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Address correspondence to Michel Jacquet, Institut de Génétique et Microbiologie, UMR 8621 CNRS Université, Bat 400, Université Paris-Sud, 91405 Orsay cedex, France. Tel.: 33-169-15-7963. Fax: 33-169-15-4629. E-mail: michel.jacquet{at}igmors.u-psud.fr
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Abstract |
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Key Words: stress response; signaling; nuclear localization; imaging; computational model
J. De Mey's present address is École Supérieure de Biotechnologie de Strasbourg, CNRS-UMR 7100, 1, Bld. Sébastien Brant, F-67400 Illkirch-Graffenstaden, France.
* Abbreviation used in this paper: STRE, stress response element.
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Introduction |
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Using high-resolution time-lapse video microscopy on single cells, we followed the kinetics of the nuclear translocation of an Msn2GFP fusion protein. Here we show that yeast cells sense as a stress light emitted by the microscope, as evidenced by Msn2 migration into the nucleus. Moreover, and unexpectedly, the populations of Msn2 and Msn4 molecules display an oscillatory behavior, shuttling repeatedly between the nucleus and cytoplasm with a periodicity of a few minutes. The DNA-binding domain of Msn2 is not essential for this periodic behavior. The cAMPPKA system interferes with the nucleocytoplasmic shuttling by controlling the sensitivity of the stress-dependent transport of Msn2 into and out of the nucleus. In the absence of PKA, oscillatory shuttling of Msn4 continues while Msn2 is permanently localized in the nucleus. The occurrence of repetitive nucleocytoplasmic shuttling raises the possibility that Msn2 and Msn4 participate in autoregulatory loops, producing oscillations in their subcellular localization. A computational model based on such a regulation provides a theoretical framework accounting for the main observations on stress-induced oscillatory shuttling of the transcription factors.
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Results |
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Light triggers migration of Msn2GFP to the nucleus
To assess whether the illumination of cells under the microscope was responsible for the transfer of Msn2GFP to the nucleus, we set the intensity of light to a minimum and took pictures every 2 min instead of 10 s. Under these conditions, as shown in Fig. 2 A, Msn2GFP remained cytoplasmic during the 1-h period of observation, in contrast to the oscillatory shuttling observed under usual conditions with a higher light intensity (Fig. 2 C). However, some abortive episodes of shuttling, occurring in <2 min, were observed in a subset of the cells in the conditions of Fig. 2 A. We also determined the proportion of labeled nuclei in a cell population fixed with formaldehyde before examination, and found Msn2GFP in the nucleus of <3% of several hundred cells. This is to be compared with an average occupancy of the nuclei ranging from 30 to 60% under illumination at higher light intensity during regular time-lapse recording. Thus, we may conclude that light induces nuclear localization of Msn2. The observation of a delayed cell division cycle in most experiments is another indication of a cellular stress response to illumination (Belli et al., 2001).
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Uncoupling oscillations from transcription activation
Entry into the nucleus per se is not sufficient for Msn2 activation. Indeed, as described by others and confirmed by us (unpublished data), despite nuclear localization of Msn2 in msn5 exportin mutants, transcriptional activation occurs only if a stress signal is applied to the cell. Once in the nucleus, activated Msn2 must interact with the regulatory elements of responsive genes through the transcription machinery. By removing the zinc finger domain of Msn2, we investigated whether the fixation of Msn2 to the STREs was required for oscillatory behavior. The truncated construct continues to respond to stress in regard to its nuclear localization and oscillates with a period similar to that observed for the complete protein (Fig. 3; see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200303030/DC1). To eliminate the possibility that the truncated Msn2 was driven through association with Msn2 or Msn4, we checked that the oscillatory behavior was maintained in a strain with a double deletion of these two genes (unpublished data). Therefore, the mechanism underlying the oscillations involves only the control of nuclear localization and not the STRE-dependent gene activation process.
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Differential sensitivity of Mns2 and Mns4 oscillations to cAMPPKA
Both the NLS and the putative nuclear export or cytoplasmic retention signal of Msn2 contain PKA-dependent phosphorylation sites whose substitutions alter nuclear import or export, respectively (Gorner et al., 1998, 2002). Thus, Msn2 nucleocytoplasmic shuttling is modulated by the activity of the cAMPPKA pathway. In the absence of PKA, in a cell deleted for the three genes TPK1, TPK2, and TPK3, which encode the kinase catalytic subunits, Msn2GFP stays permanently in the nucleus of all the cells (Fig. 4 C). To perform these experiments, the growth defect due to the lack of PKA was rescued by a deletion in the gene of kinase YAK1 (Garrett et al., 1991); we controlled that this deletion does not significantly alter the oscillatory behavior of Msn2GFP (Fig. 4 B). Conversely, high levels of cAMP in the phosphodiesterase mutant rca1/pde2 (Wilson et al., 1993) maintain Msn2GFP in the cytoplasm of most cells (Fig. 4 A). However, the shuttling mechanism was still operative in these cells. Oscillations indeed resume when an additional stress, such as osmotic or oxidative shock, is applied (unpublished data). Thus, the cAMPPKA system increases the threshold level of the Msn2 response without disrupting the mechanism of oscillations. The role of PKA in the control of translocation of Msn2 was further demonstrated by the use of a mutant of BCY1 (BC-N4), which encodes the regulatory subunit of PKA, deleted from its NH2-terminal part involved in its nuclear localization (Griffioen et al., 2001). In this mutant strain, Msn2GFP enters the nucleus upon stress but never leaves it (Fig. 4 D), which indicates a key role for nuclear PKA in the export.
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The dynamic behavior predicted by the model is illustrated in Fig. 6 (BD) for increasing values of parameter VKS, which measures the maximum activity of the stress-activated enzyme that acts on Msn2 by catalyzing the conversion of M into M* in the scheme of Fig. 6 A. We assume that the value of parameter VKS increases with stress in the cytosol, but a similar model could be generated with an effect of stress within the nucleus. In the absence of stress, for a low value of VKS (Fig. 6 B), the system reaches a stable steady state in which Msn2 is located predominantly in the cytoplasm; this stable steady state is reached after damped oscillations. For a large stress corresponding to a high value of VKS (Fig. 6 C), the situation is reversed; close to 80% of Msn2 now resides in the nucleus while some 20% of the transcription factor is in the cytosol. It is at intermediate values of VKS (Fig. 6 D) that sustained oscillations occur, with Msn2 shuttling back and forth between the cytosol and the nucleus. In the case illustrated in Fig. 6 D, the total nuclear and cytosolic fractions of Msn2 oscillate in antiphase between 10 and 90%, with a period of
6 min.
The model predicts the existence of a range of VKS values producing Msn2 oscillations, bounded by two critical values of the stress enzyme activity (Fig. 6 E). As observed in the experiments, outside this domain, a stable steady state is found in which Msn2 is predominantly either in the cytosol, in the absence of stress, or in the nucleus, when stress is strong. The variation of the period in the oscillatory domain remains less than a factor of two (Fig. 6 F). Interestingly, when sustained oscillations begin as VKS passes its first, lower bifurcation value, the magnitude of Msn2 oscillations is relatively reduced (Fig. 6 E). Such behavior could account for the "aborted" oscillations, which are sometimes observed in the experiments. The oscillations predicted by the model are much more regular than those shown in Fig. 1. Fluctuations due to molecular noise could be included in the model by resorting to stochastic simulations. Such simulations yield good agreement with the predictions of deterministic models, as previously shown for circadian oscillations (Gonze et al., 2002; Goldbeter, 2002).
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Discussion |
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The analysis of a computational model shows that, as in the case of other oscillatory phenomena such as Ca2+ oscillations (see Box 1 figure in Goldbeter, 2002), such periodic behavior occurs only in precise conditions, in a range bounded by critical parameter values. At low stress intensities, the transcription factors remain in the cytoplasm, whereas at high stress levels, they remain in the nucleus. Only at intermediate strength of the stress do they oscillate between the cytoplasm and nucleus. These observations, accounted for by the model, explain why oscillations occur only for sufficiently high light intensity and why moderate ethanol stress triggers oscillations, whereas at higher levels, ethanol sends Msn2 permanently to the nucleus. The occurrence of aborted translocations, observed in some cells, is consistent with the existence of thresholds in the molecular mechanism underlying oscillations. Aborted transitions can also be viewed as corresponding to the low-amplitude oscillations predicted by the computational model at low stress intensity (see the envelope of the oscillations as a function of stress in Fig. 6 E).
Our results demonstrate the ability of light to trigger nuclear localization of Msn2 and Msn4. The shuttling behavior of these stress-sensitive transactivators was not induced at very low intensity but occurred readily with stronger illumination. The time spent in the nucleus by Msn2 also correlated with the intensity of light (unpublished data). The excitation of GFP fluorescence is likely to release reactive oxygen species that could then produce an oxidative stress. More generally, these observations suggest that the state of the cells containing GFP molecules can be modified by simple observation with a fluorescent microscope.
The sensitivity of the cells is controlled by the activity of the cAMPPKA pathway. In contrast to normal cells, in which oscillations were induced by illumination under the microscope, in cells defective in phosphodiesterase Pde2, additional stress was required to trigger nuclear migration of Msn2. Thus, the cAMPPKA system controls the sensitivity of Msn2 oscillations by affecting the kinetics of import and export of Msn2. Indeed, PKA-dependent phosphorylation negatively regulates the NLS of Msn2 (Gorner et al., 1998). In addition, we showed here that delocalization from the nucleus of the PKA with a truncated regulatory subunit prevents exit of Msn2 from the nucleus. As a consequence, without PKA, Msn2GFP accumulates in the nucleus (Fig. 4) whereas Msn4GFP still oscillates in the same strain. Thus, Msn2 and Msn4 present differences in regulation by, or sensitivity to, cAMP, as previously reported at the level of phosphorylation (Garreau et al., 2000). With a narrow window for transcription activation due to thresholds in transport to the nucleus, it might be useful for the cell to have two sensors with high and low sensitivity. Msn2 and Msn4 are involved in lower and higher stress response, respectively.
The large variability in shuttling behavior observed among cells in the same microscopic field (Fig. 1) likely reflects cellular heterogeneity in sensitivity to stress. In line with a recent study that demonstrated, by FACS® analysis, the heterogeneity of gene expression in response to stress in a yeast population (Attfield et al., 2001), the present data highlight the variability at the single-cell level of a so-called homogenous population. In spite of this heterogeneity, the time required for Msn2 and Msn4 to enter the nucleus is quite constant among cells, irrespective of the triggering conditions. Such transfer time is similar to that measured in a mammalian cell for the entry of a GFP-fused protein possessing an NLS (Ribbeck and Gorlich, 2001).
In the cases where an oscillatory expression program has been described (Darlington et al., 1998; Lev Bar-Or et al., 2000), an autoregulatory loop involving the expression of some controlling element is involved. Thus, periodic nucleocytoplasmic shuttling of the transcriptional regulators PER and TIM occurring on a circadian time scale in Drosophila involves autoregulation of gene expression (Sehgal et al., 1995; Young, 1998). Autoregulation of protein synthesis and degradation underlies oscillations of the tumor suppressor p53, which occur with a period of 3 h (Lev Bar-Or et al., 2000). On a similar time scale, oscillations have recently been described for NF
B (Hoffmann et al., 2002). The phenomenon involves synthesis and degradation of an inhibitor and has been accounted for by a computational model. Yet another example of oscillatory gene expression is provided by the segmentation clock that controls somite formation in embryogenesis with a periodicity of
90 min (Maroto and Pourquie, 2001). The latter oscillations, based on negative autoregulatory feedback on transcription (Dale et al., 2003), have recently been shown to occur with a 2-h period in serum-treated cultured cells (Hirata et al., 2002).
We have ruled out the possibility that the mechanism of Msn2 oscillations involves the control of protein synthesis because oscillations are not affected by the addition of cycloheximide (unpublished data). Moreover, the fact that oscillations occur with an Msn2 truncated from its DNA-binding domain, which is not capable of transcriptional activation, eliminates an effect directly mediated by STRE-dependent gene expression. The molecular mechanism underlying the oscillations remains to be further characterized. The fact that protein synthesis and DNA binding of Msn2 are not required suggests a control at the level of import or export of the transcription factors.
To our knowledge, the present experimental observations provide the first report of rapid oscillatory shuttling of a transcriptional activator between the cytoplasm and nucleus. Because they are not based on transcriptional regulation or de novo protein synthesis, oscillations in the subcellular localization of Msn2 inaugurate a new class of periodic processes controlling gene expression. The proposed computational model provides a unifying explanatory framework for the observations and should help to unravel the molecular mechanism of the phenomenon. It predicts the existence of one or more novel components in an autoregulatory loop that primes Msn2 and/or Msn4 for nuclear export. These components are likely to be kinases and/or phosphatases. In regard to the physiological significance of oscillations, shuttling between the cytoplasm and nucleus permits the sensing by Msn2 of the state of the cell and its adaptation in both compartments to the level of stress. Oscillations allow the generation of maximum responses during brief episodes during which the transcription factor reaches high levels that could have detrimental effects if maintained over an extended period of time. Furthermore, stress-dependent oscillations in the nuclear localization of transcription factors could play a role in fine tuning the cellular response to stress by modulating the level and the order of expression of genes after stress. Such a view is consistent with the results of Gasch et al. (2000), which showed that the number of genes activated, the level of induction, and the duration of the effect depend on the strength of the stress.
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Materials and methods |
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To delete YAK1, the gene was cloned in a PRS314 plasmid from -609 to 2840 relative to the ATG, the internal fragment 2472293 was replaced by an EcoRISal1 fragment of pMPY.ZAP containing the URA3 gene, and a Pvu2Pvu2 fragment encompassing the yak1 gene with its flanking regions was transferred to the OL583 yeast strain (cyr1
, rca1/pde2) (Mallet et al., 2000), resulting in OL595-1. To delete CDC25, a PCR fragment containing the HIS3 gene between two CDC25 sequences, respectively, from -60 to -26 and 4818 to 4853 relative to ATG was transferred by homologous recombination to the OL549-3 strain isogenic to FY16792 (Thierry et al., 1995) except the integration in trp1 of the pGR113 containing the RAS2ile152 and the TRP1 gene.
The plasmid pAMG (gift from C. Schüller, Institute of Biochemistry, Vienna, Austria) encodes the fusion MSN2GFP (GFP at the COOH-terminal part) downstream of an ADH1 promoter in a centromeric LEU2 plasmid (Gorner et al., 1998). PJL42 (gift from L.J. Parrou, INSA Toulouse, Toulouse, France) is a plasmid where the SalI MSN2 fragment is inserted at the SalI site of pGFP-N-Fus (Niedenthal et al., 1996). The plasmid pGR247 is derived from pAMG in which the MSN2 ORF, cut out by Sal1 and Nco1, has been replaced by the MSN4 ORF amplified from a plasmid containing it (pAL3245) with oligos containing, respectively, a Sal1 and an Nco1 site. To construct the pAMG-Z plasmid, the flanking region of the zinc fingercoding region (amino acids 642698) had been first amplified by PCR with overlapping oligos and then amplified together with the external oligos containing the Sal1 site at the 5' end and the HindIII site at the 3' end. The resulting fragment, Msn2
ZnGFP, replaced, after restriction cutting and ligation, the Sal1HindIII Msn2GFP fragment in pAMG.
Time-lapse microscopy
Time-lapse microscopy was performed using the set-up previously described (Sibarita et al., 2002). It consists of an inverted microscope, Leica DM IRBE, mounted on an antivibration table and placed in an incubator at constant temperature. The objective was a 100x PL APO z-positioned by a piezo-electric driver. Fluorescence was recorded by a CCD detector (Roper MicromaxTM RTE-782-YHS; Roper Scientific). For illumination, we used a Sutter Instrument Co. DG4 instrument with a 175-W Xenon lamp. The experimental set-up was steered by MetamorphTM 4.6 Software (Universal Imaging Corp.).
Imaging
Every 10 s, during a 1-s period, a stack of 21 frames along the Z-axis (every 0.3 µm) was recorded. Images were deconvoluted using an algorithm developed by Sibarita et al. (2002). For each stack, a brightest point z projection was done using the MetamorphTM program to obtain the final reconstructed image. Pixel values in the nucleus and the cytoplasm, after removal of background noise, were used to monitor the distribution of Msn2GFP over time. Normalization was based upon the lowest and the highest pixel values in the nucleus when complete transfer to the cytoplasm and into the nucleus was observed. For cells showing incomplete translocation, the values were calculated with respect to the size of the cell and the nucleus.
Computational model for oscillatory shuttling of Msn2
One possible implementation, among others, of the delayed activation mechanism (schematized in Fig. 6 A) involves the switching on, by the nuclear form of Msn2, of a bicyclic cascade of phosphorylationdephosphorylation reactions, leading to the activation of a protein (e.g., a kinase or phosphatase) that would elicit Msn2 export from the nucleus (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200303030/DC1). Oscillations can also be obtained with a monocyclic cascade, but their amplitude is more reduced because the delay in the feedback loop is shorter. We assume that within the nucleus, activated Msn2 (MN*) promotes the conversion of an enzyme, X, from an inactive form, XI, into an active form, XA. The latter activates a regulator of Msn2, R, from RI into RA. The active form, RA, triggers the conversion of MN* into the form, MN, that leaves the nucleus. Both R and X, as well as Msn2, could be controlled by the antagonistic interplay of kinases and phosphatases. An example of a related mechanism that generates oscillations is provided by a cascade model for the periodic activation of cdc2 kinase during early mitotic cycles in amphibian embryonic cells (Goldbeter, 1991).
The model is governed by the following system of kinetic equations describing the time evolution of the fractions of M, M*, MN*, MN, XA, and RA:
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The curves of Fig. 6 (BF) were obtained by numerical integrations of equations 1a1f using the Berkeley MadonnaTM software for the following parameter values: VP = 0.3, VKSN = 0.5, VPN = 2, VKX = 1.3, VPX = 0.6, VKR = 1.6, VPR = 0.9, k1 = 6.6, k2 = 5 (all these parameters are in min-1), Ki = 0.01 (i = 1, ... 8), and Ka1 = Ka2 = 0.2. Initial conditions in panels BF of Fig. 6 are M = XI = RI = 1, and M* = MN = MN* = XA = RA = 0. These values correspond to the situation where, before stress is applied, the transcription factor is in its cytosolic state M, while regulators X and R are in their inactive form.
Online supplemental material
The supplemental material (Videos 13; Fig. S1) is available at http://www.jcb.org/cgi/content/full/jcb.200303030/DC1. The videos show nucleocytoplasmic shuttling of Msn2GFP in W303 cells, shuttling of Msn2ZnGFP in W303 cells, and a montage of Msn2GFP and Msn4GFP in the cell deleted for the three TPK genes and YAK1. Fig. S1 represents a scheme for the autoregulatory loop of the model.
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Acknowledgments |
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This work was supported by the CNRS, the University of Paris-Sud, Association pour la Recherche sur le Cancer (ARC) grant 5693 to M. Jacquet, ARC grant 9725 to J. De Mey, and grant 3.4607.99 from the Fond de la Recherche Scientifique Médicale (Belgium) to A. Goldbeter.
Submitted: 5 March 2003
Revised: 3 April 2003
Accepted: 3 April 2003
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