Under certain conditions, ice with a cubic stacking sequence can also be formed, but ideal ice Ic without stacking-disorder has never been formed until recently. Here we demonstrate a route to obtain ice Ic without stacking-disorder by degassing hydrogen from the high-pressure form of hydrogen hydrate, C2, which has a host framework isostructural with ice Ic. The stacking-disorder free ice Ic is formed from C2 via an intermediate amorphous or nano-crystalline form under decompression, unlike the direct transformations occurring in ice XVI from neon hydrate, or ice XVII from hydrogen hydrate. The obtained ice Ic shows remarkable thermal stability, until the phase transition to ice Ih at 250 K, originating from the lack of dislocations. This discovery of ideal ice Ic will promote understanding of the role of stacking-disorder on the physical properties of ice as a counter end-member of ice Ih.Water freezes below 0 °C at ambient pressure ordinarily to ice I Metastable cubic ice has been identified in several conditions relevant to geo and astrochemistry, but was always characterized by stacking disorder. Here the authors synthesize a hydrogen hydrate and degas hydrogen, obtaining pure non-defected cubic ice, observed by X-ray and neutron diffraction. However, it is also known to produce “ice Ic” nominally with a cubic stacking sequence under certain conditions1, and its existence in Earth’s atmosphere4, or in comets6 is debated. “Ice Ic”, or called as cubic ice, was first identified in 1943 by König7, who used electron microscopy to study the condensation of ice from water vapor to a cold substrate. Subsequently, many different routes to “ice Ic” have been established, such as the dissociation of gas hydrates, warming amorphous ices or annealing high-pressure ices recovered at ambient pressure, freezing of μ- or nano-confined water (see ref. 1). Despite the numerous studies on “ice Ic”, its structure has not been fully verified, because the diffraction patterns of “ice Ic” show signatures of stacking disorder9, and ideal ice Ic without stacking disorder had not been formed until very recently10. This is the reason why “ice Ic” is double-quoted1, and it is recently proposed that the stacking-disordered ice should not be termed as ice Ic, but as ice Isd8.Water freezes below 0 °C at ambient pressure, ordinarily to ice Ic” (ice Isd) is known as a metastable form of ice at atmospheric pressure. But, recent computer simulations suggest that even ice Isd could be the stable phase for crystallites up to sizes of at least 100,000 molecules11. The stability of stacking-disordered ices is extremely important because of the ubiquitous nature of ice. Stacking-disordered ice can be characterized by the degree of ice cubicity, χ, which is defined as the fraction of cubic stacking13. Until very recently, the highest cubicity was limited to ~80%14, but it has been reported that ideal ice Ic with 100% cubicity has been obtained by annealing ice XVII10.“Ice I15 and ice XVII17 are obtained by degassing gas molecules from neon and hydrogen hydrates, respectively. From these findings, we hypothesized that ideal ice Ic could be obtained by degassing hydrogen from hydrogen hydrate, C2. Five different phases in the H2–H2O system have been reported to date (see ref. 18): Among them, neutron diffraction experiments have never been conducted for the higher-pressure phases, C1 and C2, probably due to the technical difficulty in loading hydrogen into a pressure vessel, or compressing it to pressures in the giga-pascal range. To synthesize ideal ice Ic, decompression under low-temperature conditions for degassing is necessary, which is also not straight-forward using conventional pressure-temperature controlling systems. We have developed a Mito system19, and have overcome these technical difficulties (see details in Methods).Recently new ice polymorphs, ice XVIc without stacking disorder, obtained from degassing hydrogen hydrate C2. We also report an unexpected amorphous-like state in the transformation from C2 to ice Ic, and the thermal stability of ice Ic.Here we present the neutron and X-ray diffraction results showing ice I2O and MgD2, which is an internal deuterium source, to synthesize hydrogen hydrate, C2. After loading the mixture into a pressure-temperature controlling system, MgD2 was decomposed by heating at 403 K and at ca. 0 GPa for 1 h through a nominal reaction of MgD2 + 3D2O → Mg(OD)2 + 2D2 + D2O , and the host structure was identical to ice Ic9 , D at the 32e site ). The calculated diffraction pattern was in good agreement with the observed one, as shown in Fig. The neutron diffraction pattern for the C site x, /8, 1/8, d → e). In the diffraction pattern taken at e in Fig. 20. This observation indicates that fluid deuterium coexisted with C2 through the path from b to d.The sample was then cooled from 300 K to 100 K at around 3 GPa (path 2 phase persisted at pressures at least as low as 0.5 GPa on decompression at 100 K (path e → f). However, surprisingly, the Bragg peaks of C2 mostly disappeared at 0.2 GPa  = −0.001(1)). This shows that the guest hydrogen molecules are below the detectable limit at 130 K under evacuation. The peak profile around 111 peak of ice Ic has neither the feature of stacking disorder nor the peaks from ice Ih, as shown in the diffraction pattern in the region at around d = 3.9 Å, where the strongest 100 reflection of ice Ih is expected 13, as clear as the recent discovery of ideal ice Ic by annealing ice XVII10.We conducted a separate run in order to obtain a neutron diffraction pattern for the structure refinement of the ice Ic surprisingly persists up to at least 240 K until ice Ih started to appear at 250 K 1. However, in stacking-disordered ice, the cubic stacking sequence starts to change into a hexagonal stacking sequence at a much lower temperature, and the phase transition to ice Ih is completed at 240 K. The notable stability of the ice Ic would be derived from the lack of stacking disorder. The stacking-disordered ice has more dislocations, which promote the phase transformation from ice Isd to ice Ih by reducing the activation energy required to change the stacking sequence22. This is also supported by a recent mesoscopic-size calculation23. Note here that the critical temperature of 240 K has been identified as the temperature above which ice Ih without cubic stacking faults forms spontaneously, which is the reason for the anomalous self-preservation regime of natural gas hydrates24.It is also noteworthy that the ice I0 K Fig. . The temc and Ih, rather than stacking-disordered ice with many stacking faults, judging from “stackogram” reported in the literatures13. At 250 K, crystal growth would be dominant, rather than crystal nucleation. Therefore, once a crystallite nucleates, it quickly grows before other crystallites nucleate, resulting in the mixture of ice Ic and Ih, rather than stacking-disordered ice. This observation also suggests a smaller number of dislocations in the ice Ic observed in this study. On the contrary, the remarkable stability of the ice Ic and the bulk mixture of ice Ic and Ih at 250 K strongly support the conclusion that the obtained ice is not stacking-disordered, and it can therefore be called ice Ic without the need for quotation marks.The diffraction pattern observed at 250 K looks a mixture of bulk ice Ic will allow us to research the real physical properties of ice Ic without stacking disorder. For example, accurate heat capacity or vapor pressure measurements from low temperature will provide the free energy of ice Ic, which settle the long-standing argument for the thermodynamic stability of ice Ic compared to ice Ih. The physical properties of the ideal ice Ic are also important to understand how stacking disorder plays a role in the physical properties of ice Isd. For instance, since the thermal conductivity of ice Isd is significantly smaller than ice Ih25, the difference of thermal conductivities between ices Ih and Ic will emboss the effect of stacking disorder. It is also interesting what will be happened when ice Ic is compressed under low temperature; whichever ice Ic will be transformed into HDA (High Density Amorphous ice26) or not? In any case, it is worth conducting what we previously did for ice Ih, to this ideal ice Ic as well.The discovery of ideal ice I2, used as the starting material in this study, was synthesized from reagent-grade MgH2 as follows. MgH2 powder  was purchased and further ground in an agate motor to increase the surface area, after which it was placed in a copper tube with a diameter of 4 mm and a length of 40 mm. The tube was mechanically sealed and but not welded, allowing the transfer of hydrogen gas. The copper tube was inserted into a 1/4” Inconel tube and connected in parallel to a deuterium gas cylinder and a turbo-molecular pump (TMP) with 1/16” stainless tubes and stop bulbs. The Inconel tube, including the sample copper tube, was heated to 773 K for 1 h using a tube furnace under evacuation using the TMP. Under these conditions, MgH2 completely decomposed to Mg and H227, and the degassed H2 was evacuated. Then, the D2 gas was introduced up to 4 MPa, and the temperature was cycled at the rate of 1 K/min between 673 K and 773 K while keeping the pressure at 4 MPa, which represents stable and unstable conditions for MgH227, and this temperature cycle was repeated for 20 times. This activation process is necessary for the reaction Mg + D2 → MgD2. Finally, the p-T conditions were maintained at 673 K and 4 MPa for 3 days to complete the reaction. The recovered sample was analyzed by powder X-ray diffraction  and identified to be MgD2 with a trace amount of MgO. Both MgD2 and MgO react with D2O and produce Mg(OD)2, so this small amount of MgO does not affect the conclusion of this study.MgD28 in the Material and Life Science Experiment Facility (MLF) of J-PARC, Ibaraki, Japan. The incident beam consists of 25 Hz pulsed spallation neutrons produced from a liquid Hg target via a decoupled moderator and traveled through collimators, choppers and supermirror guides to the sample positioned at 25.0 m from the moderator28. Approx. 20 mg of MgD2, synthesized as described above, was filled into TiZr null scattering gaskets, and D2O water  was dropped on the MgD2 powder, resulting in the molar ratio of MgD2:D2O~1:3. The gaskets were sandwiched between a pair of tungsten carbide anvils, and loaded by using a hybrid Mito system, which is a modified version of an original pressure-temperature variable Mito system19. The hybrid Mito system uses both flowing liquid nitrogen and a 4 K cryostat , which allows us to control temperature rapidly, owing to the large latent heat of liquid nitrogen and efficient thermal insulation by zirconia and GFRP seats. The hybrid Mito system also allows us to achieve temperatures below 77 K, and reach a minimum temperature of ~35 K, owing to the cryostat. Another remarkable feature of the hybrid Mito system is that it affords pressure control, even at low temperature, as well as the original Mito system, which is indispensable for this study. Flexible copper cloths were attached on the support rings of the anvils, and the cloths were placed in contact with the cold head of the cryostat for thermal conduction. The accessible minimum temperature of the hybrid Mito system is ~35 K, which may be the current technical limitation due to an unavoidable influx of heat from the surrounding cell. The sample pressure was estimated from the observed lattice parameter of Mg(OD)2 brucite using the equation of states29 and the observed unit cell volume of brucite at 0 GPa, assuming the temperature derivative of the bulk modulus of brucite, dK/dT, was ~0. Although this assumption may cause some error in the pressure estimated at low temperature, we placed emphasis on avoiding unwanted Bragg peaks from additional sources of pressure marker. Moreover, the error would be too small to affect the conclusion. The sample position was aligned by scanning to maximize the sample scattering intensity. The obtained intensities from the sample in the cell was subtracted by the intensity of the empty cell, and subsequently normalized by the attenuation corrected intensity of vanadium pellet in the cell, which was also subtracted by the intensity of the empty cell30. The Rietveld analyses were performed using the GSAS31 with EXPGUI32, and the crystal structure was drawn with the VESTA program33. The GSAS TOF profile function 331 is used as the profile function in the Rietveld analyses.Neutron powder diffraction experiments were conducted at the beamline PLANET2O (Milli-Q) and MgH2  mixture as starting materials were performed at the beamline BL-18C in the Photon Factory . Samples were exposed to 0.6134 Å monochromatized synchrotron radiation, and the diffracted scattering was detected by an imaging plate (IP). The pressure was generated using CuBe alloy diamond-anvil cells and the temperature was controlled using a 4 K GM cryostat  equipped with a temperature controller . Sample pressure was estimated from the difference in the R1 line wavelengths of rubies inside and outside the sample chamber34. The temperature was monitored using a Si-diode sensor inserted in the cold head edge. We confirmed that the measured temperature was almost the same as that of the diamond anvils after temperature stabilization. The experimental p-T path was basically identical to the case of neutron diffraction, as shown in Fig. d was 4.1 GPa.Powder X-ray diffraction measurements using a H35 with a 0.6 mm culet was placed in the direction of the detector with an opening angle of 2θ < 40°, whereas a conventional anvil with a 0.8 mm culet was positioned in the direction of the X-ray source. A CuBe plate with a hole of diameter 0.3 mm and an initial thickness of 0.2 mm was used as a gasket. This gasket was not subjected to pre-indentation. The load was applied by driving the piston by bellows using a He gas cylinder. The bellows allow us to control pressure at a few kbar more precisely than conventionally used membranes.One conically shaped Boehler-Almax type diamond-anvil36 was used for the DFT calculations38. We used Perdew-Burke-Ernzerhof  type nonempirical exchange-correlation functions39 for this study. The pseudopotentials were derived using projector augmented-wave approximation40. The dispersion effects were taken into account using the exchange-hole dipole moment method (XDM), which calculates coefficients for polynomial of DFT-D dispersion energy41 from the exchange-hole dipole moment calculated from simulated electron wave function43. XDM damping function parameters are taken from44. The enthalpies of four possible configurations for the ordered form of ice Ic were calculated within a unit cell with a kinetic energy cutoff of 70 Ry and a Brillouin zone k mesh of 8 × 8 × 8. The cell parameters and atomic coordinates were optimized using BFGS quasi-Newtonian methods at atmospheric pressure.Quantum EspressoSupplementary Information


Observational records starting in the 1950s show West Antarctica is amongst the most rapidly warming regions on the planet. Together with increased intrusions of warm circumpolar deep water (CDW) onto the continental shelf due to local wind forcing (the primary mechanism in recent decades), this has resulted in enhanced surface and basal melting of floating ice shelves and an associated acceleration and thinning of West Antarctic outlet glaciers, increasing the rate of global sea level rise. In this study, it is shown that during the austral spring season, significant surface warming across West Antarctica has shifted westward to the Ross Ice Shelf in recent decades in response to enhanced cyclonic circulation over the Ross Sea. These circulation changes are caused by a Rossby wave train forced by increasing sea surface temperatures in the western tropical Pacific, which is tied to the springtime shift of the Interdecadal Pacific Oscillation (IPO) to its negative phase after 1992. While the local wind trends enhance warm air advection and surface warming across the Ross Ice Shelf, the strong easterly component of the wind trends reduces the likelihood for intrusions of CDW onto the continental shelf in this region. This suggests that during spring there are competing mechanisms of surface and basal melting of the Ross Ice Shelf, both of which are closely tied to natural tropical Pacific decadal variability. Moreover, that the projected transition of the IPO back to its positive phase in the coming decade, though likely to reduce surface warming on the Ross Ice Shelf, could increase the risk of disintegration of Ross Sea ice shelves due to increased intrusions of CDW and enhanced basal melting. The warming is strongest and most widespread during the austral winter and spring seasons11. In addition to the warming, there has been thinning of ice shelves in the Amundsen Sea Embayment (ASE) and an associated acceleration and thinning of West Antarctic outlet glaciers which has resulted in a negative trend in ice-sheet mass balance and a faster rate of global sea level rise18. This thinning is predominately due to increased influxes of warm circumpolar deep water (CDW) along glacial troughs caused by increases in local westerly winds and upwelling over the continental shelf break23, as well as increasing SAT which causes surface melting that can lead to meltwater ponding, hydrofracturing, and ice shelf collapse27; surface melting has been the leading contributor to the disintegration of ice shelves in the Antarctic Peninsula30, and is likely to increase dramatically in importance in the ASE region by the end of the century32.Observational records beginning in the late 1950s recorded an increase in surface air temperature (SAT) over West Antarctica that was more than twice as fast as the global average34. Multiple gridded temperature datasets suggest that spring warming has continued and even intensified across West Antarctica in recent decades, but it has shifted westward to the Ross Ice Shelf36. While no major surface melting has been documented in this region during spring, surface melting is commonly observed over the Ross Ice Shelf during summer27, and it is likely to become more frequent by the end of the century32. This westward shift in spring warming is confirmed by the sparse station observations in the region, which show over recent decades a reduction in warming at Byrd station in central West Antarctica3, while McMurdo station located on the western Ross Ice Shelf exhibits a large springtime warming trend that is stronger than any Antarctic station during any season37. The cause of the shift in spring warming is suggested to be tied to anomalous meridional winds3 due to an increase in cyclonic circulation offshore over the Ross Sea39, resulting in local changes to thermal advection36 and sea ice concentration10. However, anomalous easterlies over the continental shelf break resulting from the strengthened cyclonic circulation would be expected to decrease intrusions of CDW onto the inner continental shelf and thus reduce basal melting of ice shelves in the Ross Sea region40, i.e. there are competing impacts on the stability of the ice shelves in this region as an increase in surface melting (and related ponding and hydrofracturing) due to warming may be offset by a reduction in basal melting. See Fig. However, a closer examination reveals that SAT trends across West Antarctica exhibit considerable decadal variability and, despite their strong magnitude, lie within the bounds of natural variability44 and observational36 studies have suggested that during spring the increased cyclonic circulation is linked to tropical variability, namely the transition of the Interdecadal Pacific Oscillation (IPO)47  to its negative phase after the 1990s43 as well as increasing sea surface temperature (SST) in the tropical North Atlantic48. The IPO is a naturally occurring mode of decadal variability in the El Niño-Southern Oscillation (ENSO)51, meaning that these changes may be part of natural variability52. Despite these findings, the precise mechanism by which the negative IPO phase causes an increase in cyclonic circulation over the Ross Sea remains controversial; previous studies suggest a variety of mechanisms are responsible, including negative SST anomalies over the eastern tropical Pacific43, increased deep convection in the South Pacific Convergence Zone36, increased wave activity from the central tropical Pacific35, and a wave train emanating from the tropical Atlantic48. Our present work addresses this important gap by analyzing gridded datasets and station observations in conjunction with atmosphere-only climate model simulations to show a robust mechanism linking western tropical Pacific SST variability associated with the IPO to the recently observed spring circulation and temperature changes across the Ross Sea and Ross Ice Shelf, as well as local zonal winds over the continental shelf break, and by extension potential atmospheric drivers of ice shelf thinning.Previous modeling−1 over the western tropical Pacific as well as the tropical Indian and North Atlantic basins, while the eastern tropical Pacific cooled 0.1–0.2 °C decade−1  observed at McMurdo and Marble Point (located on the western Ross Ice Shelf), while weaker and statistically insignificant warming is measured at Byrd and Gill .During the post-1979 modern satellite era, spring tropical SSTs increased by up to 0.3 °C decadee−1 Fig. . Contempion Fig. . Additioion Fig. ), but reion Fig. . The rea3. We therefore use the reanalysis 2 m temperature at the grid cell nearest to McMurdo station to form a continuous temperature record at McMurdo for further analysis (termed McMurdo temperature hereafter). This site is chosen because McMurdo has the most complete record in the region, which is used to constrain the reanalysis, and it also shows strong autocorrelation with reanalysis 2 m temperatures throughout the Ross Ice Shelf and western West Antarctica , enhanced cyclonic circulation in the Ross Sea, and western tropical Pacific SSTs that are strongly influenced by the IPO.The tropical Pacific SST trends shown in Fig. ing Fig. . Closelying Fig.  transitiing Fig. , as welling Fig. . McMurdoing Fig. . It is ning Fig. , but cloing Fig.  and ther65 for 30 years (following a 10-year spin-up) forced by pre-industrial SSTs. We then performed a perturbation experiment by repeating the control run, but with the addition of a 2 °C SST anomaly in the western tropical Pacific . To test this, we re-calculated the spring trends after removing years when La Niña occurred in isolation without a negative IPO event  in order to isolate the negative IPO forcing without La Niña. The new reanalysis trends  is the primary driver of the recent spring changes in circulation, SAT, and local wind forcing of CDW across the Ross Ice Shelf.The model difference also shows the tropically-forced wave train is associated with an anti-cyclonic circulation in the Amundsen Sea, which is not apparent in the reanalysis trends Fig. . This isnds Fig.  depictinThis study demonstrates strong covariability of spring circulation and temperature over the Ross Ice Shelf with remote forcing from the western tropical Pacific tied to natural variability in the springtime IPO. Performing a sensitivity experiment with a climate model shows that positive SST anomalies in the western tropical Pacific results in a Rossby wave train that reproduces the observed increase in cyclonic circulation in the Ross Sea and the associated westward shift of West Antarctic spring warming to the Ross Ice Shelf. While the circulation causes surface warming on the Ross Ice Shelf, the anomalous easterlies over the continental shelf break would likely be associated with a reduction in CDW intrusions and basal melt in this region. Therefore, the post-1979 springtime circulation and temperature trends across the Ross Ice Shelf are primarily a result of the post-1992 transition of the IPO to its negative phase, and future springtime changes, including both surface melt (via increases in SAT) and potentially basal melt , will be modulated by natural tropical Pacific SST variability, though more work is needed to quantify its influence on ice shelf stability.35), which is also likely tied to the springtime reduction in sea ice concentration in the southeast Ross Sea and southwest Amundsen Sea  that produces cold air advection over continental West Antarctica east of 105°W  and surface melt events on the Ross Ice Shelf become more common31. The results presented here along with ref.24 indicate that multiple aspects of natural tropical Pacific variability play important roles in modulating surface melting of the Ross Ice Shelf and potentially influxes of CDW onto the continental shelf due to local zonal wind forcing. In the coming decades, as the IPO likely shifts to its positive phase73, an increase in anti-cyclonic circulation over the Ross Sea is expected during spring, which would reduce the surface warming but increase local westerly wind anomalies over the continental shelf. Such changes in surface melt and ocean-driven basal melt therefore oppose each other in this region, and the relative importance of these competing mechanisms for future Ross Ice Shelf stability will require further cross-disciplinary study involving glaciologists and oceanographers to fully understand the influence of the IPO on the stability of the Ross Ice Shelf (which is outside the scope of this investigation). In particular, more work is needed to quantify the magnitude of basal melt one would expect from shifts in the IPO, and if a reduction in surface warming would offset any of the potential ice shelf thinning. Similar cross-disciplinary work has been proven successful in the ASE40, though similarly more work is needed to fully quantify the magnitude of basal melt resulting from remote tropical forcing of local zonal winds in these regions74. To fully understand this threat will also require improving the projection of natural modes of decadal tropical variability and their associated teleconnections over the South Pacific.Lastly, recent work by ref.75 (ERA-Interim). Other reanalyses yield similar results during spring (as discussed by ref.35), but ERA-Interim has been shown to be most reliable over high southern latitudes78. Sea surface temperature data are from the National Oceanic and Atmospheric Administration (NOAA) Extended Reconstructed SST version 4 (ERSSTv4) dataset81, sea ice concentration data are from the Hadley Centre Sea Ice and SST dataset82, and outgoing longwave radiation data are from the NOAA Interpolated Outgoing Longwave Radiation dataset83. Antarctic weather station temperature data are from the Antarctic READER project84. We monitor variability in the IPO using the tripole index47.Atmospheric circulation and SAT data are from the European Centre for Medium-Range Weather Forecasts (ECMWF) Interim reanalysist test.Linear trends were calculated using a linear least squares regression analysis and relationships were investigated using linear Pearson’s correlation analysis. Statistical significance of trends, correlations, and differences in the model climatologies were calculated using a two-tailed Student’s 85 (HadGEM3A) utilizes the Global Atmosphere 4.0 and Global Land 4.0 configurations of physics and dynamics65. The model experiments were run at N96 resolution , with 85 vertical levels up to a height of 85 km, partitioned with 50 levels below 18 km and 35 levels between 18 km and 85 km65. Both experiments were forced by pre-industrial concentrations of greenhouse gases, representative of the 1860s. The SST and sea ice concentration fields for both experiments are prescribed and also representative of pre-industrial conditions, which are generated from the HadGEM2-CC coupled climate model historical simulation submitted to phase 5 of the Coupled Model Intercomparison Project86. Both experiments were run for 30 years in response to a repeating annual cycle of climatologically averaged SSTs and sea ice to remove sources of natural variability, such as ENSO.The atmosphere-only third generation Hadley Centre Global Environmental ModelSupporting information


In many Actinobacteria it is the sole annotated serine threonine protein phosphatase to counter the activity of multiple serine threonine protein kinases. We used transcriptional knockdown, electron microscopy and comparative phosphoproteomics to investigate the putative dual functions of PstP as a specific regulator of cell division and as a global regulator of protein phosphorylation. Comparative phosphoproteomics in the early stages of PstP depletion showed hyperphosphorylation of protein kinases and their substrates, confirming PstP as a negative regulator of kinase activity and global serine and threonine phosphorylation. Analysis of the 838 phosphorylation sites that changed significantly, suggested that PstP may regulate diverse phosphoproteins, preferentially at phosphothreonine near acidic residues, near the protein termini, and within membrane associated proteins. Increased phosphorylation of the activation loop of protein kinase B (PknB) and of the essential PknB substrate CwlM offer possible explanations for the requirement for  Phosphoserine and phosphothreonine are chemically stable, and the transient, reversible nature of the signal relies on the activity of protein phosphatases as well as protein turnover. The conserved Actinobacterial regulator GarA is an example of a stable phosphoprotein that is replaced by protein turnover since its buried phosphothreonine resists phosphatase activity2, while the autophosphorylation sites in the activation loops of protein kinases are examples of short-lived phosphorylation signals, since these exposed sites are dephosphorylated rapidly by protein phosphatase4.Serine/threonine phosphorylation in bacteria is a ubiquitous reversible signal that controls cell cycle events, virulence, and responses to environmental cues6. Studies of purified bacterial S/T phosphatases suggest that these are also multi-specific7, for example recombinant Mycobacterium tuberculosis PstP dephosphorylates itself, protein kinases PknB and PknA and phosphoproteins PbpA and GarA8. These in vitro studies necessarily lack any regulatory proteins and use non-physiological protein concentrations. Phosphatase knockout in Enterococcus faecium has provided evidence of substrate selectivity in cells9.Serine threonine (S/T) phosphatases are greatly outnumbered by S/T kinases in bacterial and eukaryotic genomes. Human protein phosphatases tend to have lower substrate specificity than protein kinases, and more substrates. Substrate selection is driven by subcellular localisation and interaction with regulatory partnersM. smegmatis has 18 S/T kinases, one transmembrane and one soluble S/T phosphatase. The function of the soluble S/T phosphatase MspP (MSMEG_1928) is unknown, although its activity has been demonstrated in vitro10. Only the transmembrane S/T phosphatase PstP is conserved between M. smegmatis and M. tuberculosis (91% identity in the phosphatase domain), and this genetic locus is conserved throughout the Actinobacteria , regulate many events in the cell cycle, as demonstrated by diverse phenotypes caused by gene disruption in Bacillus subtilis, Staphylococcus aureus, Corynebacterium glutamicum, Streptomyces coelicolor, M. tuberculosis and Streptococcus pneumoniae21. The mechanisms by which PknB regulates cell division are being elucidated and some may be organism-specific, for example PknB regulates peptidoglycan biosynthesis in M. tuberculosis by phosphorylation of several key enzymes and regulators including the regulatory protein CwlM23 and proposed lipid II flippase18.The gene cluster ria Fig. . PknB ho24, S. pneumoniae knockout had defects in cell size and location of peptidoglycan synthesis25, S. aureus knockout had thickened cell wall26, and C. glutamicum knockout was slow-growing with pleiomorphic bulging cells. While this study was in progress, PstP was shown to be essential in M. tuberculosis and Mycobacterium smemgatis because depletion of PstP led to cell wall defects27. However, the cognate substrates of PstP and the mechanism by which it regulates cell cycle processes were unknown.PstP phosphatase function is also implicated in regulation of cell wall biosynthesis or cell division: a group B Streptococcus knockout had increased phosphorylation of cell division proteinsM. smegmatis during PstP depletion, measuring changes in growth and viability, cell morphology and the phosphoproteome. These changes clarified the phosphoproteins regulated by PstP and support both roles.We hypothesised that PstP might play two roles: as a global negative regulator of protein S/T phosphorylation, and as a specific regulator of PknB and PknA by dephosphorylating enzymes and regulators of cell wall biosynthesis and cell division. We characterised pstP promoter was replaced with the pristinamycin inducible promoter . Only five proteins showed significant changes in abundance . Protein kinases PknA and PknB decreased to a lesser extent, confirming the presence of additional promoter(s) downstream of pstP that contribute to their expression. The conditional mutant strain pstP-CM is therefore considered a triple knockdown of pstP, rodA and pbpA. Reintroduction of pstP restored growth  of PstP might encompass cell wall synthesis or cell division. Other aspects of the phenotype were partially complemented  were significantly more abundant in the knockdown condition  was the most abundant phosphosite Fig. , as seenown Fig. , fittingin vitro specificity of M. tuberculosis protein kinases31. Furthermore, amongst the phosphosites changed during knockdown there was a bias towards phosphorylation sites near the N-terminus . The greater frequency and more labile nature of phosphorylation near the protein termini is expected from the more exposed nature of the termini compared to residues in defined protein folds12, making them more accessible to kinases and phosphatases.Motif analysis of the global phosphoproteome revealed a preference for acidic residues on either side of the phosphorylation site Fig. , which iProtein localisation is also likely to influence substrate access to PstP. Membrane proteins and membrane associated proteins were significantly enriched amongst the differentially phosphorylated proteins, fitting with the membrane localisation of PstP Fig. .The functional classes of proteins that are differentially phosphorylated could reveal specific cellular processes that are regulated by PstP. However, half of all phosphopeptides were increased upon knockdown, representing all functional classes (supporting information). The ten phosphoproteins with the greatest increase in abundance Table  include 32, and this is likely the case for other kinases33. Conversely, PknB and PknA phosphorylate PstP to increase its activity13, suggesting a possible homeostatic mechanism , the transporter MSMEG_1642 (Rv1747-homologue), FabG, SigH and EspI were increased >16-fold during knockdown. CwlM, MviN and Rv1747 are involved in synthesis, regulation and export of cell wall components. Phosphorylation-dependent changes in their activity could contribute to the cell wall thickening seen by electron microscopy  and which is essential in both M. tuberculosis (Rv0227) and M. smegmatis35. It is tempting to speculate that this reflects a role for PstP in control of cell wall processes, fitting with the changes in morphology observed during prolonged PstP knockdown  and FhaA. Further work will be required to characterise the specific effects of PstP dephosphorylation on the activity of each. The soluble S/T phosphatase MspP, which shares 38% amino acid identity with the catalytic domain of PstP, remained undisrupted in the knockdown (mspP is in a distant genetic locus and there was no significant change in protein abundance) thus MspP cannot substitute for the function of PstP. No MspP phosphosites were identified during phosphoproteome analysis.PstP is required for normal cell wall synthesis and cell division, as demonstrated by the cell wall and morphological changes with loss of viability upon knockdown of the pstP operon surpassed those observed in previous phosphoproteomic comparisons of gene disrupted bacteria in the number and proportion of proteins that were differentially phosphorylated and in the magnitude of the fold-change in the phosphopeptides. The outnumbering of PstP by 18 S/T kinases implies that PstP depletion could affect the concentration of many phosphoproteins but the high fold-change values was unexpected given the mild and brief period of PstP depletion: by 6 hours PstP concentration fell 5.7-fold while 52 phosphopeptides increased >32-fold and globally 50% of phosphosites were significantly changed with 22% increased by over 8-fold. By comparison, knockout of S/T kinases or phosphatase in other bacteria led to fewer significant changes and lower fold-change: 8 out of 64 sites in B. subtilis S/T phosphatase knockout, 55/603 in Mycobacterium bovis pknG knockout36 and 19/456 in M. tuberculosis pknB knockdown23. A possible explanation for the large, extensive and rapid changes in phosphorylation emerged from examining differential phosphorylation of protein kinases. PknB had the greatest increase in phosphorylation, including at threonines in and near the activation loop (49-fold increase in pT166 and 36-fold increase in pT179). In vitro evidence suggests that PknB and PstP regulate each other8. Loss of PstP would lead to prolonged PknB activation32, and potentially to positive feedback from uncontrolled autoactivation of PknB  albeit with possible confounding effects from the growth medium, growth phase and polar effects12. Similarly PstP overexpression and PknB depletion both result in longer cells27. Overall, the non-uniform increase in protein phosphorylation upon knockdown of the pstP operon demonstrates that the role of PstP is more complex than general dampening or reversal of the effects of protein kinases. Phosphorylation changes upon knockdown were enhanced at certain motifs and on certain substrates. A study of the PstP homologue in Enterococcus faecium also found evidence of substrate selectivity when recombinant phosphatase was used to dephosphorylate whole cell extracts9.The phosphoproteomic and genetic evidence linking the function of PstP and PknB provides a mechanism by which PstP can regulate cell wall synthesis by dephosphorylation of the substrates of PknB (and PknA) and by dephosphorylation of PknB itself to reduce activation. Supporting this explanation are similarities between the morphological defects that result from PstP-depletion and PknB overexpression: both result in widened, bulging, pleiomorphic cells Fig.  and17,27In summary, PstP is a regulator of cell wall synthesis and cell division and is also a global regulator of protein S/T phosphorylation thus influencing all cellular processes. The first role in cell division may occur directly by the action of PstP of the enzymes and regulators of cell wall synthesis or indirectly by specifically controlling the activity of PknB, or by both mechanisms. The general role in global regulation may also occur directly through substrate dephosphorylation, or indirectly by specific dephosphorylation of the activation loops of kinases, or by both routes.M. smegmatis mc2155 was routinely cultured at 37 °C on Middlebrook 7H9  liquid media supplemented with 10% (v/v) Albumin-Dextrose Complex , 0.2% (v/v) glycerol and Tween 80, 0.05% (w/v). Antibiotics were used as follows: the conditional mutant pstP-CM was routinely cultured in the presence of hygromycin (50 μg/ml) and pristinamycin (2 μg/ml), and pstP-CM transformed with pMV306 or derivatives was routinely cultured in the presence of hygromycin, pristinamycin and kanamycin (30 μg/ml).pstP with the cognate promoter was PCR amplified from M. smegmatis genomic DNA and cloned in the KpnI and HindIII sites of pMV30637. pstP was PCR including the ribosome-binding site (402 bp) was PCR amplified from M. smegmatis genomic DNA  and cloned under the control of the pristinamycin inducible promoter in the NcoI and SpeI sites of pAZI947928 in order to disrupt the genomic pstP locus by homologous recombination. The sequenced plasmid was transformed into M. smegmatis by electroporation and selection with hygromycin. The site of recombination was confirmed by PCR .The 5′ end of M. smegmatis pstP-CM was cultured in Middlebrook 7H9 broth with appropriate antibiotics . Cells were washed in 7H9 without inducer, serially diluted, and spotted in parallel onto 7H10 plates with/without inducer. Plates were incubated at 37 °C for three days and used to take photographic images. Images are representative of 3 independent experiments.pstP operon on growth in liquid medium, cultures were prepared and washed as above, and used to inoculate parallel cultures with/without inducer. The initial optical density was 0.05. Cultures were grown at 37 °C with shaking in a BioScreen  and the remainder were pelleted in 3% agar, cooled, and cut into 1 mm3 cubes. Agar cubes were treated with 1% w/v aqueous osmium tetroxide, dehydrated through an ethanol series, and embedded in Spurr’s Resin. Samples were sectioned to a thickness of approximately 70 nm using a Reichert Ultracut E ultramicrotome and collected onto copper mesh grids. Dry grids were submerged briefly in methanol before staining in 2% (w/v) uranyl acetate for 30 minutes followed by Reynolds lead citrate for 5 minutes. Samples were viewed on a JEOL JEM-1400 TEM with an accelerating voltage of 100 kV. Images were collected using a Megaview III digital camera with iTEM software.An aliquot of glutaraldehyde-fixed cells (10 µl of each strain) was applied to a Poly-L-lysine treated glass slide, then further treated with 0.5% (w/v) aqueous osmium tetroxide and dehydrated through ethanol and anhydrous hexamethyldisilazane. Dried slides were mounted onto aluminium stubs and sputtercoated with gold/palladium in a Quorum Q150TES coating unit. Bacteria were viewed on a Hitachi S3000H SEM with an accelerating voltage of 10 kV. The length of 100 cells of each strain was measured using ImageJ and differences between the strains were analysed by Mann Whitney test.pstP-CM was washed and used to seed parallel 200 ml cultures with and without inducer at initial optical density of 0.1. Cultures were incubated at 37 °C with shaking and harvested after 6 hours. Replicate cultures were used for Western blotting and for proteomic analysis. Cells were resuspended in extraction buffer ). Cells were disrupted using a Minilys personal homogenizer (Bertin instruments) using glass beads (Sigma) and cell debris was removed by centrifugation at 20,000 g for 30 minutes. The supernatant was concentrated using 1000 kDa molecular weight cut-off filters and protein concentration estimated using Qubit assay (Thermofisher). Protein concentration was normalized before Western blot analysis, and normalization was verified by parallel SDS PAGE with Coomassie blue staining. Polyclonal phosphothreonine antibody was purchased from Cell Signalling and used according to the manufacturer’s instructions.Exponential phase M. smegmatis pstP-CM cell extracts were reduced and alkylated in extraction buffer containing 5 mM Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and 10 mM chloroacetamide (CAA), for 30 min at room temperature. Proteins were precipitated using the methanol-chloroform method38 then dissolved in 20 mM HEPES-Trypsin-LysC (1:100) and digested for 16–17hrs.The tryptic peptides concentration was determined using Qubit. Part of the peptide sample (95%) was used for phosphoproteomics analysis and the remainder (5%) for proteome analysis. The peptides were dried using a SpeedVac concentrator and then resuspended in 80% acetonitrile, 5% trifluoroacetic acid (TFA). For the phosphoproteomics analysis, phosphopeptides were enriched using Titansphere  according to the manufacturer’s instructions. Dried peptides were resuspended in a buffer containing 2% acetonitrile and 0.1% TFA prior to analysis by mass spectrometry.pstP-CM, 2 µm, 100 Å (Thermo Scientific) using 0.1% formic acid and acetonitrile containing 0.1% formic acid. Eluted peptides were converted to gas-phase ions by means of electrospray ionization and analysed on a Thermo Orbitrap Fusion (Thermo Scientific).Reversed phase chromatography used an Acclaim PepMap µ-precolumn cartridge 300 µm i.d. × 5 mm, 5 μm, 100 Å and an Acclaim PepMap RSLC 75 µm i.d. × 50 39 and peptide lists were searched by the Andromeda Search Engine40 against the M. smegmatis Uniprot database . Bioinformatics and statistical analyses were performed in the Perseus software (version 1.5.5.3). Confidently assigned phosphorylation sites with localization probabilities greater than 75% and Score difference greater than 5 were defined as Class I sites and were used as site localization cutoff from the Phospho (STY)Sites table generated by MaxQuant analysis. Missing data points were imputed by creating a normal distribution with a width of 0.3 and a downshift of 1.8. Student’s T-tests for statistical significance were performed with permutation-based FDR correction threshold of 0.05. PRIDE accession number: PXD011805.MS raw files were analyzed by MaxQuant software version 1.5.5.3Supplementary figures S1-S9Table S1


Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.Aschantin is a bioactive neolignan found in  Magnoliaflos and Hernandia nymphaeifolia -mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with IC50 values of 27.8, 40.5, 22.7, and 57.5 µM, respectively , indicating that it should be used carefully by patients taking CYP2C9 substrates such as celecoxib, diclofenac, glyburide, losartan, tolbutamide, torasemide, and S-warfarin, to avoid drug interactions -mephenytoin  were obtained from Corning Life Sciences . SN-38 was the product of Santa Cruz Biotechnology . 13C2,15N-acetaminophen , chenodeoxycholic acid-24-acyl-β-glucuronide , mycophenolic acid-β-d-glucuronide , and SN-38 glucuronide  were obtained from Toronto Research Chemicals . Acetonitrile and methanol (HPLC grade) were obtained from Burdick & Jackson Inc. ; all other chemicals were of the highest quality available.Acetaminophen , amodiaquine hydrochloride , coumarin , diclofenac sodium , 7-hydroxycoumarin , midazolam , 50 values) of aschantin toward CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were evaluated using pooled human liver microsomes employing a cocktail of CYP substrates and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The incubation mixtures were prepared in total volumes of 100 µL, as follows: pooled human liver microsomes (0.2 mg/mL), 1.0 mM NADPH, 10 mM MgCl2, 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin in DMSO , and a cocktail of seven CYP probe substrates as defined in our previous report -mephenytoin, 5 µM bufuralol, and 2.5 µM midazolam. After 3 min pre-incubation at 37 °C, the reactions were initiated by addition of NADPH and incubation proceeded for 15 min at 37 °C in a shaking water bath. The reaction was stopped by placing the tubes on ice and adding 100 µL amounts of ice-cold methanol containing internal standards . Then the incubation mixtures were centrifuged at 13,000× g for 4 min at 4 °C. All assays were performed in triplicate and the average values were used in calculations. To measure the mechanism-based inhibition of CYP activities, various concentrations of aschantin (0.1–100 µM) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. Each reaction was initiated by adding the seven-CYP probe substrate cocktail. The degrees of inhibition . The metabolites formed from the seven substrates were simultaneously quantified using our previously described LC-MS/MS method . To this2, 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin (0.1–100 µM), pooled human liver microsomes (0.2 mg/mL), and a CYP2B6-selective substrate (50 µM bupropion), as reported previously -mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. It weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively. The compound negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A3, UGT1A4, and UGT2B7. Aschantin should be examined in terms of potential in vivo pharmacokinetic drug interactions attributable to inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.Aschantin exhibited potent mechanism-based inhibition of CYP2C8-mediated amodiaquine


Background: Cross-reactive carbohydrate determinants (CCDs) as they occur on natural allergens from plants and insects influence the measurement of antigen-specific IgE-antibodies in the context of in vitro allergy diagnosis. When positive results are based solely on the reaction of CCDs with anti-CCD IgE, results must be rated as false-positive. A generally applicable solution to this problem has not yet been presented. Methods/Patients: Sera of patients for whom an assumed allergy should be verified or ruled out were tested with three methods for specific IgE determination  in the absence and presence of a novel, semi-synthetic CCD-blocker. The study was not prospective and for many patients unequivocal clinical data were missing; the data section thus focusses on few, well-defined patient sera. Results: More than 20% of all patients were tested positive for IgE-anti-CCD antibodies and hence against a multitude of similarly glycosylated allergen extracts in a strip-based multiallergen test. Incubation of these positive sera with the CCD-blocker led to significant reductions of read-out values and in many cases to negative test results. The inhibitory efficiency was highest for the allergen strip test and for the component array. Results remained positive for relevant allergens for which a true sensitization had been indicated by skin tests or other means. The CCD-blocker did not alter the read-outs for unglycosylated allergens or – with CCD-negative sera – for all allergens. Conclusion: Elimination of CCD-specific IgE antibodies by means of a synthetic CCD-blocker drastically reduced the number of false-positive in vitro test results without compromising the sensitivity for relevant IgE interactions. Thus, the herein described CCD-blocker constitutes a valuable tool for increasing the test specificity of routine in vitro allergy diagnosis.  A positive test merely confirms the presence of such antibodies in serum, but does not permit a reliable statement as to whether the reactivity with a specific allergen source is exclusively due to CCDs or whether protein-specific IgE antibodies are additionally involved. The AWMF guidelines known as In vitro Allergiediagnostik  [Specific IgE antibodies to cross-reactive carbohydrate determinants (CCDs) in the serum of persons with pollen allergy are responsible for cross-reactivity with several inhalant and food allergens as well as insect venom and latex , 6, 7, 8 allergy , 10. The allergy , 6, 11,  allergy . The preagnosis)  state thagnosis) , 15, theThus, there is a need for a simple procedure to separate the chaff of anti-CCD IgE antibodies from the wheat of clinically relevant antigen-specific IgE. Irrelevant or false-positive test results should be suppressed while relevant ones should not be influenced. The technical handling of the procedure should be simple, the substance stable and unproblematic, the costs low, the results reproducible, and the validity high. A CCD blocker to resolve exactly this problem was developed recently (www.proglycan.com). In the present article we describe the application of this CCD inhibitor using three different measuring systems for allergen-specific IgE. At a referral laboratory, all serum samples received for sIgE testing from July 2011 to December 2012 were tested for the presence of anti-CCD IgE using the multi-allergen strip test named AllergyScreen, provided by Mediwiss Analytic Company . The samples had been sent for investigation of suspected inhalant and/or food allergies and insect venom allergy. From January 2012 onward, all samples showing evidence of IgE antibodies to CCDs were re-tested with inhibition by pre-treatment with the CCD  in order to compare the presence of specific IgE antibodies to inhalant or food allergens with and without the blocker. Both results were sent to the referring physician or site with appropriate comments. The validity of the study is limited by the fact that, for the majority of samples, the patients’ symptoms were not known with certainty at the laboratory. Therefore, only random samples could be used to make statements concerning the frequency of anti-CCD antibodies and their susceptibility to blocking. The CCD blocker is a synthetic glycoprotein made from human serum albumin  and a highly purified vegetable glycopeptide. Bromelain is first isolated from pineapple stem extract (Sigma-Aldrich) and digested with a protease. Any existing protein epitopes are destroyed by this process. The glycopeptide thus obtained is purified with the so-called MUXF structure   and a diThe CCD blocker can be added to sera before determination of specific IgE antibodies using any type of IgE testing system. Based on dilution series and experiments in the ELISA format with a pooled serum sample containing ascorbate oxidase, a final concentration of 20 mg/l was selected. This corresponds to the addition of 10 µl of the CCD inhibitor (= 10 µg) to 0.5 ml of serum. Subsequent experiments showed that markedly lower concentrations achieve sufficient inhibition in a large majority of serum samples. Pre-incubation before further use of the serum is not required. The ready-to-use CCD blocker solution was divided into portions, deep-frozen at –20° C, defrosted, and could be used for 4 weeks thereafter at 4 – 8°C. The final preparation is equivalent to the ProGlycAn CCD blocker described in the Internet (www.proglycan.com). Serum samples were tested for the presence of IgE antibodies using three commercially available test procedures. The Mediwiss multi-allergen strip test was used for the large majority of samples. Two strips were used for adults, i.e. one for inhalant  and one for food allergens . In children we used a mixed panel . The specially prepared strips contained pathways for CCD allergens . Alternatively, or additionally, single-allergen testing was performed with ImmunoCAP of ThermoFisherScientific (previously Phadia). Anti-CCD IgE was determined here using the CCD test allergen MUXF3 (o214). The serum samples were divided and then subjected to the respective IgE testing method either in untreated form or together with the CCD inhibitor  without preincubation. Some serum samples were subjected to component-specific diagnostic investigation using ImmunoCAP ISAC (ThermoFisherScientific). Of approximately 6,000 serum samples tested in 2012, which were investigated with the multi-allergen strip test , 22% showed IgE antibodies to CCD’s; 86 % of these had anti-CCD IgE antibodies of RAST classes 2 or higher. Classification of positive CCD reports according to age showed maximum levels in the age group of 10- to 20-year-old persons (35% were affected). On the strip test for inhalant as well as food allergens, these serum samples revealed a number of clearly positive signals, which were by no means demonstrably related to the patients’ medical history. In order to achieve a meaningful laboratory diagnosis for these patients as well, we started to use the synthetic CCD blocker. In many CCD-positive serum samples, pre-incubation with the CCD blocker resulted in the complete disappearance of many bands or at least a drop in signals to below the reference value. In a random sample of 43 fully evaluable patients , complete inhibition of CCDs as well as specific allergens was achieved in 18 (41.9%). In 22 patients (51.2%) the inhibition was partial . In 3 patients (7%) the inhibition failed . As the study was not prospective, and a large part of the clinical data could not be verified, a general evaluation of data according to these criteria was not performed. This will be tested in the exploratory phase of the system and presented on the basis of unequivocal patient data. Encouraged by the prospect of being able to eliminate a large number of irrelevant reactions by means of the CCD blocker, a few cases were subjected to more exact testing with various IgE test methods while taking clinical reports into account. The case of a 16-year-old girl serves as a typical example . WithoutThe case of a 70-year-old woman was even more convincing. Her serum tested strongly positive for a large number of vegetable allergens on the test strip, but her skin-prick test was not positive for any pollen extract , w 70. OThe serum of a 46-year-old man was sent for investigation of sensitization, although he had no identifiable symptoms of allergy . A bee or wasp venom allergy was also suspected. The serum was first tested on a strip test, which showed wide polysensitization to plant allergen extracts. Of 25 strongly positive reactions , none remained positive after inhibition . i.e. the positive CAP value to rApi m1) as an unexplained artifact. It is more important to note that a few recombinant allergen components were measured on ImmunoCAP and ISAC in the presence of a CCD inhibitor as well. The results show that the inhibitor caused no undesirable changes apart from statistical fluctuations. These unequivocal results clearly raise the question as to whether, and to what extent, the CCD blocker suppresses reactions that may actually indicate relevant sensitization. We also wished to determine whether this type of CCD inhibition could be used in other test procedures as well, especially ImmunoCAP and ISAC. A notable aspect of the non-inhibited values are the much more moderate data in regard of sIgE concentrations, which were retained on ImmunoCAP when compared to the semiquantitatively treated multi-allergen strip test. Nevertheless, inhibition below the threshold value was only achieved in 5 of 8 cases. Especially the grass mixture and bee venom remained very high (above 1 kU/l). In the case of bee venom, however, it is not certain whether this was a protein-based cross-reaction because the patient was markedly sensitized to wasp venom. Reactions to wasp venom as well as its components remained clearly positive even with the CCD inhibitor. The MUXF3 test allergen was not measured in this patient, but was measured in a few others. We observed partial inhibition in many cases. Accordingly, the mild persisting reaction to grass or ragweed pollen may still be rated false-positive, especially because all grass components and Art v 1 tested negative on ISAC. One exception was nPhl p4, which is a natural component and apparently tested positive here in the non-inhibited approach because of its CCD glycans. This is also true of a few other natural components. Since an insect venom allergy was suspected in this patient, CAP and ISAC tests for the venom and its components were performed. A rather confusing aspect is the discrepancy of reactions to rApi m1 on the ImmunoCAP and the ISAC, and the apparent inhibition of this recombinant allergen on ImmunoCAP by the CCD blocker. As IgE binding to rApi m1 was not influenced by the blocker in some cases (data not shown), we regard this doubly questionable test outcome  to a specific allergen the loss of sensitivity due to dilution of the sample is negligible. Binding to recombinant CCD-free allergens persists in the presence of a CCD blocker, thus corroborating its specificity (refer, for instance, to the ISAC values of Patient m 12 in In specific cases the CCD blocker tested here failed to achieve sufficient inhibition. This was especially true of a few ImmunoCAP tests, such as those for grass, common ragweed, or insect venom, and appears to correlate with the level of anti-CCD IgE. Although the relevant allergen is clearly identified even in cases of incomplete inhibition in the presence of apparent double sensitization to insect venom (see Patient m 46 in in vitro allergy diagnosis. Several other factors lurk in their vicinity, such as poor IgE specificity, competition from IgE or other “mimickers of allergy” [The fact that not all relevant questionable reports could be investigated by CCD inhibition was because CCDs constitute just one potential source of error in allergy” . HoweverIn the ideal case the clinician should be able to justifiably assume that all IgE antibodies remaining after inhibition are actually directed towards peptide epitopes and hence potentially of clinical relevance. This is undoubtedly a high demand of a test and may not always be achievable in actual practice. This demand of a CCD blocker appears to be exaggerated in view of the substantial divergence of various sIgE test procedures, regardless of the CCD problem. However, it may be stated that a breakthrough has been achieved here with relatively simple resources and without significant technical effort. The entirely justified recommendation to use a flow chart for obtaining diagnostic evidence of anti-CCD IgE in serum, described as a desirable measure in a recent overview, is quite difficult to put into practice in clinical routine – which includes the medical office and the laboratory for routine diagnostic investigation . in vitro antibody binding. However, we have data to show that, in persons with an insect venom allergy, the basophil activation test may be disturbed by anti-CCD IgE [Prospective studies in specifically defined patient populations using different IgE test systems will have to follow now. Basically CCD inhibition should be investigated in test methods based on -CCD IgE . We regard the present data – of which only a part have been shown here – as sufficient to establish that, even now, the mere suspicion of false-positive in vitro IgE reports should be followed by a subsequent test using an CCD inhibitor. The Mediwiss strip test and the ISAC multi-component array may even be generally used for the initial analysis. An official recommendation as to whether the CCD blockade should be routinely used in the future with some or even all methods in order to avoid such results from the very start will obviously require a broad-based prospective investigation. The results obtained with this CCD blocker might explain why different IgE test methods systematically yield diverse results and the products of the various manufacturers are therefore not comparable – a point of criticism that has been expressed for several decades now, but has received little attention from manufacturers thus far .


Saturating concentrations (10 µmol/L) of either FKBP12 or 12.6 significantly reduced the frequency, spread, amplitude and Ca2+ spark mass relative to control, while the tomograms revealed both proteins shifted the tetramers into a largely side-by-side configuration. Phosphorylation of immunophilin-saturated RyR2 resulted in structural and functional changes largely comparable to phosphorylation alone. dSTORM images of myocyte surfaces demonstrated that both FKBP12 and 12.6 significantly reduced RyR2 cluster sizes, while phosphorylation, even of immunophilin-saturated RyR2, increased them. We conclude that both RyR2 cluster size and the arrangement of tetramers within clusters is dynamic and respond to changes in the cellular environment. Further, these changes affect Ca2+ spark formation.The effects of the immunophilins, FKBP12 and FKBP12.6, and phosphorylation on type II ryanodine receptor (RyR2) arrangement and function were examined using correlation microscopy (line scan confocal imaging of Ca They arrcolemma . The smae (CICR) . This are (CICR) .2+ influx through Cav1.2, RyR2 open stochastically but with a very low probability , and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail.The images in 2+ spark parameters were not normal, so they were displayed as box plots , possibly reflecting their differing effects on RyR2 gating (−4). The number of sparks analyzed, cells examined, and animals used are in The immunophilins significantly reduced the spark frequency and most of the Ca2 gating . The spa2+ sparks.Given these results, and those from our previous study, we hypothesized that changes in the tetramers’ distribution would accompany the functional changes and used correlation microscopy to examine dyads in the same cells from which we had recorded the CaRyR2 is a homotetramer resembling a square-capped mushroom about 27 nm wide and 12 nm deep . Its sizUsing Amira, a single plane from a dual-tilt tomogram is displayed in Measurement of the tetramers’ centre-to-centre nearest neighbor distances (NND), also derived from RyR_fit, is displayed in Tetramer arrangements recorded in each of the indicated experimental conditions using the RyR_fit program which is available in Comparable images of a myocyte treated with the phosphorylation cocktail are shown in Permeabilized myocytes treated with saturating concentrations of either FKBP12  or FKBP1Exposing immunophilin-treated tetramers to cell-wide phosphorylation partially reversed the proteins’ effects , but seeTo determine which, if any, of the tetramer distributions could have been derived from the same population, the histograms were expressed as cumulative distribution functions CDF;  to enablTomograms currently provide the highest possible resolution with which to view the tetramers, but to determine their position and orientation the images must be limited to a small area within a single dyadic cleft. To determine whether the results were truly representative of wide-spread changes in the cells we imaged the distribution of RyR2 near the myocyte surface using our custom-built superresolution microscope in near-TIRF mode . The resImages of tetramer clusters near the cell surface were placed into an array with columns designating the treatment , and rows indicating whether the cells were (bottom) or were not (top) exposed to the phosphorylation cocktail. The images demonstrate dramatic, widespread, changes in the appearance and shape of the clusters. The immunophilins appeared to have fractured the clusters into smaller pieces, while all of the phosphorylated cells appeared to have larger clusters than their non-phosphorylated counterparts.As demonstrated in To quantify the dSTORM images, we used the higher resolution tomographic images to guide our analysis. We first drew alpha shapes around the tetramer arrays in the tomograms  to calcuMedian mean cluster areas and number of clusters examined. The alpha shapes and areas were generated using CGAL.Median tetramer coverage per cluster recorded from the indicated number of tomograms using the RyR_fit program . Mean anWe have provided compelling evidence that inhibitory factors, such as the immunophilins, are associated with the RyR2 tetramers moving into a largely side-by-side configuration and decreasing the cluster size. In direct contrast, excitation due to phosphorylation, even in the presence of the inhibitory immunophilins, is associated with the tetramers moving apart into a checkerboard-like configuration and increasing the cluster size. These findings show that our understanding of RyR2 function has been incomplete and a full understanding should incorporate these dynamic effects. The results confirm and extend our earlier findings, and demonstrate that normal operation of the mammalian ventricular dyad involves movement and rearrangement of the tetramers and changes in the cluster size.The distribution of nearest-neighbor centre-to-centre distances (NND) identified in control myocytes was bimodal, comparable to our previous results, with peaks at 28 and 34 nm. The median of the two modes varied little  with a rRecently published superresolution data appeared to show mostly single and isolated tetramers in a unimodal distribution with a mean NND of 40 nm . In cont2+, which also inhibits RyR2 . All chemicals were purchased from Sigma-Aldrich  unless otherwise stated.2+ sparks were recorded on a Zeiss LSM 700 confocal microscope in line scan mode (63x objective) using 30 µmol/L Fluo-4 (Invitrogen). Sparks were analyzed using SparkMaster  1.8 FKBP12.6/RyR2 (45% occupancy) and no FKBP12 ; (ii) Atd spread .2+ sparks had been recorded, and they were fixed, embedded, sectioned and ready for tomographic analysis The 2D EM images demonstrate that the ultrastructure was well preserved regardless of the experimental protocol  using our established protocols. We used Inspect 3D to align and Amira 5.3  to visualize the data sets, and employed no image processing steps other than a contrast stretch . 2D elecprotocol . AdditioAn individual tetramer’s position relative to its neighbors was classified using Amira and a custom-written C++ program, ‘RyR_fit’, as previously described . BrieflyE. coli Rosetta (DE3) cells with 0.4 mmol/L isopropyl-β-D-thiogalactoside (IPTG) at 0.6 optical density at 600 nm. After growth for 4 hr at 37°C, the cells were collected, re-suspended in the buffer A  with 100 μM PMSF, 25 μg/ml DNaseI and 25 μg/ml lysozyme and 10% glycerol, and disrupted by sonication. Cell debris was removed by centrifugation at 40,000 g for 30 min. Cell lysate was applied to a PorosMC column (Thermo Fisher Scientific), were washed with five column volumes (CV) of buffer A and 5 CV of buffer A plus 10 mmol/L imidazole, and were eluted with buffer A plus 300 mmol/L imidazole [pH 7.4]). The protein was dialyzed overnight against buffer A and was cleaved simultaneously with recombinant TEV protease. The samples were then run on another PorosMC column in buffer A, and the flow-through was collected and dialyzed against buffer C , applied to a Q Sepharose column , and eluted with a gradient from 0% to 50% buffer D . Finally, the samples were run on a Superdex 200 16/60  column in buffer A plus 5 mmol/L 2-mercaptoethanol, which separated dimeric and monomeric fractions. The monomeric fractions were pooled, aliquoted and flash-frozen for further use.FKBP12 and FKBP12.6 were cloned to obtain a fusion protein with N terminal histidine and maltose binding protein (MBP) tag. FKBPs were induced in To visualize and better understand the 3D architecture and arrangement of the t-tubules, jSR and ryanodine receptors, we manually segmented these compartments on the dual tilt tomograms using Amira (FEI). Using the segmentation module, the contours were hand drawn, creating a 3D model, by tracing the individual membranes in every slice of the tomograms using a Wacom Cintiq 27QHD drawing tablet. In each dyad, a single RyR2 tetramer was traced, then copies of it were placed in the proper position and orientation; this simplified the drawing. As displayed in the supplementary videos, t-tubules were traced in blue, jSR in red and RyR2 tetramers in green. Automatic smoothing was applied to the surface models to reduce the number of polygons and improve the models’ appearance. When drawing, the tomogram cannot be rotated, so to ensure that we identified the correct position and orientation of the tetramers we used the Slice option to superimpose the tomographic data onto the model. This simultaneous visualization ensured accurate positioning of the tetramers in the 3D data set. Movies of the complete models were created using Amira’s MovieMaker module.2 for storage at −80°C until use. Protein  was separated on 12% SDS-polyacrylamide gels and transferred to Immobilon-P transfer membrane  overnight at 4°C. Membranes were then blocked in skim milk in Tris-buffered saline with 0.1% Tween (TBS-T Sigma) at room temperature for one hour. Primary antibody incubation was performed at 4°C overnight and secondary antibody incubation at 37°C for one hour. Protein bands were visualized using the SuperSignal West Femto Chemiluminescent Substrate kit .We used a standard Western Blot protocol to confirm that exogenous FKBP12 or FKBP12.6 raised the cellular levels beyond control values . The 6X We used the following antibodies: Anti-FKBP12  to detect both FKBP 12 and FKBP12.6, anti Calsequestrin  and anti-RyR2  were used as loading controls. Secondary antibodies: HRP-Goat anti-mouse IgG  and HRP-Goat anti-rabbit IgG .2+ load was normally distributed, virtually none of the other parameters were, so, similar to Sikkel et al., we used the log transformation to analyze the data. We treated the p value generated by the R-program differently from Sikkel et al, using the Holm-Bonferroni correction .To interpret the images, we defined a cluster as consisting of pixels where no pixel was more than 50 nm distant from its nearest neighbor. The area of each cluster was determined by defining the boundary with an alpha shape . Cluster In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:2+ spark properties and CRU configurations are altered in myocytes when exposed to saturating concentrations of FKBPs and/or a phoshporylation cocktail. Ca2+ spark properties change as well as RyR orientations within clusters. The RyR orientations are determined from EM tomograms. Additional data on peripheral cluster outlines is provided by optical super-resolution microscopy.Asghari et al. describe new results concerning the variable/changeable distribution of RyRs in response to a number of stimuli and chemical modifications. The manuscript shows that both CaDecision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kenton Swartz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Bjorn Knollmann (Reviewer #3). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "Cardiac ryanodine receptor distribution is dynamic and changed by auxiliary proteins and post-translational modification" for consideration by Summary:This manuscript by Asghari et al. reports the distribution, arrangement, and cluster size of the type II ryanodine receptor (RYR2) tetramers under various conditions. This group has previously reported that phosphorylation and low concentrations of magnesium moved the tetramers into a predominantly checkerboard arrangement, whereas high concentrations of magnesium induced a dense side-by-side configuration. The present work confirms and extends their early findings and shows that FK506 binding proteins (FKBP12 and FKBP12.6) moved the tetramers into a largely side-by-side configuration and decreased the cluster size. In contrast, phosphorylation even in the presence of FKBPs moved the tetramers into a checkerboard-like configuration and increased the cluster size. By correlating the effects of FKBPs and phosphorylation with their tetramer arrangements, the authors propose that the side-by-side configuration of tetramer arrangement is associated with channel inhibition, whereas the checkerboard-like configuration is associated with channel activation.Essential revisions:1) The authors should show cluster rearrangements together with changes in Ca sparks in intact cells in response to strong phosphorylation. This would go a long way to demonstrate that the structural rearrangement modulate RyR function in a more physiologic setting. Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.2+ spark experiments and RyR distribution experiments. Recently, Sikkel et al. [1] published an analysis of Ca2+ spark data and included a discussion of general experiments with myocytes. They concluded that it may be required to conduct a hierarchical analysis to draw valid conclusions from myocyte experiments. The authors should show that their results hold up to such a hierarchical data analysis.2) Statistical evaluation of Ca3) Determination of tetrad position and orientation in tomograms. The procedures used to determine these parameters are not clear to this reviewer. In the Materials and methods the authors say "An individual tetramer's position relative to its neighbors was classified using a custom-written C++ program, 'RyR_fit', as previously described .". I consulted Asghari et al., 2014, and could not see any reference to this or a similar software in the main text or supplementary of Asghari et al., 2014. I downloaded the C++ source of RyR_fit from the supplementary of this manuscript to learn more but had difficulty to decipher answers to my questions from the lengthy source listing. In particular, is the extraction of tetrad positions and orientations fully automatic? Alternatively, does the software require the user to choose positions and orientations? A mixture of the two? In addition, with what precision/error can these parameters be determined? For example, looking at Figure 2F: for several tetrads it is not clear how the "smudges" shown can be related to a precise orientation? A more detailed description of the procedures used, particularly the role of a human operator or the robustness of an automatic procedure, should be added – including a procedure to estimate the precision with which the orientation and centre of a tetramer can be determined.4) Actual resolution in dSTORM data. There are now a set of tools, in particular Fourier ring correlation, that can estimate the actual resolution in super-res images. This would be important to carry out to establish at what resolution level this data provides reliable information. In my experience dSTORM data often has resolution worse than 50 nm which limits conclusions that can be drawn about detailed cluster shape changes. It is important to add such estimates and then possibly re-evaluate the information that is accessible from this data.5) "Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.6) The major novelty of the work is the discovery of the tremendous plasticity of the RyR2 cluster. The authors should quantify the degree of movement in more detail. What is the time course? Distances?7) Determining the mechanism responsible for the RyR2 movements is beyond the scope of the work but the authors should discuss possible mechanisms in more detail. For example, what is the role of Bin-1 or other molecules postulated to be important for dyad formation/assembly. Essential revisions:1) The authors should show cluster rearrangements together with changes in Ca sparks in intact cells in response to strong phosphorylation. This would go a long way to demonstrate that the structural rearrangement modulate RyR function in a more physiologic setting.We are preparing a manuscript to address the hypothesis that phosphorylation of RyR2 alone, rather than other targets, is required for the functional and structural changes that we’ve observed. We used 300 nM isoproterenol as the stimulus, rather than the phosphorylation cocktail, and the data will be in that paper. The results are identical to what we have reported in this manuscript and the Asghari et al., 2014 paper.Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.Multiple pieces of information indicated that RyR2 phosphorylation, or hyperphosphorylation, takes precedence. Neither protein dissociated from RyR2 to a significant extent as demonstrated by our Western Blot , and by our spark analyses and superresolution results. These are discussed in the subsection “Phosphorylation”. They agree with others who have performed co-IP experiments and have examined fluorescent FKBP derivatives concluding there was no significant dissociation.2+ spark experiments and RyR distribution experiments. Recently, Sikkel et al. [1] published an analysis of Ca2+ spark data and included a discussion of general experiments with myocytes. They concluded that it may be required to conduct a hierarchical analysis to draw valid conclusions from myocyte experiments. The authors should show that their results hold up to such a hierarchical data analysis.2) Statistical evaluation of Card author on the Sikkel et al. manuscript), and he agrees with us that the modified correction has greater statistical power. The statistical analysis is discussed in the subsection “Statistical Analyses”. There were minor changes in the results and these are reflected in Figure 1B and its source data, and in various places throughout the text.Our results do hold up and the conclusions were unchanged. We reanalysed our data using the Sikkel et al. methodology as requested, but we differed in our handling of the probability with multiple comparisons and used the sequential Holm-Bonferroni rather than the simple Bonferonni correction. We have been in contact with the R-program’s author, Dr. James Howard (the 33) Determination of tetrad position and orientation in tomograms. The procedures used to determine these parameters are not clear to this reviewer. In the Materials and methods the authors say "An individual tetramer's position relative to its neighbors was classified using a custom-written C++ program, 'RyR_fit', as previously described . ". I consulted Asghari et al., 2014, and could not see any reference to this or a similar software in the main text or supplementary of Asghari et al., 2014. I downloaded the C++ source of RyR_fit from the supplementary of this manuscript to learn more but had difficulty to decipher answers to my questions from the lengthy source listing. In particular, is the extraction of tetrad positions and orientations fully automatic? Alternatively, does the software require the user to choose positions and orientations? A mixture of the two? In addition, with what precision/error can these parameters be determined? For example, looking at Figure 2F: for several tetrads it is not clear how the "smudges" shown can be related to a precise orientation? A more detailed description of the procedures used, particularly the role of a human operator or the robustness of an automatic procedure, should be added – including a procedure to estimate the precision with which the orientation and centre of a tetramer can be determined.We have made adjustments to Figure 2 and to the text in the Results to clarify the procedure.RyR_fit is used to manually place a square of the appropriate size and orientation over a tetramer once the operator has identified it using Amira. RyR_fit is essential for classifying the tetramers’ positions , for generating the NND and for determining the area occupied by the tetramers using α shapes .We estimated that the positioning error for a tetramer was ~3 nm; the basis for this estimate, and other possible errors, is discussed in detail in Asghari et al., 2014, pg 258. We have added this information to the subsection “Classifying RyR2 Positions”.2+) and those that brought them together (immunophilins and high Mg2+). These are large changes, and an error of a few nm in determining the centre of the tetramer, or of degrees in their angle, has no impact on our conclusions.New high resolution cryo-EM structures of RyR2 show that the tetramers are 27 nm on a side , rather than 29 nm, which was the best estimate at the time Asghari et al. was published. It is notable that this difference has not affected our conclusions. Comparing the results from Asghari et al., 2014 to the results presented here, Control cells still show a bimodal distribution in their NND, and we see the same changes in distribution in response to agents that moved the tetramers apart (phosphorylation and low Mg4) Actual resolution in dSTORM data. There are now a set of tools, in particular Fourier ring correlation, that can estimate the actual resolution in super-res images. This would be important to carry out to establish at what resolution level this data provides reliable information. In my experience dSTORM data often has resolution worse than 50 nm which limits conclusions that can be drawn about detailed cluster shape changes. It is important to add such estimates and then possibly re-evaluate the information that is accessible from this data.Thank you for this suggestion. Fourier ring correlation indicated that the resolution of our images was between 16 and 25 nm. This is stated in the first paragraph of the subsection “Super-resolution immunofluorescence microscopy”, and an image of the program’s output is included in Figure 6—figure supplement 1.5) "Importantly, these results show that hyperphosphorylation takes precedence over excess FKBP12 or 12.6." How do you reconcile this statement with some reports that phosphorylation itself leads to reduced binding of FKBPs to RyR2? Is it phosphorylation itself or is it consequent unbinding of FKBP's. How much FKBP is still bound to phospho-RyR2? I think this is shown in Supplementary Figure 6, but that figure is not referenced in the text. Is it a co-IP? This is a critical issue that needs to be addressed. Basically, does phosphorylation remove the immunophilins from the RyR2 tetramers or not? In addition to co-IP, this could/should be done with fluorescent-labelled FKBP.Please see the answer to question 1 and the subsection “Phosphorylation”.6) The major novelty of the work is the discovery of the tremendous plasticity of the RyR2 cluster. The authors should quantify the degree of movement in more detail. What is the time course? Distances?Comparisons of tetramer and cluster positions before and after treatment aren’t possible because we fix the myocytes for examination by both tomography and superresolution immunofluorescence microscopy. Each of the experimental treatments involves different cells, so we cannot track individual tetramers with either technique, making it impossible to quantify the distance and time course of their movements.Since the first time point we examined was 10 minutes after the application of the treatments we must assume that the changes occurred within this period. This is now stated in the last paragraph of the Discussion.7) Determining the mechanism responsible for the RyR2 movements is beyond the scope of the work but the authors should discuss possible mechanisms in more detail. For example, what is the role of Bin-1 or other molecules postulated to be important for dyad formation/assembly.A brief examination of the role of BIN1 as a possible mechanism of tetramer translocation has been added to the last paragraph of the Discussion.