(42). These foci occur in the absence of radiation and can

lead to overestimation of foci numbers. The recent advent of

fluorescent ubuiquitination-dependent cell cycle indicator

(Fucci) probes (43) enables the dynamic monitoring of cell

cycle phase (G1 compared to S/G2/M) in live cells, and

makes it possible to better correlate radiation responses with

cell cycle phase. For instance, the Fucci probes have been

recently used to sort cells within various stages of M phase,

and to show that cells in early M phase are more

radiosensitive than those in late M phase (44). The Fucci

probes should also prove to be valuable in accurately

quantifying the dynamics of radiation-induced cell cycle

arrest. Original studies, which visualized track structure,

used cloud chambers of etchable plastics. Now it is common

to use protein specific antibodies to visualize track structure

directly in the cell nucleus (Fig. 2). Using an irradiation

geometry with a small incidence angle of the beam (,58),

heavy-ion tracks are easily visualized when staining to

markers such as c-H2AX and MRE11 (45). A similar

approach was used for the visualization of a-particle tracks

staining for c-H2AX, MRE11 and RAD51 (46). Tracks do

not show a homogenous staining for the selected repair

protein, and many publications report gaps in between the

protein aggregates and a granular structure in the tracks (45,47). The mechanism behind this remains under discussion,

but clustering of DNA damage has been proposed (48). In a

live cell imaging experiment with Ni-ions the track showed

similar inhomogenity, proving that the presence of gaps in

the track structure is not an artifact of the staining protocol,

but represents the true biological behavior of the repairproteins (49).

Besides c-H2AX, phosphorylated ATM (pATM) is usedto study DSB after irradiation. In exponentially-growingcells the background level of c-H2AX foci was higher thanthat of pATM foci (50–51). This indicates that pATM couldbe a suitable marker for radiation-induced DSB after low-dose irradiation. In the same study, cells showed equalnumbers of pATM foci in G1 and G2, but higher numbers inS phase (51). This could mean that the ATM backgroundlevel of foci is independent of the DNA content and onlydependent on the transcriptional activity of the cell.

Defining the critical players in DNA repair proteincomplexes has historically been done with immuno co-precipitations. Although this is a sensitive method, it doesnot provide information on the spatial distribution ofcomplexes or their dynamic recruitment to the site ofdamage. Colocalization studies of repair proteins in double-labeled samples show overlapping of signals for the variousproteins (Fig. 3). To give some examples, the colocalizationof c-H2AX with XLF (52), ATM (51, 53–54), 53BP1 (49,54–56), XRCC1 (49), NBS1 (57), and Mre11 (57), wasshown as well as the colocalization of ATM with 53BP1(53–54), NBS1 (53), MDC1 (53), and RPA (51). Moreover,53BP1 colocalizes with MDC1 (54), RPA (47), and XRCC1(56), and CDKN1A has overlapping signals with Mre11B(36, 45). Recently, the colocalization of c-H2AX, phos-phorylated ATM, 53BP1, and MDC1 in X-ray irradiatedhuman 3D skin tissue equivalents was shown after paraffinembedding (54). The authors report compromised c-H2AXsignals as well as diminished or invisible pATM foci in

FIG. 3. Nuclear repair protein co-localization. Normal human diploid cells synchronized in G0/G1 wereexposed to 0.25 Gy of X rays and fixed at each time point indicated. Cells were stained with anti-phosphorylatedATM at serine 1981 and anti-53BP1 antibodies. The primary antibodies were recognized by Alexa488- (forATM-P) and Alexa594- (for 53BP1) conjugated secondary antibodies. The images were taken with afluorescence microscope and z-stack images were obtained by maximum intensity projection. The detailedmethodology can be found in (54). (Image courtesy of K. Suzuki.)


some of the tissues, while 53BP1 and MDC1 were notaffected by the paraffin embedding. They conclude that53BP1 and MDC1 are more suitable biomarkers for DSB invivo (54).

In performing protein colocalization measurements, itshould be noted that there are several potential sources ofartifacts and false positives. An important issue inmultispectral imaging that is often neglected is the need tomerge both images with a resolution at the sub-pixel level.Figure 4 shows the ratio of two spectral images that have, orhave not, been registered. With the commonly used CCD-based systems, all image pixels are simultaneously capturedand must be registered in a post-processing step. Accuratelyregistering such images can be further complicated bydifferences in factors such as tilt, focal length, and opticaldistortions between the two optical paths. Perrine and co-workers have developed a multispectral method, whichallows for real-time image registration (58–59). Thismethod provides a pixel-for-pixel match between imagesobtained over physically distinct optical paths. Theregistration algorithm uses an online calibration methodand allows for successful registration of multispectralimages at rates exceeding 15 frames/s (58–59).

Factors such as sample preparation, use of uncharac-terized antibodies, and the improper selection of probes,image acquisition parameters, and filter sets can also serveto confound colocalization results. These artifacts andguidelines for the proper design, acquisition, and analysisof colocalization measurements have been extensivelydescribed in several reviews (60–62). Further, theinterpretation of colocalization experiments needs toaccount for the fact that the resolution of a typicalfluorescence imaging experiment is ;200 nm in the focalplane, with the depth (z axis) resolution being ;2.5 timesless. Given that the typical protein size is in the order ofnanometers, the observation of colocalization does notimply that the proteins are in fact interacting, eitherdirectly or indirectly. Although more sophisticated meth-ods such as Fluorescence Resonance Energy Transfer(FRET) can enable the identification of protein-proteininteractions at length scales less than ;10 nm, FRETsignals are usually small, and the experiments requirecareful planning, interpretation and several controls (63).FRET is also not well suited to resolve complex structures,and to identify interactions that occur at distances .10 nm(e.g., indirect binding in a large multiprotein complex).The recent advent of super-resolution microscopy offersthe promise of extending the power of traditionalcolocalization measurements (64), thereby providingadditional insight into DNA repair processes. For example,the use of 4Pi microscopy to study the colocalization ofH2AX and c-H2AX has revealed that the inherentclustering of endogenous H2AX constrains the spreadingof c-H2AX foci following DNA damage (60, 65).

Typical studies of foci formation and DNA repaircomplex formation involve static measurements, which

infer dynamics through the study of multiple time points infixed cells. Although live cell imaging would bepreferable, such studies require the expression of fluores-cent fusion proteins (FFPs). Constructing FFPs whileensuring that the protein of interest retains its normalbehavior and localization is a non-trivial task. Greenfluorescent protein (GFP) is a bulky moiety (27 kDa) that,in some cases, can inhibit protein complex formation. Forexample, if the carboxy terminus of the target proteincontains an important protein-protein interaction domain,attaching GFP to the C-terminus may interfere with thisdocking site, which, in turn, could influence proteinfunction and/or localization. The N- and C-termini of GFPare closely located to each other enabling this protein to beinserted in the middle of a fusion partner (66). This can beadvantageous in cases where the fusion target is intolerantof tagging at both N- and C-termini. A detailed discussionof the various factors to consider in proper construction ofFFPs can be found in (67–68). Constructing FFPs can beparticularly challenging if the protein of interest isextremely large as is the case for several proteins involvedin the DNA repair process. There are additional technicallimitations in performing live cell imaging at radiationfacilities. Often the time between sample exposure andfixation is prolonged so that very quick radiation-inducedreactions cannot be detected.

Both c ray and local laser irradiation produce limitedmovement of DSBs and chromatin decondensation (57, 69).The clustering of DSBs following a-particle irradiation hasbeen proposed (46). However, contrary results for ultrasoftX rays and ions have found DSBs to be immobile (49, 70).Live cell imaging can help answer questions regardingchromatin movement following exposures to ionizingradiation. And, despite the inherent technical challengesnoted above, such studies are becoming increasinglycommon. Live cell imaging results observed no tendencyto form repair clusters after ion exposures (49). Recently in-line beam microscopy has been used to detect aprataxinaggregation within a few seconds after exposure to heavyions (71). As real time microscopy is used more extensivelyto study the kinetics of DNA repair and the recruitment ofmultiple proteins to the site of damage, a clearer picture ofthe repair process will take shape.

FIG. 4. Importance of image registration for proper imageinterpretation. False color image of the ratio of red and green emissionchannels of a histological section stained with hematoxylin and eosin.All images were obtained with a spinning disk confocal microscope.Details of the image registration procedure can be found in (59, 122).


Radiation-Induced Calcium Transients

Intracellular calcium is an important second messengerthat regulates many cell functions including activation of theERK/MAPK pathway shown in Fig. 1 (72). A rapid andtransient release of calcium has been observed followingmechanical stress, wounding and exposure to ionizingradiation (73–75). When calcium-signaling proteins areabnormally expressed, it can lead to a high proliferativepotential observed in some cancer cells (76). Due to itsimportance as a second messenger, calcium signaling hasbeen investigated as a possible mechanism for thetransmission of the radiation-induced bystander effect.Non-exposed bystander cells are either in the vicinity ofexposed cells or are the recipient of growth media fromexposed cells (77–79). Various visible light fluorescentprobes are available for live cell calcium imaging. The mostcommonly used are based on homologs of calcium chelatingagents and include Flou3, Flou4, FuraRed, and Oregon andCalcium Green derivatives (80). In the section that follows,we will discuss transient calcium responses induced byionizing radiation and the role of calcium as a secondmessenger of exposure.

Calcium transients result when the extracellular ligandATP binds to purinergic P2 receptors (81). The involvementof P2 receptors in the cellular response to c exposures hasbeen studied over a broad dose range of 0.1 to 11 Gy (82).Exposures in this dose range were found to increase theamount of ATP in the media relative to controls within 5min of exposure. Following UV exposures, apyrase wasfound to suppress P2Y activation, indicating the involve-ment of extracellular nucleotides in propagation of thecalcium response (81). Ionizing radiation has been similarlydemonstrated to result in the release of nucleotides from thecell that activate the P2Y receptors. Anion transportinhibitors and gap junction inhibitors significantly de-creased the release of ATP, suggesting that the radiation-induced release of ATP involves both anion transporters andgap junctions (82).

Confocal time lapse imaging has been used to demon-strate that the addition of irradiated condition media tononirradiated bystander cells is capable of inducing calciumtransients, the loss of mitochondrial membrane potential,and an increase in reactive oxygen species (83–86). Thecalcium response did not appear to depend on the dosegiven to the directly irradiated cell in the range of 0.5 to 5Gy (85). It is interesting to note that production of thebystander signal persists for multiple generations and wasmaintained in the conditioned media taken from cells up toseven passages after irradiation (85). Low-dose studiesindicate that there may be a threshold for bystander-inducedcalcium transients of 2 mGy, with cells above that doseshowing decreased clonogenic survival (83). Additionally,exposure to irradiated conditioned media can reduce thesensitivity of bystander cells to subsequent exposures (86).Imaging studies have been able to suggest an involvement

of calcium as a second messenger in the propagation of thebystander response, but the exact mechanism and thebiological significance of the response remains unclear.

Studies of radiation-induced Ca2þ responses typicallyinvolve imaging with calcium-sensitive fluorescent dyes.However, several genetically encoded fluorescent proteinprobes have been developed for imaging calcium responsesin single live cells (87–88). These probes are based oncalcium-induced protein conformational changes and inter-molecular interactions, such as the binding of calmodulin tothe M13 fragment of myosin light chain kinase. Suchmolecular interactions have been used as the basis forconstructing both FRET-based biosensors (89–90), as wellas fluorescent molecules that change intensity upon calciumbinding (91–92). These probes enable the study of calciumresponses in particular cells of interest in a population; anaspect that cannot be addressed with the loading oftraditional chemical dyes. For instance, FRET-basedbiosensors have been used to image calcium transients inneurons and pharyngeal muscles of intact C. elegans (93).Further, since these probes can be targeted to particularlocations within the cell using appropriate peptide sequenc-es, they have been used to examine calcium responses atparticular intracellular locations, such as the plasmamembrane, cytosol, nucleus, ER, secretory vesicles, andcaveolae (89, 94–95). These calcium biosensors thus havethe potential to offer us a more refined picture of radiation-induced calcium responses.

Live Cell Imaging of ERK Dynamics

The ERK pathway is activated in response to a variety ofstimuli including growth factors, cytokines, ligands forheterotrimeric G protein-coupled receptors, transformingagents, and carcinogens (2, 96–97). All MAPK pathwaysincluding the ERK pathway share a conserved 3-tieredtopology (Fig. 1) involving sequential phosphorylation andactivation of a downstream kinase molecule by an upstreamkinase. In the ERK pathway, ERK is phosphorylated byMEK, which in turn is phosphorylated by Raf. ERK ispredominantly localized in the cytoplasm in resting cellsand, following activation, rapidly translocates to the nucleus(98). ERK activation leads to the phosphorylation of avariety of cytoplasmic and nuclear substrates with thepotential to affect cell migration, proliferation, differentia-tion, and apoptosis (2, 96, 99–101).

The multilevel ERK cascade structure enables responseamplification and results in sharply nonlinear dose respons-es to growth factor addition. Further, the ERK pathway isregulated extensively via a number of positive and negativefeedback loops (Fig. 1). Studies have shown that the ERKnetwork structure is dynamic, and changes even in the samecell type depending upon the stimulus (102). Transient ERKactivation, sustained activation, switch-like dose-responsebehavior, and oscillations have each been demonstrated invarious contexts (10, 102–105).


Since ERK activation leads to rapid nuclear translocationof the protein, live cell imaging can be applied to monitorERK activation dynamics in single cells. A typicalexperiment follows the nuclear translocation of an ERK1-GFP chimeric protein. Cells are co-labeled with nuclearmRFP to provide a means to automatically define celllocations for image analysis (Volocity, Improvision). Themean ERK-GFP intensity is then measured in the regionsoccupied by the cell nuclei. Using this system, nuclear ERKlevels can be monitored in cells for ;10 h withoutsignificant photobleaching and oscillatory ERK activation,if any, can visually be identified as periodic increases in thenuclear GFP signal in time-lapse movies constructed fromthe fluorescence images.

Under oscillatory conditions, nuclear ERK time-seriesdata can be analyzed further to extract quantitativeinformation regarding oscillation characteristics. Figure 5shows some of the important aspects to consider withoscillatory signals, i.e., rise time, decay time, amplitude, andfrequency. Fourier transformation can be used to estimatethe frequency component of the oscillations associated witheach cell. By inspecting the waveforms in the time andfrequency domain, cells can be classified as ‘‘clean’’oscillators, with a single, strong frequency component anda continuous train of oscillation pulses that can bevisualized in the time domain; ‘‘noisy’’ oscillators thatdisplay intermittent or very low amplitude oscillations, butwith the FT spectrum showing a peak at a time period ,30min; and ‘‘non’’-oscillators with no dominant peak in theFT spectrum. In addition to enumerating the fraction ofoscillating cells, time domain analysis of the ‘‘clean’’oscillators enables quantification of the time period, risetime, decay time, oscillation amplitude, and baseline shifts(Fig. 5).

Sustained ERK oscillations have been observed in humanmammary epithelial cells in response to EGF with a timeperiod of ;15 min (10); in mouse epidermal cells inresponse to basic fibroblast growth factor (bFGF) with atime period of ;16 min (12); in human keratinocytes inresponse to TGFa (ligand for the epidermal growth factorreceptor) with a time period of ;12 min (106). Althoughthe existence of ERK oscillations has been theoreticallypredicted based on the complex structure and feedbackregulation of the pathway (107–108), live cell imaging wasnecessary to conclusively demonstrate their existence.Further, the detailed quantification of ERK oscillationcharacteristics resulting from these studies has enabled theconstruction of mathematical models that clarify theregulatory features responsible for generation of oscillatorysignaling. It has been shown that a negative feedback loopwhere activated ERK inhibits an upstream protein in theERK cascade is necessary to generate the experimentallyobserved oscillation characteristics (10). This is an exampleof how the availability of dynamic information inconjunction with mathematical modeling can help usunderstand the encoding of signaling dynamics via specificmolecular mechanisms in the signaling pathway.

A recent study has examined how various cell stressorsincluding exposure to low doses of X rays affect ERKoscillations (106). Human keratinocytes exhibited sustainedERK oscillations in response to TGFa (Fig. 5). In contrast,10 cGy X irradiation played an inhibitory role on TGFa-induced ERK oscillations. The effect of radiation appears tobe independent of the generation of reactive oxygen species.When cells loaded with a fluorescent ROS indicator (H2-DCFDA, Molecular Probest) were exposed to 10 cGy Xirradiation, ROS levels were found to be comparable insham and irradiated cells.

FIG. 5. Radiation-induced ERK oscillations. Growth factor addition can lead to sustained oscillations innuclear ERK levels that can be observed using live cell imaging of cells expressing an ERK-GFP chimera. PanelA: Schematic depiction of oscillations in nuclear ERK levels illustrating features that can be quantified byanalyzing the time course. Since ERK oscillations are associated with several dynamic features, they canpotentially encode more information compared to steady or transient pathway activation. Panel B: Effect of low-dose X-irradiation on ERK oscillations. Sham irradiated human keratinocytes exhibit sustained oscillations innuclear ERK levels following addition of the EGFR ligand TGFa. When cells are subjected to 10 cGy Xirradiation prior to growth factor addition, oscillations are no longer observed. Representative nuclear ERKlevels are shown as a function of time for a sham and a 10 cGy irradiated cell. Arrow depicts time of TGFaaddition. [Image adapted from (106)]


While the biological significance of ERK oscillations isunclear, an oscillatory signal can potentially carry moreinformation (e.g., in the time period, amplitude rise time,decay time, baseline, etc.) compared to a transient or asustained signal (Fig. 5). Oscillations in other signalingsystems such as calcium, p53 and NF-jB have beenproposed to serve as additional layers in which informationcan be encoded (109–110). Radiation activates the p53pathway (111), which regulates DNA repair, cell cycleprogression, and apoptosis. The p53 pathway has beenshown to undergo repeated pulses of activation with a timeperiod of ;5 h following exposure to high doses (5 to 10Gy) of c irradiation (9). Detailed observation of p53activation dynamics in individual cells has been used toconstruct mathematical models that describe the biomolec-ular origins of these oscillations, and have clarified the roleof feedback in this phenomenon (112–114). The exact linkbetween p53 dynamics and cell fate decisions is still anactive area of investigation, although evidence suggests thatinformation can be encoded in the number of p53 pulsesinduced following DNA damage (115).

Only a limited number of high LET studies have observedoscillatory signaling. The GSI heavy ion microprobe has anonline imaging system for immediate imaging of cellulardynamics. A recent study using carbon and argon ions at thisfacility observed oscillations in intercellular calcium levelsfollowing exposure to heavy ions (116). However, thisresponse occurred in the absence of radiation and was linkedwith the onset of hypoxic conditions. When samples weretreated under conditions of increased gas exchange, thecalcium oscillations were eliminated. When a cell that wasalready oscillating was exposed, the kinetics and magnitudeof the oscillations did not change. This is in contrast to thedampening of ERK oscillation observed following the lowLET low-dose exposure [see Fig. 5 and (106)]. It appearslikely that the mechanism of the oscillatory responsesdepends on many factors, including radiation quality, celltype, and phase of the cell cycle (75, 106, 116).

Thus far, the oscillatory activation of signaling pathwayshas been assayed solely by monitoring changes in thesubcellular localization of FFPs. Oscillations in the ERK,p53 and NF-jB pathways were all identified by examiningthe nuclear localization of FFPs (9–12). In performing suchexperiments, care should be taken to properly construct theprobes, and to express them at levels comparable to theendogenous protein such that the behavior of the fusionprotein is truly indicative of that of its native counterpart(see 67, 68 for a guide on the construction and use of FFPs).For instance, expressing an FFP at too high a level canresult in the saturation of the translocation machinery.FRET-based biosensors that can directly detect proteinmodifications such as phosphorylation and GTP-binding areavailable for several signaling molecules including ERK,Ras, Raf, Rac, and PKC (87–88, 117). These probes can beused as an alternative to translocation measurements formeasuring the activation of effector proteins in a signaling

pathway (e.g., ERK). They are particularly useful forassaying intermediate molecules along a signaling pathwaythat may not display changes in subcellular localizationfollowing activation. Further, the biosensors can be targetedto particular locations within the cell to determine the spatialdependence of signaling pathway activation. For example,targeted versions of a FRET-based biosensor for PKCactivity have been used to show that this protein is not onlyactivated at the plasma membrane as previously believed,but also on the Golgi, mitochondrial membranes, as well aswithin the cytosol and nucleus (118).

The observation of signaling oscillations provides anexample of a scenario where traditional population-basedmeasurements fail to provide us with a true depiction of thedynamics of a signaling pathway. Oscillations betweenindividual cells are asynchronous, which makes theiridentification through population measurements difficult.Oscillations are also found to occur at low physiologicalgrowth factor concentrations that elicit very low levels ofERK phosphorylation as measured by western blots. Thus,live cell imaging is a uniquely powerful tool for monitoringsignaling pathway activation under such physiologicalconditions.

While our primary focus here has been the imaging ofradiation-induced cell signaling in vitro, there are anincreasing number of in vivo imaging studies that examineradiation-induced p53 activation in mice using downstreamgene reporters of p53 activity. Bioluminescence imaging ofknock-in reporter mice (C57BL/6 background), in which theexpression of firefly luciferace (FLuc) gene was placed underthe control of the endogenous p21 promoter, indicated thatp53-mediated activation of the downstream reporter peakedat ;8 h following 2.5 Gy total body irradiation, and steadilydeclined thereafter (119). In contrast, in transgenic mice(BALB/C background) where FLuc expression was depen-dent on the p53-responsive promoter from the MDM2 gene,5 Gy total body irradiation resulted in oscillatory biolumi-nescence with a periodicity of 5.3 h (120). The dynamics ofp53 activation observed in this study is consistent with thep53 oscillations that have been observed in vitro in culturedcells. Importantly, this study offers evidence that oscillatorysignaling occurs in vivo, and is not merely an artifact arisingfrom the use of cultured cells. Both of these studies revealsignificant differences in radiation-induced p53 activationbetween organs in mice, with the intestines typically showingmaximal induction of activity following irradiation. Theavailability of such animal models should greatly enhanceour ability to understand the spatio-temporal dynamics ofradiation-induced signaling in whole animals, and will enableus to better define the relationships between in vitroobservations and in vivo responses.

Conclusions And Future Outlook

In summary, the biological effects of radiation areregulated and implemented through the activation of cell-


signaling pathways, which control cell fate decisions.Understanding cellular decision making requires us tofigure out how information from various cell stimuli isencoded into the dynamics of cell-signaling pathways, andhow these dynamics are decoded to affect cell fate. Thedetailed spatial and temporal information provided by livecell imaging is extremely valuable in our endeavor toreverse engineer the operating principles of cell-signalingpathways and the radiation response.

ACKNOWLEDGMENTS

We would like to thank Dr. Keiji Suzuki, Nagasaki University, and Ms.

Angela Kim, Brookhaven National Laboratory, for providing additional

images for use in this review. This work was supported by the National

Aeronautics and Space Administration [NNX10AB06G] and the

Biological and Environmental Research Program (BER), U.S.

Department of Energy [DE-AC06-76RLO].

Received: May 4, 2011; accepted: January 5, 2012; published online:

March 1, 2012

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using imaging methods to interrogate radiation-induced cell signaling

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