The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the small GTPases, Gtr1/2. However, little is known about the way that other nutrient signals are transmitted to TORC1. Here we report that glucose starvation triggers disassembly of TORC1, and movement of the key TORC1 component Kog1/Raptor to a single body near the edge of the vacuole. These events are driven by Snf1/AMPK-dependent phosphorylation of Kog1 at Ser 491/494 and two nearby prion-like motifs. Kog1-bodies then serve to increase the threshold for TORC1 activation in cells that have been starved for a significant period of time. Together, our data show that Kog1-bodies create hysteresis (memory) in the TORC1 pathway and help ensure that cells remain committed to a quiescent state under suboptimal conditions. We suggest that other protein bodies formed in starvation conditions have a similar function.DOI:http://dx.doi.org/10.7554/eLife.09181.001 In humans, yeast and other eukaryotes, a group of proteins called the Target of Rapamycin Complex I (TORC1) promote cell growth and increase metabolic activity when nutrients are plentiful. Previous studies have shown how molecules that contain the nutrient nitrogen – which is needed to make proteins – activate TORC1. However, it is not clear how other nutrients regulate this complex.Bakers yeast is a simple, single celled organism that researchers often use as a model to study how cells work. The yeast TORC1 is made up of three core proteins, including Kog1 and Tor1. Kog1 selectively recruits proteins to the complex, where they are modified by Tor1 to regulate their activity. Here, Hughes Hallett et al. used microscopy to study what effect sugar starvation has on the complex.In the experiments, yeast cells were genetically engineered so that Kog1 and Tor1 appeared fluorescent under the microscope. The experiments reveal that, when sugar is in short supply, Kog1 breaks away from the rest of the TORC1 and moves to another part of the cell where it accumulates to form a cluster called a “body”. This movement is driven by a “kinase” enzyme that adds chemical groups called phosphates to Kog1, and by regions within the Kog1 protein known as prion like domains. When sugar first becomes available again, Kog1 is still in the body so Tor1 cannot immediately trigger cell growth. However, once a steady supply of sugar resumes, Kog1 rejoins the rest of the complex and the cells start to grow.Together, Hughes Hallett et al.’s findings reveal that the formation of Kog1 bodies during sugar starvation creates a “memory” that prevents TORC1 from reactivating cell growth if sugar is only temporarily available. Humans have over 100 proteins that contain prion like domains. Therefore a future challenge is to find out whether any of these proteins form similar bodies that enable our cells to remember past events.DOI:http://dx.doi.org/10.7554/eLife.09181.002 The Target Of Rapamycin Complex I (TORC1) is made up of three essential proteins, called mTOR, Raptor and mLST8 in humans and Tor1, Kog1 and Lst8 in yeast . BiochemIn the presence of pro-growth hormones and the appropriate nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins . In contIn higher eukaryotes, hormone signals are transmitted to TORC1 through the Phosphatidylinositol 3-kinase to AKT, which in turn phosphorylates the tuberous sclerosis complex . The tubS. cerevisiae) but does not appear to influence TORC1 activity in budding yeast  in key signaling and metabolic pathways to ensure that once cells commit to a stress or starvation state, they only exit when conditions are optimal.To determine if TORC1 localization is altered in glucose starvation conditions, we subjected yeast carrying Kog1-YFP, Tco89-YFP , or Tor1 with a triple GFP insertion  to acuteTo study Kog1-body formation in more detail, we next followed Kog1-YFP localization in cells transferred from rich medium (SD) to glucose starvation, nitrogen starvation, glucose + nitrogen starvation, and osmotic stress conditions. In glucose starvation and nitrogen + glucose starvation we saw rapid formation of Kog1-bodies ( τ = 11.0 ± 2.8 and 13.6 ± 2.4 min) in 57 ± 5% and 73 ± 4% of cells, respectively . Kog1-boIn separate experiments, we also followed Kog1-body formation during long periods of glucose and/or nitrogen starvation (2-–6 hrs). In all cases, we saw slow accumulation of Kog1-bodies, beyond the levels found at one hour, until 80-85% of cells had a least one, but on occasion 2 or 3, Kog1 bodies . CultureFinally, to determine if Kog1-body formation is a reversible process, we exposed cells to glucose starvation conditions for 60 min and then followed Kog1-YFP localization after adding 0.02%, 0.1%, or 2% glucose back to the medium. In both 0.1% and 2% glucose, we saw Kog1 move out of the bodies and back onto the vacuolar membrane over an approximately one-hour time-period  and reversible process, triggered by glucose and nitrogen starvation.Previous work has shown that TORC1 can localize to stress granules in both yeast and human cells . To detesnf1Δ cells. These experiments revealed that deletion of Snf1 causes a dramatic, 20-fold, increase in the time-constant for Kog1-body formation  growing in SD medium, (2) exposed to glucose starvation conditions for 5 min, and (3) exposed to osmotic stress conditions for 5 min . We then used mass spectrometry to identify the phosphorylation sites on Tor1 and Kog1 in each condition. These data showed that Kog1 is phosphorylated on Ser 491 and 494 in glucose starvation conditions, but not in osmotic stress or SD medium . A shortS491A/S494A cells also showed a reduced level of Kog1-body formation in SD medium  inhibits cell growth and/or division .During log growth, TORC1 phosphorylates and activates the S6 kinase, Sch9, to drive protein and ribosome synthesis . This keS491A/S494A, PrDm1, PrDm2 and PrDm1 + 2 strains , using standard methods and are listed in All strains used in this study were generated in a haploid 600 0.1 in 25 ml of fresh medium and allowed to grow to an OD600 of 0.4. At this point, 300 μl samples were transferred to a chamber slide (Nunc Lab-Tek II #155409) that had been treated with 2 mg/ml concanavilin A. The slides were then washed three times with 300 μl treatment medium  and loaded into a 30o°C chamber.Cultures were grown in 25 ml of SD medium (or 25 ml of SD medium missing uracil in experiments with plasmids) in 125 ml conical flasks shaking at 200 rpm and 30°C until they reached mid-log phase (OD600 between 0.3 and 0.4). The cultures were then diluted to ODImages were acquired using a Deltavision Elite Microscope equipped with an Olympus 100x, 1.4NA, objective and a sCMOS camera. We collected a Z-series of 5 images with 1 μm spacing in the YFP , GFP , and RFP  channels at each time-point to ensure that all of the fluorescent bodies in the cell were detected. Image files were then processed in ImageJ to create the maximum projection from the stacks. Glucose repletion experiments  were per∧-t/τ) + c for starvation and (A*(e^∧-t/τ) + c for repletion experiments, where A is the fraction of cells that form bodies during the time-course, τ is the apparent time-constant, and c is fraction of cells that have bodies at the start (starvation) or end (repletion) of the time-course. In cases where there was no change in Kog1-body levels during the time-course (e.g. in SD medium) the data was fit to a line. All fitting was done in Igor Pro 6.3 (WaveMetrics) and the errors reported are the estimated standard deviation from the fit.We calculated the fraction of cells containing one or more Kog1 body by manually inspecting the images for >200 cells per time-point, per experiment. The time-course data was fit to a single exponential equation; A*(1-e600 0.1 and grown (as above) in 4L flasks until they reached an OD600 of 0.6. At this point cultures were either harvested by filtration and frozen in liquid nitrogen (SD sample), or subjected to 0.4M KCl stress, or glucose starvation for 5 min, and harvested by filtration and frozen in liquid nitrogen.Yeast expressing Kog1-3xFlag were inoculated into 50 ml of SD medium (for glucose starvation experiments) or 50 ml YEPD (for osmotic stress experiments) and grown overnight in 250 ml conical flasks, shaking at 200 rpm and 30°C. These cultures were then used to inoculate 2.5L of fresh media to ODTo isolate TORC1, cells were washed off the filter with 10 ml chilled lysis buffer  and pelleted by centrifugation at 4°C. The cell pellets were then resuspended in chilled lysis buffer supplemented with a protease inhibitor cocktail , split into 100 OD unit aliquots, and lysed by bead beating . The lysate was then collected after centrifugation for 5min at 3000rpm and transferred into fresh tubes before being spun down again at 500 g for 5min at 4°C. The clarified lysate was then incubated with 100 μl protein G Dynal beads + 10 μl anti-Flag antibody for 30min at 4°C. The resin was washed 3 times for 1 min with 1 ml lysis buffer at 4°C and the protein eluted by boiling the beads in 150 μl SDS loading buffer for 5min, and loaded onto a 10% acrylamide gel (BioRad 161-1155). The gels were then stained using colloidal blue and the bands corresponding to Tor1 and Kog1 excised, subjected to protease treatment, and analyzed using Mass Spectrometry.600 between 0.55 and 0.6). At this point, a 47 ml sample was collected, mixed with 3 ml 100% Trichloroacetic acid (TCA), and held on ice for at least 30 min (and up to 6 hrs). The remaining culture was then collected by filtration, transferred to the appropriate medium after one wash with 100ml of -Glu medium, and further samples collected in TCA, as described above. The samples were then centrifuged at 4000rpm for 5 min at 4°C, washed twice with 4°C water, twice with acetone, and disrupted by sonication at 15% amplitude for 5s before centrifugation at 8000rpm for 30s. The cell pellets were then dried in a speedvac for 10 min at room temperature, and frozen until required at -20°C.Bandshift measurements were performed using a modified version of the protocol developed by Urban and Loewith . Culture3, 5 mM NaH2PO4, 5 mM p-nitrophenylphosphate, 5 mM β-glycerophosphate, 1% SDS) supplemented with complete protease and phosphatase inhibitor tablets . The lysate was then collected after centrifugation for 5 min at 3000 rpm, resuspended into a homogenous slurry, and heated at 65°C for 10 min. Soluble proteins were then separated from insoluble cell debris by centrifugation at 15,000 rpm for 5 min, and the lysate stored at -80°C until required. Samples were then subjected to cleavage by 2-Nitro-5-thiocyanatobenzoic acid (NTCB) for 12–16 hr at room temperature  before further analysis. Cell extracts were then heated to 95°C in SDS sample buffer for 5 min before they were run on an SDS-PAGE gel, transferred to a nitrocellulose membrane. Western blotting was carried out using 12CA5 (anti-HA) and an anti-mouse secondary labeled with a IRDye 800CW (LiCor) and the blots scanned using a LiCor Odyssey Scanner .Protein extraction was performed by bead beating  in urea buffer  and eluted with 70% acetonitrile/0.1% TFA. The desalted material was concentrated to dryness in a speed vac.Gel slices were washed for 15 min each with water, 50/50 acetonitrile/water, acetonitrile, 100 mM ammonium bicarbonate, followed by 50/50 acetonitrile/100 mM ammonium bicarbonate. The solution was then removed and the gel slices dried by vacuum centrifugation. Next, the dried gel slices were reduced by covering them with 10 mM dithiothreitol in 100 mM ammonium bicarbonate and heating them at 56°C for 45 min; alkylated by covering them with a solution of 55 mM iodoacetamide in 100 mM ammonium bicarbonate and incubating in the dark at ambient temperature for 30min, and washed with 100 mM ammonium bicarbonate for 10 min and 50 mM ammonium biocarbonate + 50% acetonitrile for 10 min. The gel slices were then dried again and treated with an ice-cold solution of 12.5 ng/μL trypsin  in 100 mM ammonium bicarbonate. After 45 min, the trypsin solution was removed, discarded, and a volume of 50 mM ammonium bicarbonate was added to cover the gel slices and they were incubated overnight at 37°C with mixing on a shaker. Samples were then spun down in a microfuge and the supernatant collected. The same Gel slices were then incubated in 0.1% trifluoroacetic acid (TFA) and acetonitrile, centrifuged, and the supernatant collected. At this point, the digestion supernatant and the extraction supernatant were pooled, split into two tubes, and concentrated using vacuum centrifugation. One tube was further digested with thermolysin  by resuspending the tryptically digested peptides with a solution containing 50 mM Tris-HCl pH 8 and 0.5 mM calcium chloride and adding 1 μg of thermolysin. Digestion was carried out at 75°C with mixing for 5 hr. The thermolysin was quenched by adding TFA to a 0.5% final concentration. All samples were desalted using ZipTip C18AQ  followed by peptide separations on a reversed-phase column (75 μm × 250 mm) packed with Magic C18AQ  directly mounted on the electrospray ion source. A 45-min gradient from 2% to 35% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min was used for chromatographic separations. A spray voltage of 2500V was applied to the electrospray tip and the Orbitrap Elite instrument was operated in the data-dependent mode, switching automatically between MS survey scans in the Orbitrap  with collision induced dissociation MS/MS spectra acquisition in the linear ion trap , higher-energy collision induced dissociation (HCD) MS/MS spectra acquisition in the Orbitrap  and electron transfer dissociation (ETD) MS/MS spectra acquisition in the Orbitrap . The three most intense precursor ions from the Fourier-transform (FT) full scan were consecutively selected for fragmentation in the linear ion trap by CID with a normalized collision energy of 35%, fragmentation in the HCD cell with normalized collision energy of 35%, and fragmentation by ETD with 100 ms activation time. Selected ions were dynamically excluded for 30 seconds.The proteolytically-digested samples were brought up in 20 μL of 2% acetonitrile in 0.1% formic acid and 18 μL and then analyzed by LC/ESI MS/MS with a Thermo Scientific Easy-nLC II  coupled to a Orbitrap Elite ETD  mass spectrometer using a trap-column configuration as described in . In-linewww.yeastgenome.org) that was appended with protein sequences from the common Repository of Adventitious Proteins or cRAP (www.thegpm.org/crap/). Two searches were performed corresponding to the proteolytic enzymes trypsin, and trypsin/thermolysin (no enzyme selected). The Maximum missed cleavages was set to 2. The precursor ion tolerance was set to 10 ppm and the fragment ion tolerance was set to 0.8 Da. Variable modifications included oxidation on methionine (+ 15.995 Da), carbamidomethyl (+ 57.021 Da) on cysteine, and phosphorylation on serine, threonine, and tyrosine (+ 79.996 Da). Sequest HT was used for database searching. PhosphoRS 3.1 . The data were searched against the Saccharomyces Genome Database . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "AMPK/Snf1 Promotes the Formation of Kog1-bodies to Increase the Activation Threshold of TORC1 in Budding Yeast" for peer review at The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife. However, they also noted some weaknesses, particularly the biological role of the Kog1 bodies, whether Sch9 phosphorylation status is truly a measure of TORC activity, and whether Kog1 bodies really affect TORC activity. The Reviewers made individual points and suggestions, but the overall comments are quite consistent. The most important request for the revised manuscript is to clearly connect the role of Kog1 bodies with TORC function. Instead of listing specific experiments that are absolutely required vs. others that would be useful, we request the authors address the points raised in the reviews, and do whatever experiments they judge best that will answer some of these issues (and are feasible in a reasonable timeframe). We don't expect or require the authors to address all the issues with new experiments, but we do ask that some of the "holes" be filled. We stress that the purpose of these new experiments is not simply to add more material, but rather to address specific issues raised.All the reviewers found the work interesting and, in principle, suitable for Reviewer #1: Although I'm not an expert in this area, this appears to be an interesting, well-done, and well written paper that, in principle, should be published in eLife. The authors convincingly describe a new mechanism in which glucose or nitrogen starvation results in disassembly of TORC in which the Kog/raptor subunit forms Kog1 bodies near the edge of the vacuole. This relocalization requires Snf1/AMP kinase-mediated phosphorylation of Kog1 at Ser491/494 and two nearby prion-like motifs. Finally, the authors provide evidence that the Kog1-bodies increase the threshold for TORC activation to help cells remain in the starved state unless nutrients are fully restored. This is a new mechanism of clear physiological importance in yeast. While it is unknown if this mechanism is utilized elsewhere, this should not affect its suitability for eLife. It is highly unlikely that this or a related mechanism is specific to S. cereivisae, particularly the general concept of why protein bodies might be functionally important. There is one weakness of the paper that I would like to see addressed and another experiment that would be nice.1) The experiment in 2) Of less importance, but also useful, it would be nice to examine a Ser491/494 derivative with both serines converted to either glutamate or aspartate. It is quite possible that this would lead to constitutive Kog1-body formation and hence enhance the paper. In addition, it would be interesting to know what fraction of Kog1 is incorporated into the Kog1 bodies, and whether other components of TORC1 are sequestered in these structures.Reviewer #2: This study examines the intracellular fate of two key components of yeast TORC1, namely, Tor1 and Kog1, following nutrient starvation using fluorescence microscopy. The main finding is that glucose starvation triggers a change in localization of Kog1 from a peri-vacuolar location to a punctate vacuole-associated location termed "Kog1 bodies". This change in Kog1 localization is determined to correlate to varying degrees with Kog1-dependent phosphorylation by Snf1, the presumptive ortholog of AMPK in yeast, and decreased TORC1 activity during glucose starvation, as monitored by Sch9 phosphorylation.The strengths of the manuscript include the novel finding of a change in localization of an essential TORC1 component in a nutrient responsive manner and the fact that all of the data reported are solid and well controlled. There are two major concerns that need to be addressed before publication can be considered:1) The physiological significance of these findings is unclear. Thus, the authors suggest that TORC1 activity is dampened by glucose starvation via Kog1 body formation, but the data presented do not address this directly, and some of the data using Sch9 as a readout are inconclusive, in particular with the Snf1 phosphorylation site mutants. It will be important to demonstrate that cells expressing the Kog1 mutants have reduced fitness following either prolonged glucose starvation and/or return to glucose replete conditions. One possibility would be to test phosphomimetic substitutions to see if an inability to dephosphorylate Kog1 leads to decreased viability in low glucose. In general, because the mutants examined do not have any obvious phenotype with respect to glucose starvation per se, enthusiasm for these findings is somewhat dampened.2) The relationship between Snf1 and Kog1 phosphorylation needs to be defined. The authors appear to imply that Kog1 is a direct target for Snf1 yet state in the Discussion this is likely to be mediated by another kinase. It should be noted that the yeast literature has remained quite confusing and controversial in terms of the role of Snf1/TORC1 in comparison to AMPK and mTORC1. As it stands, the present work does not clarify the yeast relationship, and it's important not to muddy the waters any further. If Snf1 is not the kinase that phosphorylates Kog1, do the authors have an idea as to the relevant kinase involved?Reviewer #3: This manuscript presents an interesting story that, if correct, seems like an important advance on an important and significant issue: the mechanisms and consequences of regulation of the nutrient-sensing protein kinase TORC1. The authors describe experiments that they claim show that:1) the TORC1 component Kog1 rapidly (in ~ 10 min) aggregates (reversibly) into "bodies"  when st2) formation of Kog1 bodies depends on Snf1 and on two Snf1-dependent phosphorylation sites in Kog1 , and on;3) two prion-like domains in Kog1 ;4) Kog1-body formation decreases TORC1 activity and ensures that cells remain in a starved state until sufficient glucose becomes available .Conclusions #1-3 seem solid and supported by unimpeachable evidence. The nub of the story is conclusion #4, supported by evidence presented in the section "Kog-1 body formation increases the threshold for TORC1 activation" . This isBut what confuses me is the observation that addition to cells of a large amount of glucose (2%) rapidly activates TORC1 (indicated by phosphorylation of Sch9 ). Doesn'In summary, the authors state their conclusions in the first sentence of the Discussion [my critique inserted]: "In this study we show that glucose starvation leads to inactivation of TORC1 signaling [only if Sch9 dephosphorylation is actually reporting TORC1 inactivation], disassociation of the Kog1-Tor complex [I don't think I saw any evidence of that in the manuscript], and the formation of Kog1-bodies ." All the reviewers found the work interesting and, in principle, suitable for eLife. However, they also noted some weaknesses, particularly the biological role of the Kog1 bodies, whether Sch9 phosphorylation status is truly a measure of TORC activity, and whether Kog1 bodies really affect TORC activity. The Reviewers made individual points and suggestions, but the overall comments are quite consistent. The most important request for the revised manuscript is to clearly connect the role of Kog1 bodies with TORC function. Instead of listing specific experiments that are absolutely required vs. others that would be useful, we request the authors address the points raised in the reviews, and do whatever experiments they judge best that will answer some of these issues (and are feasible in a reasonable timeframe). We don't expect or require the authors to address all the issues with new experiments, but we do ask that some of the "holes" be filled. We stress that the purpose of these new experiments is not simply to add more material, but rather to address specific issues raised. Reviewer #1:Although I'm not an expert in this area, this appears to be an interesting, well-done, and well written paper that, in principle, should be published in eLife. The authors convincingly describe a new mechanism in which glucose or nitrogen starvation results in disassembly of TORC in which the Kog/raptor subunit forms Kog1 bodies near the edge of the vacuole. This relocalization requires Snf1/AMP kinase-mediated phosphorylation of Kog1 at Ser491/494 and two nearby prion-like motifs. Finally, the authors provide evidence that the Kog1-bodies increase the threshold for TORC activation to help cells remain in the starved state unless nutrients are fully restored. This is a new mechanism of clear physiological importance in yeast. While it is unknown if this mechanism is utilized elsewhere, this should not affect its suitability for eLife. It is highly unlikely that this or a related mechanism is specific to S. cereivisae, particularly the general concept of why protein bodies might be functionally important. There is one weakness of the paper that I would like to see addressed and another experiment that would be nice. 1) The experiment in  This is a good suggestion and we have been trying to perform experiments like this, but have not been able to measure TORC1 activity in a single cell (and to the best of our knowledge nor has anyone else working in yeast). However, we would argue that the two-fold effect is already a quite large effect given the importance of TORC1 activity for cell growth, and is enough to establish a function of the Kog1-bodies – something that has yet to be done for other bodies/granules as we mention in the Discussion.2) Of less importance, but also useful, it would be nice to examine a Ser491/494 derivative with both serines converted to either glutamate or aspartate. It is quite possible that this would lead to constitutive Kog1-body formation and hence enhance the paper.As described above, we have now made the DD and EE mutants and find that they are not viable – indicating that Kog1-bodies are incompatible with growth. These data are incorporated into the text and supplement.In addition, it would be interesting to know what fraction of Kog1 is incorporated into the Kog1 bodies,We agree that it would be interesting to know what fraction of Kog1 molecules go to bodies, but unfortunately we have not been able to calculate this number. Kog1 levels are so low (a few hundred molecules per cell) that we cannot accurately measure the amount of Kog1 distributed around the vacuole and/or in the cytoplasm, either before or after glucose starvation – the Kog1 fluorescence levels are simply too close to the level of autofluorescence .and whether other components of TORC1 are sequestered in these structures.We have now examined localization of the other unique component of TORC1, Tco89, in glucose starvation conditions. This protein does move to bodies after 60 min, and this movement is limited in a strain where Kog1 bodies are slow to form (PriDm1+2). This is now mentioned in the text (first paragraph of the Results) and the data shown in the supplement .Reviewer #2:This study examines the intracellular fate of two key components of yeast TORC1, namely, Tor1 and Kog1, following nutrient starvation using fluorescence microscopy. The main finding is that glucose starvation triggers a change in localization of Kog1 from a peri-vacuolar location to a punctate vacuole-associated location termed "Kog1 bodies". This change in Kog1 localization is determined to correlate to varying degrees with Kog1-dependent phosphorylation by Snf1, the presumptive ortholog of AMPK in yeast, and decreased TORC1 activity during glucose starvation, as monitored by Sch9 phosphorylation. The strengths of the manuscript include the novel finding of a change in localization of an essential TORC1 component in a nutrient responsive manner and the fact that all of the data reported are solid and well controlled. There are two major concerns that need to be addressed before publication can be considered: 1) The physiological significance of these findings is unclear. Thus, the authors suggest that TORC1 activity is dampened by glucose starvation via Kog1 body formation, but the data presented do not address this directly, and some of the data using Sch9 as a readout are inconclusive, in particular with the Snf1 phosphorylation site mutants. It will be important to demonstrate that cells expressing the Kog1 mutants have reduced fitness following either prolonged glucose starvation and/or return to glucose replete conditions. One possibility would be to test phosphomimetic substitutions to see if an inability to dephosphorylate Kog1 leads to decreased viability in low glucose. In general, because the mutants examined do not have any obvious phenotype with respect to glucose starvation per se, enthusiasm for these findings is somewhat dampened.We have now tested phosphomimetic substitutions, and as described above, find that strains with Ser 491 and 494 mutated to aspartic acid or glutamic acid do not survive/grow.2) The relationship between Snf1 and Kog1 phosphorylation needs to be defined. The authors appear to imply that Kog1 is a direct target for Snf1 yet state in the Discussion this is likely to be mediated by another kinase.We did not mean to imply that Snf1 directly phosphorylates Kog1, and to make sure there was no room for confusion, directly state that we believe Snf1 acts through another kinase to phosphorylate Kog1 in the Discussion. To make sure this is clear in other parts of the paper we have now adjusted the language in several places where we refer to Snf1 activating phosphorylation of Kog1.For example in the last paragraph of the Introduction: Following up on these observations we show that the AMPK, Snf1, drives Kog1 into bodies by phosphorylating Kog1 on Ser 491 and 494.Has been changed to: Following up on these observations we show that the AMPK, Snf1, drives Kog1 into bodies by phosphorylating, or triggering the phosphorylation of, Kog1 at Ser 491 and 494.The other parts of the text where we discuss Snf1 and Kog1 phosphorylation already use appropriate wording, such as in this excerpt from Results:.S491A/S494A-YFP at the native locus and monitored Kog1 localization in glucose starvation conditions. We found that Kog1S491A/S494A forms bodies slowly , and at a rate similar to that found in the snf1△ strain  of Ser 491 and 494, and this is something we are working on. But at this point we do not know which kinase(s) act downstream of Snf1 to regulate.Reviewer #3:This manuscript presents an interesting story that, if correct, seems like an important advance on an important and significant issue: the mechanisms and consequences of regulation of the nutrient-sensing protein kinase TORC1. The authors describe experiments that they claim show that: 1) the TORC1 component Kog1 rapidly (in ~ 10 min) aggregates (reversibly) into "bodies"  when stThis is a good point, and as described above, we were too loose in our language. All we can conclude is that the rapid dephosphorylation of Sch9 shows that Kog1 body formation does not play a role in the initial inactivation of TORC1 pathway signaling, not TORC1 itself. Therefore, we have changed our language to refer to rapid inactivation of the TORC1 pathway, or rapid inactivation of TORC1-Sch9 signaling, not TORC1.This is key, because the authors' main conclusion is that Kog1-body formation regulates TORC1 function, and Sch9 dephosphorylation is their assay for TORC1 function. Is that really reporting TORC1 function?Our key conclusion (that Kog1 bodies increase the threshold for TORC1 activation) depends on experiments we did comparing the level of Sch9 phosphorylation in strains carrying mutations in TORC1 to the level of Sch9 phosphorylation in the wild-type strain. So in this part of the paper, where we show that mutations in TORC1 (Kog1) cause hyperphosphorylation of Sch9 in low glucose, the only reasonable explanation is that TORC1 itself is more active when Kog1 does not form bodies.Again, the "discovery" "…that TORC1 inhibition precedes Kog1-body formation…" depends on Sch9 dephosphorylation being an indicator of TORC1 function. Is it?As described above, this now reads TORC1 pathway inhibition precedes Kog1-body formation.Also, "Instead, it appears that Snf1 regulates TORC1 both by phosphorylating Kog1 at Ser491 and 494, and through an additional, unknown mechanism" is opaque to me.In this part of the text, we are referring to the fact that deletion of Snf1 partially inhibits the rapid dephosphorylation of Sch9 seen in glucose starvation conditions but removing the Snf1 dependent target sites in Kog1 does not. Thus, Snf1 must both promote rapid dephosphorylation of Sch9 through an unknown mechanism, and the formation of Kog1 bodies by triggering phosphorylation of Kog1 at Ser 491/494. This sentence has now been changed to… Instead, it appears that Snf1 regulates the TORC1 pathway by triggering Kog1 phosphorylation at Ser 491 and 494, and through an additional, unknown, mechanism.But what confuses me is the observation that addition to cells of a large amount of glucose (2%) rapidly activates TORC1 (indicated by phosphorylation of Sch9 ). Doesn'TORC1 is rapidly activated by saturating amounts of glucose in cells containing Kog1-bodies, but this does not suggest, or require, that Kog1 moves back into TORC1 on a rapid time-scale. In fact, as Reviewer 3 points out, we know that this does not happen – Kog1 bodies disassemble on the hour timescale. Instead, we favor the following model:Only some (let’s say 80-90%) of the Kog1 in a cell goes into bodies during glucose starvation . Thus, iIn the PriDm1, PriDm1+2, Snf1 delete, and S491/494 mutants, we have increased the number of intact TORC1 molecules by blocking Kog1-body formation, and as a result 0.02 and 0.1% glucose signals lead to low-level activation of a larger pool of TORC1 molecules – enough to activate Sch9.We already refer to this idea in the Discussion when we say:In this study we show that glucose starvation leads to rapid inactivation of TORC1-Sch9 signaling (τ<2.5 min, In summary, the authors state their conclusions in the first sentence of the Discussion [my critique inserted]: "In this study we show that glucose starvation leads to inactivation of TORC1 signaling [only if Sch9 dephosphorylation is actually reporting TORC1 inactivation], disassociation of the Kog1-Tor complex [I don't think I saw any evidence of that in the manuscript], and the formation of Kog1-bodies ."As discussed above, we agree with the first point the reviewer brings up; we cannot say that glucose starvation leads to inactivation of TORC1 signaling, and instead now say that glucose starvation leads to rapid inactivation of TORC1-Sch9 signaling (τ<2.5 min, We do not agree with the second critique. In


The biomechanical performance of the hooks and screws in spinal posterior instrumentation is not well-characterized. Screw-bone interface failure at the uppermost and lowermost vertebrae is not uncommon. Some have advocated for the use of supplement hooks to prevent screw loosening. However, studies describing methods for combined hook and screw systems that fully address the benefits of these systems are lacking. Thus, the choice of which implant to use in a given case is often based solely on a surgeon’s experience instead of on the biomechanical features and advantages of each device.We conducted a biomechanical comparison of devices instrumented with different combinations of hooks and screws. Thirty-six fresh low thoracic porcine spines were assigned to three groups (12 per group) according to the configuration used for of fixation: (1) pedicle screw; (2) lamina hook and (3) combination of pedicle screw and lamina hook. Axial pullout tests backward on transverse plane in the direction normal to the rods were performed using a material testing machine and a specially designed grip with self-aligned function.The pullout force for the pedicle screws group was significantly greater than for the hooks and the combination (p < 0.05). However, no significant difference was found between the hooks and the combination (p > 0.05).Pedicle screws achieve the maximal pullout strength for spinal posterior instrumentation. The implantation of bone screws into the pedicle was reported in the early 1960s,3. ThereHarrington’s rod for the treatment of scoliosis was implemented in 1962 as the first implant for thoracic spine surgery,6. This This study was approved by the committee of National Science Council of Taiwan. All experiments conformed to the regulations for the care and use of animals. The usage of the porcine spines was in accordance with the guidelines of replacement, reduction and refinement. Thirty-six fresh lower thoracic porcine spines were assigned to three groups (12 per group) according to their fixation configuration: (1) pedicle screws, (2) lamina hooks and 3) combination of screws and lamina hooks. In the screw group (1), pedicle screws were inserted into the neighboring vertebral bodies of one spine motion unit. In the hook group (2), a claw configuration composed of two hooks directed toward each other was used. In the combination group (3), the hooks were placed at the proximal vertebrae, while the screws were inserted into the distal vertebrae. The constructs were connected by rods. Figure combinatThe spinal constructs instrumented with the screws and with the combinations of screws and hooks are shown in FigureBecause of the relatively early application of hooks for treating scoliosis, most surgeons are familiar with this technique. There are standard size hooks available for adolescent patients and miniature hooks available for small children (body weight < 30 kg). However, there are potential disadvantages associated with hook systems-14. FirsSpinal biomechanics have been studied in several ways. Using a long segment construct to study the biomechanics of implants is time consuming and impractical. To conquer this problem, it is reasonable to use a motion unit to study spinal biomechanics. The previous literature has examined the pullout behavior of pedicles, which were loaded by tension during flexion,18. Thisin vitro analysis of specimens prepared in a laboratory environment, which does not necessarily represent clinical circumstances. There are several limitations to our study. First, we used porcine spines instead of human cadaveric spines. Although the physiological structures are somewhat different from those of human cadaveric spines, animal spines are the most convenient choice for performing the experiments when human cadaveric spines cannot be accessed. Numerous studies[Our study is an  studies,23 have The pedicle screw instrumentation achieves the maximal pullout strength for spinal posterior instrumentation. Based on this comparative study, surgeons should be better informed for selecting the optimal fixation method for the thoraco-lumbar spine before surgery.The authors declare that they have no competing interests.CLT participated in the study design, data collection, statistical analyses and drafting of the manuscript. LHC, DML and MYL participated in the study design. PLL advised and assisted with drafting the manuscript. All of the authors read and approved of the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/15/197/prepub


Cryotherapy is a widely used technique in physical therapy clinics and sports.However, the effects of cryotherapy on dynamic neuromuscular control areincompletely explained.  To evaluate the effects of cryotherapy applied to the calf, ankle and sole of thefoot in healthy young adults on ground reaction forces during gait initiation. This study evaluated the gait initiation forces, maximum propulsion, brakingforces and impulses of 21 women volunteers through a force platform, whichprovided maximum and minimum ground reaction force values. To assess the effectsof cooling, the task - gait initiation - was performed before ice application,immediately after and 30 minutes after removal of the ice pack. Ice was randomlyapplied on separate days to the calf, ankle and sole of the foot of theparticipants.  It was demonstrated that ice application for 30 minutes to the sole of the footand calf resulted in significant changes in the vertical force variables, whichreturned to their pre-application values 30 minutes after the removal of the icepack. Ice application to the ankle only reduced propulsion impulse.  These results suggest that although caution is necessary when performingactivities that require good gait control, the application of ice to the ankle,sole of the foot or calf in 30-minute intervals may be safe even preceding suchactivities.  These three regionswere chosen for their predominance of skin, joint and muscle receptors, respectively,which might differentially influence gait initiation following cryotherapy. In addition,it is known that the ankle and calf are commonly injured during sports practiceFaculdade de Medicina de Ribeirão Preto, Universidade de SãoPaulo (FMRP-USP), Ribeirão Preto, state of São Paulo - SP, Brazil. Womenwere recruited because the lack of gender differences in the effects of iceapplicationIn total, 21 young adult female university students were randomly recruited from theBD(r), Juiz de Fora, State of MinasGerais - MG, Brazil) with the cuff positioned between the ice pack and strap at aconstant pressure of 30 mmHgAn OR 6-7-1000 force plate  with a sampling frequency of100 Hz was used to record the forces applied to the ground. An ST-600 infrareddigital thermometer with laser sight (Incoterm(r), Porto Alegre, State of Rio Grandedo Sul - RS, Brazil) with a temperature reading range from -60 to 500 °C, precisionof ± 2% and resolution of 0.1 °C was used to measure the skin temperature of theevaluated regions. To standardize the pressure exerted during ice application, asphygmomanometer was used , at pre-scheduled morning appointments, and data wascollected in a climate-controlled room (23 °C±2 °C). The data were collected beforeice application (Pre), immediately after the ice was removed (T0) and 30 minutesafter the ice was removed (T30).The ice was applied using a plastic bag containing 1.5 kg of crushed ice in directcontact with the skin of the subject. To apply the ice, the subjects were seatedThe task evaluated in this study was gait initiation. For this purpose, thevolunteers remained in the initial position  and were instructed to perform thefirst step with the non-dominant limb on the force plate. Dominance was assessedthrough the task of kicking a ball. The data collected for this part of the studyrefers to the initial step performed unilaterally with the non-dominant limb. Toperform the step, the force plate was fixed between two wooden platforms of the sameheight and width at the same level. The volunteers were instructed to perform thestep and walk as naturally as possible; therefore, the speed was not standardized.After the tests, subjects remained seated with the heel resting on a wooden surfaceto prevent heat exchange.The anteroposterior (Fap) and vertical components (Fv) of GRF were analyzed in thisstudy. The GRF values were normalized by subject body weight (BW). Afternormalization, the following variables were calculated in the vertical direction: peak vaBased on the GRF, the durations of the following gait phases were also calculated:stance phase (SD), braking phase (BD) and propulsion phase (PD). The SD wascalculated as the interval between contact and removal of the foot from the forceplate. The BD was calculated as the interval between contact of the foot with theforce plate and the instant that the Fap crossed the zero axis. The PD was measuredas the interval between the instant that the Fap crossed the zero axis and removal ofthe foot from the ground.After tabulating the results, a Shapiro-Wilk normality test was applied to allvariables. For variables with normal distributions, an ANOVA test for repeatedmeasures and Tukey's test were applied. For data that were not normally distributed,a Kruskal-Wallis test and Dunn's test were utilized. The data were processed usingSPSS 20.0 software  and significance was set at a levelof 5% (p<0.05).Power was calculated by considering the vertical force with ice applied to the calf,and 95% power for T0 and 77% for T30 were obtained.The participants had the following characteristics: age of 21.7±1.9 years, body massof 57.9±7.1 kg, height of 1.65±0.07 m and body mass index (BMI) of 21.4±2.1 kg/mA significant reduction in skin temperature was observed immediately following icepack removal (T0) to the three target areas . TemperaIce application did not affect the durations of the gait phases . OverallThe application of ice to the sole and calf increased PVF1 immediately followingapplication . The samThe application of ice to the ankle reduced the propulsive impulse in the APdirection at T30 compared to Pre and T0 .,Although skin temperature poorly predicts intramuscular temperatureAn important result of this study was the observed increase in PVF1 immediatelyfollowing ice application (T0) to the sole of the foot and calf. This result suggestsincreased overload on the musculoskeletal system immediately after foot contact with theground. This overload can be partly explained by the fact that decreased temperaturesaffect neuromuscular system functioning, and both afferent and efferent nerve impulsesmight have slowed after coolingSimilarly, cooling the sole of the foot with ice has been previously described asresponsible for increased weight transfer rates following foot contact with the groundat the end of the gait cycleHowever, 30 minutes after the ice was removed from the sole of the foot and calf, PVF1values returned to baseline levels despite that temperatures remained belowpre-application levels. This result can be explained by the fact that decreased afferentimpulses following cooling are quickly compensated by the central nervous systemThe results indicate a reduction of the PVF2 peak after 30 minutes of ice application tothe ankle and calf. This result suggests that even with reduced skin temperatures, theadaptations in the central nervous system allowed learning of the gait initiation task.The same pattern was not observed when ice was applied to the sole of the foot, whichsuggests the inability of the central nervous system to learn the required task due tothe sensory losses caused by decreased afferent impulses from the plantarmechanoreceptors.,No changes in the variables studied were observed at time T0 following ice applicationto the ankle suggests that sensory information from this joint was not affected byreduced skin temperature. Although Hopper et al.33.The reduction of ankle temperature might not change the GRF at T0 due to compensationfrom afferent impulses from mechanoreceptors of other regions. This hypothesis has beensupported by observations that reduction of shoulder temperature does not change jointposition senseAt time T30, however, the application of ice to different body regions affectedpropulsion and braking values in the AP direction (BrF and PrF) and propulsive impulses.However, we can attribute these changes to learning because they were only observedduring the last repetition (T30) when the temperature, although inferior topre-application levels, does not seem to directly influence the functioning of thesensorimotor system.Regarding the limitations of this study, the fact that direct measurements of theproprioceptive system were not performed, the absence of measurement of other variablesand the assessment of other tasks do not allow conclusive statements about the effectsof ice application on the proprioceptive system. However, the changes observed in thisstudy and in the extant literature allow us to state that in the situations previouslydescribed, ice application was sufficient to cause sensorimotor changes during the gaitstance phase. Although the task chosen - gait initiation - might limit the applicationof these results to other activities that require greater neuromuscular control , in rehabilitation, one of the mostimportant tasks is the reestablishment of natural gait. Thus, clarifying whether iceapplication, which is commonly used in clinical practice for ankle and calf injuries,can change gait is of considerable importance in rehabilitation settings.Cryotherapy predominately affected the load applied to the body after foot contact withthe ground, particularly after ice application to the sole of the foot and calf insedentary young adult women. These results suggest that although there is the need forcaution when performing activities that require good gait control, ice application tothe ankle or at a 30-minute interval after ice has been removed can be safe, even whenpreceding such activities.


VARP is a Rab32/38 effector that also binds to the endosomal/lysosomal R-SNARE VAMP7. VARP binding regulates VAMP7 participation in SNARE complex formation and can therefore influence VAMP7-mediated membrane fusion events. Mutant versions of VARP that cannot bind Rab32:GTP, designed on the basis of the VARP ankyrin repeat/Rab32:GTP complex structure described here, unexpectedly retain endosomal localization, showing that VARP recruitment is not dependent on Rab32 binding. We show that recruitment of VARP to the endosomal membrane is mediated by its direct interaction with VPS29, a subunit of the retromer complex, which is involved in trafficking from endosomes to the TGN and the cell surface. Transport of GLUT1 from endosomes to the cell surface requires VARP, VPS29, and VAMP7 and depends on the direct interaction between VPS29 and VARP. Finally, we propose that endocytic cycling of VAMP7 depends on its interaction with VARP and, consequently, also on retromer. •VARP recruitment to endosomes does not depend on its interaction with Rab32:GTP•VARP contains two Zinc-binding CHPLCxCxxC motifs that bind the retromer subunit VPS29•VARP is recruited to endosomes through its interaction with VPS29•VARP, retromer, and VAMP7 are all involved in trafficking of GLUT1 to the cell surface The VAMP7-binding Rab32-effector and Rab21GEF VARP binds directly to the retromer subunit VPS29. VARP’s interaction with VPS29 recruits it on to endosomes, thereby linking the cargo sorting retromer complex with an R-SNARE involved in endosomal function. All three are shown to be involved in endosome to cell surface transport. D. melanogaster and C. elegans), as well as in some species of unicellular organisms closely related to animals, including Capsaspora and Choanoflagellates. VARP is a Rab32/38 effector, and, in line with other Rab:Rab-effector systems, it has been assumed that Rab32 would be primarily responsible for recruiting VARP on to membranes (VARP (VPS9-domain ankyrin repeat protein) is a widely expressed, multidomain protein of 1,050 residues present in most animals  folds over the nucleotide molecule and stabilizes the γ-phosphate of the GppCp. Switch-II (residues 84–97) also stabilizes the γ-phosphate but differs somewhat in conformation as compared to those in other GTP-bound Rab structures  and a third one through the interswitch of the GTPase (K546VARP-D61Rab32). The residues involved in these interactions are conserved in other organisms , as did the double mutant Q509A/Y550A, but the L513D/K546D mutant was unstable in the constantly stirring environment of the ITC cell.Four residues within VARP that contribute to the complex interface, L513or Q509A B. The L5Mutating residues M91/R93 in Switch-II of Rab32, which participate in the interaction with VARP D, result2 of solvent accessible surface area, making the total solvent accessible buried surface area on tetramer formation around 6,400Å2, which suggests that the tetramer should be stable. Existence of a dimer of heterodimers in solution is supported by analytical gel filtration, which is consistent with the existence of a concentration-dependent equilibrium of a 2:2 tetramer and a 1:1 heterotetramer in solution  confocal microscopy. WT-VARP-GFP exhibited robust colocalization with the endogenous retromer subunit VPS26 on both vacuolar and tubular domains of endosomes C and 3D.We used a combination of yeast 2-hybrid (Y2H) analysis, GST pull-downs with bacterially expressed proteins, and in vivo siRNA knockdown and rescue experiments to identify the retromer subunit VPS29 as the direct binding partner of VARP. By Y2H, strains expressing both VARP and VPS29 exhibited growth, but no growth was observed when VARP was coexpressed with retromer subunits VPS26A, VPS26B, or VPS35 A. In agrDs in the low micromolar range , which separate the first two ankyrin repeats, each contain a cysteine-rich consensus motif with the sequence CHPLCxCxxC that is conserved across all species A. Each r2+ in both Cys-rich motifs and that no other nonorganic elements are present at levels above 0.02 atoms per motif. Attempts to abrogate the interaction between VARP and VPS29 in vitro by adding EDTA in an effort to chelate the bound Zn2+ failed, presumably because of the difficulty of extracting tightly bound Zn2+, as has been previously observed for other “double Zn-finger”-containing proteins .The presence of a spatially conserved pattern of cysteines in an intracellular protein suggests the involvement of a metal: most likely, zinc. Consequently, elemental analysis by microbeam proton-induced X-ray emission (microPIXE)  was carrD affinity , or of the conserved His and Leu residues to Ser and Ala (HL1 or HL2), in the context of ∼40-residue fragments containing the first or the second Cys-rich motifs, respectively, abolished binding to VPS29 by Y2H C. In ITCaffinity , the mosWe therefore conclude that VARP is recruited on to endosomes through the direct interaction of its Cys-rich motifs with VPS29, thus placing its ability to bind and regulate the fusogenic potential of free VAMP7, and to interact with Rab32:GTP, on the surface of endosomes. In agreement with this model, GST pull-down experiments F showed Recent studies have suggested that a number of cell surface receptors and transporters recycle from endosomes to the plasma membrane by a retromer-dependent pathway . In lighTaken together, our data demonstrate that VARP and VAMP7 act within the retromer-dependent recycling pathway that traffics the glucose transporter GLUT1 from endosomes to the plasma membrane. Our rescue experiments show that the VPS29 requirement for this pathway is dependent on its binding to VARP. Given the role of retromer in mediating traffic out of endosomes, and the knowledge that VARP binds the retromer subunit VPS29 and VAMP7 at different binding sites, we decided to study further whether the intracellular distribution of VAMP7 is affected by its association with VARP/retromer. Stable HeLa cell lines expressing WT VAMP7-HA or the D69A, E71F, S73D (DES) mutant VAMP7-HA, which we have previously shown cannot bind VARP , were exRetromer drives trafficking from endosomes/endolysosomes via tubular-vesicular carriers to either the Golgi or the cell surface . Here, wThe current working model for retromer function suggests that, in mammalian cells, the VPS26/35/29 subcomplex associates with endosomal membranes via Rab7A  and Snx3VAMP7 mediates fusion of both endosomes and endolysosomes with lysosomes and fusion of endolysosomes and their derived carriers with the plasma membrane  and its associated proteins (including sorting nexins and the WASH complex) to both a key ancestral R-SNARE protein (VAMP7)  involvedE. coli Rosetta 2 pLysS. Cells were grown at 37°C, and expression was induced by the addition of 500 μM IPTG. VARP expression was carried out at 16°C for 20–24 hr, while all other proteins were expressed at 22°C for 14–18 hr. Cells were lysed in 20 mM HEPES, pH 7.4, and 500 mM NaCl (buffer A), supplemented with 5 mM MgCl2, 1 mM MnCl2, 5 mM dithiothreitol (DTT), 4 μg/ml DNase, and protease inhibitors and AEBSF. Seleno-methionine proteins were expressed in E. coli B834 using M9 media supplemented as in Protein expression was performed in 451–640His6 was further purified on Ni-NTA agarose. All proteins were purified by gel filtration on Superdex 75 in buffer A supplemented with 10 mM DTT and additionally with 5 mM MgCl2 and 10 μM GppCp or GTPγS in the case of Rab32/38. Complexes were formed by mixing VARP with a GTPase in a molar excess of 1:1.25 for 1 hr at 4°C and finally subjecting them to gel filtration on Superdex 75 equilibrated in buffer A containing 5 mM MgCl2, 10 μM GppCp or GTPγS, and 10 mM DTT. Fractions of 1:1 complex as assessed by SDS-PAGE were pooled and concentrated for crystallization. Proteins for ITC were gel filtered on Superdex 75 in 50 mM HEPES, pH 7.4, 200 mM NaCl, 5 mM MgCl2, 1 mM TCEP.Proteins were first purified on GSH-sepharose, and the GST tag was cleaved off by incubating the beads with PreScission Protease  overnight at 4°C. His-tagged VARP1–1050His10 WT and mutants were purified in 500 mM NaCl, 20mM Tris pH8.5, (buffer B) as in (2 and 10 μM of matching nucleotide.VARPB) as in . GST-tag451–640His6: Rab321–225(Q85L) were grown by sitting drop vapor diffusion at 18°C against well solutions containing 14%–19% (w/v) of polyethylene glycol 3350 and 200 mM sodium citrate, pH 8.0, at an initial complex concentration of 2.5 mg/ml. Cryoprotected crystals exhibited anisotropic diffraction at 100 K and diffracted in the best directions to 2.8 Å. The structure was solved by molecular replacement with Phaser (1YHN) and the VARP-ANKR domain 2 structure (PDB ID 4B93). Crystals of VARP-ANKR domain 1:SeMet- Rab321–225(Q85L/V100M/Q153M/V158M/I192M) and of VARP-ANKR domain 1:SeMet-Rab38 (Q69L) diffracted in their best directions to 3 Å and 4.5 Å, respectively. Their structures were solved by molecular replacement using the coordinates of native VARP-ANKR domain 1:Rab32Q85L complex as the search model. For a full description of structure determination, see Crystals of VARPh Phaser , using m2, 0.5mM TCEP, and 100 μM of the matching nucleotide was included if a Rab was present and 0.5mM ZnCl2 if a Cys-rich motif was present). The reactions were incubated for 1 hr at 4°C, and the beads were washed four times with 1 ml of buffer supplemented with 1% (w/v) NP40. Proteins were eluted from the beads using buffer containing 50 mM reduced GSH. Samples were analyzed on SDS-PAGE and/or western blots, which were probed with HisProbe-horseradish peroxidase conjugate or appropriate antibodies.Y2H for screening and analyzing specific interactions was performed using pGBT9 and pGAD-C vectors and the HF7c reporter strain as previously described . Growth D of the binding reaction, its stoichiometry, and their corresponding SD values. Analysis of results and final figures were carried out using the NanoAnalyze Software.Experiments were performed using a Nano ITC from TA Instruments. VARP constructs at 60 μM were placed in the cell at 4°C, and Rab constructs at 300 μM were titrated with 24 injections of 2 μl with injections separated by 5 min. A minimum of three independent runs that showed clear saturation of binding were used to calculate the mean KSamples were gel filtered at 10 mg/ml into 20 mM Tris, pH 7.4, 100 mM NaBr, and 0.5 mM TCEP to ensure low chlorine content and no nonprotein sulfur. The resulting spectra of X-rays were sorted into two-dimensional elemental maps from which the stoichiometric ratios of sulfur to other elements of interest were obtained.HeLaM cells stably expressing VPS29-EGFP or EGFP-VPS35 were lysed using PBS containing 1% Triton X-100. Following lysis, the samples were cleared by centrifugation at 10,000 × g for 5 min. The supernatant was transferred to a fresh tube and treated with protein-A sepharose (50 μl of a 25% slurry) for 30 min at 4°C on a rotating wheel. The lysates were then cleared again by centrifugation before being treated with a polyclonal anti-GFP antibody followed by protein-A sepharose essentially as described in 2+ and Mg2+) containing 0.05% saponin, and then immediately formaldehyde fixed. IF confocal microscopy and western blotting were carried out essentially as described elsewhere . When indicated, HeLaM cytosol was extracted prior to fixation by rinsing cells briefly with PBS, followed by incubation for 1 min in PBS (with Calsewhere . For quaficients  using ZeI.P.-D., I.B.S., G.G.H., A.J.M., and P.R.E. carried out work on the Rab32:VARP complex. G.G.H., L.P.J., L.W., I.B.S., S.R.G., M.E.H., M.N.J.S., and D.J.O. carried out work on VARP:Retromer. O.B.Z. and E.F.G. carried out the microPIXE analysis. J.P.L., M.N.J.S., and D.J.O. conceived and initiated the study. All authors contributed to the design of the study and the writing of the manuscript.


Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.—Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction. It has been estimated that nearly 800,000 hospital patients per year require controlled mechanical ventilation (CMV), and its use is especially prevalent in intensive care units (ICUs), where 33–40% of the patients require CMV for ∼6–7 d –3. Altho–4–Diaphragmatic atrophy develops with surprising rapidity during CMV, as it can be observed in as little as 16–72 h in humans and 12 h in rodent models , 10. Des13–13–To improve our understanding of the early signaling events that trigger CMV-induced diaphragm muscle dysfunction, we used a broad signaling assessment in diaphragm muscle fibers from mechanically ventilated rats. This effort led to the discovery of an upstream Janus kinase (JAK)/STAT3 signaling axis that triggers the accumulation of phosphorylated STAT3 within mitochondria and coincides with the mitochondrial dysfunction, muscle fiber atrophy, and decreased contractility that occur in the diaphragm during prolonged CMV.Animal experiments were approved by the Institutional Animal Care and Use Committees of Rigel Pharmaceuticals, Inc., or the University of Florida and were in compliance with the Public Health Service Guidelines for the Care and Use of Laboratory Animals.n=10) or to a group mechanically ventilated for 18 h (n=20). In a second series of experiments, diaphragm muscles were harvested from unfed control (n=5–6) and mechanically ventilated rats (n=5–6) at different time points  and immediately frozen in liquid nitrogen and stored at −80°C until use in biochemical and/or molecular biology assays. In a third series of experiments, the animals were randomly assigned to 1 of 3 groups (n=20/group): unfed, conscious control; 18 h mechanically ventilated and treated with a Rigel JAK1/JAK3 inhibitor (1 mg/kg/h R548); or 18 h mechanically ventilated and treated with propylene glycol (vehicle for R548). Vehicle and JAK inhibitor were delivered via venous cannula and were infused continuously during the 18 h CMV experiment. Isoflurane was used as the anesthetic. The Institutional Animal Care and Use Committee of Rigel Pharmaceuticals, Inc., approved the experiments.The JAK1/JAK3 inhibitor used in these experiments was developed as a potential therapeutic for transplant rejection, arthritis, and other systemic immune disorders in which JAK signaling plays a prominent role. Four series of experiments were conducted, and the mean mortality rate of mechanically ventilated rats was 17%. In the first 3 series of experiments, CMV studies were performed in male Sprague-Dawley rats weighing 260–280 g. Carotid artery and jugular vein cannula surgeries were performed by Harlan , and the rats were shipped after a 72-h recovery period. The rats were acclimated for ∼7 d before they were randomly assigned into groups by body weight. In the first series of experiments, the rats were assigned to an unfed and conscious control group  randomly assigned to 1 of 4 experimental groups: 12 h anesthetized, spontaneously breathing, and unfed control, treated with propylene glycol ; 12 h anesthetized, spontaneously breathing, unfed control, treated with R548  delivered at 3 mg/kg/h; 12 h of CMV treated with propylene glycol ; or 12 h of CMV treated with R548  delivered at 3 mg/kg/h. Pentobarbital was used as the anesthetic. Pharmacokinetics studies had indicated that anesthetizing rats with pentobarbital results in significantly reduced plasma levels of the active form of R548, R507. Therefore, a dose of 3 mg/kg/h R548 was used. The University of Florida Institutional Animal Care and Use Committee approved the experiments.An autonomous research laboratory with a well-established preclinical model of CMV conducted a fourth series of experiments to independently determine the efficacy of JAK/STAT inhibition on CMV-induced diaphragm wasting and contractile dysfunction. Despite differences in the sex of the rats and the duration of mechanical ventilation, this model consistently leads to diaphragm atrophy and contractile dysfunction –15. Thes–via subcutaneous administration of 1.7 ml/kg body weight/2.5 h saline. To reduce airway secretions, glycopyrrolate (0.04 mg/kg) was administered subcutaneously every 2.5 h.All surgical procedures were performed with aseptic techniques. In the first 3 series of experiments, tracheostomy surgeries and CMV were conducted as described previously –15, with–In the fourth series of experiments, tracheostomy surgeries and CMV were conducted as described in detail recently –15. Rats–ex vivo , and immersed in Krebs-Henseleit physiological solution aerated with 95% O2–5% CO2 gas. After a 15-min equilibration, the diaphragm strips were stimulated with a S88X Pulse Stimulator  and platinum wire electrodes . Contractile measurements were taken at the optimal muscle length (Lo), the length at which maximal force is obtained. Lo was determined by stimulating the muscle at a supramaximal voltage and systematically adjusting the muscle length. The force–frequency relationship was assessed by stimulating each strip supramaximally with 8 V pulses, with a train duration of 500 ms, at 15–160 Hz. Contractions were separated by a 2 min recovery period. Diaphragm force production was normalized as specific force based on muscle CSA. Total muscle CSA at right angles to the long axis was determined by the following calculation: total muscle CSA (mm2) = [muscle mass/(fiber length × 1.056)], where 1.056 is muscle density (g/cm3). Fiber length (cm) was measured at Lo. Diaphragm contractility was assessed in all experiments, except for the time course studies.Diaphragm contractile function was determined by using diaphragm strips incubated ex vivo , 13. BriSOCS3, Myf5, and interleukin 6 (IL-6) mRNA levels were analyzed in samples from the first experiment, by using the QuantiGene Plex 2.0 assay  according to the manufacturer's instructions. Briefly, muscles were homogenized in Affymetrix proprietary homogenization buffer with proteinase K , incubated at 65°C for 30 min, and centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was collected for subsequent RNA quantification. Lysates where transferred to a 96-well capture plate with target-specific probe sets, and the plate was sealed and incubated overnight at 55°C for hybridization. Next, the signals of the selectively captured target RNAs were amplified via sequential wash and hybridization steps, followed by the addition of the streptavidin-conjugated R-phycoerythrin, which emits a fluorescent signal that is proportional to the amount of target mRNA present in the sample. mRNA concentrations were normalized to the geometric average of 4 internal control genes  and expressed as median fluorescence intensity (MFI). Atrogin-1 and muscle RING-finger 1 (MuRF1) mRNA expression was measured in samples from experiment 4, with predesigned rat primer and probe sequences commercially available from Applied Biosystems , caspase-3 , calpain 1 , and α-spectrin . Atrogin-1 (AP2041) and MuRF1 (MP3401) were from ECM Biosciences . Blots were developed with an ECL Plus enhanced-chemiluminescence reagent kit obtained from Amersham Pharmacia Biotech  and detected on Kodak Bio-MAX MR film . Tubulin was used as a loading control, to confirm standardized protein loading and transfer. The bands were analyzed using the public domain ImageJ program , quantified by densitometry, and normalized to the appropriate loading controls.Total protein extracts from frozen muscle samples were prepared by homogenizing muscle tissues in the Precellys24 Tissue Homogenizer  in ice-cold RIPA buffer composed of 10 mM Tris (pH 7.2), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA, and complete protease/phosphatase inhibitor cocktail . Tissue lysates were then centrifuged on a microcentrifuge , the protein content of the supernatant  was determined with a Micro-BCA protein estimation kit , and the samples were subjected to protein normalization. The samples were then boiled in 4× NuPAGE sample buffer containing LDS/DTT, and equal aliquots (10–20 μg protein extract) were subjected to SDS-PAGE separation on premade 4–12% Bis-Tris gradient gels, with MOPS/SDS used as a running buffer system, per the manufacturer's recommendations . Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 , STAT3 , pSTAT3-S727 , phospho-p38 , myogenin , Ac-Histone H3 , tubulin , GRIM-19 , 4-hydroxynonenal , lactate dehydrogenase A , proliferating cell nuclear antigen , voltage-dependent anion channel , cytochrome Protein carbonyl levels were assessed in diaphragm muscle by using the OxiSelect Protein Carbonyl ELISA Kit , according to the manufacturer's instructions.2 EDTA tubes and centrifuged at 4000 rpm for 5 min at 4°C, and the supernatant (plasma) was collected and stored at −20°C for subsequent analysis. Plasma samples were incubated with a cocktail of beads coated with capture antibodies to specific cytokines and washed several times. A secondary biotinylated detection antibody that bound to the cytokine captured on the bead was incubated with the samples. The reaction mixture was then incubated with streptavidin-penicillin conjugate to complete the reaction on the surface of the microbead. The microbeads were then passed through a laser that excites the internal dyes marking the microbead set, a second laser that excites the reporter molecule on the surface of the bead, and high-speed digital signal processor that identify each microbead and quantify the results.Plasma IL-6 levels were determined in samples from the third series of experiments, by using the Luminex Rat Cytokine Kit , according to the manufacturer's instructions. Briefly, whole blood was collected in Kg for 20 min to remove nonsoluble proteins. The supernatant was collected, assessed for protein concentration, and standardized.Diaphragm and lung IL-6 levels were assessed in samples from the third series of experiments with an IL-6 immunoassay kit (R&D Systems). Briefly, samples were homogenized in RIPA buffer containing PhosStop Phosphatase Inhibitor Cocktail (Roche Diagnostics) and homogenization at 4°C in a PreCellys 24 Bead Homogenizer (Bertin Corp.), according to the manufacturer's instructions. The samples were then centrifuged at 13,000 Diaphragm strips from the third series of experiments were used for histologic assessment of CSA. Because diaphragm samples from that series of experiments were damaged during cold storage and subsequent tissue processing, CSA was instead assessed in samples from a previous study that used a related JAK inhibitor from the same chemical scaffold series (R545). In that study, STAT3 phosphorylation, contractile dysfunction, and rescue by treatment with the JAK inhibitor (R545) were nearly identical to results in the studies of R548. Samples from this experiment were frozen in isopentane cooled by liquid nitrogen and stored at −80°C. Frozen tissue was sectioned at 6 μm and mounted on slides, fixed in pure acetone at −20°C for 10 min, and stained with anti-laminin antibody  at 1:200 for 30 min in PBS with 5% BSA. Alexa Fluor 568 anti-rabbit secondary antibody  was applied at 1:200 for 30 min. Slides were digitally imaged with a ×4 objective to capture each entire tissue section with an IXMicro fluorescence microscope .http://cellprofiler.org). Images were analyzed separately, not as a single montage of the entire section. Thresholding was performed by using Otsu's method, minimizing weighted variance with 3 classes. The middle class was assigned to background. Morphologic techniques were applied to provide outlines for each fiber. Images with poor or incomplete thresholding were discarded from the analysis by manual inspection. Multiple image fields were usually necessary for complete coverage of the tissue sections. All image results for each section were summed, and the results for each section were averaged across sections for each animal. Muscle fiber size is reported in square micrometers.Image analysis was performed with CellProfiler . The muscle was trimmed of connective tissue and cut down to fiber bundles (4–8 mg wet weight). With the aid of a microscope and using a pair of extra-sharp forceps, we separated the muscle fibers gently in ice-cold buffer X, to maximize the surface area of the fiber bundle. To permeabilize the myofibers, we incubated each fiber bundle in ice-cold buffer X containing 50 μg/ml saponin on a rotator for 30 min at 4°C. The permeabilized bundles were then washed in buffer Z .Samples from the fourth study were permeabilized as reported recently , 19. BriRespiration was measured in samples from the fourth study polarographically in a respiration chamber maintained at 37°C  on a tabletop refrigerated microcentrifuge  to remove large particles. Low-speed centrifugation was repeated 2 times, and the clear supernatant obtained was then subjected to medium centrifugation . Both pellet (mitochondria) and the supernatant were separated carefully, and the pellet was washed ≥2 times with the washing buffer containing protease and phosphatase inhibitors, to ensure its purity. Finally, the mitochondrial pellet was dissolved in 100 to 150 μl RIPA buffer containing protease and phosphatase inhibitors. For Western blot analysis, aliquots from both the supernatant and the pellet were normalized for protein concentration by Bradford BCA assay and were loaded equally onto 4–12% Bis-Tris gels, followed by protein blotting onto PVDF membranes. VDAC served as a marker for mitochondria, and LDHA and PCNA served as markers for cytosol and nuclear fraction, respectively.All buffer reagents used for preparing mitochondria isolation buffer  and washing buffer were from Sigma-Aldrich. Mitochondria washing buffer is essentially the isolation buffer, but contains protease inhibitor cocktail (cat. no. 05892791001) and phosphatase inhibitor cocktail . RIPA buffer (cat. no. R3792) from Teknova , containing protease and phosphatase inhibitor cocktail, was used to dissolve the mitochondria pellet. All protein gels, PVDF membranes, and the Western blot system were obtained from Life Technologies, unless otherwise specified. For mitochondrial fractionation, frozen rat diaphragm tissues from the fourth series of experiments (∼100 mg wet weight) were placed in 2 ml tissue-homogenizing tubes containing 1 ml mitochondria isolation buffer and porcelain beads . The tissues were then homogenized carefully in a Precellys24 homogenizer (Bertin Corp.) at 4°C and 6000 rpm, programmed for 3 × 15 s spin with 20 s intermissions. Tissue homogenates were then immediately placed on ice for 10 min before low-speed centrifugation  and markedly increased activating phosphorylation of STAT3 (Tyr705) in the diaphragms of ventilated rats relative to nonventilated unfed controls . In addition, mRNA levels of downstream targets of JAK/STAT3 activation, such as SOCS3 and Myf5, were robustly upregulated in the diaphragm in response to CMV, lending further support to the presence of activated STAT3 in this muscle . We next examined whether the levels of total and phosphorylated STAT3 were altered at earlier time points after initiation of CMV. Diaphragm muscles were studied between 1 and 9 h after mechanical ventilation. Total STAT3 levels were not altered at any time point. STAT3 phosphorylation was unchanged after 1, 3, and 6 h, but tended to be increased after 9 h of CMV .Given the links between STAT3, muscle wasting , and facP=0.0143), as observed in either force–frequency or fatigue profiling of isolated diaphragm strips . Similar results were obtained with the JAK 1/3 inhibitor CP690550 (D). As expected, R548-mediated rescue was accompanied by significant reductions in phospho-STAT3Y705 levels within the diaphragm (E). A broad screening of diaphragm from mechanically ventilated rats, using Western blots, identified several additional proteins with notable induction patterns that were also modulated by treatment with R548, indicating that these were positioned downstream of JAK signaling and could contribute to diaphragm dysfunction (E). For example, partial attenuation of phosphorylated p38 (Thr180/Ser182) and myogenin expression was also observed, although these were reduced to a lesser extent than phospho-STAT3Y705, suggesting that at least some portion of the activation of these proteins lay downstream of JAK function (E). Since both myogenin expression and muscle atrophy have been linked to changes in the activity of HDAC proteins (E). Since mitochondrial damage is thought to play a central role in the development of ventilator-induced diaphragm dysfunction , accompanied by rescue of impaired coupling within mitochondria  and reductions in the level of ROS-mediated protein carbonyls (D) and 4-HNE-conjugated proteins (E), indicating that JAK inhibition reduces mitochondrial dysfunction and oxidative stress as part of its protective mechanism.Having demonstrated robust elevations in STAT3 activation during CMV, we hypothesized that signals transmitted through the JAK/STAT pathway are responsible for the development of diaphragm atrophy and dysfunction. To test this hypothesis directly, a second series of experiments was conducted in mechanically ventilated rats continuously infused with 1 mg/kg/h R548, the prodrug of a selective JAK1/JAK3 inhibitor, R507 . Whereas CMV did not influence atrogin-1 protein levels, MuRF1 protein levels were significantly increased in diaphragm muscles from CMV rats, and treatment with R548 successfully blocked this increase . CMV markedly increased the amount of active calpain-1 and caspase-3 , and this was accompanied by elevated levels of calpain-specific (145 kDa) and caspase-3-specific (120 kDa) spectrin breakdown products . Treatment with R548 partially attenuated protease activation and the accumulation of calpain- and caspase-3-cleaved spectrin (A–D). Collectively, these data suggest that JAK inhibition during CMV protected diaphragm from atrophy and dysfunction by blunting activation of the ubiquitin–proteasome, calpain, and caspase proteolytic systems.function , 15, 24.iaphragm A, B. Wheaspase-3 A, B, andaspase-3 A, B, andAFig. 6, CMV led to significant increases in the amount of phospho-STAT3S727, and this effect was prevented by inhibition of JAK signaling, indicating that these events were coupled, albeit indirectly, since JAKs are tyrosine kinases and are incapable of phosphorylating this site directly. Notably, the increased abundance of phospho-STAT3 (Ser705 and/or Tyr705) induced by CMV was accompanied by substantial increases in its accumulation within the mitochondria; this effect was also prevented by treatment with a JAK inhibitor . GRIM-19, a component of complex I, is critical for the import of phospho-STAT3S727 into mitochondria, and, similar to STAT3, its presence there has also been linked to increased ROS generation .Although STAT3 is best characterized by its role as a nuclear transcription factor, recent findings have demonstrated that Ser705-phosphorylated STAT3 is imported into mitochondria, where it associates with complex I of the electron transport chain (ETC) and modulates respiration –27. Sinc–neration . GRIM-19IL-6 gene expression in the diaphragm . However, IL-6 protein levels in the diaphragm were unchanged by mechanical ventilation (C), suggesting that an autocrine IL-6 signaling loop within the diaphragm does not contribute to elevated levels of plasma IL-6. Previously, ventilation-induced lung damage has been shown to result in IL-6 up-regulation locally within injured lung tissue, and we therefore investigated whether this mechanism also contributes to the observed increases in circulating IL-6 plasma levels. These experiments demonstrated that IL-6 protein levels were not increased in the lungs after 18 h of CMV, suggesting that ventilation-induced lung damage is unlikely to contribute to an increase in circulating IL-6 (D).IL-6 is a well characterized activator of JAK/STAT3 signaling, can be produced by muscle  or lung iaphragm A, B. Howiaphragm A, B. HowDespite the life-saving role played by CMV in modern medicine, one unintended consequence of its use is the rapid and progressive induction of contractile dysfunction and atrophy of the diaphragm. This CMV-induced diaphragmatic weakness is clinically important because it can substantially delay the weaning process and thereby significantly contribute to the increased cost of care, resource requirements, and medical risk for some patients who might otherwise be incrementally closer to recovery –12. Howe–The results described in this article and elsewhere  suggest We focused our efforts on JAK/STAT3 signaling for 2 reasons. First, changes in STAT3 protein levels within the diaphragm have been linked to prolonged CMV in humans , althoug25–The activation of STAT3 in the diaphragm during CMV was not limited to the canonical JAK target site at Tyr705, as robust increases in phosphorylation of Ser727 also occurred. These changes during CMV were associated with increased mitochondrial translocation of phosphorylated STAT3, suggesting a mechanistic link between activation of this pathway and the observed mitochondrial dysfunction. The import of STAT3 into mitochondria is mediated by its association with GRIM-19, a component of complex I, and depends critically on STAT3 Ser727 phosphorylation , 26. Thivia oxidant sensitive kinases or phosphatases , the absolute loss of muscle mass (atrophy) associated with CMV is also a likely contributor to the overall weakness that develops within the diaphragm. In the present study, inhibition of JAK/STAT3 signaling prevented approximately half of the atrophy associated with CMV, supporting the concept that muscle wasting during CMV is due, at least in part, to JAK signaling. Improved diaphragm mass may have been due to diminished activation of MuRF1, since CMV-mediated up-regulation of MuRF1 mRNA and protein levels were reduced by JAK/STAT3 inhibition. MuRF1 is an E3 ubiquitin ligase that marks specific contractile proteins for selective degradation by proteasome-dependent mechanisms during muscle atrophy and plays a key role in muscle wasting  is unchanged during CMV (data not shown), likely ruling out its involvement in STAT3S727 phosphorylation. CMV-induced activation (phosphorylation) of p38 was blunted by JAK inhibition, suggesting that p38 accounts for at least some portion of phospho-STAT3S727 up-regulation.Reflective of its diverse roles in normal biological and pathophysiological processes, numerous serine kinases signal through STAT3 ing mTOR , PKC . Since the extended weaning period caused by VIDD contributes to the clinical risk and cost burden for a large number of ICU patients, inhibition of JAK/STAT3 signaling provides a promising therapeutic strategy for addressing this clinically important condition.In summary, the findings presented in this article implicate JAK/STAT3 signaling in the development of diaphragm atrophy, mitochondrial dysfunction, and weakness during CMV (


IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK , Akt and mTOR , but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the  In the present paper, we report for the first time that GDF15 increases Kv2.1 expression through TGFβRII to activate the Akt/mTOR pathway, which reveals an unanticipated role of the non-Smad-dependent pathway in the previously uncharacterized signalling mediated by GDF15. GDF15 , is a novel member of the TGFβ (transforming growth factor β) superfamily that was discovered in a screen for the conserved consensus sequences of TGFβs . GDF15 hin vitro and in vivo for embryonic and lesioned dopaminergic neurons in the midbrain and the substantia nigra and is dramatically up-regulated in cryolesioned cortical neurons  on ice for 30 min. After centrifugation (12000 g for 15 min at 4°C), the supernatant was mixed with 2× SDS loading buffer and boiled for 5 min. The proteins were separated by SDS/PAGE (10% gel) and were then transferred on to a PVDF membranes (Millipore). The membrane was blocked with 10% (w/v) non-fat dried skimmed milk powder and incubated at 4°C overnight with the following primary antibodies: mouse monoclonal antibody against Kv2.1 or Kv2.2 , rabbit polyclonal antibody against phosphorylated insulin receptor β subunit , rabbit monoclonal antibody against phosphorylated ERK1/2 or total ERK1/2 , rabbit monoclonal antibody against phosphorylated Akt , rabbit polyclonal antibody against phosphorylated mTOR , rabbit polyclonal antibody against TGFβR2 , mouse monoclonal antibody against phosphorylated tyrosine , or mouse monoclonal antibody against GAPDH . After extensive washing in TBST (TBS with 0.03% Tween 20), the membrane was incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG  for 2 h at room temperature. Chemiluminescent signals were generated using a SuperSignal West Pico trial kit (Pierce) and were detected by exposure to X-ray film or using the ChemiDoc XRS System (Bio-Rad Laboratories). The Quantity One software  was used for background subtraction and for the quantification of the immunoblot data.The cells were lysed in Hepes/NP40 (Nonidet P40) lysis buffer  hexanoate (Thermo Fisher Scientific) for 45 min on ice and were rinsed in ice-cold PBS containing 1 M glycine to quench the biotin reaction. The cultured neurons were lysed and then homogenized in modified radioimmune-precipitation assay buffer . The homogenates were centrifuged at 10000 g for 15 min at 4°C, and the supernatants were collected. The supernatants were incubated with 50 μl of NeutrAvidin–agarose (Thermo Fisher Scientific) overnight at 4°C and washed three times with radioimmune-precipitation assay buffer. The total and biotinylated surface proteins were detected using quantitative Western blots as described above.Surface biotinylation experiments were performed in cultured rat granule neurons . TTX, 4-AP, DRB , CHX (cycloheximide), U0126, rapamycin, LY364947, PP2 and poly-+ currents IK and IA. GDF15 treatment for 24 h failed to modulate the IA in CGNs. Thus we examined the effect of GDF15 on IK in the present study. IK was evoked by a 100 ms depolarization to +50 mV from a holding potential of −50 mV in the presence of 5 mM 4-AP, which suppresses the IA and permits better resolution of the IK. Incubation of the CGNs with GDF15 significantly enhanced the IK densities in a concentration-dependent manner. The data obtained from 94 neurons showed that incubation of the CGNs with 10, 100 and 300 ng/ml GDF15 for 24 h increased the current densities by −2.2% (n=27), 37.7%  and 45.6% (n=26) respectively , 14.3% (n=25) and 1.2% (n=28) respectively , where GK is the membrane K+ conductance, Vm is the membrane potential and Vrev is the reversal potential for K+. The G–V curve analysis indicated that GDF15 did not alter the half-maximal activation voltage of the IK (n=17) and GDF15-treated  neurons.We then examined whether the gating properties of the channels were altered, leading to the enhancement of the f the IK A betweenIK, currents were elicited using 10 s conditioning pre-pulses from −90 mV to +10 mV before a 400 ms test pulse of +10 mV. After normalizing each current amplitude to the maximal current amplitude obtained from the −90 mV pre-pulse as a function of the conditioning pre-pulse potential and after fitting the normalization with the function IK/IK-max=1/(1+exp[(Vm1/2−Vm)/k]), we obtained an inactivation curve for the IK and calculated the voltage at which the current amplitude was half-inactivated (Vh50). The half-maximal deactivation voltage of the IK in the control neurons  was similar to that in the GDF15-treated neurons   B. These IK amplitudes might be due to the up-regulation of channel expression levels. The primary α-subunits mediating the IK in CGNs are Kv2.1 and Kv2.2 [n=4). The levels of transferrin 2, a subtype of transferrin, in the membrane fractions were not affected. Additionally, the kinetics of Kv2.1 expression induction paralleled the increase in the IK amplitudes following GDF15 treatment  or 10 μM leupeptin (a lysosome inhibitor) for 30 min before treatment with GDF15. MG-132 alone did not mimic the GDF15-induced increase in the IK amplitude after incubation for 24 h, and it actually augmented the levels of total cellular Kv2.1 protein, similar to GDF15  is responsible for degrading most of the intracellular soluble proteins, and the lysosomes degrade most membrane and endocytosed proteins. Consequently, we explored whether these two pathways assisted GDF15 in regulating the to GDF15 B–5D. Com2448 (p-mTOR) began to increase at 30 min (146.4±5.8%) and then continued to increase to 228.7±3.6% by 24 h. For Akt phosphorylated at Ser473 (p-Akt), its phosphorylation level significantly changed by 26.9±1.6% after 15 min, and phosphorylation reached its peak after 3 h (n=4)  or from 36.4% to −17.6%  respectively. Similarly, LY294002 and rapamycin decreased the expression levels of the Kv2.1 α-subunit protein (n=4) and −47.3±2.2% (n=4) respectively. These data indicate that the mTOR pathway is also required for the GDF15-stimulated up-regulation of the IK density and increase in Kv2.1 α-subunit expression.Because our data indicated that GDF15 increased Kv2.1 protein synthesis and prevented its degradation, we first tried to determine whether the Akt/mTOR pathway, which has been reported to affect protein synthesis and CGN mutation ,23, was  h (n=4) A–6C. The reduced D from 37 protein E to 0.9±n=3) respectively at 60 min  pathway. After GDF15 treatment, the phosphorylation levels of ERK1 and ERK2 (p-ERK1 and p-ERK2) were significantly increased, reaching the maximal levels of 225.7% and 235.9%  receptor and p-CREB (cAMP-response-element-binding protein), two typical markers of CGN development, were also enhanced by GDF15 treatment (results not shown). Taken together, our observations suggest that the effect of GDF15 on Kv2.1 expression may be developmentally regulated and associated with neuronal maturation. Because multiple factors are associated with the developmental regulation of CGNs, further investigations are needed to confirm this speculation.The s system . Previoumulation ,15. Indee period . We obseto 7 DIC D and 3A,DIC CGNs D and 3A.IA densities, which is also associated with the development and maturation of CGNs [IK  activity [IK densities and Kv2.1 expression was similar to that caused by TGFβ1, the mechanism and biological significance seem quite different. We observed that the IK densities and Kv2.1 expression induced by TGFβ1 are sensitive to the narrow time-window of 7–8 DIC, in which the IK properties are close to those of mature CGNs [IK density and Kv2.1 expression only occur at 4–5 DIC (IK density, and this activity was associated with the cAMP/PKA pathway rather than with the Akt/mTOR pathway. These differences between TGFβ1 and GDF15 in the temporally co-ordinated manner and post-receptor signalling of Kv2.1 expression might be related to the cell surface expression profiles of both TGFβR1 and TGFβR2 or to distinct oligomeric interactions that occur during signalling. These observations are in accordance with the hypothesis postulated by Ehrlich et al. [Our previous study reported that TGFβ1 increases the activity . Howeverure CGNs , but the 4–5 DIC , when thh et al. ,52, who IK densities by increasing the expression of Kv2.1 in CGNs. The effect of GDF15 on Kv2.1 expression might be channelled through TGFβR2 to activate the Akt/mTOR pathway. Thus the GDF15–TGFβR2–Kv2.1–IK axis may play an important role in neuronal development and the maturation of CGNs. However, due to technical limitations, certain interesting questions, such as how phosphorylated TGFβR2 activates the Akt/mTOR pathway and why the time-window of GDF15 responsiveness is extremely narrow, cannot be completely explained in the present study. The present study could serve as a foundation to guide further in-depth studies of GDF15 function and mechanism.In conclusion, we showed that GDF15 potentiates the