The next land rush p.12
News & Views
Mating in many species induces a dramatic switch in female reproductive behaviour. In most insects, this switch is triggered by factors present in the male’s seminal fluid. How these factors exert such profound effects in females is unknown. Here we identify a receptor for the Drosophila melanogaster sex peptide (SP, also known as Acp70A), the primary trigger of post-mating responses in this species. Females that lack the sex peptide receptor (SPR, also known as CG16752), either entirely or only in the nervous system, fail to respond to SP and continue to show virgin behaviours even after mating. SPR is expressed in the female’s reproductive tract and central nervous system. The behavioural functions of SPR map to the subset of neurons that also express the fruitless gene, a key determinant of sex-specific reproductive behaviour. SPR is highly conserved across insects, opening up the prospect of new strategies to control the reproductive and host-seeking behaviours of agricultural pests and human disease vectors.
The file contains Supplementary Tables 1-3; Supplementary Figures 1-3 with Legends; Supplementary Discussion; Supplementary Notes; Supplementary Methods and additional references.
There is a general consensus that planets form within disks of dust and gas around newly born stars
With the discovery of the first planet orbiting another Sun-like star
Planets form from dust and gas in circumstellar disks around young stars
One of the most important timescales for planet formation is the disk dispersal time. From observations of near-infrared excess
We acquired high-resolution spectroscopic measurements of TW Hya with the Fibre-fed Extended Range Optical Spectrograph (FEROS)
RV variations can be induced either by an orbiting companion, rotational modulation due to starspots, or nonradial pulsations. In the case of a companion, all spectral lines move simultaneously without affecting the line profile. In the case of rotational modulation and nonradial pulsations, the integral line shapes will vary and cause changes in the measured effective RV. To verify the nature of the observed RV variations, it is therefore mandatory to analyse the stellar activity indicators.
The 3.56-day RV variation appears to be regular during both observing periods. The phase-folded RV curve reveals very clearly the nearly sinusoidal variation. We find that this period is neither correlated with photometric variations nor with any stellar activity indicators. The bisector analysis of the line profile asymmetries confirms that there is no significant correlation between the 3.56-day period and the stellar activity (
Several authors have tried to derive the rotation period of TW Hya from photometric observations
We investigated all available activity indicators in the spectra, including the H&agr; emission line (
There is one activity indicator that varies with another significant period, at 9.05 days: the equivalent width of the H&agr;. The interpretation of this variability is still inconclusive, but it is probably related to a phenomenon in the disk. The corresponding keplerian radius would be ∼0.07
The detection of a young (8–10 Myr) and massive planet with 9.8MJupiter on a 0.04
In the period–mass distribution of known exoplanets
Models that assume the formation of planets beyond the snowline and include migration and accretion allow the formation of massive planets. However, it is still not clear whether a planet as massive as 9.8MJupiter could have formed through core accretion
A massive planet like TW Hya b would open a gap in the disk and undergo type II migration on a typical timescale of 10
The detection of TW Hya b opens up the possibility of directly connecting the disk evolution and planet formation processes. It is the ideal system to test numerical simulations of planet core formation, migration and accretion.
The star, the disk with a central hole, and the newly discovered planet are shown. The circumstellar disk around TW Hya is almost face-on
a, The RVs were obtained during two observing runs with 12 consecutive nights (between 28 February and 12 March 2007) and 20 consecutive nights (24 April to 13 May 2007). With typically three spectra per night, we sampled possible variability periods from about 1 to 12 days. For the RV calculations we used a cross-correlation technique, in which about 1,300 spectral lines were cross-correlated with a numerical template. The error bars are standard errors of the mean RV value. The typical accuracy of the individual RV is about 40 m s
A period analysis was performed using both a sine-fitting routine minimizing &khgr;
We used a cross-correlation technique, using several hundred spectral lines of TW Hya. We measured the bisector velocity spans (a) and bisector curvatures (b), which are well known as excellent stellar activity indicators. a, Bisector velocity span versus RV for the entire data set. There is no significant correlation (correlation coefficient ∼0.2), indicating that the 3.56-day RV variation is not caused by the line profile changes. b, The bisector curvature does not show a significant correlation with the RV (correlation coefficient ∼0.3), confirming that stellar activity is not responsible for the observed 3.56-day RV variation. The error bars are the standard mean errors of the mean bisector velocity span/curvature, computed from the bisectors of each echelle order.
We thank the 2.2 m MPG/ESO La Silla team, especially P. Francois, B. Conn, M. Stefanon, O. Schütz, M. Morell and A. Gonzales for their help during the observations. We thank W. Herbst for constructive discussion and providing the supporting data.
Author Contributions The observations were carried out by J.S. and A.M.; T.H. and R.L. were responsible for the project planning. The data analysis was done by J.S., A.M., P.W. and M.K.
The file contains Supplementary Notes with Supplementary Figures 1-2
Electrically charged particles, such as the electron, are ubiquitous. In contrast, no elementary particles with a net magnetic charge have ever been observed, despite intensive and prolonged searches (see ref.
Spin-ice materials are characterized by the presence of magnetic moments &mgr;i residing on the sites i of a pyrochlore lattice (depicted in
The distance between spins is rij, and a ≈ 3.54 Å is the pyrochlore nearest-neighbour distance. D = µ0µ
Spin ice was identified as a very unusual magnet when it was noted that it does not order to the lowest temperatures T even though it appeared to have ferromagnetic interactions
We contend that excitations above this ground-state manifold—that is, defects that locally violate the ice rule—are magnetic monopoles with the necessary long-distance properties. From the perspective of the seemingly local physics of the ice rule, the emergence of monopoles at first seems rather surprising. We will probe deeper into how the long-range magnetic interactions contained in
We consider a modest deformation of
The energy of a configuration of dipoles is computed as the pairwise interaction energy of magnetic charges, given by the magnetic Coulomb law:
We consider first the ground states of the system. The total energy is minimized if each diamond lattice site is net neutral, that is, we must orient the dumbbells so that Q&agr; = 0 on each site. But this is just the above-mentioned ice rule, as illustrated in
We now turn to the excited states. Naively, the most elementary excitation involves inverting a single dipole / dumbbell to generate a local net dipole moment 2µ. However, this is misleading in a crucial sense. The inverted dumbbell in fact corresponds to two adjacent sites with net magnetic charge Q&agr; = ±qm = ±2µ/ad—a nearest-neighbour monopole–antimonopole pair. As shown in
This interaction is indeed magnetic, hence the presence of the vacuum permeability µ0, and not 1/ε0, the inverse of the vacuum permittivity. It takes only a finite energy to separate the monopoles to infinity (that is, they are deconfined), and so they are the true elementary excitations of the system: the local dipolar excitation fractionalizes.
By taking the pictures from the dumbbell representation seriously, we may be thought somehow to be introducing monopoles where there were none to begin with. In general, it is of course well known that a string of dipoles arranged head to tail realizes a monopole–antimonopole pair at its ends
The unusual properties of spin ice arise from its exotic ground states. The ice rule can be viewed as requiring that two dipole strings enter and exit each site of the diamond lattice. In a typical spin-ice ground state, there is a ‘soup’ of such strings: many dipole strings of arbitrary size and shape can be identified that connect a given pair of sites. Inverting the dipoles along any one such string creates a monopole–antimonopole pair on the sites at its ends. The associated energy cost does not diverge with the length of the string, unlike in the case of an ordered ferromagnet, because no domain walls are created along the string, and the monopoles are thus deconfined.
We did not make reference to the Dirac condition
Indeed, the monopoles in spin ice have a magnitude qm = 2µ/ad = 2(µ/µB)(&agr;&lgr;C/2&pgr;ad)qD ≈ qD/8,000, where &lgr;C is the Compton wavelength for an electron, and &agr; is the fine-structure constant. The charge of a monopole in spin ice can even be tuned continuously by applying pressure, because this changes the value of µ/ad.
The monopoles are sources and sinks of the magnetic field H, as is appropriate to the condensed matter setting. More precisely, as in other instances of fractionalization
Our magnetic monopoles would in principle show up in one of the best-known monopole searches, the Stanford experiment to detect fundamental magnetic monopoles from cosmic radiation. This experiment is based on the fact that a long-lived current is induced in a superconducting ring when a monopole passes through it
The above observations are the central qualitative results of our work: ice-rule-violating defects are deconfined monopoles of H, they exhibit a genuine magnetic Coulomb interaction (see
We re-emphasize that the ice rule alone does not permit a consistent treatment of the excited states of the physical problem: crucially, the energetic interaction between our defects is absent altogether. Also, in previous discussions of the purely ice-rule problem and related short-range problems
The most satisfactory way to demonstrate the presence of a monopole would be to measure the force on magnetic test particles, say by a Rutherford scattering experiment or by clever nanotechnological means. Unfortunately, given the lack of elementary magnetic monopoles, we would have to use dipoles as test particles, which significantly weakens such signatures.
An alternative strategy is to look for consequences of the presence of magnetic monopoles in the collective behaviour of spin ice. This is most elegantly achieved by applying a magnetic field in the  crystallographic direction. Such a field acts as a (staggered) chemical potential (see
We thus have a tuneable lattice gas of magnetic monopoles on the diamond lattice. The basic structure of the phase diagram as a function of magnetic field and temperature can be inferred from work by Fisher and collaborators
To confirm this scenario, we have demonstrated by Monte Carlo simulations that the actual phase diagram of dipolar spin-ice model has precisely this structure. To rule out the appearance of the liquid–gas transition being due to effects introduced by the approximations leading to
This scenario is indeed observed experimentally in spin-ice materials
The presence of a liquid–gas transition was noted to be very remarkable because there are few, if any, other experimentally known instances in localized spin systems
The existence of magnetic monopoles in a condensed matter system is exciting in itself. (The monopoles appearing in the interesting work on the anomalous Hall effect are not excitations and do not involve the physical magnetic field
The magnetic moments in spin ice reside on the sites of the pyrochlore lattice, which consists of corner-sharing tetrahedra. These are at the same time the midpoints of the bonds of the diamond lattice (black) formed by the centres of the tetrahedra. The ratio of the lattice constant of the diamond and pyrochlore lattices is
The dumbbell picture (c, d) is obtained by replacing each spin in a and b by a pair of opposite magnetic charges placed on the adjacent sites of the diamond lattice. In the left panels (a, c), two neighbouring tetrahedra obey the ice rule, with two spins pointing in and two out, giving zero net charge on each site. In the right panels (b, d), inverting the shared spin generates a pair of magnetic monopoles (diamond sites with net magnetic charge). This configuration has a higher net magnetic moment and it is favoured by an applied magnetic field oriented upward (corresponding to a  direction). e, A pair of separated monopoles (large red and blue spheres). A chain of inverted dipoles (‘Dirac string’) between them is highlighted in white, and the magnetic field lines are sketched.
Comparison of the magnetic Coulomb energy
The location of the monopole liquid–gas transition from numerics (blue line) compared to experiment (black line; ref.
We thank S. Bramwell, J. Chalker, C. Chamon and S. Kivelson (especially for pointing out ref.
Author Contributions All authors contributed equally to the manuscript.
The file contains Supplementary Notes and Figures.
An unambiguous determination of the three-dimensional structure of nanoparticles is challenging
Scanning transmission electron microscopy (STEM), in the mode where incoherently scattered electrons are collected by a high-angle annular dark field (HAADF) detector, is appealing as a method of probing three-dimensional structure of nanoparticles (via an analysis of the intensity map from a single HAADF-STEM image) because its intensity is strongly dependent not only on the atomic number Z of the observed atoms but also on the number of atoms in a column
We demonstrate this for size-selected AuN (where N = 309 ± 6) clusters, where Au309 is known to be a possible ‘magic number’ nanocluster (see
To establish a foundation on which to analyse quantitatively the structure of the Au309 clusters, we carried out a series of integrated HAADF intensity measurements on size-selected Au clusters in the size range N = 55–1,500 atoms. For each sample, the HAADF intensity integrated over each cluster shows a narrow distribution (see
The linearity shown in
Close comparison between
The observed structures of the Au clusters can be understood from their calculated total potential energies for different polyhedral geometries (icosahedral, Ino-decahedral and cuboctahedral) as a function of the number of constituent atoms. After local energy minimization, it was found that, for very small Au clusters (N < 100), the icosahedral structure is much more stable than the Ino-decahedral and the cuboctahedral structures. The total potential energy is in the order of: icosahedral < Ino-decahedral < cuboctahedral. However, for the larger clusters (N ≈ 500–1,000), the order of stability begins to change, with the Ino-decahedral structure becoming more stable than the icosahedral geometry: Ino-decahedral < icosahedral < cuboctahedral. Further increasing the cluster size results in the order of stability changing to: Ino-decahedral < cuboctahedral < icosahedral. For Au309, the difference in total energy between different geometries was less than 1.2 eV (that is, less than 3.88 meV per atom) from the most stable to the least stable. Moreover, there are many local energy minima and the energy barriers between these structures are small. These results support our experimental findings that no one structure dominates. For Au309, we see a similar proportion of clusters with Ino-decahedral (32%) and cuboctahedral (25%) structures and a much lower population of icosahedral structures (8%). In the remaining population, some clusters show irregular facets and some do not show any ordered geometry, possibly because of significant rearrangement of the outer-shell atoms, akin to the solid–liquid phase coexistence predicted for other systems
In conclusion, we have demonstrated the suitability of high-angle annular dark-field imaging in the aberration-corrected STEM for detailed structural and stability analysis of size-selected metallic clusters on solid supports at atomic resolution. The multiplicity of cluster geometries revealed by our detailed study of the atomic arrangement of soft-landed Au309 clusters on amorphous carbon supports is consistent with many local energy minima predicted for clusters of this size by cluster simulations. Evidence for increased fluctuations and motion of cluster surface atoms relative to the core atoms within the Au309 clusters may reflect an inherent property of the nanometre-sized gold clusters that could be related to their enhanced catalytic properties through much reduced coordination. Vertical depth information can be extracted from a single projection, with single-atom sensitivity, opening up the possibility to use the technique as a routine three-dimensional structural characterization tool for small nanoparticles at the atomic-scale level, with the help of image simulation based on ab initio cluster modelling including dynamical effects. The experimental approach has practical advantages, such as the more relaxed constraints on cluster stability
The gold clusters are formed by gas-phase condensation of sputtered atoms in a rare-gas atmosphere
Gold cluster beams were produced by a source based on radio-frequency magnetron plasma sputtering and gas aggregation
Systematic measurements of integrated HAADF intensities for a wide range of size-selected Au clusters were carried out using a Tecnai F20 200 kV STEM at the Nanoscale Physics Research Laboratory, University of Birmingham. High-resolution imaging of Au309 clusters was performed using a dedicated VG HB501 STEM at the UK SuperSTEM facility at Daresbury
To simulate HAADF images of the Au309 clusters for comparison with experiments, we first generated idealized icosahedral, Ino-dechedral and cuboctahedral geometries with the magic number N = 309. The structures, which were modelled by the Gupta many-body potential
This was done by locating each trial cluster within a two-dimensional grid of dimensions 50 Å× 50 Å, with an interval spacing of 0.25 Å in both the x and y coordinates. A probe of diameter 1 Å visited each grid point and checked for atoms within the probe radius of this grid point in the two-dimensional x–y plane. The quantitative contribution of an atom to the intensity was related to the actual distance between the probe centre and the centre of the atom by a gaussian distribution. All the atomic contributions within the probe radius were then summed and used to calculate the total intensity. Finally, we examined the calculated image for various alignments of each cluster relative to the electron beam probe. The above procedure was repeated for high-symmetry clusters with the magic number N = 309, including the icosahedron, the cuboctahedron and the Ino-decahedron structures.
We have used an approach described by Kirkland
Typical images show various outline shapes, that is, cluster projections: pentagon (a), square (b) and hexagon (c). The intensity variation within the clusters clearly demonstrates atomic column resolution. Single atoms can be seen in the vicinity of the clusters, as indicated by the circle in c, and occasionally some distance away, as indicated by the circle in a. Resolution of the mass selector is ±2%.
Integrated HAADF intensity of size-selected Au clusters on amorphous carbon film plotted as a function of the number of atoms they contain, showing a linear relationship. The line is drawn as a guide to the eye. Each data point is obtained from a statistical intensity distribution analysis over a large number of clusters with a given number of atoms. The standard deviation is used for estimating the error bars. An example of such a distribution for Au309 is shown in the inset.
a, Three-dimensional atom density profile of Au309, derived from
We thank Y. Chen for assistance with the electron microscopy work in Birmingham. We gratefully acknowledge the UK Engineering and Physical Science Research Council (EPSRC) and the EU for their financial support of the cluster work. The EPSRC funded the UK SuperSTEM facility at Daresbury Laboratory. N.P.Y. and B.C.C. acknowledge the EPSRC and the University of Birmingham for PhD funding, respectively. The work at Tsinghua University was supported by the Ministry of Science and Technology and Ministry of Education in China.
The file contains Supplementary Notes, Supplementary Tables S1-S4 and a Supplementary Figure S1 with Legend.
The carbon balance of terrestrial ecosystems is particularly sensitive to climatic changes in autumn and spring
The carbon balance of terrestrial ecosystems is highly sensitive to climate changes at the edges of the growing season
The seasonal cycle of atmospheric CO2 concentrations provides an integrated measure of the net land–atmosphere carbon exchange (net ecosystem productivity; NEP) and its temporal characteristics
We verified that the strong negative correlation between upward CO2 zero-crossing date and temperature predominantly reflects climate-driven fluctuations in NEP, rather than interannual fluctuations in atmospheric transport. To do so, we prescribed either variable NEP or climatological NEP fluxes from ORCHIDEE to the global transport model LMDZt driven by variable wind fields (see Methods). With the exception of the Mt Cimone (CMN) and Cape Kumukahi (KUM) stations, we found that the fluctuations in upward zero-crossing dates are driven mainly by changes in NEP, and only partly by interannual wind changes (see Methods,
There is also a long-term trend in the autumn upward zero-crossing date of atmospheric CO2 superimposed on interannual fluctuations. At Point Barrow, for instance, we determined a systematic advance of -0.40 day yr
Next, we analysed 108 site-years of eddy-covariance CO2 measurement data from 24 northern ecosystem sites (
The large-scale atmospheric concentration records, taken together with the ecosystem-scale eddy-covariance flux measurements (about 1 km
Simulated September to November NEP shows a trend towards increasing carbon losses in the Northern Hemisphere (north of 25° N) at a rate of 13 Tg C yr
Our results suggest that net carbon uptake of northern ecosystems is being decreased in response to autumnal warming. The spatial distribution of the response of carbon flux to temperature, as projected by the ORCHIDEE model, is shown in
Observed historical climate data
Applying the future Northern Hemisphere warming of 3.8–6.6 °C predicted by a climate model
We analysed the effects of autumn temperature on the carbon balance of northern ecosystems at different scales, using three different methods.
First, we used smoothed flask CO2 data from the NOAA/ESRL network
Second, we analysed the net CO2 flux data measured by the eddy-covariance technique from 24 northern ecosystem sites (
Third, hints on the processes that control the integrated autumn NEP response to temperature, through the individual sensitivity of photosynthesis and respiration, were provided by integrating the ORCHIDEE vegetation model
We used flask data from the NOAA/ESRL network to characterize trends in the CO2 zero-crossing dates (spring downward and autumn CO2 upward) that correspond roughly to the time of maximum NEP uptake in spring and maximum release in autumn. Following the approach described in ref.
The downward and upward CO2 zero-crossing dates were correlated with spring (March to May) and autumn (September to November) air temperature
The eddy-covariance CO2 flux observations were performed in accordance with the routine procedures established by regional networks (for example, Fluxnet-Canada and CARBOEUROPE
The global vegetation model called ORCHIDEE (‘ORganizing Carbon and Hydrology In Dynamic Ecosystems’)
With the use of 1901 climate data and the 1860 atmospheric CO2 concentration of 286 p.p.m., a first model spin-up was performed to bring carbon pools to equilibrium. A second spin-up was performed with interannually variable climate data over 1901–1910 to define the initial condition of a run covering 1901–2002. The monthly climate data sets were supplied by the Climatic Research Unit, University of East Anglia, UK
The modelled NEP over 1980–2002, prescribed in an atmospheric transport model (LMDzt), was found to faithfully reproduce the interannual variations in the spring drawdown date and autumn build-up date at high-latitude (north of 50° N) stations that are predominantly affected by the fluxes of the Northern Hemisphere (
We used the three-dimensional eulerian transport model LMDzT derived from the general circulation model of the Laboratoire de Météorologie Dynamique, LMDz
To separate the effects of transport and terrestrial carbon fluxes on the zero-crossing date signal, we performed two simulations. The first one used interannual daily NEP fluxes calculated during the period 1980–2002 by ORCHIDEE. The second simulation (referred to as ‘transport only’) used climatological but daily variable NEP. The contribution of interannually varying fluxes to the variability in zero-crossing date is assessed by the difference in simulated atmospheric CO2 between the first and the second simulations, which is referred to as ‘flux only’. In addition, we computed the contribution to CO2 concentrations from air–sea exchange and fossil fuel emissions and their increase (annual increase per group of countries) by following estimates from refs
a, Interannual variability in anomaly of upward zero-crossing date (red) observed at Point Barrow, Alaska, and the corresponding autumn (September to November) temperature (black) over the region between 51° and 90° N over the past two decades. Upward zero-crossing date is strongly anti-correlated with autumn temperature (slope = -5.4 days °C
A total of 108 site-years have been aggregated in this figure. The average (blue) and median (green) anomaly of ending date of net CUP is shown for different autumn temperature anomalies binned into 0.5 °C intervals. The top horizontal axis labels correspond to the number of site-years and sites (in parenthesis) in each temperature bin. The bottom left inset shows the relationships between ending date of CUP and temperature anomalies. There is a marginally negative correlation between autumn CUP ending date and temperature anomalies (y = -1.7x - 0.0087, P = 0.07). If we exclude the four site-years with the most extreme cold anomalies (&Dgr;T < -2 °C), the negative correlation between CUP ending date and temperature becomes highly significant (P = 0.03) and the slope is steeper (y = -2.4x + 0.3007), suggesting that below a certain threshold of cold anomaly there is no further decrease in respiration. The top right inset shows the relationships between autumn NEP and temperature anomalies. A positive NEP value indicates an increased carbon uptake. Autumn was defined as the 60-day interval around the average CUP ending date for each site. Eddy-covariance data show increased carbon losses under warmer conditions, with a temperature sensitivity of NEP of -3.2 g C m
a, ORCHIDEE model-derived autumn GPP. b, ORCHIDEE model-derived autumn NPP. c, ORCHIDEE model-derived autumn NEP. d, Sum of satellite-derived autumn normalized difference vegetation index (NDVI). The sensitivity is expressed as the linearly regressed slope of autumn carbon flux or of NDVI against autumn temperature for each pixel over the past two decades. A positive slope of NEP indicates that terrestrial carbon uptake is increasing with warmer temperatures, and vice versa. Areas with a low sensitivity or insignificant (P > 0.05) relationships between the variables are coloured in grey.
We thank all the people and their respective funding agencies who worked to provide data for this study, and specifically B. Amiro, M. A. Arain, T. A. Black, C. Bourque, L. Flanagan, J. H. McCaughey and S. Wofsy for providing some of the flux data from the Canadian sites, and M.-A. Giasson and C. Coursolle for their help in compiling the data. We also thank A. Friend, P. Rayner and N. Viovy for helpful comments and discussions. This study was supported by European Community-funded projects ENSEMBLES and CARBOEUROPE IP, and by the National Natural Science Foundation of China as well as by Fluxnet-Canada, which was supported by CFCAS, NSERC, BIOCAP, MSC and NRCan. The computer time was provided by CEA. We thank the NOAA-ERSL global air sampling program for collecting and analysing the long-term CO2 flask data, and K. Masarie at NOAA-ERSL for generating each year the GLOBALVIEW-CO2 collaborative data product, which formed the basis of our atmospheric data analysis. The ongoing exchange of ideas, data and model results in the international research community on the carbon cycle is facilitated by the Global Carbon Project.
Author Contributions P.C., S.P., P.F. and P.P. designed the research. S.P., P.C. and P.F. performed ORCHIDEE modelling analysis. P.P. and S.P. performed transport analysis. S.P., S.L., M.R., H.M. and P.C. performed eddy-covariance data analysis. S.P., P.C. and J.F. performed satellite data analysis. All authors contributed to the interpretation and writing.
The file contains Supplementary Discussion, Supplementary Figures 1-3 with Legends and additional references.
Near-surface warming in the Arctic has been almost twice as large as the global average over recent decades
The recent warming of the Earth’s surface is most probably due to an increase of atmospheric greenhouse-gas concentrations
The Arctic amplification can also be caused by other processes. Idealized experiments with models that have no surface-albedo feedback also reveal a polar-temperature-amplification response to a doubling of CO2 concentration
The linkage between Arctic warming and changes of atmospheric circulation has been investigated by studying various Northern Hemisphere circulation indices, such as that associated with the Arctic Oscillation
The vertical structure of the Arctic warming during the 1980s and 1990s, based on the ERA-40 reanalysis (see Methods), exhibits trends throughout large parts of the troposphere that are comparable in magnitude to those at the surface (
It is also notable that during the same period observations solely from Arctic land stations reveal an amplification of the temperature trend during the dark months, November–February (
So what are the mechanisms giving rise to the vertical structure of the Arctic warming? Changes in the advection of atmospheric energy into the Arctic region might imply Arctic warming with a maximum not necessarily located at the surface. Mid-tropospheric temperatures in the Arctic are sensitive to advection of energy across the Arctic boundary: this is evident from linear regressions of the Arctic 500 hPa temperature field on the atmospheric northward energy transport (ANET) across 60° N (
The ANET across 60° N has increased during recent decades, except in January and February. For the summer half-year, April through to October, the ANET can explain a substantial part of the Arctic temperature trends (
Other processes that might be important contributors to the warming above the surface include changes in cloud cover and the atmospheric water vapour content. In the Arctic, except possibly for a short summer period, persistent low clouds are believed to induce surface warming
Our results do not imply that studies based on models forced by anticipated future CO2 levels are misleading when they point to the importance of the snow and ice feedbacks. It is likely that a further substantial reduction of the summer ice-cover would strengthen these feedbacks and they could become the dominant mechanism underlying a future Arctic temperature amplification. Much of the present warming, however, appears to be linked to other processes, such as atmospheric energy transports.
The ERA-40 reanalysis
The atmospheric energy can be divided into four components
The sensitivity of the Arctic temperature field to the ANET across 60° N is estimated as follows: first, the ANET across 60° N based on 6-hourly data is determined using
For estimations of the Arctic temperature trends, which are linked to the ANET across 60° N, monthly mean rather than daily data are used as the warming response lags the ANET by around 5 days (
The shading in
Trends are shown for winter (a, December–February), spring (b, March–May), summer (c, June–August) and autumn (d, September–November). The linear trends are estimated from monthly mean data using a least-squares fit.
Data were obtained from land-station observations. In the main figure, the symbols represent means from individual years, whereas the lines show the temporal evolution when variability over timescales smaller than 20 years has been removed using a wavelet filter. Solid line and open circles are based on observations north of 65° N, while the dashed line and dots are for the entire Northern Hemisphere. Inset shows the smoothed temperature time series for the full instrumental period. The data were provided by the Climate Research Unit (CRU) as a 5° × 5° gridded data set
a, Regressions averaged around latitude circles as a function of latitude and time lag; b, regressions for 5-day lag as a function of longitude and latitude. Solid and dotted contours indicate positive and negative regressions, respectively. In each point the regression has been scaled by the spatial standard deviation of all regressions. Light- and dark-grey shading shows areas where regressions differ significantly from zero at the 99% and 99.9% level, respectively. The regressions indicate temperature anomalies associated with an ANET anomaly at lag zero. For instance, a positive ANET anomaly is followed 5 days later by warming and cooling north and south of 60° N, respectively.
a, Total trends; b, trends that are linked to the ANET across 60° N. The shadings indicate trends, and the white contours indicate areas where trends differ significantly from zero at the 99% and 99.9% level, respectively.
We thank P. Lundberg for comments on the manuscript. The ERA-40 data were obtained from the European Centre for Medium-Range Weather Forecasts (ECMWF) data server, whereas the Climate Research Unit (CRU) at the University of East Anglia provided the observational data used for
Author Contributions The analysis was performed and the manuscript written by R.G.G., and to some extent T.M. The original idea to use ERA-40 data to study Arctic warming was due to R.G.G., M.T. and E.K. All authors contributed with ideas, discussions and text.
The file contains Supplementary Figures S1-S2 with Legends.
It remains unknown how the small strains induced by seismic waves can trigger earthquakes at large distances, in some cases thousands of kilometres from the triggering earthquake, with failure often occurring long after the waves have passed
Laboratory studies of granular friction have emerged as a powerful tool for investigating tectonic fault zone processes and earthquake phenomena, including post-seismic slip, interseismic frictional restrengthening and earthquake nucleation
Experiments on sheared layers of glass beads (like those shown in
The top curve of
Vibration perturbs the recurrence period of inelastic stress increase before the failure of major events and induces small-amplitude stick–slip events. In many cases one or more small stick–slip events occur during vibration, as well as cascades of delayed, small-amplitude stick–slip events (
We also apply acoustic pulses, rather than the longer-duration waves described above. Pulses are more analogous to a single seismic wave in Earth, whereas vibration may be more analogous to the near-source region where quasi-continuous-wave energy may exist for significant periods of time in the form of aftershocks. Our data show that continuous and pulse modes of dynamic triggering yield similar behaviour. See
When we apply vibration or pulsed sound at stresses below ∼95% of the failure strength there is little or no effect on stick–slip. This implies that the system must be in a critical state to be susceptible to dynamic triggering, which is consistent with seismic data on earthquake triggering
Analysis of the primary stick–slip recurrence intervals for otherwise identical experiments with and without vibration shows that failure becomes progressively more erratic and, on average, lengthens with time in experiments with wave excitation (
We have described three primary experimental observations: (1) acoustic waves disrupt recurrence intervals and, to a lesser degree, stress drops of large magnitude events; (2) acoustic waves trigger immediate and delayed small-magnitude events, some aseismic; and (3) strain memory of acoustic perturbation is maintained through successive large-magnitude stick–slips. We assess the implications of these results for dynamic earthquake triggering by considering that the primary slick–slip events represent tectonic earthquakes and that the vibration-induced events represent triggered earthquakes.
The overall trend of increasing stress drop (and maximum frictional strength) with recurrence interval is consistent with a large body of previous laboratory and field observations
We find that vibration has measurable effects only when the system is in a critical state, approaching failure (for example, see
One mystery regarding dynamic earthquake triggering is that it can take place minutes, hours or days after the seismic perturbation. Our experiments show delayed failures following acoustic perturbations, frequently manifesting as cascades of small events. We do not yet understand the physics responsible for this observation; however, we speculate that triggered events, as well as the recurrence and stress-drop disruptions are manifestations of frictional contact mechanics coupled with granular processes. Previous work shows that stick–slip initiates as failure of a contact junction between beads in highly stressed chains of particles
We posit that acoustic waves disrupt granular force chains, leading to material softening and simultaneous weakening (granular flow), similar to what is described in a recently proposed phenomenological model
Our previous work shows that permanent damage to the grains themselves is negligible
The origin of dynamic earthquake triggering by transient seismic waves is a complex problem. Our results show that granular-friction processes are consistent with two as-yet-unexplained observations in earthquake seismology: (1) small-amplitude waves can trigger both immediate failure and delayed failure relative to the strain transient, and (2) earthquake recurrence patterns are complex. Understanding the role of vibration-induced disruption of earthquake recurrence could have significant implications for seismic hazard assessment and reliable forecasting of earthquakes.
In our experimental study of acoustic waves interacting with a laboratory-scale fault system, we employ a double-direct shear configuration to shear 4-mm layers of glass beads at constant normal stress (1–18 MPa), using shearing rates of 1–100 µm s
We use a double-direct shear configuration in a biaxial load frame, which applies a horizontal stress to three steel forcing blocks that contain symmetric layers of glass beads at the block interfaces (
Vibration is applied via an acoustic source (
In the pulse experiments, a toneburst of 10–20 cycles with frequency ranging from 6.1–8.67 kHz is applied. We use tonebursts of approximately 3.3 ms duration and a centre frequency of 6,100 Hz for the results shown. In general, sound is applied every third stick–slip cycle after steady-state conditions are reached, but experiments were also conducted in which we applied sound at shorter and longer stick–slip intervals.
The strain amplitudes we apply range from about 5 × 10
a, Apparatus, showing horizontal piston applying constant normal stress, and vertical piston applying a constant (vertical) displacement rate, which drives shear. The dashed circle shows the sample assembly. b, Sample assembly showing three-block arrangement of the double-direct shear configuration (front and side views). We note the location of the acoustic wave source and accelerometer in relation to the glass bead layers and normal stress (horizontal).
a, Shear stress versus experiment time for a typical run. Note that maximum stick–slip stress drops are ∼30% of the shear strength. Over the total duration of the experiment, there is a small but progressive compaction of about 1% of the glass bead layer thickness (not shown). b, Detail of the stick–slip cycles (top) and change in layer thickness (bottom). The layer thickness has had the overall trend removed. We note consistent failure strength, recurrence interval, and creep before stick–slip. p1108 refers to experiment number.
a, Stick–slip behaviour under constant shearing rate, with vibration. Shear stress versus experiment time (upper curve); and measured, rectified strain amplitudes of the detected acoustic waves (lower curve). The letter ‘V’ denotes times and thick black horizontal bars indicate the durations of vibration. Vibration has a marked influence on the stick–slip behaviour. For instance, the applied vibration at ∼2,050 s produces an immediate, small-magnitude stick–slip. The two successive major stick–slips that follow exhibit longer recurrence times as well as multiple small stick–slip events in between—these are triggered events. Regions of triggered events are shaded light grey. Similarly, irregular cycles occur following the applied vibration at 2,155 s. Vibration applied at ∼2,255 s produces an immediate small-magnitude stick–slip event and an increased major-event recurrence interval. b, Comparison of non-vibration versus vibration, emphasizing increased recurrence and irregular behaviour, including triggering, due to acoustic waves. p870 and p1108 refer to experiment numbers.
a, Recurrence versus experiment time for runs with vibration (solid circles) and without. The shaded region and dashed lines show the mean recurrence interval of ±1 standard deviation. Data trend removed. Compared to the non-vibration experiments, both the scatter and average recurrence interval increases progressively in experiments with vibration. b, Stress-drop variation versus recurrence for experiments conducted with and without vibration. We cannot compare stress-drop amplitudes directly owing to minor differences from one experiment to the next; however, when we compare the variation of stress drop to the experimental mean, we see a clear trend of longer recurrence interval for a given change in stress drop.
Funding was provided by Institutional Support (LDRD) at Los Alamos and the DOE Office of Basic Energy Science (P.A.J.), by the National Science Foundation (C.M., H.S., M.K.), and by the United States Geological Survey (J.G.). We thank E. Brodsy, B. Behringer, N. Beeler and X. Jia for comments and reviews.
Author Contributions P.A.J., M.K., H.S. and C.M. designed the study. M.K., P.A.J. and C.M. designed and carried out the data collection procedure. P.A.J. and H.S. did most of the data analyses. P.A.J. and C.M. did most of the writing. P.A.J., H.S., M.K. and C.M. did the laboratory work and J.G. and C.M. did much of the writing interpretation. All authors contributed to the interpretation and writing.
Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice p.61version="1.0" encoding="utf-8"?> ]>
The file contains Supplementary Methods, Supplementary Figures 1-7 with Legends and Legends to Supplementary Movies 1-2.
The file contains Supplementary Movie 1.
The file contains Supplementary Movie 2.
Electrical microstimulation can establish causal links between the activity of groups of neurons and perceptual and cognitive functions
We used in utero electroporation
We next characterized the responses of ChR2–GFP-expressing neurons to photostimulation in anaesthetized mice. To sample from the entire population of ChR2–GFP-expressing neurons, unbiased by ChR2–GFP expression level, we recorded from red fluorescent neurons using two-photon targeted loose-patch recordings
When stimulated with 1 ms light pulses, ChR2–GFP-expressing neurons were able to follow frequencies up to 20 Hz (
We next determined the relation between photostimulus intensity and the probability of spiking of ChR2–GFP-expressing neurons. During cell-attached recordings we stimulated with 1 ms light pulses while varying the photostimulus. With decreasing light intensity, neurons switched abruptly from firing action potentials with high probability to firing no action potentials. The photostimulus intensity required to trigger action potentials varied substantially across the population of ChR2–GFP-expressing neurons (
Can awake mice learn to report photostimulation of layer 2/3 pyramidal neurons in the barrel cortex? To address this question we delivered light pulses to ChR2–GFP-expressing neurons in freely moving animals (
How many action potentials triggered by photostimulation are necessary for perception? To address this issue we further trained five mice to respond to one, two and five photostimuli at 20 Hz (example in
To determine the relation between performance and the number of neurons directly activated by light, we measured behaviour as a function of light intensity (
We counted the number of ChR2–GFP-positive somata and measured their positions (
For each animal we then estimated the number of active neurons as a function of normalized intensity (Io = intensity/Imax) as:
Here rk is the horizontal position of the kth ChR2–GFP-positive cell and f is the fraction of ChR2-positive cells activated at intensity Ioi (
Two factors make us believe that our estimates of the number of active neurons should be interpreted as an upper bound. First, the measured spatial distribution of light in the tissue is likely broader than the actual distribution of light (see
Not surprisingly, triggering more action potentials yields better detection accuracy (
Activated ChR2–GFP-positive neurons were distributed over most of the barrel cortex, with a smattering in adjacent sensory areas. The activated cortical region contains at least 40,000 layer 2/3 neurons (approximately 2,000 per barrel column, unpublished data) implying that synchronous action potentials in less than 1% of layer 2/3 neurons can be robustly perceived. These data imply that mechanisms exist to read out extremely sparse codes from primary sensory areas
We have shown that ChR2-based optical microstimulation can be used to dissect the impact of precisely timed action potentials in a few genetically defined neurons on mammalian behaviour. Our data show that the favourable characteristics of ChR2 reported previously in vitro
Photostimulation of genetically defined neurons
DNA solution (ChR2–GFP and either mCherry or DsRedexpress (‘RFP’); 4:1 molar ratio; final concentration 2 µg µl
An imaging window was implanted on the electroporated mice
After completion of behavioural experiments, the brain from each animal was cut into coronal or tangential sections (40–60 µm thick) on a cryostat (
All experimental protocols were conducted according to the National Institutes of Health guidelines for animal research and were approved by the Institutional Animal Care and Use Committee at Cold Spring Harbor Laboratory and HHMI Janelia Farm Research Campus.
Venus or GFP was fused to the carboxy (C) terminus of the first 315 amino acids of channelrhodopsin-2 (gift from G. Nagel). The construct (‘ChR2–GFP’) was inserted into pCAGGS vector modified for in utero electroporation
A chronic imaging window was implanted on the electroporated mice at postnatal age 40–50 days. The mice were anaesthetized by using an isoflurane-oxygen mixture (2% isoflurane/O2 by volume) delivered by an anaesthesia regulator (
The distribution of light intensity at the surface of the brain was measured using a beam profiler (
For targeted cell-attached recordings, similar surgery was performed as described above except that the skull opening was only 1.5 mm and a custom-shaped semilunar coverglass was sealed in place using dental acrylic, leaving the lateral edge of the exposed dura accessible to the recording electrode. To monitor the level of anaesthesia, an electrocorticogram was recorded by inserting a thin Teflon-coated silver wire between the dura and skull in the contralateral hemisphere. A reference wire was inserted above the cerebellum. In vivo imaging was performed by using a custom-made two-photon laser-scanning microscope controlled by ScanImage software
Mice with implanted LEDs had free access to food but had restricted access to drinking water to maintain 80–85% of their pre-training weight. Water was only available during and immediately after the behavioural sessions, with a minimum of 1.5 ml per day. Body weight was monitored daily before the training. The mice were kept at a reversed 12 h light/dark cycle and sessions were performed during the dark cycle. The behavioural box consisted of a white Plexiglas chamber (200 mm × 140 mm × 200 mm) with three ports mounted on one wall. The ports were conical and equipped with an infrared phototransistor–photodiode pair that signalled the interruption of the beam when the mouse entered his snout. The floor was a washable plastic kitchen cutting board. Both box and floor were cleaned with 70% ethanol after each animal. The box was placed in a sound- and light-proof cabinet that was constantly illuminated with bright white light. The box was covered with a transparent Plexiglas plate in which an infrared camera (for monitoring) and a bright masking light (high-power blue LED 470 nm,
Training consisted of several phases. Transitions from one phase to the next were triggered by performance at 65% correct or above.
1. Mice were habituated to the behavioural box and trained for one week to get water from either left or right water port by breaking the light beam inside the port. The availability of water in a port was signalled with a white noise click from a loudspeaker (synchronized with the 5 ms valve opening). Water was delivered with a gravitational system and the drop size was controlled with solenoid valves (
2. The snout of the mouse had to enter the centre port (trial initiation) to make water reward available in either the left or the right port for 10 s.
3. The LED was connected to the behavioural control system before the mouse was placed in the box. The cable to the LED controller ran through a hole in the Plexiglas cover and was mounted on a rotating hook 60 cm above the mouse. Reward was only available in the left port after photostimulation, whereas water was available on the right port in the absence of photostimulation (
4. Animals were trained to respond to fewer stimuli and decreased light intensity.
After completion of behavioural experiments, the mice were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain was carefully removed from each animal and cut into coronal or tangential sections ranging from 40 to 60 µm on a cryostat (
a, Coronal section through the electroporated mouse somatosensory cortex after immunohistochemical staining for ChR2–GFP. b, Individual layer 2/3 neuron, side view. c, Maximum value projection (top view) of an image stack in vivo (see
a, Schematic of the photostimulation setup (see Methods). b, Schematic of the behavioural apparatus and reward contingencies. The mouse initiates a trial by sticking its snout into the central port. Photostimuli are applied during a stimulation period (300 ms) accompanied by a series of bright blue light flashes delivered to the behavioural arena (30 Hz, 300 ms) to mask possible scattered light from the portable light source. The mouse then decides to enter either the left or the right port for a water reward. If a photostimulus was present, the choice of the left port was rewarded with a drop of water (hit, green star) whereas the choice of the right port lead to a short timeout (4 s, miss, red star). If the stimulus was absent, only the choice of the right port was rewarded with reward (correct reject, green circle) whereas the left port lead to a timeout (4 s, false alarm, red circle). c, Data from one session (200 trials) with a single stimulus (1 ms) with decreasing light intensities. Each horizontal line delineates 20 trials at fixed light intensity. Blue dots indicate the presence or absence of a photostimulus. Stimulated and non-stimulated trials were presented pseudo-randomly with a probability of 0.5.
a, Comparison of the performance ((hits + correct rejections)/total trials) in mice expressing ChR2–GFP (n = 9) and control mice (n = 6) after training with five photostimuli (P < 0.001, t-test). b, Performance as a function of light intensity (as percentage of Imax = 11.6 mW mm
We thank B. Burbach, D. Flickinger, H. Kessels, D. O’Connor, T. Sato, R. Weimer and A. Zador for help with experiments, and D. O’Connor for comments on the manuscript. This work was supported by the Swiss National Science Foundation (to D.H.), the National Institutes of Health and the Howard Hughes Medical Institute.
Author Contributions D.H. and K.S. designed the experiments. D.H. performed the behavioral and in vivo physiological experiments. L.P., D.H. and K.S. performed the brain slice measurements. N.G. performed histology. S.R., T.H., Z.M. and K.S. provided advice and equipment. D.H. and K.S. wrote the paper. All authors discussed the results and commented on the manuscript.
This file contains Supplementary Figures S1-S8 with Legends illustrating additional analysis of single cell stimulation experiments in somatosensory cortex.
Understanding how neural activity in sensory cortices relates to perception is a central theme of neuroscience. Action potentials of sensory cortical neurons can be strongly correlated to properties of sensory stimuli
Based on its volume
Once animals responded consistently to low microstimulation currents, we approached a cortical neuron closely with a glass pipette and evoked short (200 ms) trains of action potentials by juxtacellular stimulation, a technique developed to label individual neurons
Microstimulation and single-cell stimulation trials were randomly interleaved with ‘catch’ trials (with no or subthreshold current injection) (
A population analysis revealed, however, that single-cell stimulation biased animals towards responding.
Further observations show that the animals’ responses were caused by the stimulation of single and not multiple neurons. (1) Juxtacellular stimulation currents were approximately three orders of magnitude lower (3–43 nA) than those required for evoking motor or sensory responses with microstimulation (2–200 µA). (2) Although we occasionally observed the inadvertent stimulation of a second neuron by the appearance of a second large action potential waveform in our recordings, such inadvertent stimulation was rare (accounting for only about 1% of evoked action potentials across experiments;
Because microstimulation in barrel cortex can evoke whisker movements, we combined stimulation experiments with whisker tracking to assess if rats sense single-cell stimulation indirectly by detecting movements. Our data argue against such an indirect mechanism: near detection threshold microstimulation did not evoke movements even though it was reported (
The bias towards responding evoked by single-cell stimulation was weak on average (approximate 5% effect size: single-cell stimulation hit rate – catch trial response rate). As illustrated in
Reaction times for single-cell stimulation were long and variable (
The combination of single-cell stimulation and control experiments shows that the activity of single sensory cortical neurons can lead to a behaviourally reportable effect. It has been estimated that a single barrel cortical column contains approximately 8,500 excitatory cells that generate about 1,550 spontaneous action potentials in a 200 ms period and about 4,000 action potentials in response to a small (3.3°) whisker deflection
Our finding that stimulation of putative interneurons (which project locally) can lead to strong sensory effects suggests that (1) local circuits are involved in the read-out of single-cell activity and that (2) read-out mechanisms are sensitive to suppression of action potentials. Cortical microstimulation evokes pronounced and long-lasting inhibition (
Animals were trained to report microstimulation (40 cathodal pulses at 200 Hz, 0.3 ms pulse duration) applied to the barrel cortex through a tungsten microelectrode at a depth of 1,500 µm. In the first training session, current intensities no greater than 200 µA were applied; subsequently current intensity was decreased according to detection performance. During training animals were put on a water restriction schedule with daily access to water ad libitum for one hour after the experiment. Once the animal performed at detection thresholds no greater than 5 µA in at least one block of trials on two consecutive days, we switched to the single-cell stimulation report task; here microstimulation currents were on average 5.0 ± 1.6 µA. To stimulate single neurons close to the microstimulation site, we glued a tungsten microelectrode close to the tip of a glass pipette (average tip separation approximately 75 µm). The construct was inserted through the intact dura to a mean depth of 1,400 ± 271 µm, whereby the actual depth from the cortical surface was less because of dimpling and oblique penetrations. From the histologically identified neurons it appears that most single-cell stimulation experiments were performed in cortical layers 4, 5A and 5B. Cells were classified as fast spiking neurons (putative interneurons) if the action potential width was no greater than 0.4 ms (peak to trough) and/or if they responded with at least 50 action potentials (that is, at least 250 Hz) during at least one 200 ms current injection (see
We used standard surgical and electrophysiological techniques to prepare animals (n = 15 Wistar rats, about P35 at the day of surgery) for chronic, head-fixed recording of the barrel cortex (P3, L5 relative to bregma)
Cells were included if at least five catch trials and five single-cell stimulation trials had been presented that satisfied our inclusion criterion. All single-cell stimulation and catch trials were included for which the animal responded to both the preceding and the succeeding microstimulation. We also included all trials where the animal responded only to either the immediately preceding or immediately succeeding microstimulation. All numbers on single-cell stimulation experiments refer to these included trials. For our barrel cortex experiments an average of 30.2 single-cell stimulation trials and 17.7 catch trials were included per cell. Applying the inclusion criterion led to the exclusion of many trials with low response rate (on average 14.0 single-cell stimulation trials and 7.7 catch trials). When all trials were considered, animals still responded significantly more often in single-cell stimulation trials than in catch trials (data not shown). From these trial numbers it becomes clear that it is difficult to obtain a significant result on the single neuron level. Monte Carlo simulations show that approximately 480 single-cell stimulation trials (about 16 times more than our average) are required to obtain a significant outcome with 90% probability (one-sided binomial test by normal approximation, &agr; = 0.05) assuming a 10% effect size (30% hits, 20% false positives).
As we trained animals to report stimulation of the barrel cortex, we tested the prediction that single-cell stimulation led to responses (hits). Thus, differences between hit rates and false-positive rates were evaluated by using a one-sided, paired t-test, and differences between single-cell stimulation experiments and control experiments by using a one-sided, unpaired t-test. However, all results presented here were also significant when applying two-sided t-tests or when using Monte Carlo simulations of the statistical distributions. To test non-parametrically if the variance of sensory effects was greater for putative interneurons than excitatory neurons (see
It is our impression that the interneuron inclusion criteria (action potential width no greater than 0.4 ms and/or a response of at least 50 action potentials during at least one 200 ms current injection) were conservative and may have led to the false classification of potential interneurons as excitatory cells. The difference in sensory effects between putative interneurons and excitatory cells persisted when more inclusive interneuron inclusion criteria were chosen (data not shown). Further observations support our classification scheme. First, morphologically identified excitatory neurons were correctly classified as putative excitatory cells (
On the last day of experiments with an animal, we included biocytin (1.5%) in the stimulation pipette and processed brains as described previously
a, Stimulation experiments were performed in the barrel cortex of awake rats. Animals responded to stimulation by interrupting a light beam (dashed line) with multiple tongue licks. The time of the first lick was taken as the reaction time and reward was delivered for correct responses (right). Top, single-cell stimulation pipette with stimulation current wave form (upper) and tungsten microelectrode with stimulation pulse train (lower). b, Three types of stimulus were presented at random intervals (Poisson process, mean 3 s): microstimulation (2–8 &mgr;A) (40% probability), juxtacellular single-cell stimulation (40%) and no (or subthreshold) current injection ‘catch’ trials (20%). Licks within the interstimulus interval led to an additional 1.5 s delay to presentation of the next stimulus (left box) and were rewarded after a stimulus (right box) for all three trial types. c, Single-cell stimulation trial by juxtacellular current injection. Triangles indicate stimulation onset and offset artefacts. d, Evoked action potentials (open circles) in a series of stimulation trials. Spontaneous action potentials (solid circles) were quantified for 1 s before each stimulation. The left y axis label applies to both spontaneous and evoked action potentials; the right y axis label applies to evoked action potentials.
a, Reconstruction of the stimulated neuron with dendritic tree (red) and axon (blue, incompletely filled). Superimposed is a micrograph of a stimulation pipette and a tungsten microstimulation electrode aligned along the electrode track. Barrel rows (brown) are labelled with letters. L, layer; WM, white matter. b, Action potential (ticks) raster plots and first lick responses (red squares) during juxtacellular single-cell stimulation trials (top), no-current-injection catch trials (middle) and 19 randomly selected microstimulation trials (bottom). The neuron was inhibited during and after microstimulation (stimulation current, 4 µA). c, Quantification of responses to single-cell stimulation, catch trials and microstimulation.
a, Response rates for single-cell stimulation trials (hits) versus no-current-injection catch trials (false positives) (n = 51 neurons; note several points coincide). Fast spiking, putative interneurons (filled circles); non-fast spiking, putative excitatory neurons (empty circles). b, Response rates for single-cell stimulation trials (hits) versus subthreshold current injection catch trials (n = 19 neurons). Conventions as in a. c, Response rates for trials in which we applied 25 nA (twice the average juxtacellular stimulation current) into extracellular space (hits) versus no-current-injection catch trials (false positives) (n = 90 stimulation sites). d, Response rates for single-cell stimulation trials (hits) versus no-current-injection catch trials (false positives) for visual cortex neurons (n = 21), while microstimulation was applied in barrel cortex. Animals had been trained to report microstimulation in barrel cortex. e, Distribution of sensory effects (single-cell stimulation hit rate - catch trial response rate) across putative interneurons and putative excitatory neurons in barrel cortex single-cell stimulation experiments (conventions as in a).
a, Cumulative distribution of reaction times for microstimulation (dashed), single-cell stimulation (solid) and catch trials (dotted). b, Difference of the cumulative distributions of reaction times for single-cell stimulation and catch trials. This isolates the contribution of single-cell stimulation from false-positive responses. The vertical line marks the time where 50% of the peak difference is reached; the grey area marks the time from 25% to 75% of the peak difference. c, Difference of the cumulative distributions of reaction times for microstimulation and catch trials. Conventions as in b.
We thank B. Sakmann for suggesting the juxtacellular stimulation approach, J. van der Burg, E. Haasdijk and G. Maas for technical contributions, P. den Iseger and A. Lee for discussions, and G. Borst, M. Frens, C. Hansel, L. Herfst, A. Lee, B. Voigt and J. Wolfe for comments on the manuscript. This work was supported by the Bernstein Center for Computational Neuroscience and Humboldt University Berlin, Erasmus MC, and VIDI (NWO) and HFSP grants to M.B.
The file contains Supplementary Methods, Supplementary Figures 1-14 with Legends, Supplementary Results and Discussion and additional references.
Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor
TRPC5 is markedly activated by extracellular lanthanide ions
Pairs of cysteine residues may be covalently linked by a disulphide bridge that can be cleaved by reduction. We therefore applied the chemical reducing agent dithiothreitol (DTT) to HEK-293 cells expressing TRPC5 (refs
To test the hypothesis further, we expressed TRPC5 mutants containing alanine in place of cysteine. Such mutants were constitutively active and were not stimulated by reducing agent or lanthanide (
Thioredoxin is an important redox protein with established biological roles including those in cancer, ischaemic reperfusion injury, inflammation and ageing
Thioredoxin concentrations up to a mean of 0.41 &mgr;g ml
There have been no previous reports on the expression of TRPC channels in synovial joints, so we explored synovial tissue biopsies from patients with rheumatoid arthritis. TRPC5 and TRPC1 proteins were detected and localized together with CD55 (
The I–V relationship of the rTRX-evoked current in FLS cells was similar to that of the TRPC5–TRPC1 heteromultimeric channel (
Further evidence that TRPC5 and TRPC1 contribute to the endogenous rTRX-responsive channel of FLS cells came from studies with anti-TRPC5 (T5E3) and anti-TRPC1 (T1E3) antibodies, which target the predicted extracellular loop region and specifically block the functions of TRPC5 and TRPC1, respectively
One of the functions of FLS cells is to secrete matrix metalloproteinases (MMPs), which are associated with tissue remodelling and the progression of arthritis
The data of this study indicate that secreted TRX is a type of ion channel agonist that acts through its reduced form to break a restraining intra-subunit disulphide bridge between cysteine residues in TRPC5, thereby stimulating the channel either as a homomeric assembly or as a heteromultimer with TRPC1. A transduction mechanism is therefore revealed that can directly couple cell activity to extracellular reduced thioredoxin. This mechanism may have particular relevance in conditions such as rheumatoid arthritis, in which TRX concentrations are strongly elevated, but the broad distributions of TRX and the channels indicate that the mechanism could be widely used.
Synovial tissue biopsies were obtained with informed consent from patients diagnosed with rheumatoid arthritis at the Academic Unit of Musculoskeletal Disease, Chapel Allerton Hospital, Leeds. Ethical approval was given by the local ethics committee. Human synovial tissue biopsies were washed with PBS and digested in 0.25% type 1A collagenase for 4 h at 37 °C, after which FLS cells were cultured in
Whole-cell patch-clamp recordings were performed
Ionic currents are shown as positive values when they increased in response to a treatment, and as negative values when they decreased. Data are expressed as means and s.e.m., where n is the number of individual experiments. Data sets were compared by using paired or unpaired Student’s t-tests, with a significant difference indicated by P < 0.05 (asterisk) and no difference by n.s. All human tissue or cell data are derived from, or are representative of, at least three independent experiments on samples from three patients.
HEK-293 cells stably expressing tetracycline-regulated human TRPC5 have been described
A salt-agar bridge was used to connect the ground Ag–AgCl wire to the bath solution. Signals were amplified with an Axopatch 200B patch clamp amplifier and controlled with
Sections 4 &mgr;m thick were obtained from snap-frozen synovial tissue biopsy samples of patients suffering from rheumatoid arthritis, fixed with acetone and stored at -80 °C until use. Staining was in accordance with standard protocols. In brief, sections were incubated with primary antibody overnight at 4 °C and with secondary antibody (
FLS cells were cultured for 24 h in 24-well plates and serum-starved for 24 h; fresh serum-free medium was then added that contained a TRX cocktail, which included TRX (0.4 &mgr;g ml
All salts and reagents were from Sigma or BDH.
Whole-cell recordings from HEK-293 cells. a, In a cell expressing TRPC5, response to bath-applied 10 mM DTT and 75 &mgr;M 2-APB. b, I–V relationship from a. c, As for b but with 1 mM TCEP. d, Currents at -80 mV evoked by 10 mM DTT (n = 8), 1 mM TCEP (n = 5) or 5 mM MTSET (n = 6) in cells expressing TRPC5. DTT had no effect without TRPC5 (n = 5). e, Inhibition of current at -80 mV by 0.1 mM Cd
a–c, Whole-cell current data from HEK-293 cells expressing TRPC5 alone (a, b), TRPC1 alone or TRPC5 plus TRPC1 (c). a, Effect of 4 &mgr;g ml
a, Tissue sections from joints of patients with rheumatoid arthritis stained with T1E3 or T5E3 (green) or anti-CD55 (red) antibodies. Controls were omission of anti-CD55 antibody, and T1E3 or T5E3 preadsorbed on its antigenic peptide. b, Normalized rTRX-evoked I–V relationships for rabbit FLS cells (n = 3) and HEK-293 cells expressing TRPC5 and TRPC1 (n = 5). c, Changes in currents at -80 mV in response to 10 &mgr;M La
a, Zymogram showing MMP-9 (pro and active) and MMP-2 from rabbit. Ab, antibody. b, As for a but mean data after normalization of rabbit MMP-9 band intensity to the control group without antibody (n = 3 for each). c, ELISA data for human MMP-2 (n = 4). d, Effect of T5E3 (n = 4) on inhibition of human MMP-2 secretion by exogenous TRX cocktail. For each group, secretion in TRX was normalized to that in its absence (control). e, f, As for c, d, but for secretion of human pro-MMP-1. Results are expressed as means and s.e.m. Asterisk, P < 0.05; n.s., no significant difference.
This work was supported by Wellcome Trust grants to D.J.B. and A.S., and a Physiological Society Junior Fellowship to C.C. P.S. has an Overseas Research Scholarship and University Studentship, J.N. has a Biotechnology and Biological Sciences Research Council PhD studentship, Y.M. a university studentship and Y.B. a scholarship from the Egyptian Ministry of Higher Education.
The file contains Supplementary Tables 1-2 and Supplementary Figures 1-4 with Legends.
Epidemiological studies spanning more than 50 yr reach conflicting conclusions as to whether there is a lower incidence of solid tumours in people with trisomy 21 (Down’s syndrome)
The most widely used model of Down’s syndrome is the Ts65Dn mouse, which is trisomic for orthologues of about 100 Hsa21 genes and recapitulates in detail several phenotypes of Down’s syndrome
Female Ts65Dn mice were crossed to Apc
We reanalysed these data considering the inheritance of susceptible or resistant alleles of the modifier of Min 1 (Mom1) locus that result in higher or lower tumour number (Mom1
We analysed Ts1Rhr mice to narrow the candidate region for the gene or genes responsible for reduced tumour number. These mice have segmental trisomy for 33 of the genes that are triplicated in Ts65Dn (
The 33 genes at dosage imbalance in Ts1Rhr and Ms1Rhr mice include several possible candidates for the tumour number effect (
Mice that carried a single copy of Ets2 in a euploid background showed a substantial, 20% increase in tumour frequency (P = 0.075), reminiscent of the increase in tumours in Ms1Rhr mice, which carry a single copy of this gene. These mice developed severe disease much earlier than mice of other genotypes and several did not survive long enough for tumours to be counted. Thus this difference in tumour number is probably under-represented. Ets2 messenger RNA and protein levels corresponded directly to gene copy number in all of the genotypes (
The size of tumours in a given genetic background provides one indicator of tumour initiation and growth rates. We compared the size of tumours between trisomic and euploid Apc
To determine whether this difference was evident earlier in the course of tumour formation, intestines of trisomic and euploid mice carrying the Apc
In contrast to Ts65Dn mice, tumour size was not different from euploid in either Ts1Rhr or Ms1Rhr mice (data not shown). The absence of a tumour size phenotype even though tumour number is reduced in Ts1Rhr mice indicates that multiple genes on Mmu16 (and Hsa21) may contribute to different aspects of tumour repression caused by trisomy.
For 50 yr, epidemiological studies examining rates of solid tumours in individuals with Down’s syndrome have reached discrepant conclusions about whether trisomy is protective against cancer
Notable among the Hsa21 genes that have been implicated in pro- or anti-tumorigenesis is endostatin, an inhibitor of angiogenesis that has been shown to be a potent inhibitor of tumour growth in model systems
Two general implications that stem from the observation that trisomy and specifically Ets2 dosage can repress or promote tumour growth are worth special note. First, repression of tumorigenesis when Ets2 expression is elevated may in fact be a characteristic of many genes identified previously as oncogenes or tumour suppressor genes. Natural variation in average expression levels of ETS family (or other) repressor genes may exist in tumour-prone families without a known molecular basis for a high cancer frequency (reduced expression of Ets2) or in cancer-resistant families (elevated expression). This phenomenon might be exploited to identify a pharmacological-based approach to tumour protection.
Second, previous observations about the role of the ETS2 proto-oncogene in cancer could not have predicted that elevation of expression beyond euploid levels would provide a natural repression of tumour formation and growth. If trisomy for Hsa21 was not viable, the correlation of increased gene expression with lower solid tumour frequency would not occur in a systematic manner and may not have been observed for some time. The implication for promoting tumour resistance in all people on the basis of gene dosage of ‘oncogenes’ is thus a product of the genetic heritage of those with Down’s syndrome.
For Mom1, PCR primers were designed to amplify the wild-type (Mom1
PCR was used to type Ets2
All animals were assessed blind to genotype in all assays. Groups of littermate mice from closely related mothers (and inbred fathers) were euthanized at 120 ± 2 days of age. Intestines were removed and rinsed then cut longitudinally and placed in fresh PBS. Tumours were counted under 20× magnification across the entire length of the small intestine. For
For visible tumours (at 20× magnification), tumour size was determined for the longest axis of the tumour using an eyepiece reticule. Statistical significance was determined using a Student’s t-test. For microscopic tumours, intestines were recovered from Apc
Mouse embryo fibroblasts (MEFs) were established from fetuses at E13.5. Fetuses were removed and the visceral tissue separated. Remaining tissue was minced in Trypsin/EDTA and incubated at 37 °C for an hour. Trypsin was neutralized by addition of medium (DMEM plus 10% serum and antibiotics) and cells collected and plated, taking care to avoid transfer of larger pieces of tissue. The next day, cells were re-fed, then passaged as they reached confluence. For these experiments, cells were used between 6–8 passages.
Total RNA was isolated from mouse small intestine or MEFs with TRIzol reagent (invitrogen) and RNeasy Mini Kit (Qiagen), including a DNase I treatment step. RNA concentration was determined by UV spectrophotometry and 1 &mgr;g was reverse transcribed with GeneAmp RNA PCR kit (Applied Biosystems). After dilution, 10 ng of complementary DNA was amplified by real-time PCR with SYBR Green PCR master mix (Applied Biosystems) using specific primers for Ets2 (Forward, AGAGAAGGGAGCACAGCAAA; Reverse, AAGAACATGGACCAAGTGGC) (
For western blots, whole cell lysates were prepared by lysing MEFs with RIPA buffer, and 100 &mgr;g of protein from each sample was separated by 8% SDS PAGE. The membrane was blotted overnight with anti-Ets2 (ref.
Average tumour number at 120 days is measured for the four genotypes, error bars indicate s.d. Number of mice analysed, P value and the gene copy number of Ets2 in each strain are indicated. *, statistical significance by Student’s t-test of the designated pair. Although the increased tumour number in euploid Ets2
Distribution of tumour sizes for trisomic (open bars) and euploid (closed bars) mice. a, At 120 days of age, tumour number is reduced and tumours are significantly smaller in Ts65Dn, Mom1
The authors thank L. Siracusa for advice regarding the Apc
Author Contributions T.E.S. and R.H.R. designed the experiments. T.E.S. and A.Y. managed husbandry and collected tumour data, which were analysed by T.E.S., A.Y. and R.H.R.; F.L. and M.C.O. designed the Ets2 conditional knockout mice; and A.Y., F.L. and M.C.O. analysed Ets2 expression. R.H.R. wrote the paper with substantial input from all authors.
This file contains Supplementary Discussion and additional references; Supplementary Table S1 and Supplementary Figures S1-S6 with Legends.
NUMB is a cell fate determinant, which, by asymmetrically partitioning at mitosis, controls cell fate choices by antagonising the activity of the plasma membrane receptor of the NOTCH family
p53 is one of the major tumour suppressor proteins
Previous work has shown interaction in vivo between overexpressed NUMB and HDM2 (ref.
We analysed the effects of NUMB knockdown (NUMB-KD) on p53 by targeting two different sequences in NUMB using short interfering RNA (siRNA) or short hairpin RNA (shRNA), respectively. Both methods yielded 80–90% decrease in NUMB levels and an approximately twofold decrease in the p53 steady-state levels (
In NUMB-KD cells, p53 messenger RNA levels were not altered (
The simultaneous silencing of NUMB and HDM2 restored p53 to levels indistinguishable from that of control cells or HDM2-KD cells (
HDM2 regulates p53 turnover through its E3 activity. In NUMB-KD cells, we detected enhanced ubiquitination of p53, which was inhibited by nutlin (
NUMB inhibits NOTCH activity. Thus, it was important to prove that the observed effects were not a consequence of deregulated NOTCH activity. We treated NUMB-KD cells with inhibitors of presenilin/&ggr;-secretase (known as &ggr;-secretase inhibitors, GSI) to abolish NOTCH activity. GSI had no significant effect on p53 levels in NUMB-KD or control cells (
We tested whether the NUMB–HDM2 counteraction was relevant to p53-dependent transcriptional activity. HDM2 significantly inhibited the trans-activating ability of p53 on a luciferase reporter gene. However, NUMB restored, albeit not completely, this ability in a dose-dependent fashion (
Perturbation of NUMB levels should result in alterations in p53-mediated responses to DNA damage. To monitor DNA damage, we analysed the levels of phosphorylation at serine 139 of histone H2AFX (&ggr;-H2AX) in cells treated with cisplatin, because prolonged persistence of &ggr;-H2AX is considered to be a marker of persistent DNA damage
We also tested the effects of NUMB or p53 ablation on perturbations of the cell cycle induced by genotoxic drugs. In MCF10A cells, cisplatin induced an S-phase block; this was, however, independent of p53 or NUMB, and thus not informative for our purposes (
After treatment and washout with doxorubicin or SN38, MCF10A cells did not efficiently re-enter the cell cycle for at least 20 h (
We then performed experiments under conditions of NUMB overexpression. Expression of NUMB–GFP (green fluorescent protein) in MCF10A cells increased the levels of p53 in both unstressed cells and cisplatin- or doxorubicin-treated cells (
The above results demonstrate that NUMB overexpression increases p53 stability and activity, predicting enhancement of p53-mediated responses to genotoxicity, such as apoptosis. Thus, we monitored activation of caspases
In breast tumours, loss of NUMB expression is frequently detected
In class 1 compared with class 3 cells, the steady-state levels of p53 were reduced (
Deficient p53 activity is associated with resistance to the cytotoxic effects of chemotherapy
Finally, we analysed a cohort of 443 breast cancer patients who received adjuvant chemotherapy. We found that NUMB status was inversely correlated with the major clinical and pathological parameters indicative of biologically aggressive neoplastic disease (
Many questions await answers. It remains to be established where NUMB, HDM2 and p53 interact. This is not trivial because HDM2 and p53 are by-and-large nuclear proteins whereas NUMB is in the cytoplasm, mostly associated to biomembranes
Finally, because NUMB is involved in binary fate decisions
Cultivation of primary epithelial cells was performed as described previously
pG13–Luc and expression vectors for p53 and HDM2 were a gift of K. Helin. p53 shRNA pSUPER was extracted from a shRNA library (a gift from R. Bernard). The retroviral PINCO–NUMB–GFP vector was as described
Specific siRNA for NUMB and controls were as described previously
Real-time PCR for p53, HDM2, p21 (also known as CDKN1A), p53R2 (also known as RRM2B), PUMA (also known as BBC3) and FAS was performed using the TaqMan Gene Expression Assays Indentification: Hs00153349-m1, Hs00242813-m1, Hs00355782-m1, Hs00153085_m1, Hs00248075_m1 and
Cultivation of primary normal and tumour human mammary epithelial cells was as described previously
For survival assays (
Cells were washed with PBS and were subsequently fixed in 70% ethanol and either stored at 4 °C or directly used for staining with propidium iodide. For propidium iodide staining, cells were washed once in PBS supplemented with 1% BSA and were counterstained overnight with 5 &mgr;g ml
pG13–Luc, a luciferase reporter controlled by multimeric p53 binding sites, and expression vectors for p53 and HDM2 were a gift from K. Helin. A retroviral vector to silence p53 (p53-shRNA pSUPER) was extracted from a shRNA library (a gift from R. Bernard). The retroviral PINCO–NUMB–GFP vector was as described
Specific siRNAs for NUMB and the corresponding control were described previously
The relative quantity of mRNA transcripts for p53, HDM2, p21, p53R2, PUMA and FAS was determined by real-time PCR with TaqMan Gene Expression Assays Identification: Hs00153349-m1, Hs00242813-m1, Hs00355782-m1, Hs00153085_m1, Hs00248075_m1 and
Procedures for immunofluorescence, immunoblotting and immunoprecipitation were also performed as described
For the binding assays in
For the in vitro ubiquitination assay in
For sequential immunoprecipitations, total cellular lysates from HEK293 cells overexpressing HDM2, Flag–NUMB and p53 (
The GSI DFP-AA (also known as Compound E) and DAPT (
To establish a possible correlation between NUMB status of breast tumours and patient outcome, we used data from 443 breast cancer patients enrolled in a surgical trial
The 443 patients were followed for a median of 54.8 months (range 4.3–60). During the follow-up period, a total of 40 new events were registered. Ten patients developed a loco-regional event, 9 developed a controlateral carcinoma, and 21 developed a distant metastasis. Association between the clinical/pathological features of the tumours and NUMB expression was evaluated by the Pearson chi-squared test (see
a, MCF10A lysates (3 mg) were immunoprecipitated (IP) and immunoblotted (IB). The control was an irrelevant antibody. b, Pure HDM2, GST–p53 and NUMB were mixed (3.2 nM each) and the solution was immunoprecipitated and immunoblotted as shown. c, Left, lysates (40 mg) from U2OS cells were immunoprecipitated with anti-NUMB (control, irrelevant antibody) and an aliquot (one-twenty-fifth) of the immunoprecipitate and immunoblot as shown. Right, the immunoprecipitate was eluted with the immunogenic NUMB peptide (amino acids 537–551); the immunoprecipitate and immunoblot were as indicated. Inp, one-fortieth of the eluate; Lys., lysate. d, MCF10A lysates, transfected as shown, were immunoblotted as indicated. e, f, MCF10A cells, transfected as shown, were exposed to cisplatin (24 h). The immunoblot was as indicated. Right, quantification (mean of three experiments) of p53 induction in control (open circles) and NUMB-KD cells (filled circles). g, Quantitative PCR with reverse transcription (RT–PCR) in control siRNA (open bars) and NUMB-KD (filled bars) MCF10A cells. Values represent mean (control siRNA/no cisplatin = 1) ± s.d. from two experiments. Cisplatin, 8 h.
a, p53 mRNA levels in control-siRNA and NUMB-siRNA MCF10A cells. Values represent the mean ± s.d. (control siRNA = 1) from two experiments. b, c, MCF10A cells, transfected as shown, were treated with cycloheximide (CHX). Immunoblot was as indicated. In c, quantification of p53 and HDM2 levels in control-siRNA (open circles) and NUMB-siRNA (filled circles) cells are shown; values are expressed relative to time 0 (normalized to vinculin), and represent, in the case of p53, the mean ± s.d. of three experiments. d, e, f, Lysates from MCF10A cells, transfected and treated as indicated, were immunoprecipitated and immunoblotted as shown. In f, p53 levels were normalized by loading proportionally different amounts of cell extracts. g, GST–p53 was subjected to in vitro ubiquitination assay as indicated. Detection was in the immunoblot (Ub, anti-ubiquitin antibody). h, Lysates from MCF10A cells, transfected and treated as shown, were immunoblotted as indicated. i, Luciferase assay in U2OS cells transfected as indicated. Results represent mean ± s.d. from three experiments.
a, b, MCF10A cells, in which p53 (p53 shRNA, a) or NUMB (NUMB shRNA, b) had been silenced, were treated with cisplatin (6 &mgr;g ml
a, Class 1 and class 3 cells were treated with MG132 (+). Immunoblot was as indicated. b, p53 transcripts. Results represent mean (normalized to class 3) ± s.d. from four tumours. c, Class 1 and class 3 cells were transduced as shown. Immunoblot was as indicated. d, Class 1 and class 3 cells were transfected with HDM2 siRNA (+) or control siRNA (-). Immunoblot was as indicated. e, Class 1 and class 3 cells were transduced as indicated, treated with cisplatin and nutlin, and analysed for cell viability. Results represent mean ± s.d. from triplicate points. In a, c, d and e, results are representative of four class 1 and four class 3 cultures, from different patients. f, NUMB status (as evaluated by immunohistochemistry) and prognosis (as evaluated by cumulative probability of any secondary event) in patients with breast cancer. Kaplan–Meier curves were compared by the Log-rank test. Hazard ratio before adjustment for clinical and pathological features (Cox proportional hazard method), 0.610 (P, 0.0068); hazard ratio after adjustment, 0.651 (P, 0.0291); Log-rank, 0.00387.
We thank K. Helin for the p53 and HDM2 reagents; L. Van Parijis for the pLL3.7 lentiviral vector; R. Bernard for the p53 shRNA pSUPER vector; G. Matera for technical assistance; P. Maisonneuve and G. Goisis for statistical analysis; the Imaging Service at IEO; and the Real Time PCR Service at IFOM. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and MIUR to S.P. and P.P.D.F., from the European Community (VI Framework), The Ferrari Foundation, the Monzino Foundation and the CARIPLO Foundation to P.P.D.F., and from the G. Vollaro Foundation to S.P.
Author Contributions I.N.C., D.T., F.S.-N. and S.P. performed experimental work. P.N., V.G. and G.V. performed the clinical part of the work (patient selection, histology and data analysis of the patient’s case collection). S.P. and P.P.D.F. planned and supervised the project, performed data analysis and wrote the manuscript.
This file contains Supplementary Methods, Supplementary Figures 1-2 and Supplementary Table 1.
This file contains Supplementary Video 1.
This file contains Supplementary Video 2.
This file contains Supplementary Video 3.
Post-translational modification (PTM) of proteins plays an important part in mediating protein interactions and/or the recruitment of specific protein targets
Damaged DNA and the mitotic apparatus (mitotic spindle, centromeres and centrosome) represent major sites of poly(ADP-ribose) accumulation
CHFR is a ubiquitin ligase that functions in the antephase checkpoint by actively delaying passage into mitosis in response to microtubule poisons
The putative C2H2 zinc-finger is separated by a 6–8 amino acid spacer and has the consensus [K/R]xxCx[F/Y]GxxCxbbxxxxHxxx[F/Y]xH (
Mutation of the conserved cysteine residues in the single putative PBZ motif within CHFR (
Depletion of zinc, by incubation of wild-type CHFR and APLF with the metal-chelating agent EDTA, resulted in a severe reduction in the ability of each protein to bind PAR (
Interactions between PAR and recombinant CHFR and APLF were further analysed by surface plasmon resonance (
To investigate whether APLF and CHFR were themselves substrates for poly(ADP-ribosyl)ation, they were incubated with PARP1 in the presence of
Mutational analysis of the PBZ motif revealed that the conserved arginine preceding the zinc finger was required for PAR-binding in both APLF (APLF*R1) and CHFR (CHFR*R1) (
We next determined whether APLF and CHFR associate with PAR in vivo. When Flag-tagged APLF and CHFR proteins were transiently expressed in HEK293T cells, we found that the Flag pull downs of each protein contained PAR, as detected by western blotting (
To examine the functional importance of the PBZ motif, we analysed its requirement for the CHFR-dependent antephase checkpoint in Ptk1 cells. Following treatment with microtubule poisons (such as colcemid), late G2 and prophase cells with an intact checkpoint delay entry to mitosis and decondense their chromosomes, while the nuclear envelope remains intact
HeLa cells do not express CHFR and therefore lack an intact antephase checkpoint
Finally, a direct link between PAR metabolism and the antephase checkpoint was established by treating Ptk1 cells with the specific PARP inhibitor KU-0058948
In this work, we have defined a novel PAR-interaction motif present in a number of proteins associated with the DNA damage response and checkpoint regulation. Although two functionally equivalent domains have previously been reported, this is the first example of a zinc-dependent motif implicated in PAR binding and poly(ADP-ribosyl)ation. Zinc fingers were originally identified as nucleic acid recognition elements
The use of the PBZ motif is widespread amongst eukaryotes, and is particularly prominent in Dictyostelium discoideum (
Using CHFR, we established the functional importance of the PBZ motif, demonstrating that specific PBZ-targeted mutations abrogate CHFR function in the antephase checkpoint and that treatment with a PARP inhibitor abolished this checkpoint in CHFR-proficient cells. Thus, PAR assumes a major role in modulating CHFR activity, and consequently in regulation of the antephase checkpoint in response to microtubule poisons. The physiological importance of the PBZ motif is further supported by observations that APLF localizes at sites of DNA damage, by a mechanism dependent on the region of APLF containing the PBZ motif and on PAR synthesis
In general, PAR modifications regulate a dynamic network of intermolecular associations. It has been estimated that PARP1-associated PAR constitutes the major fraction of PAR within the cell
All proteins were purified after expression in Escherichia coli. Modification by PARP1 was carried out using a
APLF and CHFR clones were obtained from the RZPD German Resource Centre for Genome Research, whereas a C. elegans
Rabbit anti-APLF polyclonal antibodies were raised against purified recombinant APLF protein prepared from E. coli. Mouse monoclonal and rabbit
Recombinant proteins were modified using a
Proteins (2 pmol) were spotted onto a nitrocellulose membrane, which was subsequently blocked with TBS-T buffer (Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween) supplemented with 5% milk. Radioactively labelled PAR was prepared from 200 ng of automodified PARP1 as described
For the zinc-depletion experiments, proteins were incubated with 20 mM EDTA overnight at 4 °C. For zinc rebinding, the proteins were desalted and incubated with 1 mM ZnSO4.
Biotinylated PAR was coupled to streptavidin-coated BIACORE sensor chips, and assays were carried in 20 mM HEPES, pH 7.2, 150 mM NaCl and 0.005% P20 surfactant. Off rates were measured in the presence of 5 µM PAR (defined in monomeric units) using the coinject function of the Biocore. Biotinylated PAR was produced using 10 µM
Human embryonic kidney 293T cells were transiently transfected using
HEK293T cells grown on glass coverslips were transfected with GFP-fusion constructs of the wild-type and mutant APLF and CHFR proteins. Lipofectamine 2000 was used as a transfecting agent. Post transfection (24 h), the cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS and blocked with 2% BSA in PBS. Following incubation with the monoclonal anti-PAR antibody, and Alexa Fluor 546 goat anti-mouse
Auto-ubiquitylation was performed essentially as described
Ptk1 cells were cultured on 0.15 mm
HeLa cells were transfected with CFP-tagged wild-type or mutant CHFR by electoroporation (250 V, 1,500 mF,
In the PARP inhibition experiments, Ptk1 cells were pre-treated with 1 µM KU-0058948 or PBS for 1 h and challenged with 15 µM colcemid. Prophase cells that decondensed their chromosomes were scored as returning to interphase, and cells that broke down their nuclear envelopes were scored as continuing to mitosis.
a, Schematic diagram of the PBZ motifs in CHFR and APLF. Mutations are indicated in red. b, PAR binding by APLF, CHFR and C. elegans DNA ligase III (lig3) as determined by dot-blot analysis. XRCC1 and BSA were used as controls. c, PAR-binding is abolished by mutations in the PBZ motif. WT, wild type. d, PAR-binding is dependent on zinc. e, Analysis of PAR binding by surface plasmon resonance. f, In vitro poly(ADP-ribosyl)ation of APLF and CHFR by PARP1. g, PAR binding by the APLF and CHFR PBZ mutants.
a, Immunoprecipitation of Flag-tagged APLF and CHFR from HEK293T extracts. Inputs (10%) and Flag-precipitates were blotted using PAR, APLF and CHFR antibodies. Ubi-CHFR indicates ubiquitylated CHFR. b, Association of PAR with wild-type and PBZ-mutated Flag-tagged APLF. c, Co-localization of wild-type APLF and CHFR proteins with PAR in HEK293T cells transfected with GFP-tagged APLF and CHFR wild-type and mutant constructs. Scale bar, 10 µm.
a, Ptk1 cells expressing CFP-tagged CHFR&Dgr;FHA (top panels) or CHFR*PBZ&Dgr;FHA (lower panels) were treated with 15 µM colcemid (0 min) and their behaviour was monitored by time-lapse DIC and fluorescence microscopy at 3-min intervals. Scale bar, 10 µm. b, Quantification of the data from a. c, HeLa cells expressing wild-type or mutant CHFR and untransfected cells were treated with colcemid and the mitotic indices (±s.d., n = 3) were determined at two-hourly intervals. d, Auto-ubiquitylation of CHFR does not impair PAR-binding. Wild-type and mutant CHFR were ubiquitylated in vitro and analysed for PAR binding (upper panels) or by western blotting (lower panels). e, Analysis of Ptk1 cells pre-treated with the PARP inhibitor KU-0058948 (1 µM), or PBS for 1 h, and challenged with 15 µM colcemid. Their behaviour was monitored by time-lapse DIC.
We thank T. Lindahl (LRI, CRUK) for XRCC1, G. Smith for the PARP inhibitor KU-0058948, and J. Gannon for assistance with the Biacore. This work was supported by Cancer Research UK, the EU DNA Repair Consortium and the Louis-Jeantet Foundation. I.A. and D.A. are supported by EMBO fellowships.
Author Contributions I.A. and D.A. discovered the PBZ motif and performed most of the experiments. A.J.C. carried out supporting analyses. T.M. and J.P. defined the role of PBZ in the antephase checkpoint. S.J.B. and S.C.W. are joint senior authors who managed the project and helped write the manuscript.
The file contains Supplementary Figures 1-4 with Legends and Supplementary Tables 1-4.
Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources
Ethanol is not an ideal fuel because it has a lower energy density than gasoline, and its hygroscopicity poses a problem for storage and distribution. Higher alcohols (C4 and C5), on the other hand, have energy densities closer to gasoline, are not hygroscopic, and are less volatile compared with ethanol. Except for 1-butanol
Here, we devised a synthetic approach to produce the above-mentioned longer chain alcohols as next-generation biofuels. This strategy was implemented in E. coli, although other user friendly hosts such as Saccharomyces cerevisiae are readily applicable. These host organisms have fast growth rates and are facultative anaerobes, allowing for a flexible and economical process design for large-scale production
2-Keto acids are intermediates in amino acid biosynthesis pathways. These metabolites can be converted to aldehydes by broad-substrate-range 2-keto-acid decarboxylases (KDCs) and then to alcohols by alcohol dehydrogenases (ADHs). Using this strategy, only two non-native steps were needed to produce biofuels by shunting intermediates from amino acid biosynthesis pathways to alcohol production (
A critical enzyme in this alcohol production strategy is KDC, which is common in plants, yeasts and fungi but less so in bacteria
The existing E. coli metabolic pathways were then genetically modified to increase the production of the specific 2-keto acid so that the desired alcohol could be produced. To produce isobutanol, the ilvIHCD genes under the control of the PLlacO1 (ref.
To improve isobutanol production further, the alsS gene from Bacillus subtilis was used instead of ilvIH of E. coli. AlsS of B. subtilis has high affinity for pyruvate, whereas E. coli IlvIH has higher preference for 2-ketobutyrate
To demonstrate the generality of this approach, the same strategy was also applied to 1-butanol production. Some clostridial species produce 1-butanol during fermentative growth and many of the enzymes in this pathway are oxygen-sensitive and CoA-dependent
Therefore, to produce 1-butanol, the operon encoding the ilvA–leuABCD pathway under the control of PLlacO1 (ref.
To improve 1-butanol production further, the ilvD gene was deleted. This gene encodes dihydroxy-acid dehydratase
Because strains of E. coli that hyperproduce
Non-native hosts such as E. coli lack tolerance to high alcohols. Isobutanol is slightly less toxic to microorganisms than 1-butanol. The native 1-butanol producers can tolerate concentrations of 1-butanol up to about 2% (w/v) (ref.
The strategy described above opens up an unexplored frontier for biofuels production, both in E. coli and in other microorganisms. This strategy takes advantage of the well-developed amino acid production technology, and channels the amino acid intermediates to the 2-keto acid degradation pathway for alcohol production. The strategy avoids CoA-mediated chemistry, which is commonly used in alcohol production in native organisms, and enables the synthesis of other higher and complex alcohols on large scales. Specific strategies for producing other alcohols can be readily devised based on the synthetic pathways and metabolic physiology. These strategies can also be implemented in yeast or other industrial microorganisms. In the case of isobutanol production, the complete pathway is CoA-independent and requires only pyruvate as a precursor. This feature avoids the mitochondria compartmentalization issue of acetyl-CoA when implementing the strategy in yeast.
The JCL16 strain is BW25113 (rrnBT14 &Dgr;lacZWJ16 hsdR514 &Dgr;araBADAH33 &Dgr;rhaBADLD78) with F′ transduced from XL-1 blue to supply lacI
Unless stated otherwise, M9 medium containing 0.2 M glucose and 1,000th dilution of Trace Metal Mix A5 (2.86 g H3BO3, 1.81 g MnCl2·4H2O, 0.222 g ZnSO4·7H2O, 0.39 g Na2MoO4·2H2O, 0.079 g CuSO4·5H2O, 49.4 mg Co(NO3)2·6H2O per litre water) was used for cell growth. Ampicillin (100 &mgr;g ml
A list of the oligonucleotides used is given in
To clone ADH2, genomic DNA of S. cerevisiae (ATCC) was used as a PCR template with a pair of primers A67 and A68. PCR products were digested with SphI and XbaI and cloned into pSA46 cut with the same enzyme, creating pSA49.
To clone kivd, genomic DNA of Lactococcus lactis (ATCC) was used as a PCR template with a pair of primers A96 and A97. PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme, creating pSA55.
To clone ARO10, we used genomic DNA of S. cerevisiae (ATCC) as a PCR template with a pair of primers A98 and A99. PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme, creating pSA56.
To clone THI3, we used genomic DNA of S. cerevisiae (ATCC) as a PCR template with a pair of primers A100 and A101. PCR products were digested with Acc65I. pSA49 was digested with SphI and blunted with Klenow fragment, followed by digestion with Acc65I. This backbone was ligated with PCR products, creating pSA57.
To clone the pdc gene of Clostridium acetobutylicum, we used genomic DNA of C. acetobutylicum (ATCC) as a PCR template with a pair of primers A102 and A103. PCR products were digested with Acc65I and SphI and cloned into pSA49 cut with the same enzyme, creating pSA58.
To replace PLtetO1 of pZE21-MCS1 (ref.
To clone ilvC, genomic DNA of E. coli MG1655 was used as a PCR template with a pair of primers A71 and A72. PCR products were digested with SalI and XmaI and cloned into pSA40 cut with the same enzyme, creating pSA45.
To clone ilvD, genomic DNA of E. coli MG1655 was used as a PCR template with a pair of primers A74 and A84. PCR products were digested with BspEI and MluI and cloned into pSA45 cut with SalI and MluI, creating pSA47.
To clone ilvI and ilvH, genomic DNA of E. coli MG1655 was used as a PCR template with a pair of primers A70 and A83. PCR products were digested with BsaI and SalI and cloned into pSA40 cut with Acc65I and SalI, creating pSA51.
To clone ilvC and ilvD downstream of ilvH, pSA47 was digested with SalI and MluI. The shorter fragment was purified and cloned into plasmid pSA51 cut with the same enzymes, creating pSA52.
To replace replication origin with p15A, pZA31-luc (ref.
pSA66 includes the 3′ fragment of an alsS sequence. The alsS sequence was obtained using the genomic DNA of Bacillus subtilis as a PCR template with a pair of primers A123 and A124. PCR products were digested with Acc65I and SalI and cloned into pSA40 cut with the same enzyme.
pSA67 includes alsS sequence. The 5′ fragment of the alsS sequence was obtained using the genomic DNA of B. subtilis as a PCR template with a pair of primers A125 and A126. PCR products were digested with BsrGI and XbaI and cloned into pSA66 cut with Acc65I and XbaI.
pSA68 includes ilvC and ilvD sequence downstream of alsS. pSA47 was digested with SalI and MluI. The shorter fragment was purified and cloned into plasmid pSA67 cut with the same enzymes.
pSA69 was created by transferring the p15A replication origin from pZA31-luc, digested with SacI and AvrII, to plasmid pSA68.
To clone leuABCD, genomic DNA of E. coli MG1655 was used as a PCR template with a pair of primers A106 and A109. PCR products were digested with SalI and BglII and cloned into pSA40 cut with SalI and BamHI, creating pSA59.
To clone ilvA, genomic DNA of E. coli MG1655 was used as a PCR template with a pair of primers A104 and A105. PCR products were digested with Acc65I and XhoI and cloned into pSA59 cut with Acc65I and SalI, creating pSA60.
To replace replication origin with p15A, pZA31-luc (ref.
Alcohol compounds produced by our strains were identified by GC–MS. The system consisted of model
The produced alcohol compounds were quantified by a gas chromatograph equipped with flame ionization detector. The system consisted of a model
a, Various 2-keto acid precursors lead to corresponding alcohols through 2-ketoacid decarboxylase and alcohol dehydrogenase. b, The synthetic networks for the non-fermentative alcohol production in engineered E. coli. Red arrows represent the 2-keto acid decarboxylation and reduction pathway. Blue enzyme names represent amino acid biosynthesis pathways. The double lines represent a side pathway leading to norvaline and 1-butanol biosynthesis.
The cells were grown in M9 medium containing 36 g l
This work was partially supported by UCLA-DOE Institute for Genomics and Proteomics. We are grateful to H. Bujard for plasmids, and members of the Liao laboratory for discussion and comments on the manuscript.
Author Contributions S.A. and J.C.L. designed experiments; S.A. and T.H. performed the experiments; S.A. and J.C.L. analysed the data; and S.A. and J.C.L. wrote the paper.
The file contains Supplementary Figures 1-6 with Legends.
Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions
Mistranslation results from insertion of amino acids at wrong codons
For class II tRNA synthetases such as ThrRS (encoded by thrS) and AlaRS, a special insertion or fusion provides the centre for editing
Genome-encoded, active free-standing fragments homologous to editing domains of tRNA synthetases, including AlaRS, are widely distributed in nature
To separate out the functional components of AlaRS required for editing and tRNA recognition, deletion mutants lacking the catalytic site for aminoacylation were created. The design of these mutants was roughly based on the sequences of AlaXps, either type I (E. coli AlaRS(438–730)) or type II (E. coli AlaRS(438–875)) (see
Both E. coli AlaRS(438–730) (homologous to type I AlaXp) and E. coli AlaRS(438–875) (homologous to type II AlaXp) were tested for their ability to deacylate Ser–tRNA
Still unclear was whether fragment E. coli AlaRS(438–875), which lacks the ĠU-specific determinants of the aminoacylation domain, would be specific for tRNA
Thus, specific determinants for the recognition of tRNA
To investigate further the specificity of the newly found determinants for tRNA recognition in the region outside the domain for aminoacylation, we installed the G3˙U70 base pair into Ser–tRNA
This work shows that AlaRS contains two protein motifs for specific recognition of tRNA
Constructs described were prepared by PCR of the targeted sequence and cloning of the PCR product. Recombinant protein was produced by E. coli overexpression and Ni-NTA purification. The concentration of purified proteins was determined by Bradford assay. Transfer RNA was produced by either in vivo overexpression
Assays were performed at 25 °C (pH 7.5) with assay buffer (50 mM HEPES (pH 7.5), 20 mM KCl, 2 mM DTT and 10 mM MgCl2) in 96-well plates as described
The enzyme concentration in
Plasmids for expression of E. coli alaS deletion mutants were constructed through PCR amplification of the targeted region of the alaS gene with oligonucleotides containing either an Nde1 or an Xho1 site and ligated into pET21b as described above to generate pET21b-EcAlaRS(438–875), pET21b-EcAlaRS(438–730) and pET21b-EcAlaRS(438–808). The plasmid for expression of M. mazei AlaXp was constructed through PCR amplification of
Transfer RNA (E. coli tRNA
E. coli AlaRS, all deletion mutants, and M. mazei AlaXp were prepared by gene expression from plasmid pET21b in
Deacylation assays are described in the Methods Summary. Binding of tRNA
An open BLAST search was initiated using the sequence of M. mazei AlaXp. From all of the hits obtained in this search, known AlaRS and ThrRS sequences were discarded. Next, type II AlaXps, which contain the tRNA-binding C-domain, were manually removed. Finally, sequences lacking the two known catalytic motifs (HxxxH and CGGxxH) were discarded. This left approximately three groupings of sequences (group one representing ∼70%; group two, ∼20%; and group three, ∼10%). To select approximately ten sequences for a representative alignment, six sequences of group one were randomly selected, two sequences from group two were selected, and M. mazei (group one) and P. horikoshi (group three) sequences were added. The AlaRS sequences of the corresponding organisms were then selected. The sequence alignments in
For generation of an AlaRS–tRNA docking model, coordinates of a model of E. coli AlaRS were obtained through the Phyre server (Imperial College, London,
a, Domains of E. coli AlaRS and type I and II AlaXps. The domain for amino acid activation and G3˙U70 tRNA
a, Deacylation of Ser–tRNA
The first checkpoint occurs in the N-terminal domain in recognition of tRNA
We thank P. O’Maille for his gift of plasmid pH8GW and M. Sokabe for discussions about the model of the tRNA complex with Pyrococcus horikoshi AlaXp. This work was supported by grants from the National Institutes of Health, the Skaggs Foundation, and the National Foundation for Cancer Research.
Author Contributions K.B., M.M. and E.M. performed experiments and produced all materials. K.B., M.M. and P.S. conceived ideas, designed experiments, and wrote and edited the manuscript. All authors reviewed and approved the final manuscript.
This file contains Supplementary Notes on structure validation, Supplementary Tables 1-3, Supplementary Figures 1-11 with Legends, and additional references.
The ‘RNA world’ hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter’s domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts (‘RNPzymes’). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure
The group I intron catalytic core has a conserved three-dimensional structure consisting of two extended RNA domains, P4–P6 and P3–P9, which interact to form the intron’s active site (
We recently determined a crystal structure of CYT-18/&Dgr;424–669, which lacks the flexibly attached C-terminal domain but still promotes the splicing of most group I introns
Here we determine by molecular replacement a co-crystal structure of CYT-18/&Dgr;424–669 bound to the bacteriophage Twort orf142-I2 group I ribozyme, using data extending to 4.5 Å resolution. At this resolution, interacting regions of the protein and RNA are clearly discernible, but local conformational changes could go undetected (
The structure shows that the Twort RNA binds across the two CYT-18 subunits (denoted A and B), with the protein contacting both the P4–P6 and P3–P9 domains of the catalytic core of the intron but not peripheral RNA structures. The intron RNA-binding surface is electropositive
The P4–P6 domain of group I introns is a rod-like structure formed by the stacking of the P4 and P6 helices (
In addition to contacting the P4–P6 domain, biochemical studies indicated that CYT-18 also contacts the P3–P9 domain to stabilize the two domains in the correct relative orientation to form the intron’s active site
The J3/4 junction region is seen in the structure to interact with the N-terminal extension H0-B and Ins1-B of CYT-18 (
In J6/7, the first two nucleotides (G117 and C118) form major-groove triples with the first two base pairs of P4, and the last nucleotide (A119) interacts with P7 to help in forming the guanosine-binding site
The P3–J6/6a interaction may be promoted by CYT-18 in two ways. First, the previously described docking of Ins1-B in the major groove of P6 and P6a (
Finally, the interaction between the L9 tetraloop and the minor groove of P5 seems to be stabilized by direct contacts with the protein. The L9 tetraloop is positioned by the binding of Ins2-B within the minor groove of P9 and by contacts to H5-A, whereas P5 is oriented by the previously described interaction between subunit A and P5a (
The N. crassa mitochondrial group I introns that are natural substrates for CYT-18 form by themselves most of the conserved secondary structure, but they form little tertiary structure even at high Mg
The structure suggests that CYT-18 interacts almost exclusively with the intron’s phosphodiester backbone, with the only potential base contacts between residues in Ins2-B and P9, and between H0-B and J3/4. These findings agree with previous chemical footprinting experiments, which showed similarly positioned phosphate-backbone protections in the N. crassa ND1, mitochondrial large subunit rRNA (LSU) and yeast bI5 introns, and few if any base contacts (
The co-crystal structure provides a striking snapshot of how structural functions of RNA can be assumed by proteins. Previous work showed that CYT-18 could replace the peripheral RNA structure P5abc to promote the splicing at low Mg
The structure also shows how the unique structural adaptations of the N. crassa mitochondrial TyrRS are related to splicing activity. Thus, the CYT-18-specific insertions H0, Ins1 and Ins2, which had been implicated previously in splicing activity
Finally, analysis of genome sequences showed that H0, Ins1 and Ins2 are uniquely characteristic of mitochondrial TyrRSs of fungi belonging to the same subphylum as N. crassa and P. anserina (Pezizomycotina), and we confirmed that several of these other mitochondrial TyrRSs have group I intron splicing activity (P.J.P. and A.M.L., unpublished observations). These findings suggest that these mitochondrial TyrRSs adapted to function in splicing after the divergence of the Pezizomycotina and the Saccharomycotina about 360 million years ago
The Twort ribozyme and CYT-18/&Dgr;424–669 protein were synthesized and purified as described
Data were collected at beamline 23-ID-D at the Advanced Photon Source, and indexed and integrated with HKL2000 (ref.
A gene encoding a ribozyme derivative of phage Twort orf142-I2 was synthesized by PCR amplification of pTHS17 (ref.
CYT-18 protein was added to the RNA such that the molar ratio of dimeric CYT-18/&Dgr;424–669 to RNA was 1:1. Before crystallization, the buffer was exchanged to 10 mM potassium cacodylate pH 6.5, 15 mM MgCl2, 50 mM KCl and concentrated to 100 &mgr;l with a
Molecular replacement was performed with Phaser
The model was refined with CNS
a, Ribbon diagram. CYT-18 subunits A and B are coloured magenta and violet, respectively, and CYT-18-specific insertions H0, Ins1 and Ins2 are coloured cyan. b, Secondary structure of the Twort orf142-I2 intron ribozyme showing nucleotide residues within 4 Å of the protein in the co-crystal structure (circled). Boxed nucleotide residues correspond to phosphodiester-backbone positions protected by full-length CYT-18 in the N. crassa ND1 intron
a, CYT-18 binds the length of the P4–P6 domain, with the CYT-18-specific insertions (cyan) contributing to the formation of binding pockets for different regions. CYT-18 is drawn as a surface model, with subunits A and B coloured magenta and violet, respectively. b, c, Putative contacts between CYT-18 and the P4–P6 domain. The surfaces of protein atoms within 4 Å of the RNA are coloured red, atoms within 4–5 Å yellow, atoms within 5–10 Å peach, and atoms farther away than 10 Å orange. The views in b and c are rotated by 90° around the vertical axis.
RNA and protein are coloured as in
a, Orthogonal ribbon diagrams of the CYT-18/&Dgr;424–669–Twort ribozyme co-crystal structure. b, The corresponding views of the T. thermophila LSU intron crystal structure
We thank C. Correll, M. Hermodson, R. Russell and J. Tesmer for comments on the manuscript; T. Cech for discussions; the staff of SER-CAT and GM/CA-CAT, N. Sanishvili, D. Khare and M. Oldham for assistance with crystallographic data collection; and H. Kim for performing kinetic assays. Data were collected at GM/CA-CAT and SER-CAT beamlines at the Advanced Photon Source, Argonne National Laboratory. This work was supported by a grant from the National Institutes of Health to A.M.L.
Author Contributions P.J.P. and E.C. prepared materials for crystallization. E.C. crystallized the CYT-18–Twort RNA complex. J-H.C. collected and processed diffraction data. P.J.P. solved the structure. P.J.P., B.L.G. and A.M.L. interpreted data and wrote the paper.
The file contains Supplementary Table S1, Supplementary Figures S1-S9 with Legends and additional references.
Typical 2-Cys peroxiredoxins (Prxs) have an important role in regulating hydrogen peroxide-mediated cell signalling
Reactive oxygen species, such as hydrogen peroxide (H2O2) and peroxynitrite, have been recognized as compounds capable of modifying protein, DNA and lipids, especially when present at elevated levels
Structural studies on 2-Cys Prxs have revealed that the active site region can exist in fully folded and locally unfolded states
Using X-ray crystallography, we determined the structure of human Srx in complex with PrxI to 2.6 Å resolution after screening many engineered constructs. This complex contained one PrxI dimer (
A superposition of the model of human Srx with ATP bound to it
A comparison of the Srx-PrxI structure to Prx molecules present in two different oxidation states further supports the necessary flexibility of the GGLG motif, the active site helix containing the Cys-SPH residue, the YF motif, and Cys-SRH movements. The Srx and the YF motif of the adjacent Prx monomer cannot occupy the same space at the same time (
The binding of the PrxI C terminus onto the backside of Srx was surprising (
The Srx variants were analysed by circular dichroic spectroscopy (
The necessity for the C terminus of 2-Cys Prxs to bind and embrace Srx highlights its expanding cellular roles. For example, the interaction of the human PrxI C terminus with the PDZ domain of Omi/HtrA2 is necessary to promote protease activity
The importance of Srx is likely to extend beyond the repair of the decameric form of 2-Cys Prxs. The association of Prx decamers into stacks of toroids has been observed via electron microscopy and within the crystal structure of human PrxII-SO
In summary, the embrace observed in the Srx-Prx complex represents an unexpected structural rearrangement fundamentally important for the repair of Prxs in higher organisms. A structural basis is now available for designing future biochemical and cellular studies to dissect additional aspects of the Srx reaction mechanism and the roles of Srx and Prxs in modulating cell signalling.
One key to stabilizing the Srx–Prx crystals was to mimic the proposed thiosulphinate intermediate (
Several approaches were used to obtain crystals of the human Srx–Prx complex. Both His-tagged and untagged recombinant PrxI and human PrxII constructs were simultaneously manipulated to obtain crystals. Attempts to crystallize hyperoxidized PrxI and PrxII decamers in association with Srx and the presence or absence of ATP/ADP were not successful. In order to facilitate crystallization, a covalent disulphide bond between the active site Cys 99 of Srx and the peroxidatic Cys in Prx (Cys 52 in PrxI and Cys 51 in PrxII) was generated as described below. The dimer–dimer interface of the Prx decamer was manipulated by mutating Cys 83 to Glu/Ser/Val in PrxI or the corresponding Thr 82 in PrxII to Glu or Val. Some of these mutant constructs were also truncated at the C terminus (position 170 or 185 in PrxI and the corresponding positions in PrxII). Three different forms of Srx were used in the crystallization attempts as well: full length (residues 1–137), ET-Srx (32–137) or TT-Srx (38–137)
Human Srx was expressed using
A single wavelength (1 Å) data set was collected on beamline X8C at NSLS. Diffraction intensities were integrated using
In order to generate the hyperoxidized form of PrxI for Srx to repair, WT PrxI was added to a solution containing 50 mM Tris pH 7.5 and 100 mM KCl. Hyperoxidation was achieved by four step-wise additions of 5 mM H2O2 and 10 mM DTT with incubation for 30 min at 37 °C. Excess DTT and H2O2 were removed by extensive buffer exchange (50 mM Tris-HCl pH 7.5 and 100 mM KCl) via ultrafiltration (
The N-terminal amine of ET-Srx (present in 75 mM NaHCO3 adjusted to pH 8.3) was derivatized by incubation with a twofold molar excess of the fluorophor
a, In the typical 2-Cys Prx catalytic cycle (violet), the peroxidatic Cys is depicted as a thiol (SPH) or sulphenic acid (SPOH). The disulphide bond between Cys-SP and the resolving Cys, Cys-SRH, from the adjacent monomer is reduced by Trx
a, ATP modelled (translucent) into the active site of the Srx–PrxI complex containing the disulphide bond between Cys 99 of Srx (cyan) and Cys 52 of PrxI (violet). b, Ribbon diagram of the locally unfolded human PrxI active site in complex with Srx. c, Human PrxII-SO
a, The C-terminal tail of PrxI binds to Srx. Conserved residues of PrxI are coloured orange, and the conserved surface of Srx is coloured grey. b, Surface residues of Srx that interact with the C terminus of PrxI. c, Srx–PrxI-SO
We thank M. Murray for contributions to the structure determination, L. B. Poole, P. A. Karplus and N. H. Heintz for discussions, the staff of the NSLS and beamline X8C for their assistance during data collection and the RapiData course, and R. R. Hantgan for help with the circular dichroism and fluorescence anisotropy experiments. This work was supported by an NIH grant (W.T.L.) and an American Heart Association Postdoctoral Fellowship (T.J.J.). NSLS is supported by the US Department of Energy and NIH.
Author Contributions T.J.J. and L.C.J. performed all biochemical and crystallization experiments. T.J.J. and W.T.L solved the structure. T.J.J. and W.T.L. wrote the paper. All authors discussed the results and commented on the manuscript.
Coordinates and structure factors have been deposited with the Protein Data Bank under the accession number