Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells

BRAIN RESEARCH ELSEVIER

BrainResearch738(1996)1-7

Research report

Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells Carla B. Green “*, Joseph C. Besharse a, Martin Zatz b aDepartment of Anatomy and Cell Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7400 USA b Laboratoq of Cell Biology, National Institutes of Mental Health, Bethesda, MD 20892, USA

Accepted11June1996

Abstract Chick pineal cells contain a circadian oscillatorthat drives rhythmic synthesisand secretionof melatonineven in dispersedcell culture. Here, we demonstratethat the mRNA encodingtryptophm hy~oxylase (TpH), the first enzyme in the melatonin synthetic pathway,is expressedrhythmicallyunder the controlof the circadianclock. TPH messagelevels doubledbetweenearly day and early night, under both cyclic lightingand constantlightingconditions.The amplitudeof the TPH mRNArhythmwas increasedto 4-foldby culturingthe cells at 43.3°Cfor 48 h instead of 36.7”C.Additionof forskolinto the culturesin early day produceda modest increase (50%) in TPH message levels but had no effect at other times. Because TPH mRNA levels are regulated by the endogenouspineal circadianclock, this providesa valuablesystemin whichto studythe molecularmechanismof clock controlof gene expression. Keywords:

Melatonin;Forskolin; Circadianrhythm

1. Introduction The chicken pineal gland synthesizes melatonin rhythmically, producing high levels during the night and low levels during the day [3]. These rhythms are controlled both by light and by a circadian clock. Because pineal cells can be dispersed and grown in static culture, where they maintain rhythmic melatonin synthesis and a functional and entrainable circadian oscillator [9], they have provided a valuable in vitro system in which to study the acute and clock controlled aspects of melatonin regulation. Melatonin synthesis is regulated in two distinct ways: phase shifting agents (such as light or temperature) impinge on the circadian clock, resulting in a stable change in the phase of the melatonin rhythm, while acute regulators act on the melatonin pathway distal to the clock mechanism and alter the melatonin output without causing phase changes of the rhythm in subsequent cycles. Light acts on melatonin both by phase shifting the rhythm (in a phasedependent manner) and by acutely inhibiting melatonin synthesis [3,16,30]. Temperature increases can both phase shift the rhythm and acutely increase melatonin output *Corresponding author.KansasCity,KS66160-7400. Fax:(1)(913) 588-2710; e-mail:[email protected]

[26]. Other effecters, such as cAMP [10], act only acutely, to increase melatonin output, but do not alter the phase of the rhythm [27]. Although the molecular components of the clock and the mechanisms by which the clock exerts its control are not well understood in any system, it is becoming clear that macromolecular synthesis plays an important role. Both clock-controlling genes and clock-controlled genes have been identified in invertebrate systems [11]. In vertebrates, tryptophan hydroxylase (TPH), the first enzyme in the biosynthetic pathway converting tryptophan to melatonin, was recently identified as a clock-controlled gene in Xertqws retina [12,14]. There, TPH is regulated transcriptionally by a retinal circadian clock; TPH mRNA levels are low early in the subjective day and increase to a peak in early subjective night. Like chick pineal, Xenopu.s retina contains an endogenous circadian oscillator that regulates rhythmic melatonin synthesis and is functional and entrainable in culture [2,5]. Within the retina, TPH is expressed in photoreceptors [13], the cells that contain the circadian oscillator responsible for regulating rhythmic melatonin synthesis [6]. In the experiments described here, we tested the hypothesis that pineal TPH mRNA is regulated rhythmically in a manner similar to that in Xenopus retina. Our results

0006-8993/96/$15.00 Copyright01996 ElsevierScienceB.V.Allrightsreserved. PZZS0006-8993(96)00743-

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illustrate that TPH mRNA levels are expressed rhythmically under the control of a circadian clock. We also tested the effects of several other known regulators of melatonin synthesis, including light, temperature, and forskolin.

2. Materials and methods

dissolved in alcohol, and used at a final concentration of 0.1% alcohol or less. Norepinephrine bitartrate was purchased from Sigma (St. Louis, MO), dissolved in 10 mg/ml ascorbate and used at a final concentration of 0.1 mg/ml ascorbate. 2.3. Analysis of TPH mRNA levels

2.1. Pineal cell culture White leghorn chicks were received 1–2 days after hatch from Clay’s Hatchery (Blackstone, VA). Pineal cells were dispersed in trypsin and plated in modified McCoy’s 5A Medium (GIBCO 380-2230) containing 25 mMHEPES buffer, L-glutamine, penicillin, streptomycin, 10% heat-inactivated fetal bovine serum, and IO~ochicken serum as described previously [30]. Experiments used cells from up to 125 glands in 20 wells (6-well plates) or 42 wells (12-well plates). Cells were fed by exchange of medium at least daily. Days in culture are numbered successively from the day of plating (day 1). Cells were fed with the plating medium (modified by omission of fetal bovine serum) through day 3. On day 4 they were switched to the same medium without added serum and with an additional 10 mM KC1 added. The effects of feeding schedule, media, sera and potassium on melatonin production were described previously [30]. 2.2. Light cycles, temperature, and drugs Cells were maintained under 57. C02 in air in tissue culture incubators containing red lights, white lights and timers as described previously [30]. They were all exposed to a cycle of 12 h white light (L) and 12 h red light (R) in either ‘straight’ (LR 12:12) or ‘reversed’ phase (RL 12:12). In these schedules, L acts as ‘day’ and, by convention, starts at Zeitgeber Time (ZT) zero; R acts as ‘night’ and started at ZT 12. Thus, in this schedule, ZT 6 is ‘midday’ and ZT 18 is ‘midnight’. Lighting at harvest, however, need not correspond to that used for entrainment. In some experiments, cells were maintained in LR or RL throughout the experiment; in others, cells were switched from LR or RL to constant red light (RR) or white light (LL) on day 5. Similarly, cells were all entrained at 36.7°C (98”F). In some experiments, cells were maintained at 36.7°C throughout the experiment; in others, cells were switched from 36.7 to 43.3°C (110”F) on day 4 or 5. Exposure of various groups to different lighting conditions and\or temperatures required the simultaneous use of several incubators. Up to 5 identically configured Napco 4100 incubators were used. Cells were harvested in L or R as indicated. Media was removed from the wells; cells were washed in place, scraped into phosphate-buffered saline, transferred, and pelleted by centrifugation. Cell pellets were frozen rapidly in solid COZ and stored at –70”C. Forskolin was purchased from Sigma (St. Louis, MO),

Isolation of RNA from chick pineal cells was done as previously described for Xenopus retinas [14]. A typical yield of RNA from a single well of a 6-well plate was 4–5 pg. Routinely, one fourth of the total RNA yield was used for northern blot analysis of TPH mRNA levels. Northern blot hybridization was done as described in De et al. [8]. Briefly, RNA was denatured for 5 min at 65°C in a solution of MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA, pH 7.0) containing 50% formamide and 2.2 M formaldehyde, and then separated by electrophoresis in a 1% agarose formaldehyde gel [19]. RNA was transferred to Nytran filters (Schleicher and Scheull, Keene, NH) [22] and the filters were then prehybridized and hybridized as described in Andrews et al. [1] with 2 X 106 cpm/ml probe at 65°C for 16 h. Filters were washed for 1 h at 65°C in 1X SSPE, 0.1YoSDS, followed by a second l-h wash at 65°C in 0.3X SSPE, 0.1% SDS. Filters were then exposed to Kodak X-OMAT X-ray film. Filters were then treated briefly with RNaseA by first incubating in 2X SET (20X SET is 3 M NaCl, 0.6 M Tris-HCl, pH 8.0, 0.04 M EDTA) at 37°C for 20 rein, followed by treatment with 1 pg/ml RNaseA (Sigma, St. Louis, MO) in 2X SET and 100 p,g/ml BSA at 37°C for 5 min. This was followed by a final wash in 2X SET, 0.590 SDS for 30 tin at 37°C and the filters were then re-exposed to film Samples were normalized by determination of the levels of ~-actin message or 18S rRNA. Filters were stripped by boiling twice for 10 min in O.OIX SSPE, 0.1% SDS. They were re-exposed to X-ray film to insure complete removal of probe, and were then prehybridized and hybridized, as described above, with riboprobes made from chicken (+ actin cDNA [7] or 18S rRNA clones. Washing conditions were as described above. Anti-sense riboprobes were synthesized using the RNA Transcription Kit from Stratagene and were purified with NucTrap gel filtration columns (Stratagene, La Jolla, CA). Quantitation of message levels was done directly from the radiolabeled filters using the Phosphoimager (Molecular Dynamics, Sunnyvale, CA). Total counts per TPH band (minus background) were divided by the total counts per band of ~-actin or 18S rRNA (minus background) to get numbers that were normalized for differences in lane loadings. Final results are usually expressed as relative to the control sample, which in most cases is the untreated ZTO sample. Statistical analyses were done by one factor analysis of variance (ANOVA).

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3. Results

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A portion of the Xerzopus retinal TPH cDNA clone [12] corresponding to the coding region, was used to generate riboprobes for detection and quantitation of chick pineal

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Zeitgeber Time (hours) Fig. 1. TPH mRNA expression is rhythmicin diurnallighting.Chick pinealcellswereculturedfor5 daysin cycliclightandharvestedat the timesindicated.RNAisolatedfromthesecellswereanalyzedforTPH mRNAby Northernblot hybridization(A) followedby subsequent phosphoimager analysisfor quandtation(B). A: Northenblots were as describedin themethods. hybridized with a Xerropus TPHriboprobe Thechickpined laneseachcontain1 ~g of totalRNA.TheNorthern blot containsa laneof Xenopus retinal RNA (1 Kg totrd RNA) as a positive control and a lane of chicken liver RNA (5 Kg total RNA) as a negative control to show specificity of the Xenopus probe for chicken TPH. Xenopus TPH mRNA normally is present as two bands of similar size. Blots were afso probed with chicken ~-actin riboprobes for normalization of the lanes. B: TPH message levels were quantified as ratios of TPH mRNA to actin mRNA. ZT O is arbitrarily set to 1.0 and the other time points are expressed as ratios relative to this point. The dark hatched histogram bars indicate harvest after and in red light, white bars indicate harvest after and in white light. ZT 6 is mid-day and ZT 18 is mid-night. The data shown are the means of samples collected from three separate experiments (n= 7). Error bars represent the S.E.M. TPH mRNA levels at ZT 12 are significantly different from those at ZT O, ZT 6 and ZT 18 (P< 0.01, ANOVA, Student-Newmar-Keuls).

Fig. 2. TPH mRNA expression is controlled by a circadian lock, Chick pineat cells were cultured in cyclic light for 5 days and then transfened to constant red light. Cells were harvested at the times indicated and TPH mRNA levels determined by Northern blot anrdysis and phosphoimager quantitation. Bar at the top of the histogram illustrates lighting condition throughout this time period. White bar indicates culture in light, dark hatched bars indicate red light in subjective night and light hatched bar indicates red light in subjective day, The white histogram bar indicates harvest after and in white light at the end of the day, the dark hatched histogram bar indicates harvest after and in red light at the end of the night (ZT O) or the end of the day (ZT 12). Message levels were normalized by measuring chick ~-actin mRNA levels and expressed reIative to the ZT Otime point which was arbitrarily set to 1.0. These data represent averages from three separate experiments (total n = 7). Error bars represent standard error of the mean. TPH mRNA levels at both ZT 12 times are significantly different from ZT O (P <0.001, ANOVA, Student-Newmar-Keuls)

TPH rrtRNA. Initial experiments tested varying hybridization and washing conditions to determine optimal conditions for specific hybridization to the chicken TPH message (data not shown). All blots included a lane of Xenopus retinal RNA as a positive hybridization control and a lane of chicken liver RNA, which served as a negative control. This tissue was chosen because it does not express TPH, but should express the message for the hepatic enzyme phenylalanine hydroxylase which is homologous to TPH [15]. Conditions were identified that allowed detection of a single band in the chick pineal RNA lane, and no band in the chicken liver RNA lane, even though this lane contained five times more RNA (Fig. 1A). It is crucial to verify specifity when using heterologous probes and these controls provide evidence that the observed hybridization is to the authentic chicken TPH rnRNA. Northern blot analysis was carried out on RNA isolated from chick pineal cells, cultured in 12 h white: 12 h red, cyclic light (LR), harvested at 6-h intervals throughout the day (Fig. 1A). Quantitation of the hybridization signal

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Experimental Treatment Fig, 3. Effect of increased temperature on TPH MRNA levels, Chick pineal cells were all cultured in cyclic light for 4 days at 36.7 ‘C and then some of the cultures were transferred to 43.3 ‘C for 24 or 48 h before the time of harvest on day 6. Cultures were harvested at either the end of the night (ZT O)or at the end of the day (ZT 12). RNA from the cultures was isolated and TPH mRNA levels were determined as previously described. The dotted horizontal lines represent the average values for the ZT Oand the ZT 12 controls. These data are from two experiments. The ‘error bars’ shown represent the range of values obtained (n = 2–4).

from these blots revealed a 2-fold rhythm in TPH mRNA levels (Fig. IB). The TPH mRNA rhythm shows an amplitude and temporal pattern similar to the TPH message rhythm observed in Xenopus retina [12,14], with low levels early in the day and peak levels in early night. When chick pineal cultures were transfemed into constant red light, the TPH mRNA rhythm was maintained (Fig. 2), indicating that this rhythm is under the control of a circadian clock and not driven by changes in ambient lighting. A recent report showed that increasing the culture temperature of the chick pineal cells from 36.7°C (98”F) to 43.3°C (110”F), results in increased melatonin output [26]. We tested the effects of increasing culture temperature on TPH message levels. RNA was isolated from cultures exposed to temperatures of 43.3°C for 24 h or 48 h (Fig. 3) prior to harvest and TPH mRNA levels were compared to those of controls maintained at 36.7°C. Harvests were done at both ZT O (dawn) and ZT 12 (dusk). Increased TPH message levels were observed with the higher temperatures in all cases. The increases after 24 h of heat were smaller (23% at ZT O, 30% at ZT 12) than those seen after 48 h of exposure to 43.3°C (48% increase at ZT O and 9790 increase at ZT 12). Also, morning (ZT O) increases were smaller than evening (ZT 12) increases (Fig. 3). Similar results were observed in another experiment. These data are consistent with the effects of 43.3°C on melatonin synthesis [26], where smaller increases in melatonin production were seen after 1 day at 43.3°C and greater increases were observed after 2 days at the higher tempera-

ture, and with most of the increases seen at night, resulting in nocturnal melatonin production about twice as high as in cultures maintained at 36.7°C. Studies of cultured chick pineal cells have identified cAMP as an acute effecter of melatonin production [10,27,28]. We tested the effect of forskolin on TPH mRNA levels. Initially, 12-h pulses of forskolin (1 PM) were given to the cultures, followed by harvest at ZT Oand ZT 12 (Fig. 4A). TPH mRNA levels were unchanged at ZT O. On average, there was a small increase at ZT12 (32%; significant by 1 factor ANOVA, Student-NewmanKeuls, P < 0.05),but the magnitude of the response varied considerably among experiments. We then tested the effects of 6-h pulses of forskolin, given at four different times of day (Fig. 4B). No changes in TPH rnRNA levels were observed when forskolin was given in either the first half of the night (ZT 12–ZT 18) or in the last half of the night (ZT 18–ZT O). However, when forskolin was given in the morning, from ZT O to ZT 6, TPH levels increased 50%, rising to levels normally seen at ZT 12. We verified this result in an experiment testing the effects of forskolin pulse duration at different phases. TPH mRNA levels in cells harvested at ZT 6 after treatment with 2, 4, or 6-h pulses of forskolin increased more, relative to controls, than did levels in cells harvested at ZT 12 after 2, 4, or 12 h forskolin pulses (data not shown). As before, 6-h pulses from ZT O to ZT 6 produced increases of 50% in TPH message levels as compared to the ZT 6 control. Since increasing cAMP levels with forskolin had rela-

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were in constant red light (Fig. 4A). Twelve-h pulses of norepinephrine also produced no change in TPH message levels at ZT O or ZT 12 (a single experiment, data not shown). These results indicate that the expression of TPH is not much affected by conditions that decrease the levels of cAMP.

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4. Discussion

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Experimental Treetment Fig. 4. Effects of forskolin, white light, and red light on TPH mRNA levels. A: chick pineal cells were cultured under cyclic red and white light as described in Materiafs and methods and then subjected to pulses of the indicated treatment prior to harvest. Cells were exposed to forskolin (1 wM) under control lighting conditions for 12 h prior to harvest at ZT O or ZT 12. For the perturbed lighting conditions, some samples harvested at the end of the night (ZT O) received light at night (24 h white light prior to harvest), and some samples harvested at the end of the day (ZT 12) received red light in the day (24 h red light prior to harvest). TPH mRNA levels were determined as previously described and are represented relative to ZT O, whicb was arbitrarily set to 1.0. The data shown were collected in three separate experiments. Sample number for each treatment group rangesfrom 5 to 8. The dotted lines represent the average vafues for the ZT O and the ZT 12 controls. Error bars represent standard error of the mean. TPH mRNA levels at ZT O are significantly different than ZT12 (P < 0.01). None of the treatments harvested at ZT O were significantly different from the ZT O control. Perturbed lighting conditions did not affect message levels at either ZT Oor ZT 12. mRNA levels after forskolin treatments harvested at ZT 12 were significantly different from control (P < 0.05; ANOVA, Student-Newman-Keuls). B: chick pineal cells were cultured in cyclic light for 5 days and then subjected to 6-h pulses of medium containing 1 WM forskolin (FSK) or control medium during the 6 hours prior to harvest. TPH rnRNA levels were determined as previously described. The data shown are from one experiment with 3 weIls per group. TPH mRNA levels after forskolin treatment were significantly different from control only at ZT 6 (P < 0.05; ANOVA, Student-Newman–Keuls).

tively small and restricted effects on TPH mRNA levels, we tested the effects of decreasing cAMP levels with white light or norepinephrine [29]. Differences between ZT O and ZT 12 were maintained in constant white light just as they

The mRNA encoding TPH, the first step in the melatonin biosynthetic pathway, is expressed rhythmically in chick pineal cells in culture. The message levels are low early in the day and peak in early night with a 2-fold amplitude in both white: red cyclic light and in constant red or white light (Figs. 1 and 2 and 4A). This indicates that the expression of TPH message is controlled by an endogenous circadian clock. The rhythm of TPH mRNA expression is similar, both temporally and in amplitude to that observed in Xerzopus retina [12,14]. Additionally, although RNA levels were not examined, TPH enzyme activity was found to be regulated by a circadian clock in a similar manner in chicken retina [21]. All three systems, Xenopus and chick retina and chick pineal, contain an endogenous circadian clock complete with photoentrainment pathways which are maintained in vitro. Since TPH is part of the melatonin biosynthetic pathway, we began our analysis of TPH message regulation by assessing the effects of perturbations known to change melatonin output. For example, it is known that changing the culture temperature of chick pineal cells alters the levels of melatonin production. Maintaining the cells at 43.3°C instead of 36.7°C doubles the amplitude of the melatonin rhythm [26]. Melatonin production was increased at all times of day, but the increases during the day (melatonin troughs) were small, while the increases at night (melatonin peaks) were large, resulting in melatonin levels twice as high as seen at 36.7”C. The increased melatonin production was seen after one day at the higher temperatures, but were not maximal until cultures had been held at the higher temperatures for 2 days [26]. TPH message levels increase in reponse to culture at 43.3°C in a similar manner to the changes in melatonin production (Fig. 3). Like melatonin, small increases in TPH message are seen after 24 h, and much larger increases after 48 h. TPH message levels were twice as high at ZT 12 after 48 h at 43.3°C as they were at 36.7”C, while only small increases were observed at ZT O. This resulted in a change of amplitude from about 2-fold at 36.7°C to about 3-fold at 43.3”C. The mechanism of the temperature effect on TPH message levels (or on melatonin production) in these cells is unknown. Although the increase in TPH mRNA levels (over 48 h) seems too slow to be the direct result of a heat shock response, incubation of chick pineal cells at 43.3°C has been shown to induce several heat shock proteins [23]. It is possible that the effect of temperature on TPH mRNA

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levels is due to an indirect effect of heat shock on message stability. It is also possible that increasing the culture temperature raises the overall metabolism of the cells resulting in generally higher transcription levels; however, this seems an unlikely explanation since the TPH mRNA levels reported here change relative to actin mRNA. Exposure of chick pineal cells to light for 24 h resulted in no change in TPH mRNA levels measured at ZT O(Fig. 4A), although these cells are well known to respond to this light pulse by significantly decreasing melatonin production. The lack of effect of light on message levels is difficult to interpret since they were only measured at the low point, but this result is consistent with data from Xenopus retina and from chicken retina. In Xenopus retina, the effects of light throughout the night were not examined, but a 3-h exposure to light in early night produced no measurable acute effects on TPH message levels [14]. In chicken retina, TPH message levels were not examined, but TPH enzyme activity was only marginally affected by constant light exposure (30% decrease at night compared to 85% decrease in N-acetyltransferase activity) [21]. The role of cAMP in acute regulation of melatonin is well established in the chick pineal and in Xenopus retina. Agents that increase cAMP levels such as forskolin, 8-BrcAMP, and calcium increase melatonin production [18,24,27]. Agents that decrease cAMP levels such as light or norepinephrine (pineal) or dopamine (retina) decrease melatonin levels [16,17,28,29]. cAMP appears to be working to regulate melatonin downstream of the circadian oscillator, since altering levels of cAMP do not result in phase-shifts of the melatonin rhythm [27-29]. cAMP appears to have a phase-dependent effect on TPH mRNA levels in chick pineal cells (Fig. 4). Forskolin given at night appears to have little effect on TPH message levels, but TPH message levels do appear to increase when forskolin is given during the day in the morning hour. At the concentrations used in these experiments, forskolin specifically and directly activates adenylate cyclase [20] and results in sustained increases in cAMP in cultured chick pineal cells [25]. Lowering cAMP levels (light at night or norepinephrine treatment) produced no measurable effect on TPH message levels. Likewise, in Xertopus retina, dopamine treatment in early night (which decreases cAMP levels) also has no effect on levels of TPH mRNA [14]. This analysis of TPH is based on steady state mRNA levels. Thus, we do not know if changes in TPH mRNA levels reflect changes in synthesis or changes in degradation. However, regulation of TPH at the level of synthesis is observed in both Xenopus retina and chicken retina. In Xenopus retina, the rhythmic change in TPH message levels can be accounted for by changes in transcription initiation [12]. In chicken, no data are available concerning transcription, but night time increases in TPH activity require new protein synthesis [21].

Very little is known about regulation of the TPH gene in any system. However, a recent report suggests that cAMP may play a role in activation of the human TPH promoter; 5-fold increases in promoter activity were measured in transected rat pinealocytes after addition of 8-brcAMP [4]. In chick pineal cells, forskolin treatment produced only a modest effect (at best, 50%) on endogenous TPH message levels (Fig. 4), smaller than that obtained with changes in either temperature or time of day. Perhaps the importance of cAMP-mediated regulation of TPH gene expression varies among systems and\or species. It is not known whether the rhythmic expression of TPH mRNA contributes to the regulation of rhythmic melatonin synthesis in chick pineal cells. However, the levels of TPH mRNA are clearly under the control of the pineal circadian clock. Because this chick pineal cell culture system contains all the elements of a functional circadian system, it may provide the means for future studies of the mechanisms of clock regulation of rhythmic messages, such as TPH, at the level of transcription.

Acknowledgements We thank James R. Heath, III for expert technical assistance and Dr. Donald Cleveland for providing the chicken (3-actin clone. This work was partially supported by NIH grants EY06489 (CBG) and EY02414 (JCB).

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