We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring  when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography,  However, describing the conformational changes of proteins within these complexes, especially when the proteins are in their native cellular context, has proven challenging. In this paper, we establish a technique to visualize the orientations of domains of proteins The scaffold of the NPC is a cylindrical channel composed of proteins that form an eight-spoked core with an axis perpendicular to the nuclear envelope . NPCs arin vivo. Using polarized-total internal reflection fluorescence microscopy (pol-TIRFM), we monitored the orientation of mEGFP-based sensors incorporated into different domains of individual NPCs in living cells. Previously, to measure the organization of various Nups with respect to the NPC we used fluorescence anisotropy. We determined the organization of the Y-shaped complex with respect to the NPC , where Θ is the angle between the excitation light and the excitation dipole. To monitor the orientation of the mEGFP, we excited the fluorophore sequentially with light polarized in two orthogonal directions. To restrict excitation to the basal surface of the cell we created the two polarized fields using excitation by total internal reflection (TIR). Because the basal nuclear envelope is parallel to our coverslip, the NPCs embedded in the nuclear envelope are oriented relative to the optical axis of our microscope. Therefore, we could monitor the orientation of Nups within individual NPCs in living cells. Our results show a rearrangement of the structural core of the NPC, in particular of the inner ring Nups, in response to manipulations of transport through the NPC.In this study, we examine the orientation of components of the scaffold and establish that the conformation of the inner ring of the NPC is modulated by both transport state and specific transport factors. To monitor conformational changes in the scaffold of the NPC, we built orientational sensors by rigidly attaching mEGFP to different Nup domains. We conjugated the alpha helix at the amino terminus of mEGFP to the carboxyl terminus of a Nup domain. Thus, the orientation of mEGFP is fixed to that of the Nup. The orientation of mEGFP can be monitored because the excitation dipole is fixed within the molecule. The strength of mEGFP excitation is proportional to cosWe alternated the polarization of the excitation field between mination .We probed for conformational changes in the NPC with orientational sensors in Nup133, Nup93, Nup54, and Nup58. Nup133 is a member of the Y-shaped complex, which forms two reticulated rings at both the nuclear and cytoplasmic faces of the NPC . Nup93 iPlasmids encoding putative orientational sensors were engineered to encode each Nup fused via an alpha helix to mEGFP. We removed the first four amino acids of the mEGFP because they are not resolved in the crystal structure, suggesting that they are not rigid. We transfected these plasmids into HeLa cells . With an:s ratio . If the If the mEGFP is held rigidly relative to a Nup, then changing the length of the alpha-helical linker by a single amino acid should rotate the mEGFP ~103° around the axis of the alpha helix. Therefore, if our reporter is a proxy for the orientation of the Nup, the p:s ratio should shift with the number of amino acids in the linker. This hypothesis was tested by inserting rigid or flexible linkers of different lengths between mEGFP and the terminal structured alpha helix of Nup54 , which iUsing this criterion, we confirmed that we have orientational sensors with mEGFP conjugated to Nup133, Nup93, Nup58, and Nup54 . We saw In yeast, starvation inhibits the import of cargo with nuclear localization sequences, or NLS-tagged cargo . We testAfter starvation there were no detectable changes in the orientations of Nup133 and Nup93 . These r494, or Nup54494) exhibited significant shifts post-starvation , the carboxyl terminus , an evolutionarily conserved export factor . We treaf Nup133 .To disrupt both import and export, we expressed a dominant-negative Ran. Ran is a GTPase that mediates nuclear transport . Ran und494 was rearranged once again with respect to the NPC but the Nup133 did not change orientation after cells were transfected with a dominant-negative Ran  remain associated with the NPCs for a period of hours . These k494 in one direction and restoration of kaps reversed this shift . We engineered a homozygous cell line where Nup62 was replaced with a copy of Nup62 with FRET sensors (mEGFP290 and mCherry321) on opposite sides of an alpha-helical domain of Nup62 , Hap1 , and Hap1-derived Nup-mEGFP cell lines (this paper) were cultured in Dulbecco’s Modified Eagle’s Medium , supplemented with l-glutamine and sodium pyruvate (from here-on referred to as DMEM) and 10% (vol/vol) fetal bovine serum  in humidified incubators at 37C and in a 5% pCOFor starvation experiments, cells were imaged, then washed 3x with PBS and placed in 1x Hank’s Balanced Salt Solution with calcium and magnesium  for 24 hr and imaged. For leptomycin B experiments, cells were either treated with 25 nM leptomycin B (Sigma) or vector (methanol) for 15 hr, at which point both were imaged.Cells were seeded onto MatTek dishes with no. 1.5 coverslips. For HeLa cells, the dishes were uncoated, but for Hap1 cells  the dishes were coated with fibronectin (Gibco). During imaging, the media was replaced with cell imaging media , supplemented with 10% FBS .Cells were imaged on a custom-built microscope, based on an inverted IX-81 frame  and equipped with a custom-built through-the-objective  polarized TIRFM illuminator equipped with a 405 nm laser , a 488 nm laser , a 594 nm laser , and a 647 nm laser  . For livThe excitation TIR light was azimuthally scanned at 200 Hz with mirror galvanometers . An electro-optic modifier  and a quarter-wave-plate  before the galvanometers controlled the polarization of the 488 nm laser.The galvanometers, EOM, camera shutter, and 488 laser shutter were all driven by a multifunctional data acquisition board  and controlled from custom written software in LabView  . All emiImages were collected on a CMOS camera  connected with Hamamatsu Camera Link interface to a workstation  running image acquisition software  .image had an exposure time of 5 ms (laser power: 100 mW), and a new image was collected every 15 ms.A sequence of 20 images was taken with alternating  and 10 All image analysis was automated with author-written analysis algorithms written in MATLAB. 10 lgorithm  using thCells were imaged with 488 laser (laser power: 5 mW) and 594 laser (laser power: 5 mW) for 200 ms.pCO2. Cells were fixed with 4% (wt/vol) paraformaldehyde in PBS for 15 min at room temperature. They were then washed 3 times for 5 min in PBS, and then permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 10 min. Cells were then blocked for 1 hr in blocking buffer (0.1% vol/vol) Triton X-100, 2.5% normal donkey serum, 2.5% normal goat serum, and 1% BSA . Primary antibodies were then added in blocking buffer and incubated overnight at 4C in a humid chamber. Cells were then washed 3 times for 5 min in PBS and then incubated with secondary antibody in 0.1% (vol/vol) Triton X-100 in PBS for 1–2 hr at room temperature. Dishes were dried and mounted with SlowFade Diamond Antifade Mountant . Antibodies used: monoclonal rat-α-karyopherin α1/6  at 1:500, monoclonal mouse-α-kap1β/impβ−1  at 1:1000, goat α-rat  at 1:2000, goat α-mouse  from Invitrogen (1:2000). Images were quantified using FIJI software   . The nucCells were imaged every 20 s with 594 laser  for 2 min, then 594 laser acquisitions  were interleaved with pulses of 488 acquisitions  every 20 s for 13 min, and then cells were imaged every 20 s with 594 excitation light  for 15 min with no 488 excitations. All excitations were done in a ‘semi’-TIRF excitation mode, which restricts fluorescence to a few micrometers near to the coverslip to illuminate more of the cytosol. Regions of the nucleus and the cytosol were manually selected in FIJI , avoidinCells were seeded onto fibronectin-coated MatTek dishes as described above. For starvation conditions, cells were grown 1 day then washed 3x with PBS and placed in 1x Hank’s Balanced Salt Solution with calcium and magnesium  for 24 hr and imaged. For control condition, cells were grown 2 days then placed in cell imaging media as described above. Cells were imaged with the 488 laser  and the 594 laser . The mCherry was then bleached by imaging every second with the 594 laser  for 1 min. Cells were then imaged again with the 488 laser  and the 594 laser .NPCs are manually identified in pre-bleached images in Metamorph and then tracked to a post-bleach image. The maximum value pixel is quantified. Apparent FRET efficiencies are calculated by: as previously described re)Ipost .Nups and LANS constructs were transfected 48 hr before imaging, with Fugene6 . DN-Ran was transfected 16 hr before imaging. Cells were transfected with 1 µg DNA (for LANS and Nup133-mEGFP), 500 ng DNA (BFP-DNRan and Nup93-mEGFP), or 250 ng DNA (Nup54-mEGFP) and 3 µL FuGENE6 (Promega) in Opti-Mem I (Gibco) to a final volume of 100 µL according to the manufacturer’s instructions.Cells were permeabilized as previously described . Cells w600 and the fractions with the highest concentrations were pooled, buffer exchanged, and concentrated on Amicon Ultra Centrifugal Filter . Samples were stored in aliquots of 10 mg in 50 mM TRIS pH 8, 150 mM NaCl, and 10% glycerol at −80C. Purification was confirmed by running the protein on a 4–12% Bis-Tris gel  and performing a Coomassie .His-tagged Ran in the pET28 vector (a gift from Dr. Günter Blobel) was transformed into BL21 (DE3) RIL-competent cells . Expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside  and cells were grown for 3 hr at 37C. Cells were spun at 6000 x g for 10 min at 4C  and the pellet was frozen overnight. The pellet was resuspended in 50 mM TRIS pH8, 150 mM NaCl with benzonase endonuclease  and rLysozyme  and 1x EDTA-free cOmplete protease inhibitors . The resuspended pellet was passed through a high-pressure homogenizer  for lysis. The lysate was spun at 30,600 x g at 4C for 30 min . Imidazole was added to the lysate to a concentration of 10 mM. The supernatant was added to Ni-NTA beads  and nutated at 4C for 1.5–2 hr. This supernatant was then loaded on a column (Qiagen) and washed 6x with 50 mM Tris pH8, 150 mM NaCl, 20 mM imidazole. Ran was then eluted with 50 mM TRIS pH8, 150 mM NaCl, 300 mM imidazole in 500 µL fractions. The concentration of Ran was estimated by ODhttp://n2t.net/addgene:91767; RRID:Addgene_91767). This protein was purified as above except it was grown overnight and no IPTG was added, because expression is constitutive.His-tagged human Kap1α, Kap1β, and NTF2 in pET28 expression vectors were purified as described above with the following exceptions. These cultures were induced with 0.3 mM IPTG. Kap1α was grown overnight (~16 hr) at 18C following IPTG induction. Kap1α was stored in 200 µM aliquots, Kap1β in 20 µM aliquots, and NTF2 in aliquots of 11.4 mg/mL. The SV40 NLS was cloned into pNCStdTomato , 2 µM Ran, and 1 µM NTF2 in TB + pvp as described above. Both conditions also contained an ATP regenerating system, which includes 1 mM ATP (VWR), 1 mg/mL creatine phosphate (Sigma), and 15 U/mL creatine phosphokinase (Millipore). Finally, the two conditions either did or did not contain a receptor mix (+/- Kaps), consisting of 1.5 µM Kap1α and 1 µM Kap1β.After the incubation period, we subsequently washed the cells to remove excess cargo and transport elements from the nuclear periphery. Cells were incubated with live-cell Hoechst . Cells were then imaged to quantify the nuclear NLS-tdTomato. All excitations were done in a ‘semi’-TIRFM excitation mode, which restricts fluorescence to a few micrometers near to the coverslip to illuminate more of the nucleoplasm. Nuclear regions were determined by constructing a mask in FIJI using the Hoescht staining and finding the average intensity in arbitrary units of the tdTomato signal .2 such at the final concentration of MgCl2 was 25 mM. The solution was then dialyzed in TB overnight.Ran was loaded with GDP using published methods . 1 mM RaTo remove kaps, cells were treated with Ran mix using published methods . Cells w494-mEGFP(0) and Nup133-mEGFP(−9) were synthesized by GenScript as dsDNA. This DNA was then A’-tailed and placed in a TOPO-TA Cloning Vector . Repair templates for Nup54494-mEGFP(1) and Nup54494-mEGFP(2) and Nup133-mEGFP(−8) were cloned from the repair template plasmids constructed above via the QuikChange Lightning Site-Directed Mutagenesis Kit . For each CRISPR cell line, cells were transfected with linearized repair templates and the PX459 plasmid containing the appropriate guides using FuGENE HD (Promega) with a ratio of Reagent : DNA of 3 : 1 according to manufacturer’s protocol. Transfected cells were selected via antibiotic selection with puromycin  for 48 hr. Cells were released from antibiotic selection and allowed to recover for 24 hr. GFP positive cells were collected in the Rockefeller University Flow Cytometry Resource Center using a FACSAria II flow cytometer . Cells were resuspended in PBS , 0.5% (vol/vol) bovine serum albumin , 5 mM EDTA (Gibco), and 15 mM HEPES (ThermoFisher). GFP positive cells were sorted and screened via live-cell microscopy for signal at the nuclear rim . Cell lines were screened for homozygosity via PCR. Two bands were seen for heterozygotes and one band for homozygotes, both fragments were sequenced to confirm amplification of correct region and proper incorporation of the mEGFP. In order to confirm that the mEGFP was not incorporated elsewhere and producing another labeled protein within our cells, western blots were performed. Protein samples were run on a 4–12% Bis-Tris gel (Novex) and Western blots were performed with the following antibodies: α-GFP (Clontech Living Colors 632381 (JL-8); RRID:AB_2313808, mouse monoclonal, 1:1,000) and goat α-mouse–HRP . Membranes were then striped with Restore Western Blot Stripping Buffer (ThermoFisher) and western blots were performed with the following antibodies: α-actin  and goat α-mouse–HRP . All blocking and incubations were done in 5% (wt/vol) nonfat milk powder in Tris-buffered saline with Tween 20.Guides and repair templates were constructed by using the Benchling CRISPR design tool . Guides were cloned into the pSpCas9(BB)−2A-Puro (PX459) V2.0 plasmid following standard protocols as developed by the Zhang lab . The repStatistical analyses were performed using MATLAB Version 2019b and are described in the figure legends and in the Method Details. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Experimental approaches capable of revealing the conformations of protein assemblies in living cells are likely to provide new insights into a host of biological questions. Here, the authors combine optical methods to develop just such a technique. This work leverages this new approach to reveal how the transport state of individual nuclear pore complexes is coupled to changes in its conformation, highlighting the flexibility of this structure.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen Vivek Malhotra as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Cargo modulates the conformation of the nuclear pore in living cells" for consideration by 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 but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:In this manuscript Pulupa et al. develop the application of "pol-TIRFM" applied to individual nuclear pore complexes in living cells. Technically, this work builds on their past published papers :1706-17), now extended to single NPC resolution. After exploring suitable fluorescent protein fusions of nucleoporins in the Y-complex (Nup133), and two aspects of the inner ring  that are sensitive to the orientation of the fluorophore, they then examine how different perturbations to transport affect these orientation sensors. Numerous treatments expected to alter nucleocytoplasmic transport change the orientation of Nup54 and Nup58, including starvation, inhibition of Crm1-mediated nuclear export, and expression of a dominant-negative form of the Ran-GTPase. Another observation is that permeabilizing cells alters the orientation of Nup54 (mimicking the perturbations listed above), but this can be restored by incubation with Kapa1 and Kapb. Consistent with the interpretation that the Kaps alter the conformation of Nup54, the orientation change upon permeabilization can be augmented (and sped up) by addition of Ran-GTP, which was previously shown to strip Kaps from the NPC. Last, the authors use an orthogonal approach (a FRET reporter in Nup62); the Nup62 protomers show increased FRET upon starvation.Overall there was enthusiasm for the approach, particularly its potential to provide a quantitative analysis of conformational changes in the NPC, as well as an appreciation for the technical feat of detecting anisotropy changes on the single NPC level. In terms of the biology, while there is a rich understanding of the architecture of the NPC from structural biology, a major unanswered question is the dynamic conformations of the nuclear pore complex that are sampled, under what conditions such structural changes occur, and the physiological relevance. In this respect, the reviewers found the evidence that the reporters reflect changes in the inner ring of the NPC in response to starvation and other perturbations to be largely convincing. There were, however, several aspects of the work that the reviewers felt could be improved. A particular concern was how the orientation changes relate to the structural states of the NPC. A second was on the ability to fully interpret the changes in the orientation of Nup54 and Nup58 with regards to large scale changes in the NPC. There were also several areas that the reviewers felt could use further clarification, as outlined below. Lastly, the reviewers expect that the authors will want to comment on their results in the context of the recent work from the Beck lab on starvation and NPC structure.Essential revisions:1) There was a consensus that getting beyond a "yes/no" of whether a change occurred to some concept of the kind of orientation change that took place in the context of the NPC would greatly strengthen the work. As presented, it was not clear how much Nup orientation changed nor how this relates to the state of the NPC itself  or its engagement with components like transport factors. The reviewers made several suggestions for how this might be achieved. First, the reviewers felt that the authors should address/interpret/discuss the quantitative differences/similarities in the p:s behavior across test conditions, particularly when this could be put into greater context of the NPC structure. Another approach that the reviewers considered is whether the authors can better leverage the extensive prior structural studies on the NPC and nucleoporins, the known orientation of the NPC relative to the evanescent wave and the polarization in this experimental set-up, and the differences in the observed p:s ratio upon stepping the linker by single amino acids, to interpret more detail about how the perturbations are influencing the position of the tagged nups in the NPC. Combining such data with a model-based theory could constrain the possibilities. Last, and related to the prior two points, was the possibility that the new work from the Beck lab might better position the authors to bridge their observations with more specific changes in the NPC (see next point).2) While this paper has been under review a related paper  from Martin Beck's group  was posted to the bioRxiv. The reviewers felt it important that a revision address this work in their interpretation and discussion. Specifically: 1) How do the observations compare about the response to starvation ?; 2) Can the authors draw any parallels with the Allegretti paper, particularly the increase of FRET in Nup62 after starvation? Can the authors suggest if this implies constriction or dilation?; 3) the Beck paper suggests that the observed changes are distinct from those proposed in the "Ring Cycle" – this should be taken into account in the Discussion here.3) The reviewers felt it important to discriminate between the effects of Kap binding to the FG nucleoporins in the act of transporting cargo versus effects solely of Kap binding (independent of transport). This point could be addressed using Kap separation-of-function mutants such as the N-terminal truncation of Kap-β.4) There were several questions related to the observation that the specific nucleoporins that showed changes in orientation in response to transport properties of the NPC directly associate with karyopherins through their FG-domains, which are part of the same polypeptide with the structured domains (where the fluorophore is fused). Thus, the major movements could be restricted to within the same polypeptide. This begs the question of whether transport alters the inner ring in a concerted manner beyond the state of the individual FG nucleoporin. One suggestion is to monitor, in the transient transfections, Nup54 truncations lacking the FG-repeat domain still respond in the same manner, which would be strong evidence propagation of the inner ring complex independent of engagement of its FG region. Ideally this would be achieved by titrating in the FG-less Nup54.5) Further explanation/discussion is needed for the observed effects of adding Ran mutant/leptomycin B. Specifically, when hydrolysis is blocked, RanQ69L-GTP-importin complexes should not dissociate. If so, importins would no longer be available for undertaking another round of import in the cytoplasm. Likewise, NES-cargo-RanQ69L-GTP-exportin triple complexes should not disassemble. In this event, transiting pools of RanQ69L-GTP-importin and NES-cargo-RanQ69L-GTP-exportin ought to remain at NPCs due to binding to the FG Nups. How is it the p:s ratio in Figure 4L shares a similar trend with Figure 4H ? How might this compare to what happens in the NPC after permeabilization ? Leptomycin B inhibits NES-cargo binding to Crm1, thereby decreasing the amount of NES-cargo being exported through the NPC. Can the authors please attempt an explanation as to how the p:s ratio in Figure 4J shares a similar trend to Figure 4H and L ?6) Can the authors please comment on the difference in observed behavior reported in Figure 5F/H and Figure 4H? The Nup54 p:s ratio increases upon removing endogenous karyopherins by +Ran mix . Adding exogenous karyopherins then returns the p:s ratio towards pre-Ran mix values (blue). Moreover, no change to the p:s ratio is seen when Ran mix is absent . Again, Nup54 p:s ratio decreases "in a buffer containing no transport factors or cargo"  when these experiments do not seem so different. What might bring about these differences?7) The reviewers were concerned that some statements in the manuscript were either over-sold or over-interpreted. Specifically, please revise to avoid "first time statements" including: "we have developed the first tools to directly observe the orientational dynamics of Nups within a single NPC in living cells and to track these dynamics over time 1 with different cargo conditions", which is dismissive of the various prior studies in the literature showing that the NPC can change its structure. While the authors employ live-cells (clearly an advance), their "dynamics" are in reality snapshots at steady state, at least several minutes or hours apart. In addition, the reviewers would ask the authors to revise the manuscript to acknowledge that they cannot rule out that a conformational change occurs outside the inner ring because it could not be observed using this approach. Lastly, the reviewers suggest that the title "Cargo modulates the conformation of the nuclear pore in living cells" is overstated. Suggested: "Transport state modulates the conformation of the nuclear pore in living cells"8) The reviewers would like the authors to report not just orientation (the p:s ratio in the manuscript) but also rigidity – that is, the anisotropy. This was seen as particularly important because it is unclear whether rigidity of inner ring Nups will vary with nuclear transport state. Essential revisions:1) There was a consensus that getting beyond a "yes/no" of whether a change occurred to some concept of the kind of orientation change that took place in the context of the NPC would greatly strengthen the work. As presented, it was not clear how much Nup orientation changed nor how this relates to the state of the NPC itself  or its engagement with components like transport factors. The reviewers made several suggestions for how this might be achieved. First, the reviewers felt that the authors should address/interpret/discuss the quantitative differences/similarities in the p:s behavior across test conditions, particularly when this could be put into greater context of the NPC structure.Thank you for this suggestion. We are strong believers in doing quantitative analysis of the results, especially across different test conditions. We have analyzed the quantitative differences across different treatments of the same probe. However, we are also cautious about over-interpreting our results. In the absence of additional structural information (see below), we think it is inappropriate to try to calibrate the physical magnitude of the change of the orientation of the Nup.Therefore, we have added further interpretation to both the analysis of Figure 4:“The effects of starvation and permeabilization appear to be more pronounced than that of the drug treatment and the dominant-negative Ran. This may be due to a more severe reduction in cargo flux, and therefore a more complete assumption of the NPC of the state adopted when cargo flux is reduced.”Another approach that the reviewers considered is whether the authors can better leverage the extensive prior structural studies on the NPC and nucleoporins, the known orientation of the NPC relative to the evanescent wave and the polarization in this experimental set-up, and the differences in the observed p:s ratio upon stepping the linker by single amino acids, to interpret more detail about how the perturbations are influencing the position of the tagged nups in the NPC. Combining such data with a model-based theory could constrain the possibilities.We appreciate this suggestion! In the past, we have used fluorescence polarization measurements to determine the orientation of Nup133 and the y-shaped complex within the NPC. We did this by comparing results of measurements of anisotropy to computational predictions based on the crystal structure of Nup133 and the cryo-EM structure of the y-shaped complex. These experiments were easier to perform with Nup133 because we were simply testing how the y-shaped complex fit into the NPC, and there was much established structural knowledge of the y-shaped complex.We have been intrigued by the possibility of combining the orientational changes we are able to measure with structural data of the NPC. We believe that this is a potential next step. Determining the precise angle of the change of orientation of the inner ring nups would require additional structural information about how exactly these alpha helices are incorporated into the NPC, as well as considerable computational work. Since August 2019 we have been speaking with Jan Kosinski about joining forces to use his structural measurements with our fluorescence. Alas, the structural information is still not resolved enough to join these data sets so this kind work will have to be outside of the scope of this project. We look forward to building, in the future, a computational model to calculate the angle changes of the alpha helices to which our probes are conjugated.Last, and related to the prior two points, was the possibility that the new work from the Beck lab might better position the authors to bridge their observations with more specific changes in the NPC (see next point).2) While this paper has been under review a related paper  from Martin Beck's group  was posted to the bioRxiv. The reviewers felt it important that a revision address this work in their interpretation and discussion.Thank you. We have added discussion of this paper to our manuscript. This beautiful work with an orthogonal technique is an independent confirmation of our data showing conformational changes in the main scaffold of the NPD that are associated with transport state. We started an active discussion with Kosinski and Beck over a year ago, once we felt we had a robust, reproducible story. We have been trying to coordinate with them both the kinds of perturbations on transport that we use as well as the Nups we focus on. However, we want to be cautious about not overinterpreting our data. We are confident in the measurement of the change of FRET and the change in the relative excitation by p and s light. Consequently, we are confident in changes of orientation that correlate with the change of transport. However, we feel as if it could be a disservice to the reader to overinterpret the results at this point in terms of a specific model.Specifically: 1) How do the observations compare about the response to starvation ?;We have added the following section to the Discussion:S. cerevisiae cells in exponential growth phase were ~20 nm larger in diameter than isolated NPCs from S. cerevisiae, underscoring the potential flexibility of the NPC .““While this manuscript was in review, cryo-EM tomograms revealed in situ NPCs taken from And revised our analysis as follows:“The conformational changes we observe in the NPCs of living cells may be coincident with cargo translocation or may be an indication that the scaffold of the NPC serves as a dynamic gate that can regulate nuclear trafficking. […] Further studies will be needed to determine the exact conformations we are monitoring in this study, and to what extent the conformations we observe align with these models. ”2) Can the authors draw any parallels with the Allegretti paper, particularly the increase of FRET in Nup62 after starvation? Can the authors suggest if this implies constriction or dilation?;Yes, we have added the following to the Discussion:In addition to changes in orientation, we also observe increases in FRET in the FG-regions of Nup62 upon starvation, which is also consistent with a spatial rearrangement and constriction of the NPC diameter.3) the Beck paper suggests that the observed changes are distinct from those proposed in the "Ring Cycle" – this should be taken into account in the Discussion here.We have made the above changes to the Discussion to explain the difference.3) The reviewers felt it important to discriminate between the effects of Kap binding to the FG nucleoporins in the act of transporting cargo versus effects solely of Kap binding (independent of transport). This point could be addressed using Kap separation-of-function mutants such as the N-terminal truncation of Kap-β.We are very interested in distinguishing between the effect of Kaps binding on their own from Kaps transporting cargo (including specific cargo such as large cargo or inner nuclear membrane proteins).In order to test the effect of Kaps devoid of cargo on the NPC, we introduced karyopherins alone in Figure 5H. Although this is not the Kap separation-of-function mutant, there was no additional cargo introduced in this experiment. In the absence of cargo, we did observe that the Kaps alone caused the NPC to be in an alternate conformation. In the future, we hope to track individual cargos transiting through the pore while monitoring the orientation of Nups to see the how transiting cargo affects the conformation of the NPC.4) There were several questions related to the observation that the specific nucleoporins that showed changes in orientation in response to transport properties of the NPC directly associate with karyopherins through their FG-domains, which are part of the same polypeptide with the structured domains (where the fluorophore is fused). Thus, the major movements could be restricted to within the same polypeptide. This begs the question of whether transport alters the inner ring in a concerted manner beyond the state of the individual FG nucleoporin. One suggestion is to monitor, in the transient transfections, Nup54 truncations lacking the FG-repeat domain still respond in the same manner, which would be strong evidence propagation of the inner ring complex independent of engagement of its FG region. Ideally this would be achieved by titrating in the FG-less Nup54.Thank you for this suggestion. We have performed this experiment, and we show that Nup54 orientational sensors lacking an FG-domain experience the same conformational changes as Nup54 orientational sensors containing a wild-type FG-domain. We appreciate the suggestion and agree that these results provide evidence that the conformational change is propagated throughout the inner ring, independent of the engagement karyopherins with individual Nup whose orientation is being altered.We have added this figure to our manuscript as Figure 3—figure supplement 2. We have also added the following statement to our Results:“The orientation shift of Nup54 was also seen in Nup54 orientational sensors where the FG-Nup domain was eliminated. This result suggests that these orientational changes are propagated throughout the inner ring, independent of whether the individual Nup54 polypeptide containing orientational sensor is bound to a kap.”5) Further explanation/discussion is needed for the observed effects of adding Ran mutant/leptomycin B. Specifically, when hydrolysis is blocked, RanQ69L-GTP-importin complexes should not dissociate. If so, importins would no longer be available for undertaking another round of import in the cytoplasm. Likewise, NES-cargo-RanQ69L-GTP-exportin triple complexes should not disassemble. In this event, transiting pools of RanQ69L-GTP-importin and NES-cargo-RanQ69L-GTP-exportin ought to remain at NPCs due to binding to the FG Nups. How is it the p:s ratio in Figure 4L shares a similar trend with Figure 4H ? How might this compare to what happens in the NPC after permeabilization ? Leptomycin B inhibits NES-cargo binding to Crm1, thereby decreasing the amount of NES-cargo being exported through the NPC. Can the authors please attempt an explanation as to how the p:s ratio in Figure 4J shares a similar trend to Figure 4H and L ?All methods of decreasing cargo flux through the NPC resulted in the same orientational change in Nup54.  all showed the same trend. One possible explanation that would explain this shift is that in all of these cases, the cargo flux is decreased. It is possible that the conformational plasticity we observe is necessary for the transport of large cargoes or inner nuclear membrane proteins, transport of all of which would be reduced in cells under starvation, permeabilization, leptomycin B, or dominant-negative ran treatment. Thus, the conformational change is permissive for transport, but is neutral as to the direction of transport. Specifically, in the text we state:“These results suggest that attenuating the transport of NLS-driven cargo shifts the orientation of select alpha-helical domains of inner ring Nups. The direction of the p:s ratio shift of Nup54-mEGFP494 is consistent among all mechanisms of reducing cargo flux.”6) Can the authors please comment on the difference in observed behavior reported in Figure 5F/H and Figure 4H? The Nup54 p:s ratio increases upon removing endogenous karyopherins by +Ran mix . Adding exogenous karyopherins then returns the p:s ratio towards pre-Ran mix values (blue). Moreover, no change to the p:s ratio is seen when Ran mix is absent . Again, Nup54 p:s ratio decreases "in a buffer containing no transport factors or cargo"  when these experiments do not seem so different. What might bring about these differences?We apologize for the confusion and we have sought to clarify these points in text. We see a change in the orientation of Nup54 upon permeabilization of the cells. This, we believe is the result of the reduction of karyopherins and cargo. In the case of Figure 4H, the control  is an unpermeabilized cell in complete media. The experiment  is a permeabilized cell. Thus, the right column of Figure 4G is equivalent to the t=0 (left most column) of Figure 5F-I.We have modified text when describing Figure 4H and Figure 4G to help clarify this point:494 compared to the orientation of Nup54494 in unpermeabilized cells grown in complete media. We saw no change in the orientation of Nup133 in permeabilized cells compared to Nup133 in unpermeabilized cells grown in complete media .”“When we permeabilize cells and incubate in transport buffer plus 1.5% (wt/vol) 360kD polyvinylpyrrolidone to mimic cytosolic conditions, we observed a distinct shift in the orientation of Nup547) The reviewers were concerned that some statements in the manuscript were either over-sold or over-interpreted. Specifically, please revise to avoid "first time statements" including: "we have developed the first tools to directly observe the orientational dynamics of Nups within a single NPC in living cells and to track these dynamics over time 1 with different cargo conditions", which is dismissive of the various prior studies in the literature showing that the NPC can change its structure. While the authors employ live-cells (clearly an advance), their "dynamics" are in reality snapshots at steady state, at least several minutes or hours apart. In addition, the reviewers would ask the authors to revise the manuscript to acknowledge that they cannot rule out that a conformational change occurs outside the inner ring because it could not be observed using this approach. Lastly, the reviewers suggest that the title "Cargo modulates the conformation of the nuclear pore in living cells" is overstated. Suggested: "Transport state modulates the conformation of the nuclear pore in living cells"Thank you for this response. We agree with these comments and have made the suggested changes in text and in the title. In addition to eliminating “first time statements” and replacing the word “dynamics,” we also added the following statement to our Discussion:“It is possible that those regions are moving translationally and thus escaping our detection, which is sensitive only to orientation changes with respect to the nuclear-cytoplasmic axis.”8) The reviewers would like the authors to report not just orientation (the p:s ratio in the manuscript) but also rigidity – that is, the anisotropy. This was seen as particularly important because it is unclear whether rigidity of inner ring Nups will vary with nuclear transport state.We have been struggling to report anisotropy values using pol-TIRF. However, we do not yet feel comfortable reporting absolute values for two reasons. First, anisotropy will always be a convoluted measurement, containing information about both orientation and rigidity. In a system where the Nups are organized with dependence around an optical axis, it is highly challenging to separate the two. Another obstacle to reporting anisotropy is the mixing of light of different polarizations when they are collected using a high-NA TIRF objective . Thus, as the light is collected, parallel and perpendicular emissions are mixed, and at the high-NA we are using (1.49), the correction factors for that have been developed break down. We have been working during this response to address this issue but we realize that a full reworking with proper calibrations and validations of these correction factors for lenses of high numerical aperture is its own two year project. Although, we are interested in developing methods to measure anisotropy in TIRF, we do not believe we currently are able to report anisotropy in a rigorous manner.


AP/NTS neurons and supports conditioned taste and place aversions, while the anorexia it causes can be blocked by a monoclonal antibody directed at GFRAL or by disrupting CCK neuronal signalling. The cancer-therapeutic drug, cisplatin, induces the release of GDF15 and activates GFRALAP/NTS neurons, as well as causing significant reductions in food intake and body weight in mice. These metabolic effects of cisplatin are abolished by pre-treatment with the GFRAL monoclonal antibody. Our results suggest that GFRAL neutralising antibodies or antagonists may provide a co-treatment opportunity for patients undergoing chemotherapy.The cytokine, GDF15, is produced in pathological states which cause cellular stress, including cancer. When over expressed, it causes dramatic weight reduction, suggesting a role in disease-related anorexia. Here, we demonstrate that the GDF15 receptor, GFRAL, is located in a subset of cholecystokinin neurons which span the area postrema and the nucleus of the tractus solitarius of the mouse. GDF15 activates GFRAL The cytokine, GDF15 , is expressed in several tissues throughout the body and circulates in the bloodstream of healthy humans . Plasma The GDF15 receptor, GFRAL (GDNF-family receptor α-like) is located exclusively in a small population of cells in the in the area postrema (AP) and nucleus of the tractus solitarius (NTS) of the mouse dorsomedial medulla oblongata,  a brainsSlc17a6), preproglucagon  (Prlh) , so that the double mutants expressed eYFP in discrete populations of CCK , glutamaene Gcg)  or prola) (Prlh)  neurons ) (Prlh) , as note) (Prlh) . Althoug the NTS . We conf the NTS . Thus, o the NTS . Overall the NTS . At leasmatergic . Finallymatergic , which tmatergic  or overlmatergic , respectA single injection of GDF15  at lights-out produced a significant and dose-dependent decrease in normal, night-time feeding within 2 hr (hr) of administration . CumulatTogether, these and recently published data  suggest CckCre::eYFP, PrlhCre::eYFP, GcgCre::eYFP or PomceGFP mice. A single, low-anorectic dose of GDF15 activated GFRAL+ve/CCK and TH neurons in the AP/NTS, as well as GFRAL-ve neurons at the same level of both the AP and NTS , while many in the PVH, but not the CeA and ovBNST, express corticotrophin-releasing hormone  cells , the paraventricular nucleus of the hypothalamus (PVH), the oval sub-nucleus of the bed nucleus of the stria terminalis (ovBNST) and in the central nucleus of the amygdala CeA; . Using Cone CRH; . Insteadone CRH; . CCKNTS ing CGRP ; and, CCto brain . CGRPPBNlsewhere . Therefog) cells . Thus, CNTS neurons also send direct projections to the PVH  expressing a Cre-dependent designer pro-caspase and its activator, the Tobacco Etch Protease (flex-taCasp3-TEVp) to commit CCKAP/NTS neurons to cell-autonomous apoptosis. CckCre::eYFP mice were injected into the AP/NTS with the AAV-caspase or an AAV expressing mCherry to control for viral load and transduction efficiency. Post hoc examination confirmed that the designer caspase achieved an effective ablation of CCKAP/NTS neurons, assessed by immunostaining for eYFP and also GFRAL  . Furtherhyperemesis gravidarum associated with intense nausea and vomiting in early pregnancy . We have described the generation of the PPG (GcgCre) and PrRP (Prlhires-Cre) mice , Envigo  or Janvier Labs . ires-Cre , Slc17a6re) mice . CalcairCre mice  were a kRecombinant human GDF15 was either made in house as described  or purchbregma (12 nl); ovBNST, +0.3 mm A/P, −1.0 mm M/L, −4.5 mm D/V (18 nl); PVH, - 0.7 mm A/P; −0.3 mm M/L; −5.5 mm D/V (18 nl). All animals were left to recover for 2 weeks to allow axonal transport before being transcardially perfused (see below).Fluoro-Gold  was injected into mice anaesthetised with isoflurane (2–3% in oxygen) and placed in a stereotaxic frame. The skull was exposed and holes drilled at the site of injection. Fluoro-Gold was delivered unilaterally via a glass micropipette affixed to a Nanoject II Auto Nanoliter Injector  using co-ordinates as determined in the Mouse Brain Atlas:  elPBN, −cisterna magna, and neck muscles were carefully retracted. Following dura incision, the obex served as reference point for injections with a glass micropipette. AP/NTS coordinates were approximatively 0.2 mm A/P, 0 and ±0.2 mm M/L, −0.2 mm D/V from obex. About 150 nl of virus were delivered during each of the three microinjections. Animals were administered analgesia  for 2 days post-operatively and given a minimum of 14 days recovery before night-time feeding measurement. AAV5-mCherry and AAV5-flex-taCasp3-TEVp were obtained from Addgene  and the University of North Carolina Vector Core , respectively.Viral injections into the AP/NTS were performed as described previously with minor modifications . BrieflyFor all immunohistochemical experiments, animals were deeply anaesthetised with 4% isoflurane in oxygen and transcardially perfused with heparinsed saline  followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were dissected and post-fixed overnight at 4°C and then cryoprotected in 30% sucrose. Brains were cut into 30-µm-thick coronal sections using a freezing sledge microtome  and either processed immediately or stored in cryoprotectant solution at −20°C.https://imagej.net/Micro-Manager). Specific band pass filter sets for DAPI, FITC and Texas Red were used to prevent bleed through from one channel to the next. All images were processed and analysed in Fiji ImageJ (https://fiji.sc/).Immunohistochemistry was performed on free-floating sections at room temperature unless stated otherwise. All antibodies are listed in the Key Resource Table. Brain sections were washed in 0.2% Triton X-100 in 0.1 M phosphate buffer and blocked in 5% normal serum for 1 hr, before being incubated in primary antibody  overnight at 4°C. The next day, sections were washed again and then incubated in secondary antibody for 2 hr. Sections were washed and, where biotinylated secondary antibodies were used, incubated for a further hour in streptavidin-conjugated fluorophores diluted in phosphate buffer. Finally, sections were washed in water, mounted onto glass slides, air-dried overnight and coverslipped with ProLong Gold . Sections were visualised on a Zeiss Axiomanager.D2 upright microscope  and images captured using a Coolsnap HQ1 camera  through Micromanager software v1.4.23  and Cck (cat# 402271-C1) or Cckr1 (cat#313751-C1) and Calca (cat#420361-C2), respectively. mRNA was detected using RNAscope Multiplex Fluorescent Assay reagent kits , according to the manufacturer’s instructions, at Gubra . Slides were counter stained with DAPI to identify cellular nuclei. Slides were scanned under a 20X objective in an Olympus VS120 Fluorescent scanner.Eight-week-old C57BL/6J mice (n = 3) were anaesthetised by COFor GDF15 night-time feeding experiments, food was removed from the animals for 2 hr before lights out. At lights out, mice were administered GDF15 subcutaneously and food was returned at the same time. Food intake was recorded at 0, 1, 2, 4, and 24 hr after injection of GDF15. Devazepide  was administered 45 min before GDF15. For the fast-refeeding experiment, food was removed from the mice at lights out on the night before the experiment. After a 16.5 hr fast, mice were administered GDF15 and food returned. Food intake was measured at the same time points (n = 5–7 per group).2 inhalation and blood was collected with cardiac puncture. Plasma was taken with Approtinin and DPP4 inhibitors. GDF15 levels are measured using a mouse specific ELISA, according to manufacturer’s directions . A dose-finding experiment for GFRAL mAb  was performed. Briefly, 1 day prior to GDF15 administration at 4 nmol/kg for 3 consecutive days, the mice were subcutaneously dosed once either with control antibody  at 10 mg/kg or ascending doses of antibody at 0.3, 1, 3 and 10 mg/kg of GFRAL mAb. Daily food intake and body weight were measured for three days. In a different experiment, control antibody or GFRAL mAb (both 10 mg/kg) were subcutaneously dosed once 1 day prior to intraperitoneal dose of cisplatin at 4 mg/kg.C57Bl/6J male mice  were administered a single, intraperitoneal dose of saline or cisplatin (4 mg/kg). Food intake and body weight were monitored daily. At 24, 48, and 72 hr, n = 6 mice from each group were sacrificed with COFor conditioned taste aversion, C57Bl/6J mice were housed in cages that permitted ad libitum access to water from two bottles, side-by-side, for at least 1 week before the experiment. On day 1 of the study, animals were water deprived overnight for 16.5 hr. The following morning (day 2), water-deprived animals were provided with two bottles of a novel 15% sucrose solution (dissolved in drinking water) for 30 min. At the end of the 30 min sucrose exposure, animals received an s.c. injection of either saline or GDF15 . Two water bottles were returned immediately and mice had unlimited access to water for one night. On day 3, mice were again water deprived overnight. On day 4, water-deprived animals were provided with one bottle of 15% sucrose and one bottle of water for a period of 24 hr. Volumes of sucrose and water intake were measured at 2 hr and 24 hr and used to calculate sucrose preference . Food was available ad libitum throughout the study. The positioning of the sucrose and water bottles (left or right) was randomised within treatment groups.Conditioned place aversion was performed using an apparatus composed of two chambers with distinct visual and tactile qualities, connected by a brightly lit corridor . The darker chamber consisted of a rough black floor and black spotted walls, whereas the lighter chamber consisted of a smooth grey floor and grey striped walls. Time spent in each chamber was monitored by video cameras mounted directly above the apparatus, connected to a computer running tracking software . All procedures were carried out between three and five hours after lights on. On day 1, C57Bl/6J mice (n = 12) were given free access to the full apparatus and allowed to freely explore both chambers for 30 min. Their initial pre-test preference was calculated from the time spent in each chamber. A biased design was used, whereby GDF15 was associated with the most-preferred chamber, which was the darker chamber for all mice. On days 2 and 3, a conditioning session was performed, where mice were restricted to one chamber following administration of either GDF15  or saline (0.9% NaCl injected s.c. on day 3). Following each injection, mice were returned to their home cage for 10 min and then placed in the relevant chamber for 30 min. On day 4, a test session was performed in identical fashion to day 1, and their post-conditioning preference was calculated from the time spent in each chamber. Food and water was available ad libitum throughout the study, except for when mice were in the conditioning apparatus.To measure pica behaviour, male Sprague-Dawley rats  were acclimated with kaolin pellets available ad libitum in a hopper placed continuously in the home cage. Rats were assigned randomly to three groups (n = 9–10 per group) and treated on 3 consecutive days with vehicle , human recombinant GDF15  or LiCl . Body weight, chow and kaolin intake were determined daily.N represents independent biological replicates. No statistical methods were used to predetermine sample sizes. Sample size was computed based on pilot data and published literature. Data are presented as mean ± SEM and statistical significance was set at p<0.05.Statistical analyses were performed using Prism 7 . Data were analysed using t-test, one-way ANOVA, two-way or repeated measures ANOVA with post hoc comparisons. When appropriate, non-parametric equivalents were used.  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Anorexia is a common response to illness or during cancer treatment and it can have serious consequences. The authors of this paper reveal that the cytokine GDF-15 is produced in response to the cancer drug, cisplatin and that it induces anorexia by activating cells in the hindbrain that express cholecystokinin and the GDF-15 receptor. Blockade of GDF-15 receptor or CCK signaling ameliorates the anorexic effect of cisplatin.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Richard D Palmiter as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Catherine Dulac as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Marcelo O Dietrich (Reviewer #3).Thank you for submitting your article "The cytokine GDF15 signals through a population of brainstem cholecystokinin neurons to mediate anorectic signalling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The role of GDF15 and its receptor GFRAL in signaling peripheral threats is an exciting advance in the field of interoceptive signaling. Understanding how the GFRAL-expressing neurons in the AP/NTS signal to other brain regions is important. Characterizing the signaling molecules made by GFRAL neurons and their relevant post-synaptic targets is critical to this endeavor.The reviewers are pleased with the overall thrust of the experiments described in this paper. The manuscript includes some important observations, but many details are missing (see below). Consequently, we are not sure whether your team can address these issues in a timely manner (2 months). If not, you may prefer to withdraw the paper now and submit it as a new paper when you have more definitive data.The major concerns that need to be addressed are as follows:1) The molecular identity of only about half of the GFRAL neurons was determined. What is the molecular identity of the other half? What fast transmitters do GFRAL neurons use, if any? Single-cell RNA-Seq is one way to answer these questions but is probably not feasible in 2 months unless the authors have already initiated this approach.2) Addressing the identity of GFRAL post-synaptic neurons that mediate anorexia or other phenotypes is one of the goals of this study. GDF15 activates Fos in parabrachial CGRP neurons, in agreement with previous results, suggesting that GFRAL (CCK subtype) neurons may directly innervate CGRP neurons; however, experiments should be included that reveal whether the signaling is direct or indirect. Is the projection to the PBN the only one that mediates anorexia in response to GFRAL neuron activation?3) The observation that CCK neurons and CCK itself are an important contributor to the anorexic phenotype is an interesting observation. If the GFRAL/CCK neurons are glutamatergic, it is surprising that CCK is so important. Experiments showing the CCK action in the PBN is important and determining which CCK receptors are involved are needed to solidify this interesting observation.4) Does CCK ablation in AP/NTS have long-term effects on food intake or body weight. Perhaps this experiment has already been published. If so, provide a reference.5) The conditioned place aversion experiment needs clarification. The authors say that testing was the same as day one, i.e. 30-min (1800-s) session. But Figure 2D shows only ~500 s on the preferred side which is less than half of the total time. Pairing GDF15 with the preferred side decreased that time to about 250 seconds suggesting that it is aversive. One problem is that "CPA score" is never defined.6) The number of mice used for each experiment should be included in the figure legends.The reviewers also have specific suggestions for improvement of this paper which can be found in the individual comments.Reviewer #1:The authors of this paper provide data demonstrating that GDF15 induces anorexia and nausea-like behavior in mice/rats and suggest that GDF15 is not a physiological regulator of satiety but rather a sickness signal. They also show that cisplatin stimulates GDF15 expression and the anorexia effect of this cancer-treatment drug depends on GFRAL. Many of the experiments are similar to those recently published by some of the same authors in Cell Metabolism .1) The strength of this paper is that the authors begin to characterize the signaling molecules expressed by GFRAL neurons and the neural circuits that they activate. Using a candidate-gene approach they provide evidence that about half of the GFRAL neurons express CCK and/or TH, but the remaining neurons remain unidentified. What fraction of the GFRAL neurons are glutamatergic? Using RNA-Seq of GFRAL-expressing neurons could identify the remaining GFRAL neurons and help elucidate whether GFRAL represents one or multiple populations of neurons.2) The authors show that treatment with GDF15 induces Fos expression in CGRP neurons in the parabrachial nucleus (PBN) and this induction depends on CCK-expressing neurons (ablation study) and CCK (antagonist study). The implication of these studies is that CCK/GFRAL neurons project directly to CGRP-expressing neurons in the PBN and that CCK plays a significant role in the anorexia phenotype. It is a little surprising that CCK has such a large effect considering that the GFRAL/CCK neurons are probably also glutamatergic and may express other neuropeptides. Authors should determine whether CGRP neurons in the PBN express CCK receptors and whether they are type 1 and/or type 2 receptors. Demonstrating that CCK signaling in PBN is sufficient for the anorexia phenotype is also important.Reviewer #2:The manuscript by Beebe and colleagues investigates various aspects of GDF15 signaling and its receptor GFRAL within the brainstem that extends many previous findings about this cytokine system. This current study reports five main findings: (1) GFRAL-positive neurons in the AP and NTS partially overlap with CCK. (2) GDF produces negative affect, as measured by conditioned taste aversion, conditioned place aversion, and increased pica behavior. (3) GDF15 activates CCK neurons (as measured by Fos expression), as well as several downstream brain areas including the PBN and PVN. (4) Ablating CCK neurons in the brainstem or injecting mice with a global CCK antagonist attenuates the anorexigenic effects of GDF15. (5) GFRAL receptor blockade with a monoclonal antibody attenuates the adverse side effects of a cancer therapeutic drug, cisplatin.Taken together, these results extend the findings of previous studies that reported the anorexigenic effects of GDF15 and the expression patterns of GFRAL in the brainstem. Many of the experiments in the study seem well done, however, some concerns dampen enthusiasm for this study.1) The quantitative results of the Fos studies reported in Figure 1 and Supplementary Figure 1 are not sufficient to justify the conclusion that "GFRAL-positive neurons in the AP and NTS co-localize with CCK." Indeed, the only quantification provided indicates that less than half (45%) of GFRAL cells co-localize with CCK. Could the authors provide a Table with exact measurements across many animals to fully report the quantitative details of these experiments? If 45% of GFRAL cells co-localize with CCK, then why does the Venn diagram in Supplementary Figure 1 imply that much more than 45% (looks like 2/3) co-localize? Is this Venn diagram based on data, or only a rough approximation? What is the percentage of CCK neurons that colocalize with GFRAL? The number of details missing in this analysis calls into question any major conclusion. Certainly, the claim that "GFRAL is localized to CCK-positive neurons in the AP/NTS," as highlighted in the text, is not justified by the data.2) Why is the Pica behavior performed in rats whereas all other experiments are performed in mice? Switching model organisms in the middle of a study seems odd. Wouldn't this experiment be relatively easy to perform in mice?3) The title of one of the Results sections… "The effects of GDF15 are mediated by CCK neurons" is not actually demonstrated in this section. The section that follows reports the effects of GDF15 administration on Fos staining. One cannot conclude from these studies that the effects of GDF15 are mediated by CCK neurons. Ironically, this could be the conclusion of the following section, which demonstrates the necessity of CCK neural activity for GFF15-mediated anorexia.4) Why do the authors study a loss of function of CCK neurons using two methods in Figure 4 (the Tobacco Etch Protease and CCK receptor antagonist), and then, when studying the effects of cisplatin, use a completely different method in Figure 5  to study GFRAL neurons instead of CCK neurons? This study is sometimes about CCK-expressing neurons and sometimes about GFRAL-expressing neurons, and some consistency across experiments would lend to better overall conclusions.5) Does the ablation of CCK-expressing cells in the AP/NTS cause long-term changes in food intake behavior? The results in Figure 4 imply that ablation of these cells does not alter food intake, however, they prevent the anorexia caused by GDF15 administration. Could the authors make a larger conclusion about the fact that there is no result of CCK neuron ablation in the absence of GDF15 administration?6) The histology results shown in Figure 5D are too small to discern.Reviewer #3:The paper provides novel insight into the population of brainstem neurons that express the receptor GFRAL for the cytokine GDF15 that is produced in pathological states and leads to anorexia and weight loss. In addition, they show data that suggest the effect of GDF15 to reduce food intake is in part mediated by CCK expressing neurons in the NTS and AP and by CCK signaling. Moreover, they show that GDF15 evoke conditioned taste and food aversion. These are important observations in a field of research that is rapidly expanding. Some concerns remain that would need to be addressed.1) The animal models that have Cre-recombinase can have leaky Cre-expression. At the minimum, the authors should comment on the specificity of the Cre lines used for labeling the neuropeptidergic neurons studied.2) The number of animals used in each experiment seems to be missing from all figures/experiments.3) Given the importance of the anatomical characterizations of GFRAL neurons in the brainstem, it would be useful to include quantification of cells in Figure 1.4) "Together these data suggest that GDF15 is not a natural satiety factor…". At the moment this statement is not very convincing, and the experiments were not designed to generate this conclusion.5) Conditioned place aversion . I might have missed something here, which could be clarified in the text, however it seems that in both pre-test and test conditions animals still prefer the side of the chamber in which they received GDF15, instead of switching the preference to the saline side. Can the decrease in preference be considered as a place aversion, even though the animals still prefer the same side of the chamber?6) Figure 5G: the reduction in food intake by cisplatin in the presence of control Ab seems to be small and only significant at day 2 (?) Clarify statistics and N. In Supplementary Figure 5A and B would be helpful to see the correlation between GDF15 and cumulative food intake and %BW in control mice injected saline. The major concerns that need to be addressed are as follows:1) The molecular identity of only about half of the GFRAL neurons was determined. What is the molecular identity of the other half? What fast transmitters do GFRAL neurons use, if any? Single-cell RNA-Seq is one way to answer these questions but is probably not feasible in 2 months unless the authors have already initiated this approach.Cck mRNA, though the proportion of double-labelled cells is lower in the NTS (35 ± 4 %). As 46 ± 2 % of CCK neurons in the AP contain GFRAL, this represents a significant population. In addition, using a Slc17a6Cre::eYFP mouse we were able to show that GFRAL immunoreactive cells co-express VGlut. This fits with the consensus that CCK neurons in the brainstem are glutamatergic. Thus, we feel that this is ample evidence to confirm that GFRAL cells are predominantly CCK-ergic and have reworded the manuscript accordingly.We were able to carry out two experiments before complete lockdown. We carried out some RNAScope in situ hybridisation, to further analyse the relationship between GFRAL and CCK. This showed that 69 ± 2 % of GFRAL neurons in the area postrema contain 2) Addressing the identity of GFRAL post-synaptic neurons that mediate anorexia or other phenotypes is one of the goals of this study. GDF15 activates Fos in parabrachial CGRP neurons, in agreement with previous results, suggesting that GFRAL (CCK subtype) neurons may directly innervate CGRP neurons; however, experiments should be included that reveal whether the signaling is direct or indirect. Is the projection to the PBN the only one that mediates anorexia in response to GFRAL neuron activation?Gfral-cre mouse available. However, Roman et al., 2016, have demonstrated, using channel rhodopsin-assisted mapping, that CCKNTS neurons project directly to CGRPPBN cells. We will cite this paper explicitly on this point. Bearing in mind the limitations of tracing techniques, we have shown that the majority of CCK/GFRALNTS and CCK/GFRALAP neurons, which are activated by GDF15, project to the PBN and not to other identified, presumably transynaptically activated areas. Furthermore, when we ablate these PBN-projecting CCK neurons, the anorectic response to GDF15 is lost. Importantly, 59% of CCK neurons activated by GDF15 are in the AP, whereas only 11% are in the NTS. Unfortunately, it will be difficult to separate accurately the NTS and AP GFRAL populations with AAV injections, since they span the nuclei. In the manuscript, we have also tried to highlight the population of unidentified, GDF15-activated neurons in the NTS. These too may be dependent on CCK input.We do not have a PBN neurons in the response to GDF15, using Calca-cre mice, that can be injected with a cre-dependent AAV-caspase. However, due to Covid lockdown, we cannot initiate this experiment. We have tried to be clear that, while some of the neurons activated in the PBN contain Calca-cre, not all of them do. Therefore, we are not convinced that we would necessarily block the whole effect of GDF15 by inactivating CGRP neurons . We have re-written the paragraph to make it clear that some of the activated neurons in the PBN contain CGRP, and so these are one downstream target.It may be possible to establish the role of CGRP3) The observation that CCK neurons and CCK itself are an important contributor to the anorexic phenotype is an interesting observation. If the GFRAL/CCK neurons are glutamatergic, it is surprising that CCK is so important. Experiments showing the CCK action in the PBN is important and determining which CCK receptors are involved are needed to solidify this interesting observation.Calca-cre mouse injected with AAV-mCherry for identification of CGRPPBN neurons. Although we, and some others, have attempted immunostaining for native CGRP and CCK1R, the results are not reliable and we would not trust interpretation based on this methodology. Thus, we also have performed dual label in situ hybridisation histology for Calca and Cckr1, which clearly shows a small proportion of CGRP neurons contain Cckr1 mRNA.This relates to the point above. We are reliant on the NTS neurons can be blocked with glutamate antagonists. Although establishing that input, it does not rule out an effect of CCK on the functioning of CGRPPBN or other PBN cells. Our results demonstrate that about half of the acute effects of GDF15 are dependent on CCK receptor signalling. We have rewritten the section to incorporate our new findings and to put the size of the effect of the antagonist into context. We agree that the effect of the CCK antagonist is significant, and we cannot discount an important role for synaptically activated cells in the NTS . We are keen to investigate this signalling pathway further, but cannot because of Covid restrictions.Roman et al., 2016, showed that the immediate electrical response to stimulation of CCK4) Does CCK ablation in AP/NTS have long-term effects on food intake or body weight. Perhaps this experiment has already been published. If so, provide a reference.To our knowledge, the long-term effects of ablating CCK neurons has not been published. Across the time frame we have studied in our mice, we have not seen any obvious changes in body weight. This has now been included in Figure 4—figure supplement 1.5) The conditioned place aversion experiment needs clarification. The authors say that testing was the same as day one, i.e. 30-min (1800-s) session. But Figure 2D shows only ~500 s on the preferred side which is less than half of the total time. Pairing GDF15 with the preferred side decreased that time to about 250 seconds suggesting that it is aversive. One problem is that "CPA score" is never defined.Thank you for this observation. We have not made this point clear, but there is an adjoining “corridor” between the two chambers, permitting “unforced choice.” This is considered good practice in any form of place conditioning, as described by Prus et al., . As the mice are free to explore three areas, the figures do not appear to add up. Thus, we have re-graphed the results to show the absolute time spent in the GDF15-paired chamber, which we hope is more clear. We will clarify this in the text and removed reference to the CPA score. Please see a fuller explanation in response to the reviewer.6) The number of mice used for each experiment should be included in the figure legends.Numbers of mice will be included in each figure legend.The reviewers also have specific suggestions for improvement of this paper which can be found in the individual comments.Reviewer #1:The authors of this paper provide data demonstrating that GDF15 induces anorexia and nausea-like behavior in mice/rats and suggest that GDF15 is not a physiological regulator of satiety but rather a sickness signal. They also show that cisplatin stimulates GDF15 expression and the anorexia effect of this cancer-treatment drug depends on GFRAL. Many of the experiments are similar to those recently published by some of the same authors in Cell Metabolism .This is not our paper, and it does not show the same data. However, it has been cited.1) The strength of this paper is that the authors begin to characterize the signaling molecules expressed by GFRAL neurons and the neural circuits that they activate. Using a candidate-gene approach they provide evidence that about half of the GFRAL neurons express CCK and/or TH, but the remaining neurons remain unidentified. What fraction of the GFRAL neurons are glutamatergic? Using RNA-Seq of GFRAL-expressing neurons could identify the remaining GFRAL neurons and help elucidate whether GFRAL represents one or multiple populations of neurons.VGlut. Importantly, RNAScope shows that the majority of Gfral cells contain Cck, and so they are the predominant phenotype. We do not have the opportunity, resources or time to carry out RNASeq analysis.As described above, we have now provided additional data to show GFRAL neurons contain 2) The authors show that treatment with GDF15 induces Fos expression in CGRP neurons in the parabrachial nucleus (PBN) and this induction depends on CCK-expressing neurons (ablation study) and CCK (antagonist study). The implication of these studies is that CCK/GFRAL neurons project directly to CGRP-expressing neurons in the PBN and that CCK plays a significant role in the anorexia phenotype. It is a little surprising that CCK has such a large effect considering that the GFRAL/CCK neurons are probably also glutamatergic and may express other neuropeptides. Authors should determine whether CGRP neurons in the PBN express CCK receptors and whether they are type 1 and/or type 2 receptors. Demonstrating that CCK signaling in PBN is sufficient for the anorexia phenotype is also important.PBN neurons contain Cckr1 mRNA and have stated clearly that there may be other GFRAL targets in the PBN. We have pointed out that about half of the acute anorectic effect of GDF15 is blocked by the CCK receptor antagonist. Importantly, we reiterate that GFRAL neurons appear to activate unidentified neurons in the both the PBN and in the NTS.We have now shown that a proportion of CGRPReviewer #2:[…] 1) The quantitative results of the Fos studies reported in Figure 1 and Supplementary Figure 1 are not sufficient to justify the conclusion that "GFRAL-positive neurons in the AP and NTS co-localize with CCK." Indeed, the only quantification provided indicates that less than half (45%) of GFRAL cells co-localize with CCK. Could the authors provide a Table with exact measurements across many animals to fully report the quantitative details of these experiments?We now have provided the exact measurements in a supplementary table (Supplementary file 1). This includes new data from the RNAScope study, which indicates a higher co-localisation.If 45% of GFRAL cells co-localize with CCK, then why does the Venn diagram in Supplementary Figure 1 imply that much more than 45% (looks like 2/3) co-localize? Is this Venn diagram based on data, or only a rough approximation? What is the percentage of CCK neurons that colocalize with GFRAL? The number of details missing in this analysis calls into question any major conclusion. Certainly, the claim that "GFRAL is localized to CCK-positive neurons in the AP/NTS," as highlighted in the text, is not justified by the data.Gfral-containing neurons also contain Cck mRNA. Likewise, 46 % of Cck neurons in the AP contain Gfral. These data, along with additional quantification of co-localisation of VGlut and TH in GFRAL neurons has also been included in the supplementary table (Supplementary file 1). Although we had stated that the Venn diagram was only a rough representation, we agree that it does not portray our results accurately, so we have now omitted it from the manuscript.We have now dealt with this by carrying out dual-label RNAScope (see comments to editors). This clearly shows that a majority of 2) Why is the Pica behavior performed in rats whereas all other experiments are performed in mice? Switching model organisms in the middle of a study seems odd. Wouldn't this experiment be relatively easy to perform in mice?Pica behaviour is notoriously difficult to perform in mice as they have a tendency to gnaw at kaolin and spread the powder liberally around their cages, making accurate weighing almost impossible. We think that adding data from another species adds value, but we decided only to include the figure as supplementary material.3) The title of one of the Results sections "The effects of GDF15 are mediated by CCK neurons" is not actually demonstrated in this section. The section that follows reports the effects of GDF15 administration on Fos staining. One cannot conclude from these studies that the effects of GDF15 are mediated by CCK neurons. Ironically, this could be the conclusion of the following section, which demonstrates the necessity of CCK neural activity for GFF15-mediated anorexia.AP/NTS neurons.”Strictly true, but as we have now shown GFRAL is expressed mostly in CCK neurons, it is a fair assumption. That said, we have altered the sub-section title to “GDF15 activates CCK4) Why do the authors study a loss of function of CCK neurons using two methods in Figure 4 (the Tobacco Etch Protease and CCK receptor antagonist), and then, when studying the effects of cisplatin, use a completely different method in Figure 5  to study GFRAL neurons instead of CCK neurons? This study is sometimes about CCK-expressing neurons and sometimes about GFRAL-expressing neurons, and some consistency across experiments would lend to better overall conclusions.CckCre neurons, importantly including GFRAL-expressing CCK neurons, as we have verified in post mortem cell counts . CCK neurons in the brainstem are glutamatergic and may contain other transmitters. Therefore, we wanted to assess the role of CCK itself (as opposed to other transmitters) in mediating the effects of GDF15, and so used a receptor antagonist. The last section of the paper addresses a separate question of whether the GFRAL receptor can be targeted in translational research. The GFRAL receptor antibody is an excellent tool to provide useful data, as an antagonist is not available.With respect, the reviewer will not have seen our additional results, which shows the high overlap between GFRAL and CCK neurons. Thus, we are then trying to study the relevance of that cell mediator in different ways. We believe that providing evidence using different methods can add strength to the argument. Caspase will kill 5) Does the ablation of CCK-expressing cells in the AP/NTS cause long-term changes in food intake behavior? The results in Figure 4 imply that ablation of these cells does not alter food intake, however, they prevent the anorexia caused by GDF15 administration. Could the authors make a larger conclusion about the fact that there is no result of CCK neuron ablation in the absence of GDF15 administration?To our knowledge, the long-term effects of ablating CCK neurons has not been published. Across the time frame we have studied in our mice, we have not seen any obvious changes in body weight. This has now been included as Figure 4—figure supplement 1. We are not in a position to speculate whether CCK neurons have a homeostatic role, as our experiments have not been designed specifically to answer such a question.6) The histology results shown in Figure 5D are too small to discern.We have remade this figure.Reviewer #3:[…] 1) The animal models that have Cre-recombinase can have leaky Cre-expression. At the minimum, the authors should comment on the specificity of the Cre lines used for labeling the neuropeptidergic neurons studied.CckCre and CalcaCre lines may report on more cells than express the gene in adult mice; suggesting that more cells may express significant levels of these genes during development. This is a fair point regarding the initial section, where we used a candidate-gene approach and transgenic mouse lines. However, we have now followed this up with dual-label RNAScope in adult mice, which shows a good consensus. For the experiment using CalcaCre, there is not an issue, since adult mice were injected with a viral reporter.References are provided in the Materials and methods section for all the mouse lines used, which demonstrate that each produces a true representation of endogenous gene expression. It is known that both the 2) The number of animals used in each experiment seems to be missing from all figures/experiments.Numbers have been included3) Given the importance of the anatomical characterizations of GFRAL neurons in the brainstem, it would be useful to include quantification of cells in Figure 1.Quantification has been added in the new supplementary table (Supplementary file 1).4) "Together these data suggest that GDF15 is not a natural satiety factor…". At the moment this statement is not very convincing, and the experiments were not designed to generate this conclusion.We have added reference to the recent publications and tempered the sentence by saying “Together, these and recently published data  suggest that GDF15 is probably not a satiety factor.” The following sentence in our manuscript also provides another four references which contain additional supporting evidence for this statement. In this manuscript we go on to show that GFRAL neurons are only activated by stimuli which cause gastric malaise, and not by stimuli that induce normal satiety. We also show that treatment of mice with the GFRAL monoclonal antibody itself does not affect feeding or body weight, but does block anorexia caused by a drug that causes gastric malaise.5) Conditioned place aversion . I might have missed something here, which could be clarified in the text, however it seems that in both pre-test and test conditions animals still prefer the side of the chamber in which they received GDF15, instead of switching the preference to the saline side. Can the decrease in preference be considered as a place aversion, even though the animals still prefer the same side of the chamber?difference in time spent between chambers, not the absolute time spent in each chamber. This calculation is often used in CPP/CPA experiments to account for the inherent preference of mice to one-side of a two-chamber apparatus and normalises this preference when there is a difference in separate treatment groups (which is not an issue here). Our study is further complicated by the fact that we have an adjoining corridor between two chambers, permitting unforced choice , but giving the mice three areas to explore.Thank you for this observation, as we can see how confusing this is. It highlights the issue of using a CPA “score” to denote aversion. As done by others, CPA was calculated by [time in chamber A] minus [time in chamber B] and, therefore, represents the We accept that the CPA score is misleading and perhaps not appropriate here. Therefore, we have changed Figure 2C to depict time spent in the GDF15-paired chamber and have described explicitly both our apparatus and analysis in the Materials and methods section.6) Figure 5G: the reduction in food intake by cisplatin in the presence of control Ab seems to be small and only significant at day 2 (?) Clarify statistics and N. In Supplementary Figure 5A and B would be helpful to see the correlation between GDF15 and cumulative food intake and %BW in control mice injected saline.Thank you for this comment. We agree that the result for day 1 looks small; this is because the first injection of cisplatin is given on day 2. We have altered the axis in the Figure 5G to reflect this (i.e. daily not cumulative food intake). Stats and N are now clear in the legend. We have included the correlation analysis for mice injected with control in Figure 5—figure supplement 1.