Dynamics of Depolarization andHyperpolarization in the Frontal

Cortex and Saccade GoalEyal Seidemann,* Amos Arieli, Amiram Grinvald, Hamutal Slovin

The frontal eye field and neighboring area 8Ar of the primate cortex areinvolved in programming and execution of saccades. Electrical microstimu-lation in these regions elicits short-latency contralateral saccades. To deter-mine how spatiotemporal dynamics of microstimulation-evoked activity areconverted into saccade plans, we used a combination of real-time opticalimaging and microstimulation in behaving monkeys. Short stimulation trainsevoked a rapid and widespread wave of depolarization followed by unexpectedlarge and prolonged hyperpolarization. During this hyperpolarization saccadesare almost exclusively ipsilateral, suggesting an important role for hyperpo-larization in determining saccade goal.

Electrical stimulation has been used exten-sively in brain research since its introductionmore than 100 years ago (1). Despite thepopularity of this technique and its potentialclinical applications (2, 3), many questionsremain regarding the spatial profile and thetemporal dynamics of the neural responsethat is evoked by intracortical microstimula-tion (4, 5). One of the first cortical regionsthat was systematically explored using micro-stimulation is the frontal eye field (FEF) (6–8), a cortical area situated in the anterior bankof the arcuate sulcus and known to be in-volved in planning and execution of saccadiceye movements (9–11). The FEF and neigh-boring area 8Ar (12) contain a representationof saccades to the contralateral hemifield.Microstimulation in these regions evokesshort-latency conjugate saccades that dependon the stimulation site and are very similar tovoluntary saccades (6–8).

We sought to measure and characterizedirectly the neural response that is elicited bymicrostimulation in the frontal cortex of thebehaving monkey and to determine the rela-tion of this response to the saccadic eyemovements that are evoked by stimulation.We thus combined standard electrophysio-logical techniques in awake behaving mon-keys with optical imaging using voltage-sen-sitive dyes (13). This combination provides apowerful paradigm for measuring the electri-cal activity of populations of neurons in thebrain of behaving monkeys at excellent spa-tial and temporal resolutions (14). The dyesignal measures the sum of the membrane-potential changes in all the neuronal elementsin the imaged area, emphasizing subthreshold

synaptic potentials in neuronal arborizationsoriginating from neurons in all cortical layerswhose dendrites reach the superficial corticallayers (15).

Combined microstimulation (16) and op-tical imaging were applied to the frontal cor-tex in four hemispheres of two monkeys at atotal of 39 recording sites (17) while themonkey performed a simple fixation task(18). To image the FEF and 8Ar, we openeda cranial window over the arcuate sulcus,then removed the dura and replaced it with atransparent artificial dura (19). The imaged/stimulated area included primarily a narrowstrip of cortex, lying 1 to 3 mm anterior to thearcuate sulcus, from which short-latency con-tralateral saccades could be evoked (16, 20).Figure 1A shows the frontal cortex in the lefthemisphere of one monkey 3 weeks aftersurgery; the arcuate sulcus (Ar) and the prin-

cipal sulcus (Pr) can be seen through thetransparent dura. We stained the cortex withblue voltage-sensitive dyes and imaged theresulting activation pattern at up to 400frames per second (13).

The activation pattern evoked by micro-stimulation at one stimulation site is shown inFig. 1B. A train of stimulation pulses (50 mA,500 Hz) was applied for 24 ms at the sitemarked by X. Shortly after stimulation onsetthere was a large and widespread increase influorescence, which corresponds to an overalldepolarization. This depolarization spreadover a large cortical area in a rather uniformmanner, consistent with the spread of activitymeasured in the visual cortex (21, 22). Fol-lowing this brief depolarization, the responsedropped sharply, and most of the imaged areabecame dark. This large and rapid drop in theoptical signal corresponds to an overall hy-perpolarization of the average membrane po-tential of the neuronal population to a levelwell below baseline (23).

The mean time course of the optical signalfor four different stimulation conditions wasmeasured in a small area 0.4 mm by 0.4 mmnear the site of the stimulating electrode (Fig.2). A short stimulation train of 24 ms (greencurve) elicited a rapid depolarization that wasfollowed by a large and prolonged hyperpo-larization. The hyperpolarization returned tobaseline about 230 ms after stimulation onsetand showed a small depolarization overshoot.When stimulation was applied for a longerperiod of 80 ms (dark blue curve), the re-sponse still peaked at about 25 ms and thendropped to a lower depolarization level de-spite the continuation of the stimulation.When the train of stimulation was dividedinto four short trains with short gaps in be-tween (red and cyan curves), the response

Department of Neurobiology and the Grodetsky Cen-ter for Studies of Higher Brain Function, WeizmannInstitute of Science, Rehovot 76100, Israel.

*To whom correspondence should be addressed. E-mail: eyal.seidemann@weizmann.ac.il

Fig. 1. Spatiotemporaldynamics of micro-stimulation-evoked ac-tivity. (A) The FEF inthe left hemisphere asseen through the crani-al window and thetransparent artificialdura. The dorsomedialarm of the arcuate sul-cus (Ar) can be seen, aswell as the principalsulcus (Pr). The areaimaged in the experi-ment shown in (B) ismarked by a whiterectangle. (B) Sequenceof images of the cortexduring microstimula-tion. The time intervalbetween frames is 9.6ms. The imaged area is6.8 mm by 6.8 mm. Stimulation is applied through frames 2 to 4 and is indicated by the black horizontalline. The site of penetration of the stimulating electrode is marked by an X. Stimulation at this siteevoked a 30° saccade to the right. The optical signals were low pass–filtered (s 5 250 mm) for displaypurposes only. The activation pattern is averaged across 30 repetitions.

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went through four cycles of depolarizationand hyperpolarization. This large hyperpolar-ization was typical for most stimulation sitesin the frontal cortex. In contrast, in the pri-mary visual cortex of the awake monkey, thesame stimulation parameters do not evokesimilar hyperpolarization (22).

The time course of the optical signalshows that each depolarization response inthe frontal cortex is followed by a similar,and sometimes even larger, hyperpolarizationresponse. How does this interplay betweendepolarization and hyperpolarization affectthe saccades that are elicited by microstimu-lation? The horizontal eye velocity traces

from three trials in which eye movementswere evoked by stimulation at a site in theright frontal cortex that represents a 20° sac-cade to the left are shown in Fig. 3A. At thissite and with these stimulation parametersthere was a large variability in saccade initi-ation time, and in these three trials saccadeswere initiated 50, 80, and 100 ms after stim-ulation onset. Although in all three trials sac-cades were evoked under identical stimula-tion conditions, these saccades differ mark-edly. The early contralateral saccade (redcurve) displayed the maximal amplitude andpeak velocity characteristic for this site andwas followed 130 ms later by a large ipsilat-

eral saccade. The saccade that started aroundstimulation offset (green curve) displayed asmaller amplitude and reversed to the ipsilat-eral direction at midflight. The saccade thatstarted about 20 ms after stimulation offset(cyan curve) was short and ipsiversive. All ofthese eye movements were evoked by thestimulation; on randomly interleaved trialswithout stimulation, the monkeys made nosaccades during this interval.

The changes in saccade properties overtime closely parallel the time course of theoptical signal measured near the stimulationsite. The mean time course of the opticalsignal (green curve) and the horizontal eyevelocity traces (blue curves) for all the trialsat this site and at these stimulation parameters(n 5 25 trials) are shown in Fig. 3B. Sac-cades that were initiated during strong depo-larization were contralateral and displayedhigh peak velocity. Saccade peak velocitydecreased during the drop in depolarizationand then reversed to the ipsilateral direction.Saccades that were initiated during stronghyperpolarization were almost always largereturn saccades in the ipsilateral direction.

The correlation between the measuredresponse and the properties of the evokedsaccades holds also for other stimulationconditions. Figure 3C shows eye move-ments that were evoked during three trialsof stimulation with four short trains at thesame site as in Fig. 3, A and B. In thesetrials, the first short train failed to evoke ashort-latency saccade. The first eye move-ment evoked in those trials was a shortipsiversive saccade rather than the largecontraversive saccade typical for this site.In two trials (red and green curves), ipsi-versive saccades were initiated about 35 msafter the offset of the second stimulationtrain, during the initial phase of hyperpo-larization. During this ipsiversive saccadethe third train was applied and evoked alarge contraversive saccade. It was fol-lowed by another small ipsiversive saccadethat coincided with another period of hy-perpolarization. In the third trial (cyancurve), an ipsiversive saccade was initiatedabout 40 ms after the offset of the thirdtrain and showed the same sequence ofipsi-contra-ipsi movements.

Figure 3D compares the horizontal eyevelocity traces (blue curves) and the meanoptical response (green curve) for all trialswith the same stimulation condition as in Fig.3C (n 5 26). The direction and peak horizon-tal eye velocities of the evoked saccades arehighly correlated with the optical signal mea-sured at the time of saccade initiation. Thefirst two cycles of depolarization evoked al-most no saccades. Saccades started preferen-tially during the hyperpolarization that fol-lowed the second or the third train and werealmost always ipsiversive. During the periods

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Fig. 3. Examples of the correlation between the properties of stimulation-evoked saccades and theresponse elicited by microstimulation. (A and C) Horizontal eye velocity traces for three trials with80-ms stimulation (A) or four trains of 24-ms stimulation separated by an interval of 44 mswithout stimulation (C). The horizontal lines at the bottom of each panel indicate stimulationtimes. (B and D) Comparison of the mean 6 SEM of the time course of the optical signal (greencurves) measured in a 0.4 mm by 0.4 mm area of cortex near the site of stimulation, and individualtraces of horizontal eye velocity (blue curves) for long stimulation (B) or four short trains (D). Redcurves indicate the mean 6 SEM of horizontal eye velocity on nonstimulated trials (n 5 52).

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of depolarization following the third andfourth trains the eyes moved toward the con-tralateral direction; during the hyperpolariza-tion intervals following the second, third, andfourth trains the eyes moved toward the ipsi-lateral direction.

Following microstimulation, the level ofdepolarization or hyperpolarization in FEFand 8Ar at the time of saccade initiationproves to be an excellent predictor of thedirection, size, and peak velocity of theevoked saccades across our entire data set.The results obtained from 39 stimulation sitesand 1044 stimulation-evoked saccades aresummarized in Fig. 4 (24). Each panel showsa scatter plot of normalized saccade peakhorizontal velocity against saccade initiationtime. For this analysis we combined all sac-cades that occurred shortly after stimulation,irrespective of whether these were the first

(red) or later saccades within a given trial(cyan). Under the two stimulation protocolsthere is a strong correlation between the mea-sured optical signal and the properties of theevoked saccades. Saccades that were initiatedduring depolarization were almost exclusive-ly contraversive. Saccades that were initiatedduring a period of hyperpolarization werealmost exclusively ipsiversive. Saccades thatwere initiated when the absolute level of theresponse was high tended to have a high peakvelocity and a large amplitude; saccades thatstarted when the response was low tended tohave a low peak velocity and a small ampli-tude (25). The correlation between the meanoptical signal (green curve) and peak hori-zontal eye velocity of individual saccades(red and cyan x’s) is highly significant forboth stimulation conditions [r 5 0.93 forcontinuous stimulation and r 5 0.82 for fourshort trains; P , 1026 for both conditions(26)].

Our findings raise two questions regard-ing the hyperpolarization. First, what is therole of hyperpolarization? Hyperpolarizationcould mediate saccade target selectionthrough competitive interactions betweensites that represent alternative saccades (27).It could also serve as a reset mechanismnecessary for normal saccade sequencing (9).Once a saccade of the desired amplitude anddirection had been executed, activity that en-codes this saccade should rapidly terminateso that another similar saccade would not beinitiated erroneously. Indeed, many FEF neu-rons with presaccadic activity are activelysuppressed following a saccade into their re-sponse field (28, 29), suggesting that hyper-polarization may occur also after voluntarysaccades. Hyperpolarization could also facil-itate a rapid return saccade that would bringthe eyes back to the previous eye positionafter desired and undesired saccades. Suchrapid “glances” also occur during natural vi-sual scanning behavior. A second questionconcerns the source of the hyperpolarization.In many experiments we found that hyperpo-larization and depolarization started from dif-ferent locations and displayed different spa-tial profiles (e.g., Fig. 1B). These results sug-gest that part of the hyperpolarization iscaused by a stimulation-evoked recurrent in-hibitory signal rather than by the well-knownafter-hyperpolarization. The finding that dur-ing hyperpolarization saccades are preferen-tially ipsilateral suggests that some of thedelayed inhibitory signals could be mediatedby “push-pull” inhibitory interactions be-tween sites that encode opposite saccades inthe two hemispheres (30). An exciting possi-bility, therefore, is that when the stimulationsite is hyperpolarized, the site that encodesthe opposite saccade in the other hemisphereis depolarized, contributing to the ipsilateralsaccade.

Finally, our results demonstrate that neu-ral activity with complex spatiotemporal dy-namics can be elicited by microstimulation;these dynamics depend on the stimulated areaand can have important behavioral correlates.These findings emphasize the importance offurther characterization of microstimulation-evoked activity for the interpretation of thebehavioral effects of microstimulation. Ourfinding that during hyperpolarization sac-cades are almost exclusively ipsilateral im-poses new constraints on models of the neuralmechanisms that compute saccade goal. Spe-cifically, our results suggest that suppressionof neural activity at a certain location in thesaccade motor map in the frontal cortex isinterpreted by downstream saccade circuitryas a signal in favor of an ipsilateral saccade.

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Grinvald, Soc. Neurosci. Abstr. 25, 784 (1999); Eur. J.Neurosc. 12, 126 (2000)] showed that it is possible toperform long-term voltage-sensitive dye imaging inbehaving monkeys, repeatedly from the same corticalarea, over a period of up to 1 year.

15. A. Grinvald et al., in Modern Techniques in Neuro-science Research, U. Windhorst, H. Johansson, Eds.(Springer-Verlag, Berlin, Heidelberg, 1999), pp. 893–969.

16. Biphasic current pulses (500 Hz; 30 to 100 mA) wereapplied through standard tungsten microelectrodes.At each site we used the lowest current that yieldeda reliable optical signal and evoked saccades (#50mA in most sites). These currents were around be-havioral threshold for evoking saccades when themonkey was actively fixating, but clearly abovethreshold when the monkey was freely viewing, con-sistent with previous findings (31). Only sites fromwhich clear, short-latency contralateral saccadescould be evoked during active fixation were includedin our study.

17. Saccades were evoked in 39 sites tested in a total of21 optical imaging sessions. Of these sites, four wereexcluded from analysis of saccadic eye movementsdue to poor fixation. In 22 of the 39 sites, a clearresponse to microstimulation could be measured op-tically. Failure to obtain a reliable optical signal in thepresence of a clear behavioral effect was due to poorstaining. Experiments with no reliable optical signalwere excluded from the analysis of the averageevoked response. All of the results described hereremain the same in the subset of 18 sites for which

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Fig. 4. Correlation between saccade peak ve-locity and the electrical response elicited bystimulation. (A) Long stimulation train. (B) Fourshort trains. The green curve shows themean 6 SEM of the normalized time course ofthe optical signal measured at all sites in whicha reliable optical signal was obtained (17). Thescatter plot of x’s indicates the normalized peakhorizontal eye velocity versus initiation timefor all saccades obtained during this interval inall stimulation sites (24). Red x’s indicate thefirst saccade evoked on a given trial, and cyanx’s indicate later saccades. The blue line depictsthe running average of the normalized peakhorizontal eye velocities (in a bin of 10 ms). Allof these saccades were taken from a period inwhich no saccades occurred during control tri-als without stimulation.

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reliable measurements of both optical and behavioralsignals were obtained simultaneously (26).

18. The monkey was required to maintain fixation within2° of a spot of light (0.1° by 0.1°) for 4 to 5 s toreceive a reward (a drop of juice). Stimulation wasapplied in 66 to 80% of the trials. Only on stimula-tion trials could the monkey break fixation before thefixation spot disappeared (after stimulation onset)and still receive the reward. The fixation point disap-peared immediately after the monkey broke fixation.In 77% of the stimulation sites the fixation point waspositioned at the center of the screen; in the remain-ing sites it was positioned between 5° and 13° fromthe center of the screen toward the ipsilateral direc-tion. Eye position was monitored by an infrared eyetracker (provided by Dr. Bouis Devices, Karlsruhe,Germany) and sampled at 250 Hz.

19. E. Shtoyerman, A. Arieli, H. Slovin, I. Vanzetta, A.Grinvald, J. Neurosci. 20, 8111 (2000).

20. Most stimulation sites were located in the superficialcortical layers (first 700 mm). The spatiotemporaldynamics in response to microstimulation in thedeeper layers was similar to that seen in response tostimulation in the superficial layers, consistent withprevious in vitro imaging studies (32, 33).

21. E. Seidemann, D. E. Glaser, A. Arieli, A. Grinvald, Soc.Neurosci. Abstr. 25, 784 (1999).

22. E. Seidemann, H. Slovin, A. Arieli, A. Grinvald, Soc.Neurosci. Abstr. 26, 1075 (2000).

23. The fast signals that we measure (both depolarizationand hyperpolarization) are present only in the wave-length of fluorescence emission. Thus, these signalsare not likely to be contaminated by mechanicalartifacts or fast intrinsic signals. In addition, it hasbeen shown recently, using intracellular recording invivo, that the dye signal measures membrane-poten-tial changes precisely [see figure 23 in (15); similarresults were obtained in a recent in vitro study usingthe same dye (34)]. Furthermore, an in vitro studyhad previously demonstrated that microstimulation-evoked activity measured using real-time optical im-aging is highly correlated with neural activity mea-sured intracellularly (32).

24. Eye movements that had the typical bell-shaped velocityprofile and peak velocity above 100°/s were consideredsaccades. In most stimulation sites the major componentof the evoked saccade was horizontal; we thereforefocused our quantitative analysis on the horizontal com-ponent of the evoked saccades (but see supplementaryfigures). To combine saccade peak velocity measure-ments across experiments (Fig. 4), we first normalizedthe peak velocity of each saccade relative to the maxi-mal peak velocity obtained under all stimulation condi-tions at that site. The peak velocity of saccades in thecontralateral direction was defined as positive, and in theipsilateral direction as negative. Similarly, the averageoptical signal for each stimulation site was first normal-ized relative to the maximal depolarization observed atthis site across all stimulation conditions before beingcombined to yield the grand mean shown in Fig. 4.

25. The properties of stimulation-evoked saccades ob-served here are consistent with those observed inprevious studies (6–8, 31, 35). In particular, Stanfordet al. (35) found that, following a short stimulationtrain in the superior colliculus, saccade amplitudebecomes shorter with increasing saccade latency. Noother study, to our knowledge, reported the existenceof short ipsilateral saccades that occur during earlyhyperpolarization. This may not be surprising giventhat these saccades are comparatively rare (5% ofthe first saccades in Fig. 4) and, therefore, couldeasily have been overlooked by researchers who didnot have access to the time course of depolarizationand hyperpolarization. FEF stimulation can evoke ip-silateral saccades in the “colliding saccade” paradigm(36), when microstimulation is applied during anongoing natural saccade. It remains to be determinedwhether the observed hyperpolarization plays a rolealso in the “colliding saccade” effect.

26. A correlation was computed between the normalizedpeak horizontal velocity for individual saccades (redand cyan x’s in Fig. 4) and the mean normalizedoptical response at the time of saccade initiation(green curve in Fig. 4). To compute the correlationcoefficient (Pearson’s r), we paired each saccade peak

velocity with the level of the average optical signal atthe time of saccade initiation. The statistical signifi-cance of each correlation coefficient was assessedusing an F test with 1 and (n 2 2) degrees offreedom, where n is the number of evoked saccades.The high correlation observed in Fig. 4 remains un-affected if we (i) select only the stimulation sites forwhich both behavioral and optical measurementswere obtained simultaneously (17); (ii) select onlystimulation sites in which stimulation current was#50 mA; (iii) select only stimulation sites that werelikely to fall within the FEF; (iv) compute the corre-lation between the optical signals and the normalizedsaccade amplitude (see supplementary figures); or (v)compute the correlation between the optical signalsand the normalized peak vertical eye velocity forsaccades with a large vertical component (37).

27. J. D. Schall, Rev. Neurosci. 6, 63 (1995).28. C. J. Bruce, M. E. Goldberg, J. Neurophysiol. 53, 603

(1985).29. M. E. Goldberg, C. J. Bruce, J. Neurophysiol. 64, 489

(1990).30. J. Schlag, P. Dassonville, M. Schlag-Rey, J. Neuro-

physiol. 79, 64 (1998).31. M. E. Goldberg, M. C. Bushnell, C. J. Bruce, Exp. Brain

Res. 61, 579 (1986).

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1057 (1994).34. C. C. H. Petersen, B. Sakmann, A. Grinvald, Soc.

Neurosci. Abstr. 27 (2001).35. T. R. Stanford, E. G. Freedman, D. L. Sparks, J. Neu-

rophysiol. 76, 3360 (1996).36. Reviewed in J. Schlag, M. Schlag-Rey, Trends. Neuro-

sci. 13, 410 (1990).37. Supplementary figures are available on Science On-

line at www.sciencemag.org/cgi/content/full/295/5556/862/DC1.

38. All experimental procedures were in accordancewith institutional and NIH regulations. We thank E.Ahisar, S. Barash, R. Born, C. J. Bruce, U. Zohary,M. E. Goldberg, and D. L. Sparks for critical com-ments on earlier versions of this manuscript and E.Tsabary, T. Eliahu, and D. Ettner for technical as-sistance. E.S. was supported by a Koshland Scholaraward. This work was supported by grants fromAbisch-Frankel (E.S. and A.G.) and the GorodetskyCenter and by the Koerber, Margaret Enoch, andGoldsmith foundations.

27 September 2001; accepted 13 December 2001

Chaperone Suppression ofa-Synuclein Toxicity in a

Drosophila Model forParkinson’s Disease

Pavan K. Auluck,1 H. Y. Edwin Chan,2* John Q. Trojanowski,3

Virginia M.-Y. Lee,3 Nancy M. Bonini1,2,4†

Parkinson’s disease is a movement disorder characterized by degeneration of do-paminergic neurons in the substantia nigra pars compacta. Dopaminergic neuronalloss also occurs in Drosophila melanogaster upon directed expression ofa-synuclein, a protein implicated in the pathogenesis of Parkinson’s disease and amajor component of proteinaceous Lewy bodies. We report that directed expres-sion of the molecular chaperone Hsp70 prevented dopaminergic neuronal lossassociated with a-synuclein in Drosophila and that interference with endogenouschaperone activity accelerated a-synuclein toxicity. Furthermore, Lewy bodies inhuman postmortem tissue immunostained for molecular chaperones, also sug-gesting that chaperones may play a role in Parkinson’s disease progression.

Parkinson’s disease (PD) is the second mostcommon neurodegenerative disorder and isassociated with resting tremor, postural rigid-ity, and progressive degeneration of dopami-nergic neurons in the substantia nigra parscompacta (SNpc). Characteristic pathologicalfeatures of PD include Lewy bodies (LBs),which are juxtanuclear ubiquitinated protein-aceous inclusions in neuronal perikarya, and

Lewy neurites (LNs), which are similar pro-tein aggregates found in neuronal processes(1). LBs and LNs are also characteristic ofother neurodegenerative diseases, includingthe LB variant of Alzheimer’s disease(LBVAD) and dementia with LBs (DLB).a-synuclein is a major constituent of inclu-sions found in these disorders, known assynucleinopathies (2–4). Moreover, two mis-sense mutations in the gene encodinga-synuclein are linked to dominantly inherit-ed PD, thereby directly implicatinga-synuclein in disease pathogenesis (5, 6).

In Drosophila, directed expression ofa-synuclein induces selective and progres-sive loss of dopaminergic neurons, as wellas formation of a-synuclein–positive pe-rinuclear and neuritic filamentous inclu-sions similar to LBs and LNs (7 ). Inclusion

1Department of Neuroscience, 2Department of Biolo-gy, 3Center for Neurodegenerative Disease Researchand Department of Pathology and Laboratory Medi-cine, and 4Howard Hughes Medical Institute, Univer-sity of Pennsylvania and University of PennsylvaniaSchool of Medicine, Philadelphia, PA 19104, USA.

*Present address: Department of Biochemistry, TheChinese University of Hong Kong, Shatin, Hong Kong.†To whom correspondence should be addressed. E-mail: nbonini@sas.upenn.edu

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