Cell-based regenerative medicine therapies require robust preclinical safety, efficacy, biodistribution, and engraftment data prior to clinical testing. To address these challenges, we have developed an imaging toolbox comprising multispectral optoacoustic tomography and ultrasonography, which allows the degree of kidney, liver, and cardiac injury and the extent of functional recovery to be assessed noninvasively in a mouse model of multiorgan dysfunction. This toolbox allowed us to determine the therapeutic effects of adoptively transferred macrophages. Using bioluminescence imaging, we could then investigate the association between amelioration and biodistribution. Macrophage therapy provided limited improvement of kidney and liver function, although not significantly so, without amelioration of histological damage. No improvement in cardiac function was observed. Biodistribution analysis showed that macrophages homed and persisted in the injured kidneys and liver but did not populate the heart. Our data suggest that the limited improvement observed in kidney and liver function could be mediated by M2 macrophages. More importantly, we demonstrate here the utility of the imaging toolbox for assessing the efficacy of potential regenerative medicine therapies in multiple organs. Cell-based regenerative medicine therapies (RMTs), which include pluripotent stem cells, mesenchymal stromal cells, and macrophages, have the potential to treat a variety of human diseases. However, before these therapies can be routinely used in the clinic, accurate information regarding their safety and efficacy should be obtained from appropriate preclinical models. It is also important to gain an understanding of the therapeutic mechanisms of the RMTs, such as whether their ability to ameliorate injury is dependent on their engraftment in the damaged tissues. Issues which currently prevent the generation of such data include (i) the limitations associated with commonly used blood biomarkers of organ injury, such as serum creatinine (SCr) and blood urea nitrogen (BUN) for renal function –5 and alIn this current study, we set out to develop a multimodal imaging strategy to monitor the function of the liver, kidney, and heart longitudinally in BALB/c mice in a single imaging session. We utilised multispectral optoacoustic tomography (MSOT) to assess kidney and liver function and traditional ultrasound (US) measurements to assess cardiac function. MSOT is a technique which uses multiple excitation wavelengths to resolve specific sources of absorption using the photoacoustic effect, whether they are endogenous or exogenous . It reliHere, we utilised an acute model of adriamycin- (ADR-)-(doxorubicin-) induced multiorgan injury and assessed organ function on days one and four post-drug administration in order to determine the extent of disease progression. ADR is an anthracycline antibiotic which is clinically administered as a chemotherapeutic agent; however, its use is limited predominantly due to cardiotoxicity mediated through a number of mechanisms. In rodents it can induce both chronic and acute kidney injuries, as well as cardiac and hepatic dysfunction –17. To tad libitum access to food and water. All animal experiments were performed under a license granted under the Animals (Scientific Procedures) Act 1986 and were approved by the University of Liverpool ethics committee. Experiments are reported in line with the ARRIVE guidelines.Mice were purchased from Charles River, UK, and were housed with μm) and then cultured in DMEM: F12 media containing 20 ng/ml murine recombinant macrophage colony-stimulating factor (MCSF-1). Bone marrow suspensions were cultured at 37°C and 5% CO2, and medium was replaced every other day. On day 7, macrophages were considered fully differentiated as determined by the expression of both CD11b and F4/80 (BioLegend) by flow cytometry. Mature BMDMs were then polarised towards an M2-like phenotype by the overnight addition of recombinant murine interleukin- (IL-) 4 (20 ng/ml).Primary bone marrow-derived macrophages (BMDMs) were prepared as previously described . Male BAn = 11) or saline  intraperitoneally (IP) on day 0. Adriamycin  was dissolved in warm saline (0.9%) to make a stock solution (10 mg/ml) before administration. Mice were weighed on a daily basis to monitor their well-being, and mice which received adriamycin were provided with a wet food diet.Male BALB/c mice (8–10 weeks) received either adriamycin  for liver functional measurements. The catheter was flushed with saline before administration of IRDye800 carboxylate (LI-COR) for functional kidney measurements. Mice were then allowed to recover in a heat box before being returned to their home cage. A schematic showing how the study was carried out can be found in Cardiac function was assessed using the Prospect 2.0 ultrasound system . Mice were anaesthetised using isoflurane and oxygen, and the mice were placed dorsally on a heated platform. Mice were fixed in place during imaging using a surgical tape. Ultrasound gel was applied to the chest area of the mice and the ultrasound transducer positioned above the chest area. The following parameters were measured: epicardial area and endocardial area in the long-axis view, left ventricle length, epicardial areas in the short-axis view, M-mode images of both the long- and short-axis views in order to measure heart rate, left ventricular interior diameter, and wall thickness in both diastole and systole. These parameters were used to calculate fractional shortening (FS), ejection fraction (EF), stroke volume (SV), and cardiac output (CO).μl) over a 10 s period. Data were reconstructed using a model linear algorithm (View MSOT software) and multispectral processing using linear regression for ICG, deoxy- and oxy-haemoglobin spectra to resolve the signal for the ICG dye. Regions of interest drawn around the ischiatic vessels of each mouse were used to quantify the ICG dye signal (as mean pixel intensity) in the vessels of the mice. These data were used to calculate the area under the clearance curve (AUC). Data were expressed as the change in ICG AUC in each individual mouse between days 1 and 4 (ΔICG AUC).Liver function was assessed using the inVision 256-TF MSOT imaging system . Immediately after assessment of cardiac function, the mice were moved to the MSOT imaging system. Prior to being placed into the system, the tail vein of the mice was cannulated to allow injection of the optical imaging contrast agents during photoacoustic imaging. Mice were placed in the system and allowed to acclimatise for 15 minutes prior to recording data. Imaging focussed on the ischiatic vessels close to the hips of the mice for detection of indocyanine green (ICG) and its subsequent clearance. The following parameters were used: an acquisition rate of 10 frames per second , with wavelengths of 700, 730, 760, 800, 850, and 900 nm being recorded. The half-life for ICG in the blood is approximately 3 minutes. Six wavelengths with averaging were used for ICG imaging to facilitate spectral unmixing of MSOT data which improves image specificity. The temporal resolution of this imaging protocol is less than 10 s, which is sufficient to characterise a half-life which is in the order of minutes. Data were recorded for 3 minutes prior to intravenous (IV) injection of ICG  over a period of 10 s. Data was reconstructed using a model linear algorithm and a difference protocol (775 nm–850 nm) to resolve the signal for the IRDye. Regions of interest were drawn around the renal cortex and the renal papilla/pelvis region of the right kidney in each mouse to quantify the IRDye signal in the kidney. Regions of interest were drawn on an individual animal basis to reflect the renal cortex and renal papilla/pelvis based on the visible IRDye signal resolved using multispectral processing. The mean pixel intensity data were used to calculate the AUC of both the renal cortex and papilla/pelvis. Data were expressed as the change in the ratio between the AUC of the cortex and the AUC of the pelvis regions between days 1 and 4 (ΔAUC C : P).Kidney function was assessed utilising a similar method as the assessment of liver function. After liver function assessment, the catheter was flushed with a small amount of saline. Data were recorded using the following parameters: wavelengths of 775 and 850 nm and an acquisition rate of 10 frames per second (consecutive frames averaged). The imaging protocol for kidney imaging differed from the liver in that only 2 wavelengths with averaging were used for the former, enabling the temporal resolution to be increased by a factor of 3. This was necessary because of the faster kinetics of IRDye , comparen = 6) on day 1 immediately after cardiac, hepatic, and renal imaging. BMDMs were harvested from low-adherence flasks (Corning) after maturation and polarisation by gentle agitation and scraping. Cells were then counted and suspended to a concentration of 107 cells/100 μl saline. After the mice were removed from the MSOT imaging system, 100 μl of the cell suspension was administered via the tail vein cannula and mice were allowed to recover in a heat box before being returned to their home cage. Mice which did not receive BMDMs received 100 μl saline via the tail vein catheter.M2-like primary BMDMs were administered to mice which received adriamycin , serum creatinine , and alanine aminotransferase activity  were quantified according to the manufacturer's instructions in a 96-well plate and were read using a FLUOstar Omega microplate reader (BMG LABTECH).μm) and were stained with haemotoxylin and eosin (HE) and periodic acid-Shiff (PAS) by standard methods. Kidney injury was scored 0-5 on 10 consecutive 200x microscopic fields in the outer stripe of the outer medulla and cortex on PAS-stained slides adapting the method described from Wang and colleagues [Kidney and liver tissues were fixed in 4% paraformaldehyde (4°C) for 24 hours before being washed in PBS, dehydrated with increasing concentrations of ethanol, and subsequently embedded in paraffin. The kidneys were sectioned in order to obtain two symmetrical halves, to include the cortex and medulla extending to the renal pelvis. For the liver, the large lobe was collected for analysis. Tissue sections were cut (5 lleagues . For then = 6) or saline IP (n = 6). 24 hours later, all mice received IV injections of 107 PMDMs isolated from mice expressing the CAG-luc-eGFP L2G85 transgene. All mice received luciferin  before being imaged using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). Mice were imaged in both dorsal and ventral positions using an automatic exposure time before being sacrificed. Organs were then dissected and imaged using the same protocol. The remaining mice were returned to their home cages until day 4 postadriamycin administration (the study endpoint) when they were imaged as previously described.To determine cellular biodistribution by bioluminescence imaging, male BALB/c mice (8–10 weeks) were used. The same adriamycin and macrophage dosing schedule was used as in the previous study: mice received either 20 mg/kg adriamycin IP , injured (ADR), and control mice  by measuP = 0.013, R2 = 0.39).MSOT was used to assess renal function in healthy mice, those which received ADR and those which received ADR+BMDMs, on days 1 and 4 following ADR administration . UninjurP = 0.0009, R2 = 0.59) (We assessed the liver function with MSOT as previously described  by measu = 0.59) . To inve = 0.59) .The functional data from the MSOT analyses suggested that BMDMs had a tendency to improve renal and hepatic function in ADR-dosed mice but did not improve cardiac function. To investigate whether the apparent improvement in renal and hepatic function was associated with an amelioration of tissue damage, histological analysis of the liver and kidneys of mice was carried out at the study endpoint. Kidney sections were assessed for the presence of intratubular protein casts and flattened tubular epithelium and liver sections for hepatocellular degeneration and necrosis. There was a significant difference between healthy and ADR-injured animals, but in contrast to the functional data, administration of BMDMs failed to improve the extent of histological damage in the kidney or liver. Histologically, the kidneys of control mice showed no evidence of injury  whereas In order to investigate the effect of organ injury on BMDM distribution, mice were imaged on the same day, or 3 days after administration of luciferase+BMDMs, which corresponded to the 1st and 4th day following saline or ADR administration. Bioluminescence imaging showed that on day 1, cells were mostly in the lungs of both control and ADR animals. By day 4, BMDMs were no longer detectable in the controls but had a widespread distribution in the ADR group Figures . Given tWe measured the total flux from the individual organs ex vivo to quantify the differences in organ biodistribution between the treatment groups and time points . The totIn the current investigation, cardiac function was measured by ultrasonography alongside hepatic and renal function by MSOT under the same anaesthesia session. This allowed us to determine the function of 3 separate organs utilising a multimodal imaging approach. The cardiac measurements taken easily and quickly with the ultrasound software revealed that there were no significant changes in fractional shortening or ejection fraction between days 1 and 4 in the ADR and ADR+BMDM groups when compared to the healthy mice. However, significant decreases in cardiac output and stroke volume between days 1 and 4 were detected when comparing the saline-treated mice to those which received adriamycin, regardless of the addition of M2 BMDMs. This suggests that a single dose of adriamycin significantly reduces cardiac function between days 1 and 4 of this study; however, the addition of BMDMs as a RMT shows no beneficial effect on cardiac function.We have previously used MSOT to monitor renal function in a mouse model of adriamycin-induced chronic kidney disease by measuring the clearance kinetics of IRDye . The twoIn the current study, the clearance of ICG from the blood of mice was used as a measure of liver function. ICG is a fluorescent cyanine dye which is used clinically in a number of diagnostic procedures including measurement of cardiac output, liver blood flow, ophthalmic angiography, and hepatic function –27. It ain vivo and in the liver, kidneys, lungs, and spleen, but not the heart, when imaged ex vivo. While the luminescent signal intensity decreased from days 1 to 4 in the lungs and spleen, it was still observable. On the other hand, the signal intensity in the liver and kidneys was significantly elevated between days 1 and 4. Of note, the total flux in the whole-body images on day 4 in ADR-treated mice appeared greater than on day 1.The aforementioned multimodal imaging strategy can monitor changes in the functions of the liver, kidney, and heart and identify potential efficacy of M2 BMDMs as a RMT. To investigate the relationship between biodistribution of the M2 BMDMs and any therapeutic effect, we administered M2 BMDMs expressing firefly luciferase which allowed the luminescent imaging and quantification of cells in the mice and organs (three on day 1 and three on day 4). At day 1, we observed no difference in luminescence distribution between both groups of mice, with signals only present in the lungs and spleen after IV cell administration. This pattern of biodistribution has been reported widely after IV administration of cells . HoweverWe show that ADR-induced impairment of liver and kidney function measured with MSOT correlates well with traditional serum biomarkers of injury. We also show that the addition of M2 BMDMs improves kidney and liver function, albeit not significantly so, but has no beneficial effects on cardiac function. Bioluminescence imaging showed that M2 BMDMs persisted in the kidneys and liver of ADR-dosed mice but were not detected in the heart. It is not clear if the presence of M2 BMDMs in the kidneys and liver was related to the limited functional improvement that was observed in those organs. Although we do not show any significant improvement in organ function as measured by MSOT, we do however describe and demonstrate the utility of a noninvasive multimodal imaging toolbox for assessing the efficacy and biodistribution of a potential regenerative therapy.


Epidemiological studies indicate that shift-workers have an increased risk of type 2 diabetes mellitus (T2DM). Glucose tolerance and insulin sensitivity both are dependent on the circadian timing system  and fasting duration, in rodents as well as humans. Therefore, question is whether manipulation of the circadian timing system, for example by changing the timing of feeding and fasting, is a potential preventive treatment for T2DM. Time-restricted feeding (TRF) is well-known to have profound effects on various metabolic measures, including glucose metabolism. However, experiments that directly measure the effects of TRF on glucose tolerance and/or insulin sensitivity at different time points throughout the 24 h cycle are lacking. Here we show, in rats, that TRF in line with the circadian timing system  improves glucose tolerance during intravenous glucose tolerance tests (ivGTT) in the active phase, as lower insulin levels were observed with similar levels of glucose clearance. However, this was not the case during the inactive phase in which more insulin was released but only a slightly faster glucose clearance was observed. Contrasting, TRF out of sync with the circadian timing system  worsened glucose tolerance, although only marginally, likely because of adaptation to the 4 week TRF regimen. Our results show that TRF can improve glucose metabolism, but strict adherence to the time-restricted feeding period is necessary, as outside the regular eating hours glucose tolerance is worsened. An increasing number of people are suffering from type 2 diabetes mellitus (T2DM) . T2DM isad libitum were tested with two different fasting durations. Animals were housed at a constant temperature of 22°C under a controlled 12:12 light:dark cycle, lights on at Zeitgeber Time 0 (ZT0) and lights off at ZT12. After arrival to the institute animals had an acclimation period of 1 week after which they were individually housed and randomly assigned to one of three TRF groups for 4 weeks: ad libitum feeding (AL), light phase feeding (light-TRF), or dark phase feeding (dark-TRF) (n = 14–16 per group). Light-TRF and dark-TRF animals had access to chow pellets for 10 h in the middle of the light or dark phase, respectively  were used for the TRF intravenous glucose tolerance test (ivGTT) experiments. An additional eight animals fed ectively . In autoeviously . Animalsad libitum fed animals were comparable to those of the dark fed animals, although the 17 h fasting period for the ZT16 time point is not absolute, as animals might have eaten a little during the second half of the light period before food was removed at ZT11. After a baseline blood sample (t = 0 min) had been taken animals were infused with glucose  and blood samples were taken again 5, 10, 20, 30, and 60 min after the glucose infusion. Typically, 0.25 ml of blood was drawn during a sample.After 3 and 4 weeks on TRF an ivGTT was performed at either ZT4 or ZT16, i.e., 4 h after lights-on or 4 h after lights-off, in a randomized order. During the experimental days all animals remained on their assigned TRF conditions, but in addition for all animals food was removed 5 h before the ivGTT (if applicable). Consequently, dark fed animals were fasted for 17 h during the ZT16 measurement and light fed animals were fasted for 17 h during the ZT4 measurement . During ad libitum fed rats were randomly assigned to either a 5 h or a 17 h fast before the start of an ivGTT in the dark, i.e., awake, period (ZT16). After a recovery period of 1 week the rats were tested again with the other fasting duration. The ivGTT procedure itself was identical to the one described above for the TRF ivGTT experiments.After recovery from surgery the extra eight Blood glucose was measured directly at each sampling point during the ivGTT from the untreated blood samples using blood glucose test strips with a 0.1 mmol/L accuracy . Plasma insulin was measured using a radioimmunoassay (Millipore).TRF and Sampling  as well as the Interaction (TRF*Sampling) for the insulin and glucose profiles during the ivGTTs. Tukey's post-hoc tests were performed to compare the three different TRF groups. Delta values for glucose and insulin concentrations were determined by subtracting the baseline value (t = 0) from the value of each of the subsequent time points. The net AUC  was determined using these delta values and the trapezoid rule for the duration of the entire ivGTT measurement (0–60 min). Two-way ANOVAs were used to test for effects of TRF , Fasting/Feeding (Fasting period or Feeding period) and Interaction (TRF * Fasting/Feeding) on the glucose and insulin responses (expressed as net AUC). T-tests were performed to compare the net AUC between two different time points, i.e., ZT4 and ZT16, within each TRF group. One-way ANOVAs were used to test for differences in body weight and food intake in the experimental weeks as well as to compare the net AUC of the 17 h fasted animals at ZT16 . All statistics were run by GraphPad Prism 7.All data are represented as means ± SEM. Two-way repeated measure (RM) ANOVAs were used to test for the effects of p = 0.73; one-way ANOVA).In the experimental weeks, the animals from the 3 different TRF groups ate ~19.5 g of chow/day and no significant differences in food intake were found between the groups . Also boInteraction effect between the sampling points and the TRF condition .For all three groups in the TRF experiment, the intravenously administered glucose was cleared from the circulation within 20 min at both ZT4 and ZT16, i.e., blood glucose levels had returned to baseline or were even slightly lower than baseline . During ondition , Table 1g-Period . Post-hoad libitum fed group . Additionally, only light-TRF animals showed no significant diurnal difference in insulin AUC.For all three groups and at both ZTs a sharp rise in blood insulin concentrations was found 5 min after glucose administration . The insed group . The ins animals . AUC of eraction . Post-hoad libitum animals at ZT16 of Experiment-1 (relatively fast of 17 h as they could eat until ZT11) and 17 h-fasted AL animals of Experiment-2 (absolute fast of 17 h). t = 5 min  with those of the  = 5 min  and an i = 5 min . Finally = 5 min .ad libitum showed higher glucose tolerance at the beginning of the activity period than at the beginning of their sleep period. In neither TRF group, such a significant diurnal variation was observed, clearly indicating that the well-known diurnal variation is not only due to a difference in the feeding/fasting condition. Moreover, light fed animals showed no diurnal variation in either their glucose or insulin response, another clear indication that the normal diurnal variation is not the single result of either the feeding/fasting status or the circadian timing system. Of all the ivGTTs performed, the smallest insulin response was observed in dark fed animals during their ZT16 test. This improved glucose tolerance of the dark fed animals at ZT16 was due to a combined effect of time-of-day and prolonged fasting, as it was also observed in the ad libitum animals of Experiment-2 when fasted for 17 h and tested at ZT16, but not in the ad libitum animals of Experiment-1 with a relative fast of 17 h and tested at ZT16. Improved glucose tolerance was also not observed in the day fed animals, neither when fasted for 17 h and tested at ZT4, nor when fasted for 5 h and tested at ZT16. Overall, these results show that the normal daily variation in glucose metabolism induced by the circadian timing system can be enhanced when a strict and prolonged fasting period is imposed, but only when the timing of feeding and fasting behavior is strictly in line with the circadian timing system. The latter meaning fasting during the regular sleep period and only feeding during the regular wake period, thus, respectively, light and dark period for nocturnal animals.We here present evidence that the well-known diurnal variation in glucose tolerance is due to both an intrinsic daily variation as well the preceding feeding/fasting condition. As expected animals fed Surprisingly, the disadvantageous effects of chronically feeding at the wrong time-of-day for glucose metabolism were quite limited. The most disadvantageous effect of daytime TRF being a higher insulin peak during the ivGTT in the fasting period and a slightly higher glucose response during the feeding period. Most likely, in the current experimental set-up the negative effects of feeding at the wrong time-of-day were counteracted by the positive effects of a long fasting period and the chronic condition of our TRF protocol (4 weeks) that allowed sufficient time for adaptation to the new rhythm in systemic glucose availability, through mechanisms that still need to be elucidated. One such potential mechanism that could be explored in future studies is corticosterone signaling as it is well-known that corticosterone (or cortisol in humans) affects glucose metabolism. Besides that, plasma corticosterone levels show a clear day/night rhythm with peak levels at the beginning of the active phase in both humans and rats [reviewed in ] and corad libitum animals, as indicated by the significant lower glucose levels at t = 5 as well as an overall lower glucose profile. Although the AL animals from Experiment-1 with a 17 h period of relative fasting showed an even lower glucose response  per week for a duration of 3 weeks . In rats/ob mice . Althoug/ob mice , 37, 38,/ob mice ]. Nevertad libitum fed animals. Similarly, in mice an overnight fast of 18 h resulted in enhanced insulin sensitivity as compared to a 5 h fast as measured with a hyperinsulinemic-euglycemic clamp  found that post-prandial glucose concentrations were higher during a simulated night-shift as compared to simulated day-shift, accompanied with a lower insulin response during the first hour of the meal test . Also, βSeveral studies also implemented a form of TRF in humans. In a recent study male pre-diabetic patients underwent a GTT after TRF  as well as after the control condition  . Both thad libitum fed rats acutely for 17 h are also in line with this idea. The changes seen after an acute 17 h fast period clearly differ from those of the TRF animals that were fasted for 17 h. By experimental design the TRF animals were daily fasted for 14 h, whereas ad libitum fed animals usually only have a daily fasting period of 6–8 h at the beginning of the light phase. During the TRF protocol especially glucose tolerance seems to improve again, i.e., although the insulin response is still reduced  adapt to the initial disturbances caused by circadian misalignment or shift work. The results from Experiment-2 where we fasted  reduced , glucose reduced .ad libitum animals, whereas those of light fed animals would be the reverse . On the other hand, in case the daily variation would have been completely dependent on the endogenous timing system, a similar daily variation would have been observed in all three groups as they were all housed in the same L/D condition. In fact, we observed that the daily variation in glucose responses was lost in both the dark and light fed group, whereas the daily variation in insulin responses was enhanced in the dark fed animals, but lost in light fed animals. Thus, together our results show that both time-of-day and the feeding/fasting condition modulate the effective glucose tolerance. It should be noted, however, that during ivGTTs insulin sensitivity is measured indirectly. Therefore, we cannot rule out the possibility that the smaller insulin release we find in our groups that are absolutely fasted for 17 h are due to e.g., impaired β-cell functioning, although without a change in insulin sensitivity that would conflict with the similar glucose clearance we find in these groups.As recently nicely demonstrated in humans , 30, thead libitum conditions. Probably this “lack-of-effect” is due to the chronic condition of our experiment allowing sufficient time to adapt to this new situation, as well as the positive effects of the structurally enforced prolonged fasting period. For future experiments, it will be necessary to investigate how rapid and persistent these effects are when animals are switched back and forth between TRF and ad libitum feeding conditions, as such a situation more closely resembles shift work in humans. On the other hand, feeding restricted to the active phase did improve insulin sensitivity, but only during the active phase. Most likely this effect is due to the long fasting period coinciding with the regular sleep and fasting phase. Thus, together our results show that TRF in line with the circadian timing system enhances/strengthens the normal day/night difference in glucose tolerance and improves glucose tolerance when most needed, i.e., during the regular wake and feeding period. This also means that when applying TRF for therapeutic means eating should be restricted to the prescribed eating period, as glucose tolerance is worsened outside the regular eating hours.Surprisingly, feeding restricted to the inactive phase only marginally affected glucose tolerance and the concomitant insulin response compared to The datasets generated for this study are available on request to the corresponding author.PdG and AK orchestrated the research project, performed the analyses, and wrote the manuscript. PdG, EF, WR, and NK performed the surgeries and the experiments. AK and C-XY provided both intellectual and financial support and helped writing and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Changes in the diversity of plant communities may undermine the economically and environmentally important consumer species they support. The structure of trophic interactions determines the sensitivity of food webs to perturbations, but rigorous assessments of plant diversity effects on network topology are lacking. Here, we use highly resolved networks from a grassland biodiversity experiment to test how plant diversity affects the prevalence of different food web motifs, the smaller recurrent sub-networks that form the building blocks of complex networks. We find that the representation of tri-trophic chain, apparent competition and exploitative competition motifs increases with plant species richness, while the representation of omnivory motifs decreases. Moreover, plant species richness is associated with altered patterns of local interactions among arthropod consumers in which plants are not directly involved. These findings reveal novel structuring forces that plant diversity exerts on food webs with potential implications for the persistence and functioning of multitrophic communities. Plant diversity affects ecosystem function in myriad ways, but the effect on food webs has received less investigation. Here, the authors use high-resolution food web data from a grassland diversity experiment to show that apparent and exploitative competition motifs increase with plant diversity. Concern that changes in biodiversity will threaten the services that nature provides to humans has sparked numerous experimental manipulations of diversity over the past decades2. These experiments typically manipulate plant communities, which provide food and habitat for myriad consumer species that perform important ecosystem services such as pest control and pollination. Reductions in plant diversity are associated with reduced consumer species richness4 and increased temporal variability in consumer community abundance6. These effects have been attributed to a propagation of plant diversity effects through multiple trophic levels7. However, a lack of well-resolved food webs has hindered mechanistic insight into plant diversity effects on the stability and robustness of multitrophic communities. Consequently, we are only just beginning to understand the effects of plant diversity on the structure of trophic interactions9.Global change drivers are causing pervasive shifts in the distribution of biodiversity11. One way to fingerprint the structure of food webs is to examine the distribution of the small recurring subgraphs that are the building blocks of larger networks, termed network modules or motifs13. When excluding cannibalism, there are 13 directed, connected configurations of three-node motifs, that is, triads , omnivory (s2), apparent competition (s4), and exploitative competition (s5). This non-random representation may arise due to two non-exclusive processes18. First, there may be constrains on how the network assembles such that some motifs become more common. Second, some motifs may function in a way that makes them beneficial to network stability, increasing their abundance in webs that have persisted so that they can be observed. However, current insight into patterns of network motif distributions in food webs comes predominantly from comparative studies using networks collected with different methods in a diverse range of ecosystems19.The number and arrangement of feeding links in food webs determine the ability of the network to endure perturbations that result in species loss20. Feeding links were assigned using literature- and trait-based rules to populate an interaction metaweb, which was combined with encounter probability based on species abundances on each plot22. Subsequently, we enumerated all three-species motifs in each food web and assessed their representation relative to counts in randomly rewired null-model networks to account for the relationships among plant species richness, network size, and connectance. Further, we sought to elucidate whether any effect of plant species richness on motif distribution was restricted to motifs that are grounded, that is, they contain a basal resource node (plant or other resource such as detritus)17, or whether plant species richness remotely affects the local structure of interactions among consumers at higher trophic levels. Accordingly, we examined changes in the frequency of free-floating motifs  in a sub-web for each plot containing only the arthropod consumers.Here, we examined the effect of plant species richness on the distribution of three-species motifs in replicate aboveground food webs from a long-running grassland biodiversity experiment. We constructed food webs for 80 grassland plots that were sown with a plant diversity gradient ranging from 1 to 60 plant species23. Herbivore species richness increases more rapidly with plant diversity than predator or omnivore species richness23. Thus, the herbivore:predator richness ratio increases with plant diversity4. In line with these patterns, we hypothesised that the representation of apparent and exploitative competition motifs (s4 and s5) would increase with plant species richness, with a corresponding decrease in the representation of tri-trophic chain and omnivory motifs (s1 and s2). This is because the competition motifs (s4 and s5) represent a feeding interaction that can occur between only two trophic levels. Consequently, these motifs may become relatively more common in food webs with more plant and herbivore nodes than motifs requiring an omnivore or predator (s1 and s2). Our results confirmed these hypotheses with the exception of trends in the representation of the tri-trophic chain motif (s1), providing novel insights into the assembly of food web substructures across a gradient of diversity. We contend that the observed changes in motif representation with increasing plant species richness may be due to selection against local structures that are unstable according to ecological theory.The positive effect of plant diversity on consumer species richness is strongest for consumers at low trophic levels and with a low degree of omnivoryThe total number of all motifs in the food webs increased with plant species richness Fig. , as expeFor all common configurations, the contribution of grounded motifs to the total number of motifs increased with plant species richness. This was evidenced by the number of free-floating motifs not increasing with plant species richness as quickly as the total number of motifs Fig. . However14. However, the significantly increasing overrepresentation of tri-trophic chains with plant diversity contrasted our initial hypothesis that relatively weak effects of plant species richness on predator species richness would be associated with reductions in the representation of tri-trophic chain motifs. This was perhaps due to a shift in the omnivore community towards predatory species24, or because predators could form many partially overlapping tri-trophic chains by preying on a generalist herbivore, detritivore or omnivore that was involved in numerous apparent competition interactions. We observed a similar increasing trend for both the competition motifs (s4 and s5). On average, these motifs were underrepresented at low plant diversity and overrepresented at high plant diversity  were generally overrepresented compared to the null models Fig. . This reity Fig. , the lat19. A potential reason for this inconsistency is that the role of omnivory for food web stability is highly dependent on interaction strengths and network context26. Early theoretical work suggested that omnivory would destabilise complex networks, but a range of theoretical and empirical work has more recently demonstrated the conditions under which omnivory may be stabilising, such as when embedded in larger networks or when omnivores feed adaptively27.Omnivory motifs (s2) were overrepresented compared to null models in most cases, but in contrast to the trends for tri-trophic chains s1) and competition motifs (s4 and s5), this overrepresentation significantly decreased with plant species richness Fig. . This co and comp19. However, previous work has rarely considered the species composition of motifs28, which could influence their dynamic behaviour. For instance, plants cannot engage in behaviours such as seeking refugia that can influence motif persistence29. Our experimental system revealed that plant diversity altered the trophic identity of species constituting motifs. For example, when plant nodes are few at low plant diversity, most s2 motifs were free floating . This is important because there are only limited conditions under which the intermediate consumer can coexist with the two-fold challenge of predation and competition in intra-guild predation31. In contrast, plants are generally not killed or consumed as a whole by their arthropod consumers. Consequently, future investigations will benefit from considering the traits of species in food web motifs in addition to motif prevalence.Comparative studies have shown that mixed patterns of motif representation among empirical food webs is not limited to omnivory motifs15. This exercise revealed that in isolation (i.e. when not embedded in larger networks) tri-trophic chains and competition motifs  are the most locally stable, while s3 (feed-forward loop) and all motifs with double links were unstable. Omnivory (s2) was unique in that it was moderately stable in isolation, and whether a return to equilibrium occurred depended on the sampled interaction strengths. Further, there is a correlation between the local stability of motifs and their frequency in empirical food webs; motifs s1, s4 and s5 occur more frequently than s2. Consequently, the authors suggest that these motifs are selected for because their stability offers some advantage to network function or persistence15. Based on these findings, we could make the intuitive conclusion that food webs assembling on plots with more diverse plant communities may be more robust to disturbances, as they contain relatively higher s1, s4 and s5 motif representation, and relatively lower s2 representation than the low plant diversity communities. However, the assumptions of these assessments may be violated in empirical systems because the motifs do not exist in isolation and exhibit seasonal population fluctuations rather than being at steady state.Debate currently surrounds the role and relative importance of the different three-species motifs for food web stability. Multiple recent studies have assessed the stability of three-species motifs by generating replicate Jacobian matrices based on draws of interaction strength, and assessing whether the resulting motif persists (i.e. returns to equilibrium) following a small perturbation at steady state 17. This conclusion was reached by assessing the initial number of s1, s2, s4 and s5 motifs in food webs generated with a common food web model (the niche model32) and tracking how the initial frequencies affected the long-term persistence of the entire networks with dynamic simulations. In this case, omnivory (s2) motifs contributed positively to whole network persistence, while high frequencies of both competition motifs (s4 and s5) decreased the likelihood of whole network persistence. The surprising implication of this result is, that based on network substructure, we would imply that the food webs from communities with high plant diversity are less likely to persist in the long term. We do not test any metric of network stability, so we cannot shed light on the link between our empirical results and the current state of the network stability theory. However, such a condition conflicts the fact that natural diverse communities can and do persist. Reconciliation of this potential mismatch between simulations and empirical findings will benefit from studies of food web assembly. In particular, we note that better understanding the role of dispersal, a feature not considered in the closed systems of previous modelling work, could be important. It may be that a high diversity of resources can support unstable interaction structures by being more attractive to colonists from the regional species pool. In a similar vein, omnivory motifs may be selected for at low plant diversity to enable network persistence despite these communities being less attractive to arthropod colonists.Other theoretical investigations suggest that the stability of common motifs in isolation is not directly related to their influence on whole network persistence when they are embedded in complex food webs33. We showed that our results are not sensitive to this potential scenario with an additional analysis that lowered the density-dependent probability of predator–prey interactions with increasing plant diversity  of The Jena Experiment  that were sown with 1, 2, 4, 8, 16 or 60 grass, small herbs, tall herbs and legume species in 2002 on a previously arable field20. The plant species mixtures were maintained by weeding three times per year and mowing twice annually39. The plots are arranged in four blocks corresponding to variation in soil conditions. The presence  and percentage cover of the initially sown plant species remaining on the plots in 2010 and 2012 was assessed visually on a 3 × 3 m2 area of each plot twice per year (May and August). Additional static basal resources, comprising detritus, moss, algae, fungi, dung and carrion, were assumed to be present on each plot based on previous observations.Data were collected on 80 grassland plots . This included taxa from the classes Arachnida (orders Araneae and Opiliones), Chilopoda (orders Geophilomorpha and Lithobiomorpha), Diplopoda (orders Julida and Polydesmida), Insecta  and Malacostraca (order Isopoda). Achieving a high resolution of taxonomic identification across all plots and time periods precluded the inclusion of every consumer group , so that our replicate networks represent a consistent subset of the aboveground food web. Due to the data-intensive nature of food web construction, most existing food webs do not include every species (e.g. parasites40). Traits for each consumer species were assembled through extensive literature searches24. These included body length, coarse feeding guild  and the vertical habitat stratum primarily used by the consumer .Sampling of aboveground invertebrates was conducted over two sampling periods in each of 2010 and 2012 (May/June and July/August). Ground-dwelling taxa were sampled with two pitfall traps (each 4.5 cm diameter) that were open for 20 to 29 days in each time period, and herb-layer taxa were sampled by two suction samples of an area 0.75 × 0.75 mn = 4; May/June and July/August in 2010 and 2012) by first identifying the co-occurring species from plant inventories, pitfall traps and suction samples along with the static resource nodes. However, co-occurrence does not necessarily mean that species are interacting41. Consequently, we assigned trophic interactions based on the mathematical framework for population-level interactions of Poisot et al.22. Under this framework, the probability that species i and j interact in the adjacency matrix A isN is a function defining the encounter probability of two species based on their local abundances, and T is a function giving the probability that two species actually interact based on their traits. The term ε describes higher-order effects such as the impact of the environment on interaction probability22.We constructed food webs for each plot in each time period (T) based on evidence from extensive literature searches and trait-based rules, which were used to construct a metaweb of all plausible trophic interactions21. The feeding links in the metaweb were categorised into five link types depending on how they were assigned: (1) specific feeding interactions reported in the literature; (2) generalised feeding interaction reported in the literature; (3) links based on trophic levels; (4) trait-based rules; and (5) combined trait-based rules. Generally, link types 1–3 were applied to interactions between basal resources (i.e. plants or static resource nodes) and herbivore, detritivore and omnivore consumers. An example of a generalised feeding interaction reported in the literature (link type 2) is for feeding by the beetle Chrysolina oricalcia, which is oligophagous and commonly feeds on plants in the family Apiaceae42. Link types 4 and 5 (those assigned from trait-based information) generally describe predatory (i.e. consumer–consumer) interactions. For example, the large web-building spider Coelotes terrestris preys on non-spider prey that forage on the ground44. For full details of metaweb construction see ref. 21. For each plot, the relevant rows and columns for co-occurring species were extracted, and when an interaction was identified in the metaweb, the interaction probability Tij was 1 because there is strong evidence these species will interact if they encounter each other21.We defined whether two species will interact (i.e. N depended on the link type in the metaweb and the trophic guild of the resource (i), so that interaction probability Aij isn is relative cover or abundance and α or β are scalar values. We assumed that species would always encounter one another (i.e. N = 1) in the case of species interactions that have been specifically identified in the literature  because it is likely that encounter probability operates differently for plant–consumer interactions  from ca. 2 to ca. 7 along a plant diversity gradient of 1 to 8 species8. Consequently, we selected values of α and β that maximised the median generality of omnivores in each food web at ca. 30 species  for each plot and time period, we sampled these probabilities 50 times and calculated network properties for the resulting binary food web at each iteration. Any nodes that were disconnected from a network (i.e. degree = 0) after sampling the probabilities were omitted from the calculation of network properties. The resulting plot-level trophic networks contained a mean of 20.4 to 99.0 species and mean of 36.2 to 1060.0 trophic links. The frequency of each of the 13 three-species motifs in all networks was enumerated using the triad.census function from the igraph package46 in R47 as implemented in custom functions by reference14. Each set of three species was assigned to only one motif19, the one with the highest link density. For example, three species interacting in an omnivory arrangement did not also interact as the tri-trophic chain, apparent competition and exploitative competition motifs contained within. All reported motif frequencies are the mean number from the 50 iterations of each food web. To count the number of free-floating motifs , we omitted plant and static resource nodes from the network (leaving a consumer sub-web) and repeated this process. The motif images in the figures were produced using previously published code14 and colours48.After obtaining the matrix of interaction probabilities  and degree in and out of each node (i.e. row and column sums). Thus, this process creates a null expectation by redistributing links without modifying the fundamental ecological properties of the network (e.g. plants do not eat consumers), and has previously been used in studies of food web motifs14. This null model allows us to gain insight into how the food web assembles non-randomly across the gradient of plant species richness.Network properties such as size and connectance covary with plant species richness. Therefore, we compared the number of motifs in the plot-level food webs with the number in a null model web produced by randomly rewiring the feeding links. This was achieved using the Curveball algorithmi assessed by calculating a z-score:13Xi is the frequency of motif i in the sampled web, and σi are the mean and standard deviation of the frequency of motif i in the rewired null-model networks. The z-score vector was subsequently normalised for each food web because larger networks show more extreme patterns of over- or under-representation:50j is an index over the 13 motifs. The normalised value provides information on the relative importance rather than the absolute importance of a motif to a network, which allows comparison of networks of different sizes. A positive value within the normalised profile vector (ni) indicates that the motif i was overrepresented in the observed trophic network. Z-scores are reported as the mean from the 50 iterations of each matrix of interaction probabilities.For each iteration of the probability-based food webs, we generated 250 rewired networks and calculated the representation of each of the 13 motifs 47. We analysed the effect of sown plant species richness (two-tailed test) on motif counts and normalised z-scores with linear mixed models using the lmer function of the lme4 package51. Random effects were time period (n = 4) and plot (n = 80) within experimental block (n = 4) . Motif frequencies were log transformed for normality. Lines of fit in Figs. All statistical analyses were conducted in RFurther information on experimental design is available in the Custom R scripts used to generate and analyse the data are available on GitHub (10.6084/m9.figshare.7605173.v2).Supplementary InformationPeer Review FileReporting Summary


Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells. Rapid and controlled transport and communication between the nucleus and cytosol are essential for life in eukaryotes and malfunction is linked to cancer and neurodegeneration  that GFP-tagged proteins expressed from their native promoters still localized at the nuclear envelope in old cells. In addition, we validated that changes in relative abundance of the Nups at the nuclear envelope were in line with the changes found in the proteome. We included Nup116 and Nup2 in our experiments as those Nups showed the strongest decrease in abundance . Nup133 −7 and Nup116 r = −0.56; p=6.54×10−4, see Our data contain full life histories of individual cells and, in line with previous reports , we obseTaken together, we confirmed the loss of specific FG-Nups by quantifying the localization and abundance of fluorescently-tagged Nups in individual cells during their entire lifespan. Single-cell Nup abundances at the NE can be highly variable (Nup2), while for other Nups  the loss in abundance at the NE was found in almost all aging cells and correlated with the lifespan of the cell. From the joint experiments published by A possible cause for the loss of stoichiometry could be that NPCs are not well maintained in aging. Indeed, in postmitotic cells, oxidative damage was proposed to lead to the appearance of carbonyl groups on Nups inducing more permeable NPCs . We haver < 5 nm)  in a manner that correlates with remaining lifespan . JointlyNext, we experimentally addressed the rates of transport into and from the nucleus with aging. During import and export, NTRs bind their cargoes through a nuclear localization signal (NLS) or nuclear export signal (NES) and shuttle them through the NPC. In addition to facilitating active transport, the NPC is a size dependent diffusion barrier . We measWe then looked at classical import facilitated by the importins Kap60 and Kap95, and export facilitated by the exportin Crm1. The cellular abundance of Crm1, Kap60 and Kap95 is relatively stable in aging  Figure . To testHow should we interpret the increased steady state localization of these 4 GFP reporters in aging? The steady state localization of these GFP-reporter proteins depends on the kinetics of NTR facilitated transport (import or export) and passive permeability (influx and efflux). While we cannot formally exclude that retention mechanisms appear during aging, the efflux experiments in Changes in the rates of NTR-facilitated-transport and passive permeability may be related to changes in the NPCs themselves or they may be related to an increased availability of NTRs. We measure no changes in abundance of NTRs  and findapq12  . On the apq12 . We founckground , hence w GFP-NES . To furtng aging . We founng aging . The incpetition .A previous report showed a reduction in nuclear accumulation of GFP-NLS in age 6 + yeast cells isolated from a culture , while wNext, we addressed the transport of native proteins in aged cells. We studied Srm1, the yeast homologue of Rcc1, as endogenously expressed GFP-tagged protein. Srm1 is the nucleotide exchange factor that exchanges GDP for GTP on Ran and its nuclear localization ensures that Ran-GTP levels inside the nucleus are high. The localization of Srm1 depends on Kap60/Kap95-mediated import and retention inside the nucleus via chromatin binding . While tAdditionally, we studied Msn2, a transcriptional regulator that responds to various stresses and translocates to the nucleus in pulses, a so-called frequency modulated transcription factor. Msn2 communicates information about the environment by modifying the frequency of pulses  Cai et . These pnup116, vps4pom152 and apq12 mutant strains  aging cells such as neurons and indeed a large body of data now implicate NPC function in neurodegenerative diseases. Here, we study the fate of NPCs in dividing yeast cells with the anticipation that the insights may be relevant to aging of mitotic cells such as stem cells. Overall, our data is consistent with a model where NPC assembly and quality control are compromised in mitotic aging  and wher strains . This wo strains  and 5. A strains .In a previous study, we generated directional networks to predict how the different changes observed in aging may be interdependent, and proposed that an imbalance affecting the protein biogenesis machinery is the major driving force in yeast replicative aging . The defOur work also provides the first clear example that the assembly of large protein complexes is a major challenge in aging of dividing cells. Indeed, there is data to support that loss of protein complex stoichiometry is a prominent and conserved phenotype in aging . We specSaccharomyces cerevisiae strains used in this study are listed Table 4 and were validated by sequencing. Experiments in this study were performed with BY4741 genetic backgrounds, except the deletion of apq12, which is instable in the BY4741 background. W303 apq12Δ was created by using the PCR-toolbox  (https://nl.mathworks.com/).Microscopy data was quantified with open source software Fiji (Welcome) . FluoresWelcome) . All hea8 cells were collected by centrifugation, washed in PBS and then resuspended in 500 μL of 2xPBS. To biotin-label cells, 7 mg of sulfo-NHS-LC-biotin (Pierce) was dissolved in 500 uL of ice-cold H20 and added to the cell suspension, which was subsequently incubated at RT for 20 min. The cells were pelleted by centrifugation and excess free biotin removed by washing in PBS. Biotin-labeled cells were used to inoculate 4 L of YPD and grown for ~10–12 generations. Cells were collected by centrifugation and resuspended in ice-cold PBS. The cell suspension was incubated with 250 μL of streptavidin-coated magnetic beads (Qiagen) for 20 min at 4°C. Magnetic beads with bound biotinylated cells were collected on a magnet and were washed five times with PBS. The unbound cells were used as the mixed-population (young) sample. After the final wash in PBS, cells were resuspended in a small volume of YPD.To evaluate the nuclear envelope ultrastructure of replicatively aged yeast, we cultured BY4741 in 200 mL of YPD to mid log phase. 6 × 10Yeast from either the magnetic bead sorted sample (aged) or non-binding yeast (young) were concentrated into a thick slurry by gently pelleting  and aspirating off excess media. The slurries were high-pressure frozen in a Leica HMP100. Frozen samples were freeze-substituted in a Leica Freeze AFS with 0.1% uranyl acetate in dry acetone and infiltrated with Lowicryl HM20 resin. The polymerized resin block was cut with a diamond blade into ~100 nm thick sections. Sections were collected onto a formvar/carbon coated nickel grids and stained with 2% uranyl acetate and Reynolds lead citrate for improved membrane contrast. Images were acquired on a FEI Tecnai Biotwin TEM at 80 kV equipped with a Moranda CCD camera using iTEM (Olympus) software.Following introduction of the cells to the microfluidic device, brightfield imaging was begun immediately. The process of introducing cells to the device was found to increase Msn2 activity for the first hour or two following device loading. To ensure that the baseline timecourse in young cells was representative of pulse dynamics and not affected by loading stress, fluorescence imaging was begun after cells had been allowed to acclimate for 3 hr. Brightfield images were acquired at every timepoint, with intervals of 5 min when fluorescence images were not acquired. Fluorescence images were acquired for 2 hr, at intervals of 90 s, with three z-slices of 1.5 μm. Following the fluorescence imaging, bright-field images were acquired for 8 hr to ensure that cells could be tracked and the number of daughter divisions could be scored. Cells were segmented and tracked using previously published software .Nuclear accumulation of Msn2 was quantified using a measure of skewness. Specifically, the ratio of the brightest 2% of the pixels within the cell relative to the median cell fluorescence. By normalizing to the median cell fluorescence, this is measurement is robust to photobleaching or changes in protein concentration. This measurement has been repeatedly validated and used in previous studies of transcription factor translocation dynamics . For eacIn order to model the aged NPC with the measured stoichiometry of FG-Nups from the protein abundance data see , we builwww.molinspiration.com). These software programs use the partition coefficient P  as a measure of hydrophobicity. Since the range of the ratio of concentrations is large, the logarithm of the ratio of concentrations is commonly used: Among all amino-acids, Threonine (T), Lysine (K), Proline (P) and Arginine (R) can undergo carbonylation. The change in hydrophobicity upon carbonylation of these amino-acids was calculated with the help of five hydrophobicity prediction programs    and glutamic semialdehyde (GSA), respectively, loose their positive charge and become neutral, see r-value. However, when also the charge modification is accounted for in the ‘Carbonylated’ case in r < 5 nm) is hardly affected.To explore the limited effect of carbonylation on the overall distribution of the disordered phase, we analyzed the changes in net hydrophobicity and charge. While the hydrophobicity for T and P is reduced, the hydrophobicity of K and R increases, resulting in only a 5% increase in net hydrophobicity for a maximally carbonylated NPC see . Further600 of 0.8 after which the culture was split in two portions of 50 ml; one portion was stressed by ROS by the addition of menadione (1 ml of 8 mg/ml in ethanol) for 1.5 hr, while to the other only 1 ml of ethanol was added. The cells were then harvested, washed with water and stored at - 80°C until use.100 ml of BY4741 expressing Nsp1-GFP, was grown to an ODL.lactis NZ9000 carrying a plasmid from which the yeast Heh2 ID-linker can be expressed as a GFP fusion (ID-GFP). The suspension was cleared from non-lyzed cells by centrifugation at 20,000 rcf at 4 degrees for 20 min. 1 ml of Ni-sepharose slurry was pre-equilibrated in a polyprep column (BioRad) with 10 ml of ddH2O and subsequently 10 ml of 100 mM NaPi pH7 150 mM NaCl, 10% glycerol, 5 mM DTT. The cleared lysate was mixed with the equilibrated Ni-sepharose and incubated at four degrees under mild agitation in the polyprep column. The column was subsequently drained, washed with 10 ml 10 ml 100 mM NaPi pH7 300 mM NaCl, 10% glycerol, 15 mM imidazole, 5 mM DTT and 10 ml 10 ml 100 mM NaPi pH7 300 mM NaCl, 10% glycerol, 50 mM imidazole, 5 mM DTT. The bound ID-GFP was finally eluted from the column with 100 mM NaPi pH7 300 mM NaCl, 10% glycerol, 300 mM imidazole. The buffer was exchanged to PBS with a Zebaspin desalting column (Thermo Fisher) and the protein concentration was determined using the BCA kit (Pierce). The protein was stored overnight at 4°C. (ID-GFP) was grow2O2 for 15 min at RT.Cell lysates were prepared from the cells described above in 0.5 ml lysis buffer  with 0.5 mm beads in a Fastprep machine. After bead-beating, the cells were incubated on ice for 15 min, and subsequently centrifuged at 20,000 rcf for 15 min to clear the lysates of beads and unbroken cells. The cell lysates were diluted with lysis buffer to 1 ml and 10 µl of GFP-nanotrap beads (Chromotek) were added. After 1.5 hr of incubation, the beads were washed six times with wash buffer . Bound proteins were subsequently eluted from the beads by adding 20 µl of 10% SDS to the beads and 10 min of incubation at 95°C. As a negative control a cell lysate from BY4741, not expressing any GFP-tagged nucleoporin, was treated as above. As positive controls BY4741 was spiked with 2 µg of purified ID-GFP, or 2 µg of ID-GFP which was first in vitro oxidized with 1 mM CuSO4 and 4 mM HOf the eluate, 4 µl was separated on a 10% SDS-PAGE gel, transferred to PVDF membrane and GFP-tagged proteins were detected with anti-GFP antibodies. Of the eluate, 10 µl from the immunoprecipitations was used for the oxi-ELISA. First, the SDS was removed from the sample using the HIPPR detergent removal kit (Pierce), by diluting the sample to 100 µl with PBS, and following the protocol as provided with the kit. The detergent-free protein was subsequently diluted to 1 ml with PBS and two-fold serial dilutions were prepared in PBS. 96-well Nunc maxisorp plates (Thermo Fischer) were coated with 100 µl of the serial dilutions in duplo, and incubated O/N at 4°C. Protein amounts were determined by detection of GFP-tagged bound protein with anti-GFP antibodies and a standard ELISA protocol. In a separate ELISA plate, the carbonylation state of the proteins was assessed with an Oxi-ELISA using the Oxyblott Protein oxidation detection kit (Millipore), which was essentially performed as described in Statistical parameters including the definitions and exact values of N, distributions and deviations are reported in the Figures and corresponding Figure legends. Significance of changes were determined with a two tailed Student’s t-test and with non-overlapping notches indicating 95% confidence that two samples are different. Unless mentioned otherwise, the experimental data coming from at least two independent cultures and microfluidic chip experiments were analysed together. In specific cases  the data In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michael P Rout (Reviewer #2).Thank you for submitting your work entitled "Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics" for consideration by eLife at this time.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work cannot be considered for publication in eLife would re-consider your manuscript (as a new submission and review). However, given the extend of the required work you might also want to consider to submit your manuscript elsewhere.Whereas your paper addresses an important and interesting topic, all three reviewers felt that your paper requires additional experiments in order to bolster your main conclusions. For example, this includes new result that would provide mechanistic insight into how the age-dependent changes at the NPC impact function and additional data on Nup49 and NPC assembly during aging. Since these revisions are expected to take more than 2 months to complete the editorial decision was to reject the manuscript. However, if you decided to address these concerns Reviewer #1:eLife paper. The concept that NPC function changes in aged cells presented in this manuscript also fits with previous reports of non-functional NPCs being retained in mother cells and changes in NPC transport function during ageing .Rempel et al. have applied single cell imaging technique to address age-dependent changes in NPC form and function, building off observations originally described in their 2015 eLife.In this paper, data is presented to further this correlate between ageing and NPC function, focusing in part on Nup116 and Nsp1, leading to a conclusion that aged cells show increased compartmentalization based on GFP-NLS/NES reporters and the localization of endogenous shuttling proteins. However, it remains unclear if and/or how nuclear permeability is being impacted to increase compartmentalization. For example, if the amount or types of cargo being actively shuttled changed in ageing cells, could this result in more efficient shuttling of GFP-NLS/NES cargos and the apparent increase in compartmentalization, as compared to the interpretation that permeability is changing? In the specific case of Msn2, which is seen to change shuttling dynamics, how other changes in aged cells impact these dynamics is unclear, making the interpretation of the data in Figure 4 difficult. For example, does the interaction of Msn2 with DNA change causing increased nuclear residence times? Do other modulators that contribute to Msn2 localization change in aged cells, such as cAMP/PKA that influence the sensitivity and frequency of nuclear localization (PMID 12732613). In addition, it is unclear how Nup116 and Nsp1 fit into this phenotype and ultimately if these changes in NPC function are drivers of ageing or simply the consequence of other cellular processes declining in function. These findings are interesting, but without offering direct mechanistic insights, I would not support publication of the current manuscript in Reviewer #2:In previous work, a number of the authors comprehensively surveyed changes in the transcriptome and proteome of yeast cells as they replicative age, discovering that an increase in proteins that are part of the protein biogenesis machinery relative to their transcripts seemed causally associated with other characteristic ageing phenotypes, and that one of the downstream consequences of this was a loss of stoichiometry for numerous protein assemblies, including the nuclear pore complex (NPC).Here, these findings on the NPC are extended by a detailed examination of the alterations in its composition and function that occur during aging. The abundance of a significant number of key nucleoporins decreases, both at a whole cell level and  at the nuclear envelope. Importantly, the authors establish that the impact of aging on nuclear transport is largely through an age-dependent impediment in proper NPC assembly, rather than through (as previously indicated by others) oxidative damage to NPC components. The authors present numerous lines of evidence indicating that the accumulating misassembled NPCs appear to be rendered impermeable, thus reducing the effective density of functional NPCs over time, leading to an increase in steady state compartmentalization and concomitant decrease in transport dynamics. This detailed focus on the age-related functional consequences of stoichiometric loss for one particular macromolecular assembly provides a new and key insight into the consequences of ageing of dividing cells.However, several points need to be clarified by the authors to help the readers navigate the logic of the manuscript and the technical details of the experimental work.1) The main conceptual point that is not sufficiently clear throughout the text (including the Abstract), potentially leading general readers to confusion, is the causal relationship between aging and NPC malfunction. In previous publications, the authors showed that an imbalance affecting the protein biogenesis machinery seem to be the major driving force in aging, as beautifully summarized in Figure 6B of Janssens et al., 2015. It would be interesting for the authors to put the NPC and nuclear transport machinery in context by indicating where they would localize in such a schematic based on their previous results: is the NPC ranked in the "causal" or "responsive" areas? This would help the reader interpret the results and properly position the NPC defects within the overall aging process.2) The authors decided to validate the changing Nup levels detected previously in their whole proteome analysis by quantifying the NE signal from fluorescently tagged Nups in aging mitotic cells. The approach is very reasonable, but several points are not clear, could affect the interpretation of the results and should be clarified in the manuscript:a) What were the criteria used to select the Nups tagged for the fluorescence quantification experiments? Why those and not others? Why not Nsp1, that is specifically referred to later ? This should be made clear in the text.b) Why did the authors picked Nup49 as the reference throughout the fluorescence quantification experiments? I noticed that several Nups, crucially including Nup49, seem to be missing from the original dataset and from the plot in Figure 1C. Is this a labeling issue or Nup49 was not included in such analysis? If so, why is the Nup chosen to act as a normalization reference in most of the experiments? Given that it seems the authors lack the initial information to establish it as a reliable and useful control, would it be informative to plot the raw data without normalizing to Nup49 to get an idea of what is the effect of such normalization?c) According to Figure 4—figure supplement 3, overexpression (and as such should be noted in the text) of basically any construct, seems to affect the lifespan of yeast cells. However, how does tagging of Nups affect such lifespan? It is well known that double tagging could generate synthetic effects that could potentially affect the strain fitness. I would strongly suggest that the authors perform a similar analysis as the one shown in Figure 4—figure supplement 3 for the GFP and mCherry tagged strains used in this study versus a non-tagged (wild type) strain. This way, even if some effects were detected, they could be accounted for in the final plots.Reviewer #3:In previous studies, the Veenhoff and Heinemann groups have assessed transcriptome and proteome variations in aging populations of yeast cells as a means to interrogate physiological changes in aging mitotic cells. Using data from this previous study, the authors show, in the early figures of this manuscript, that aging populations of cells appear to exhibit changes in protein levels of specific nucleoporins and nuclear transport factors. They suggest that specific changes in Nup levels and the presumed loss of stoichiometry among Nups reflect problems in NPC assembly. As supporting evidence for this conclusion, they present data showing that levels of Heh2-GFP and Apq12-GFP, two proteins that have been previously linked to NE biogenesis and NPC assembly, are reduced relative to Nup49. The authors document direct correlations between a decreased lifespan of individual cells and various parameters such as decreased levels of Nup116 and Nup100, as well as non nups including Heh2, Apq12 and Vps4. Based on these observations, they have attempted to examine nuclear transport changes in individual cells as they age. They showed that steady-state distributions of both import and export cargoes were altered as cells aged, notably showing increased compartmentalization.Overall, this is an interesting study that has applied innovative single-cell analysis approaches to interrogate NPCs and nuclear transport in aging cells. In my view, the two most significant observations reported are age-dependent variations in Nup protein levels and changes in steady-state distributions of nuclear transport cargos as cells age. However, while these are interesting observations, the study fails to provide any mechanistic insight into the interrelationship between these observations . In addition, the authors' main hypothesis that changes in nuclear transport during aging are caused by defects in NPCs assembly is not supported by direct evidence of increased NPC assembly defects in aging cells . In addition, they have not tested whether the observed alterations in the levels of specific Nups or proteins linked to NPC assembly (such as Vps4 and Apq12) are sufficient for altering NPC assembly or nuclear transport. Finally, is there a causal relationship between changes in NPCs/transport and aging? The authors suggest their studies have established such a relationship; however, I feel that, while their data have established interesting correlations, functional insight is limited.Several additional points are listed below.1) No data are presented on the levels of Nup49 during aging . As this Nup represents an important standard of comparison for various analyses in the manuscript, the authors need to discuss what the proteome analysis of aging cells revealed about Nup49.2) As suggested above, there is no direct evidence that aging is accompanied by defects in NPCs assembly nor is there any evidence that there is an increase in herniations or the sealing over of NPCs in older cells . Moreover, while null mutants of heh2, vps4 or apq12 exhibit NPC herniation phenotypes, there is no evidence that decreases in the levels of Heh2, Apq12, and Vps4 observed in the aging cells would compromise NPC assembly. This concern also applies to changes in Nup levels and their effects.3) The authors state that the "rates of NTR-facilitated-transport (import and export) and passive permeability (influx and efflux) are altered such that in old cells the kinetics of passive permeability is lowered relative to the kinetics of NTR-facilitated-transport." This conclusion is not justified by the data presented. First, no data on the kinetics (or apparent kinetics) of transport are presented in this manuscript to support this conclusion. Measuring steady-state levels of transport cargoes does not provide information on the kinetics of transport. Moreover, the authors conclude that cargo retention mechanisms are minimal contributors to the steady state distribution of cargo. It is not clear to me how the authors make this conclusion. While the data in Figure 4—figure supplement 1 suggest there is no change in efflux rates during aging, this does not address retention.4) Please provide an interpretation of results show in Figure 1—figure supplement 2C. The data presented in this figure suggest the tag (either GFP or mCh) placed on the Nup (49 or 133) can alter its relative levels during aging.5) In Figure 3B, the authors state that they exclude outliers; however, the justification for the assignment of these data points as 'outliers' is not clear.6) In a number of instances, the authors could more accurately reference their statements. For example, the authors reference Webster et al., 2014 for their statement that 'Misassembled NPCs that are induced by mutations are asymmetrically retained, and accumulated in the mother cell over time'. Prior studies performed by Makio et al., 2013, and Colombi et al., 2013, showed Nup mutants retain NPCs in mother cells. Also, regarding the statement: "there are no strains with mutations in the NPC that have been shown to lead to increased steady state compartmentalization", there are report of mutants in Nups that cause increased import in mitotic yeast cells . [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:[…] It remains unclear if and/or how nuclear permeability is being impacted to increase compartmentalization. For example, if the amount or types of cargo being actively shuttled changed in ageing cells, could this result in more efficient shuttling of GFP-NLS/NES cargos and the apparent increase in compartmentalization, as compared to the interpretation that permeability is changing? In the specific case of Msn2, which is seen to change shuttling dynamics, how other changes in aged cells impact these dynamics is unclear, making the interpretation of the data in Figure 4 difficult. For example, does the interaction of Msn2 with DNA change causing increased nuclear residence times? Do other modulators that contribute to Msn2 localization change in aged cells, such as cAMP/PKA that influence the sensitivity and frequency of nuclear localization (PMID 12732613).The reviewer is concerned that our interpretation that the increased steady state distributions of GFP-reporter proteins is related to the NPCs of aged cells is not the only possible interpretation. We agree that explicit discussion and testing of the alternatives, which had not escaped our attention, is important and we thank the reviewer for pointing this out. The alternative interpretations that we had considered and that we now address and discus  are (i) increasing levels of NTRs , (ii) retention of GFP e.g. as a consequence of aggregation , (iii) decreasing levels of native cargo .In addition, to substantiate that NPCs are indeed the problem in ageing we included new data on two more GFP-NLS reporter proteins, namely with the Nab2NLS (Kap104 import cargo), and a Pho4NLS (Kap121 import cargo) . The changes in ageing that we observe are similar to those of GFP-cNLS, namely increased steady state nuclear accumulation in aged cells. We further strengthen the link between assembly and transport by showing that Brl1 levels decrease during aging  and that NPC assembly problems can increase nuclear compartmentalization in a vps4Δheh2Δ background . Most significantly, we now provide direct evidence that aged cells have more herniations . Altogether, in the new submission we explicitly discuss or test alternative interpretations and provide important additional data supporting the interpretation that altered transport dynamics are related to the NPCs.Indeed, we agree that it is possible, that Msn2 dynamics  are influenced by other factors than active transport (most prominently retention) and this is why reporter studies provide a good complement. We state this clearly in the text . With regards to the specific suggestion of altered interactions with the DNA: if that were the case we would have expected to observe an increase in peak prominence – which we don’t observe.In addition, it is unclear how Nup116 and Nsp1 fit into this phenotype and ultimately if these changes in NPC function are drivers of ageing or simply the consequence of other cellular processes declining in function.It was unclear to the reviewer how we connect the decreases in Nup116 and Nsp1 to the transport phenotypes observed. Indeed from previous studies using mutants lacking specific FG-Nups (or FGrepeat regions), as well as from our computational modeling of an aged-NPC mimic , it is expected that NPCs are more permeable if they would miss some of the FG-Nups that we show are decreasing in abundance in aged cells. Increased permeability is indeed not what we observe. We connect both phenotypes by showing that aged cells have assembly problems  and increasing numbers of herniation with transport inert NPCs  and by proposing that aged cells consequently have fewer functional NPCs.With respect to causality we have indeed not been clear enough and we improved this in the second paragraph of the Discussion section. We now make clear that our findings support the hypothesis that, rather than initiating the ageing process, changes at the NPC further drive the aging process. This is supported by our previous system wide models predicting causal relationships as Nup116 and Nsp1  appear rather in the middle clusters of the proteome network meaning they are predicted to be consequential to earlier changes but also that they are driving later changes.These findings are interesting, but without offering direct mechanistic insights, I would not support publication of the current manuscript in eLife.With the extensive additional data now provided, we are the opinion that our data more than sufficiently supports the mechanistic model proposed which was nicely summarized by reviewer 2, “The authors present numerous lines of evidence indicating that the accumulating misassembled NPCs appear to be rendered impermeable, thus reducing the effective density of functional NPCs over time, leading to an increase in steady state compartmentalization and concomitant decrease in transport dynamics." For clarity we summarize the model in the new Figure 6.Reviewer #2:[…] Several points need to be clarified by the authors to help the readers navigate the logic of the manuscript and the technical details of the experimental work.1) The main conceptual point that is not sufficiently clear throughout the text (including the Abstract), potentially leading general readers to confusion, is the causal relationship between aging and NPC malfunction. In previous publications, the authors showed that an imbalance affecting the protein biogenesis machinery seem to be the major driving force in aging, as beautifully summarized in Figure 6B of Janssens et al., 2015. It would be interesting for the authors to put the NPC and nuclear transport machinery in context by indicating where they would localize in such a schematic based on their previous results: is the NPC ranked in the "causal" or "responsive" areas? This would help the reader interpret the results and properly position the NPC defects within the overall aging process.We thank the reviewer for this recommendation. We have indeed looked for the presence of Nups, NTRs and assembly factors in the proteome and transcriptome networks to see if they would be present in the more causal or the more responsive clusters.Apart from five exceptions, the Nups, NTR and assembly factor mRNA profiles are stable in ageing and hence they are not present in the transcriptome network (flat profiles cannot be predictive of changes). Five transcripts are found in the transcriptome network, namely, Nmd5 in cluster 2, Apq12 in cluster 3, Brl1 in cluster 5, Brr6 in cluster 7, and Mlp1 in cluster 8, where cluster 1 is most causal and cluster 9 is most responsive.Within the proteome network the Nups, NTRs and assembly factors are better represented  and they mostly appear in the more responsive clusters three to five, where cluster 1 is most causal and cluster 5 is most responsive. Specifically, eight Nups are present in proteome network, namely Ndc1 in cluster 2, Nup159 and Nsp1 in cluster 3, Nic96, Nup82 and Nup157 in cluster 4 and Nup116 and Nup2 in cluster 5. Two NTRS  and one assembly factor (VPS4) are present in proteome network in clusters 3 and 5.Altogether, the transcriptional changes in Nups, NTRs and assembly factors are small and thus unlikely important in ageing. The proteome changes in the Nups however are linked to other changes in ageing and are predicted to appear as a response to earlier more causal changes in the proteome, while also driving further changes in ageing. In the Discussion of the current manuscript we make clear that changes at the NPC are predicted to be consequential to earlier changes but also that they drive later changes . We also adjusted the Abstract.2) The authors decided to validate the changing Nup levels detected previously in their whole proteome analysis by quantifying the NE signal from fluorescently tagged Nups in aging mitotic cells. The approach is very reasonable, but several points are not clear, could affect the interpretation of the results and should be clarified in the manuscript:a) What were the criteria used to select the Nups tagged for the fluorescence quantification experiments? Why those and not others? Why not Nsp1, that is specifically referred to later ? This should be made clear in the text.We now explain why we picked those Nups specifically and why Nsp1 is not included in the analysis . Namely, we included Nup116 and Nup2 in our experiments as those Nups showed the strongest decrease in abundance. Nup133 was included because its abundance was stable in ageing and Nup100 was included because it is important for the permeability barrier. We used Nup49-mCh as a reference in all of our microfluidic experiments as Nup49 had previously been used as a marker for NPCs. The proteome data indicated that Nup49 showed a relatively stable abundance profile in aging . The tagging of the Nups with GFP and mCherry (mCh) reduced the fitness of those strains to different extents but all retained median division time under 2.5 hours . Nsp1 could not be included in the validation, because the Nsp1-GFP fusion had a growth defect and could not be combined with Nup49-mCh, Nup100-mCh or Nup133-mCh in the BY4741 background.b) Why did the authors picked Nup49 as the reference throughout the fluorescence quantification experiments? I noticed that several Nups, crucially including Nup49, seem to be missing from the original dataset and from the plot in Figure 1C. Is this a labeling issue or Nup49 was not included in such analysis? If so, why is the Nup chosen to act as a normalization reference in most of the experiments? Given that it seems the authors lack the initial information to establish it as a reliable and useful control, would it be informative to plot the raw data without normalizing to Nup49 to get an idea of what is the effect of such normalization?We now show the proteome data for Nup49 and three other NPC components that were lacking from Figure 1C . They were lacking from Figure 1C because they did not make it through the full data processing pipeline used in Janssens et al. Specifically for Nup49 the problem was that the correction that we applied to account for losses of proteins on the bead surface yielded negative values, which precluded further data processing. The raw profiles for both Nup49 replicates are however still useful and shows that Nup49 levels are rather stable in the ageing proteome.Nup49-mcherry is used as an internal reference. The dilemmas with long time lapse experiments is phototoxicity and hence we chose to only image few z-stacks. Also instrument time is an issue and hence we measured on different setups. As a consequence the absolute intensities are variable due to instrument differences and as the NE is not equally well in focus in all images. Quantifying the ratio’s between two Nups solves these issues and provides a more reliable readout. Relating everything to Nup49-mCherry also solves a third complication, which is that the fluorophore tags GFP and mCherry behave differently in ageing. This was shown in Figure 1—figure supplement 2E where the same Nup tagged with mCherry of GFP show differential absolute increases. In a separate manuscript in preparation we will detail these age-related differences in fluorophore behavior related to pH and maturation. We took out two of the panels from Figure 1—figure supplement 2 because they seem to have caused confusion.c) According to Figure 4—figure supplement 3, overexpression (and as such should be noted in the text) of basically any construct, seems to affect the lifespan of yeast cells. However, how does tagging of Nups affect such lifespan? It is well known that double tagging could generate synthetic effects that could potentially affect the strain fitness. I would strongly suggest that the authors perform a similar analysis as the one shown in Figure 4—figure supplement 3 for the GFP and mCherry tagged strains used in this study versus a non-tagged (wild type) strain. This way, even if some effects were detected, they could be accounted for in the final plots.We have indeed found that tagging of the Nups affects the fitness of our strains. Performing proper lifespan on all mutants would represent a significant effort and much microscopy time, which we do not have available. Instead, we have included a panel in Figure 1—figure supplement 2A that compares the median number of divisions during the first 15 h in the chip to give a measure of the fitness of the cells.Reviewer #3:[…] Overall, this is an interesting study that has applied innovative single-cell analysis approaches to interrogate NPCs and nuclear transport in aging cells. In my view, the two most significant observations reported are age-dependent variations in Nup protein levels and changes in steady-state distributions of nuclear transport cargos as cells age.However, while these are interesting observations, the study fails to provide any mechanistic insight into the interrelationship between these observations . In addition, the authors' main hypothesis that changes in nuclear transport during aging are caused by defects in NPCs assembly is not supported by direct evidence of increased NPC assembly defects in aging cells . In addition, they have not tested whether the observed alterations in the levels of specific Nups or proteins linked to NPC assembly (such as Vps4 and Apq12) are sufficient for altering NPC assembly or nuclear transport.In the new manuscript we better clarify and support that NPC assembly defects can connect the observations of the age-dependent variations in Nup protein levels and the changes in steady-state distributions of nuclear transport cargos as cells age. In the new version of the manuscript, we additionally show that Brl1 levels decrease during aging  and that NPC assembly problems can increase nuclear compartmentalization in a vps4Δheh2Δ background . Most significantly, we now provide direct evidence that aged cells have more NE herniations .The reviewer is further concerned that the observed alterations in the levels of specific Nups or proteins linked to NPC assembly may not be sufficient to alter NPC assembly or nuclear transport. Addressing this experimentally would require creating a strain where the expression levels of multiple Nup and four assembly factors is altered simultaneously (an aged-NPC mimic in young cells); this clearly falls beyond the scope of this project and may in fact be close to impossible to do.Finally, is there a causal relationship between changes in NPCs/transport and aging? The authors suggest their studies have established such a relationship; however, I feel that, while their data have established interesting correlations, functional insight is limited.This is also discussed in our answers to reviewer 2 point 1: we have clarified in the Discussion that the current data and the previous system wide study suggest that NPC defects rather further drive the aging process than initiating it.Several additional points are listed below.1) No data are presented on the levels of Nup49 during aging . As this Nup represents an important standard of comparison for various analyses in the manuscript, the authors need to discuss what the proteome analysis of aging cells revealed about Nup49.We have included this data in Figure 1—figure supplement 1D; please see also our explanation to reviewer 2.2) As suggested above, there is no direct evidence that aging is accompanied by defects in NPCs assembly nor is there any evidence that there is an increase in herniations or the sealing over of NPCs in older cells . Moreover, while null mutants of heh2, vps4 or apq12 exhibit NPC herniation phenotypes, there is no evidence that decreases in the levels of Heh2, Apq12, and Vps4 observed in the aging cells would compromise NPC assembly. This concern also applies to changes in Nup levels and their effects.This important point is addressed in response to reviewer 3’s initial comments.3) The authors state that the "rates of NTR-facilitated-transport (import and export) and passive permeability (influx and efflux) are altered such that in old cells the kinetics of passive permeability is lowered relative to the kinetics of NTR-facilitated-transport." This conclusion is not justified by the data presented. First, no data on the kinetics (or apparent kinetics) of transport are presented in this manuscript to support this conclusion. Measuring steady-state levels of transport cargoes does not provide information on the kinetics of transport. Moreover, the authors conclude that cargo retention mechanisms are minimal contributors to the steady state distribution of cargo. It is not clear to me how the authors make this conclusion. While the data in Figure 4—figure supplement 1 suggest there is no change in efflux rates during aging, this does not address retention.in ageing is technically very challenging and impossible at present. The efflux experiments show to the least that the GFP reporters do not aggregate in the nucleus of aged cells (and that the NE is not more permeable).We have adjusted the mentioned statement and explicitly discuss the meaning of the steady state measurements in the third paragraph of the subsection “Increased steady state nuclear compartmentalization in aging is mimicked in NPC assembly mutants”. Also we added additional data on two more GFP-NLS reporters . What is requested, direct kinetic measurements of import and export 4) Please provide an interpretation of results show in Figure 1—figure supplement 2C. The data presented in this figure suggest the tag (either GFP or mCh) placed on the Nup (49 or 133) can alter its relative levels during aging.We have removed this panel as it caused confusion, also for reviewer 2. We have no reason to think that the tag alters the actual protein levels in ageing. We do have data to show that the two fluorophores themselves have different behaviors in ageing due to differences in pH and maturation time. As mentioned before, we are preparing a manuscript to detail these effects.5) In Figure 3B, the authors state that they exclude outliers; however, the justification for the assignment of these data points as 'outliers' is not clear.th/25th percentile +/-We have clarified in the manuscript that no outliers were excluded from the analysis. We merely plotted points which were outliers individually. Outliers, are data points outside of the 751.5 times the inter quartile range.6) In a number of instances, the authors could more accurately reference their statements. For example, the authors reference Webster et al., 2014 for their statement that 'Misassembled NPCs that are induced by mutations are asymmetrically retained, and accumulated in the mother cell over time'. Prior studies performed by Makio et al., 2013, and Colombi et al., 2013, showed Nup mutants retain NPCs in mother cells. Also, regarding the statement: "there are no strains with mutations in the NPC that have been shown to lead to increased steady state compartmentalization", there are report of mutants in Nups that cause increased import in mitotic yeast cells .We apologize for these mistakes and have corrected our statements and included the reference in our manuscript.