As the novel coronavirus (SARS-CoV-2) continues to spread rapidly across the globe, we aimed to identify and summarize the existing evidence on epidemiological characteristics of SARS-CoV-2 and the effectiveness of control measures to inform policymakers and leaders in formulating management guidelines, and to provide directions for future research. We conducted a systematic review of the published literature and preprints on the coronavirus disease (COVID-19) outbreak following predefined eligibility criteria. Of 317 research articles generated from our initial search on PubMed and preprint archives on 21 February 2020, 41 met our inclusion criteria and were included in the review. Current evidence suggests that it takes about 3-7 days for the epidemic to double in size. Of 21 estimates for the basic reproduction number ranging from 1.9 to 6.5, 13 were between 2.0 and 3.0. The incubation period was estimated to be 4-6 days, whereas the serial interval was estimated to be 4-8 days. Though the true case fatality risk is yet unknown, current model-based estimates ranged from 0.3% to 1.4% for outside China. There is an urgent need for rigorous research focusing on the mitigation efforts to minimize the impact on society. The coronavirus disease 2019 (COVID-19) outbreak, which originated in Wuhan, China, has now spread to 192 countries and administrative regions infecting nearly 800,000 individuals of all ages as of 31 March 2020 . GloballPosing a significant global health threat, COVID-19 has drawn unprecedented attention from public health researchers around the globe, with more than 200 research articles published in academic journals in two months. There are also nearly 150 epidemiological and modeling preprints on COVID-19. Many of these articles seek to investigate the epidemiological parameters of the disease at different locations to disseminate critical information among both modelers and policymakers for a timely control response to be implemented. This is particularly crucial as the current outbreak involves a new pathogen (SARS-CoV-2), on which limited knowledge exists of its infectivity and clinical profile.The current estimates from this research vary widely, partly due to the differences in analytical methods and assumptions. This variance is also reflected in the estimates on the effectiveness of public health interventions implemented worldwide. As the COVID-19 outbreak is at a decisive point—as pointed out by the Director-General of the WHO on 27 February 2020—it is imperative to synthesize all existing evidence available to date and summarize the key findings to identify research gaps and to assist policymakers in evidence-based decision making for better pandemic preparedness.Based on a framework of parameter requirements and necessary modeling efforts outlined by Cowling and Leung , we perfIn this systematic review, we identified studies that describe or assess the transmission dynamics, severity, and susceptibility of COVID-19 and that examine the impact of early control measures.We selected research articles that contain estimates for at least one of the following epidemiological parameters: (i) the size of the epidemic, (ii) the epidemic doubling time, which is defined as the time it takes for the number of cases to double in the epidemic, (iii) the basic reproduction number F=1−Of 317 articles generated from the initial search on PubMed and preprint archives, we identified 41 epidemiological and modeling studies on the COVID-19 outbreak in 2019–20 through title and abstract screening. The detailed selection process is illustrated in Current estimates for the epidemic size in Wuhan largely varies, ranging from 12,400  to 75,815  for published articles and from 18,566  to 58,956  for preprints, by the 4th week of January 2020 . The epiThree published studies have estimated the final epidemic size in Wuhan. An early analysis based on a mathematical model and recent travel data estimated that more than 75,815  individuals had been infected in Wuhan by 25 Jan 2020, as the epidemic doubled in size every 6.4 days [We also reviewed three preprints that included the final epidemic size in Wuhan and China. A preprint study found that the median estimate for the total number of infections in Wuhan was 58,956  before the travel ban was implemented on 23 January 2020 . The artOverall, these estimates support an ascertainment rate of 2.22–35.58%, providing evidence of a large number of non-severe cases being undocumented by healthcare systems.Current estimates of the mean  and 3.0 . The estThe highest y period . AssuminWe identified six published articles and two preprints that reported the estimated incubation period distribution based on the epidemiological data collected from China and several other countries .All nine estimates extracted from eight published literature and preprints have the mean (or median) and uncertainty of shorter than 13 days .th percentile of the distribution at 12.5 days (9.2–18) [Current estimates for the mean or median incubation period range from 4 to 6 days, comparable to SARS-CoV (4.4 days)  and MERS(9.2–18) . Another(9.2–18) . A recen(9.2–18) .Similarly, a preprint using cases diagnosed in China (excluding Wuhan) and other countries reported a median incubation period of 5.2 days (4.4–6.0) , which iCurrent estimates of the mean serial interval for COVID-19 ranges from 5 to 8 days from two published studies, and ~4 to 5 days based on four preprints .A relatively short serial interval of ~4 to 5 days was estimated from recent preprint studies. An analysis of 468 infector–infectee pairs confirmed in China reported a mean serial interval of 3.96 days (3.53–4.39) . The stuThere is limited information from published literature on susceptibility regarding neutralizing immunity. Existing evidence suggests that everyone  who has had close contact with an infected individual is susceptible to COVID-19. According to recently published literature, which described demographic and clinical characteristics of 44,672 laboratory-confirmed patients in China , COVID-1Current evidence suggests that older individuals and those with compromised immune systems from pre-existing conditions are more likely to develop severe forms of COVID-19. To date, few modeling studies have examined the case fatality risk (CFR) mostly because the outbreak is in the early phases in most countries, and considerable uncertainty exists in regards to the ascertainment rate and asymptomatic rate.The crude CFR of COVID-19 from confirmed cases in China largely varied from 2% to 15%. Earlier analyses using a relatively small number of laboratory-confirmed patients in Wuhan in January reported an overall CFR of 11%  and 15% We identified one published study and four preprints that estimated the CFR in China. In an early analysis, an estimated confirmed case fatality rate (cCFR) of 5.3% (3.5–7.5%) to 8.4% in China (5.3–12.3%) was provided using an exponential growth spread model with case reports collected up to 24 January 2020 . A CFR oOne published study and one preprint reported modeling-based estimates of the CFR outside China ranging from 0.3%  to 1.4% We identified three published studies and four preprints of modeling interventions for COVID-19. These measures include non-pharmaceutical interventions, quarantine, and mobility reductions on the population level, and airport screening.We found a published study and two preprints that examined the impacts of the travel restriction policies, which were implemented in Wuhan on 23 January 2020 as part of the efforts to contain the further spread of COVID-19. Using a deterministic SEIR model, a published study  estimateWe found three published studies and one preprint that examined the impacts of social distancing and other non-pharmaceutical interventions  on the epidemic trajectories. A mathematical modeling study  showed tMore recently, a modeling study in Singapore which simulated the impact of different control measures found that workplace distancing was more effective in reducing the spread of COVID-19 than school closure . AccordiOne published study and one preprint examined the effectiveness of airport screening in containing the spread of COVID-19. A simulation study reported that ~46% (95% CI: 36–58) of infected travelers would not be detected through airport screening . The stuThe rapid spread of COVID-19 with pandemic potential poses one of the most significant global challenges in recent years. With more than 300 scientific reports and articles on this topic published in the past two months, it is crucial to disseminate the main findings through a comprehensive review of the existing evidence. In this review, we extracted and synthesized key epidemiological, demographic, and clinical features of COVID-19 from the published literature and preprints available to date.Our findings suggest that the true size of the epidemic is much larger than what has been reported worldwide, though these figures largely depend on the effectiveness of the control measures. The outbreak is growing fast with an infected individual infecting two to three other persons on average and doubling in size every 3 to 7 days. While the incubation period ranges from 3 to 6 days based on eight published literature and preprints, its mean (or median) is most likely to be around 5 days on average, which is similar to that of other coronaviruses, such as SARS-CoV (4.4 days)  and MERSCurrent estimates for the mean serial interval for COVID-19 range from 4 to 8 days from published articles and prints. However, it should be noted that the sample size is relatively small (six and seven pairs) for both of the published articles in which the mean serial interval was estimated to be between 5 and 8 days ,29, respWhile the true CFR of SARS-CoV-2 still remains uncertain with modeling-based estimates largely varying between studies, it is found to be less severe than other coronaviruses, such as SARS-CoV (9.6%)  and MERSControl measures such as quarantine, travel restrictions, and airport screening for travelers have been widely implemented to contain the spread of infections. The effectiveness of these containment measures in controlling the outbreak, however, remains inconclusive. Current evidence from modeling studies on COVID-19 suggests that travel restrictions leading to reduced transmissibility can be highly effective in containing the spread. While school closure is less effective than workplace distancing or quarantine of exposed individuals, a combined strategy which implements all three measures together was found to be most effective in reducing the spread. Airport screening is shown to be not as effective either, detecting only 34%  to 54%  of infecSome important limitations should be noted. In this review, we included preprints that are “in-press” or have not yet been peer-reviewed, which might impair the overall quality of the review to a certain extent. At the time of writing, however, there is still very limited information on some of the key epidemiological parameters of COVID-19. For example, we found only two published studies with an estimate for the serial interval using a small number of samples. Including five more estimates from preprints gave greater confidence of the means and distributions for the epidemiological parameters as they had bigger sample sizes with more data becoming available. It should also be noted that of preprints, we included only those that provided sound rationales for the methodology they used in the form of a complete manuscript.Another potential limitation is that most of the studies included in this review are based on data collected during the early phase of the outbreak in China. As COVID-19 is rapidly evolving, these early estimates may change as more information is collected. We believe that additional research using case reports from other countries would be extremely useful since different demographic and cultural characteristics of the population may play an important role in determining the outbreak trajectories and clinical outcomes at the population level.More rigorous research to estimate the effects of other control measures currently implemented in many affected countries, such as social distancing or school closure, could also provide important evidence for countries with sustained human-to-human transmission. As few suspected cases of reinfection have been reported in China, Japan, and South Korea, serologic studies, which examine the possibility of reinfection (or reactivation of the virus) among recovered individuals, should also be made an urgent research priority.Understanding the epidemiology and transmission dynamics of an emerging infectious disease is a key for successful outbreak control. As the COVID-19 pandemic continues to rapidly spread across continents, there is an urgent need for more rigorous research focusing on mitigation strategies (shift from containment). Here we disseminate key findings of epidemiological parameters from the literature at this time point, which can be used by modelers and policymakers for epidemic planning purposes.


The North American cheetah population serves as a reservoir for the species, and acts as a research population to help understand the unique biology of the species. Little is known about the intrauterine physiology of the cheetah, including embryo differentiation, implantation, and the development of the placenta. After mating, cheetah females frequently experience (30–65% of matings) a non-pregnant luteal phase where progestogen metabolite levels match those found in pregnant females for the first ~55 days of gestation, but parturition does not occur. Immunoglobulin J chain (IgJ) is a molecule that is involved in the activation of the secretory immune response and has been found to be indicative of pregnancy in the cheetah using fecal monitoring. In this study, western blotting was employed to track IgJ abundance in pooled weekly fecal samples following natural breeding or exogenous stimulation to ovulate, and IgJ levels were compared between individuals undergoing a pregnant (n = 12) and non-pregnant (n = 19) luteal phase. It was revealed that IgJ abundance was increased in pregnant females compared to non-pregnant females at week 4 and week 8 post-breeding, indicating the potential modulation of maternal immunity in response to sensitive events such as implantation and the increased secretory activity of the placenta. IgJ levels also tended to be higher early after breeding in females that were bred naturally with intact males compared to exogenously stimulated females with no exposure to seminal plasma, potentially indicating a response to the act of intromission or the stress of breeding, or possibly demonstrating an immune response resulting in the promotion of maternal tolerance to seminal antigens present upon embryonic implantation. Monitoring fecal IgJ may be a potential method to determine gestational status in the cheetah and will aid future conservation efforts of the species. Acinonyx jubatus) is listed as vulnerable by the International Union for Conservation of Nature (IUCN), with an estimated population of ~7100 individuals in the wild, and numbers continuously decreasing due to habitat fragmentation and human conflict [ex situ population is critical to serve as an insurance population should the wild cheetah’s numbers diminish further. The ex situ population can serve as a reservoir for the species and could potentially be used for reintroduction efforts in the future. The insurance population is also invaluable for research purposes, allowing for studies that cannot be conducted on in situ populations due to the scarcity of the species in the wild.The cheetah . The animals included in the study were female adults from 2 to 12 years of age (mean ± standard error of the mean (SEM) = 6.0 ± 0.6 y). Several females included in this analysis bred multiple times throughout the course of the study, and as a result have multiple breeding periods included. The animals in this study were fed a diet of commercial beef or horse-based meat product  a minimum of five days per week, with supplements that included whole rabbit, beef and horse bone, or organ meat. Water was available ad libitum.This study was conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The female cheetahs included in this study  were all housed at accredited Association of Zoos and Aquariums (AZA) institutions within the United States. All subjects were born Fecal samples were collected opportunistically from females that were naturally bred according to SSP breeding management recommendations and from females receiving exogenous gonadotropins to stimulate ovulation. Exogenous gonadotropin therapy was conducted under IACUC approval for separate projects according to previously published methods , 15, and3H-progesterone prior to shaking extraction. The mean extraction efficiency (±SEM) was found to be 73.6% ± 0.2% for all samples.Fecal samples were collected non-invasively from enclosures approximately 3–4 times weekly. Only fresh samples (deposited within 24 h) were chosen. Samples, about 50g in size, were collected in individual plastic bags and immediately stored in a -20°C freezer. Individual fecal samples were then lyophilized , crushed, and transferred to individually labeled tubes. Reproductive cyclicity and ovulation were confirmed by steroid hormone metabolite analysis. Fecal samples underwent a steroid hormone metabolite extraction protocol according to previously published methods , 10. Extg for 30 min. The supernatant was filtered using a 0.22 μm syringe driven filter unit (Millipore Sigma), and the proteins were then precipitated from the supernatant using a 60% ammonium sulfate saturation. The ammonium sulfate solution was shaken for 30 min and centrifuged at 7000 x g for 30 min. The protein extract pellet was collected and resuspended in 400 μL of phosphate buffered saline with protease inhibitor. This protein extract solution was then desalted using a 3 kDa Millipore spin column (Amicon Ultra-0.5) and centrifugation at 7400 x g. All extraction steps were performed at 4°C. Extracted samples were then run on a Bradford assay  to determine total protein concentration. Briefly, standards for the assay were created by serial dilution of bovine serum albumin at 0.388 mg/mL to 0.012 mg/mL. Fecal protein samples were diluted to 1:30, and 10 μL of each sample was added to a well in duplicate. 200 μL of Bio-Rad Quick Start™ Bradford Dye Reagent was added to each well, and after 5 minutes protein concentrations were determined using a Molecular Devices Filtermax F5 plate reader. Coefficients of variation for all samples in duplicate were <10%. Differences in steroid hormone and total protein concentrations between pregnant and non-pregnant groups were determined using a Student’s T-test in R (version 3.3.2) [Weekly pooled fecal samples of 0.5g were created by combining approximately 0.125g of four individual dried samples in a 15 mL centrifuge tube. Individual samples from day 1–7 post-breeding were used to create the pooled sample for week 1. Individual samples from day 8–14 post-breeding were used to create the pooled sample for week 2, etc. Because samples were collected opportunistically, some weekly time periods had insufficient samples for analysis. In these cases, no IgJ information was available for that week, making the number of replicates per week variable. Total protein was subsequently extracted from pooled dried samples as follows. 6 mL of 0.1 M phosphate buffered saline  with protease inhibitor (1:1000) was added to the pooled fecal sample, and the mixture was shaken for 30 min and centrifuged at 4600 x n 3.3.2) , with di2PO4, 3.5 M Na2HPO4, 0.3 M NaCl, H2O; pH, 7.0) and were analyzed by enzyme immunoassay (EIA). Estrogen metabolites in diluted fecal extracts were used to determine reproductive cyclicity using an estradiol EIA that has been validated for use in the cheetah [Steroid hormone neat extracts were diluted 1:20 to 1:16,000 in phosphate buffer  was used as a positive control at 16.67 μg/mL. 18 μg of each sample, and 0.15 μg of positive control were loaded into wells on a 4–20% Mini-PROTEAN TGX gel . Samples were then separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% milk, and incubated overnight at 4°C with a primary antibody (Aviva Systems Biology ARP55440_P050) diluted 1:1000 in 1% milk. This polyclonal antibody was developed in a rabbit against human recombinant IgJ and was previously found to be reactive to cheetah IgJ in western blot .Intensity of IgJ abundance was determined using GeneSys Spot Blot analysis (Syngene) of the band occurring within each lane at 18 kDa for each weekly pooled sample. GeneSys Total Lane analysis (Syngene) for each sample of the Coomassie image was used to determine the total protein in the sample as a loading control. A ratio of IgJ intensity to Coomassie intensity was calculated for each pooled sample, as well as for the positive control. Relative intensity for each pooled sample was calculated by dividing the ratio for each pooled sample by the ratio of the positive control, in order to control for inter-blot variation. A pre-breeding relative intensity value specific to each individual luteal phase was then subtracted from each weekly post-breeding relative intensity value to control for pre-breeding IgJ levels. If relative intensity values were not normally distributed, then the data was subjected to a log transformation. After transformation, 15 of 18 groups describing weekly IgJ abundance  and both groups describing early post-breeding IgJ response  were verified for normality using a Shapiro-Wilk test in R (version 3.3.2) . DiffereFecal estrogen metabolite profiles confirmed the cyclicity of monitored females. Examples of the estrogen metabolite concentrations of cycling females prior to natural breeding can be seen in Detection of IgJ by western blotting with the use of a commercially available antibody was confirmed by the use of a positive control. IgJ was confirmed in the positive control and in fecal samples at a molecular weight of ~18 kD . FemalesPregnant females tended to have higher IgJ levels (P = 0.081) in week 4 post-breeding  compared to females experiencing a non-pregnant luteal phase  . Pregnanin situ and ex situ. However, cheetahs in the wild are in decline, as habitat fragmentation and human conflict have reduced the natural range of the species [ex situ insurance population. However, creating a sustainable ex situ population with the goal of improving genetic diversity and ensuring future health and adaptability of the species has become a challenge, as many cheetahs struggle to successfully reproduce in human care. The low genetic diversity of the species as a whole, and high levels of inbreeding depression, have contributed to many health and reproductive issues that affect the cheetah, including the impairment of genes mediating immune defenses [ex situ environmental factors may be having a negative effect on reproductive capacity. Of importance to animal managers is understanding the relatively high incidence of non-pregnant luteal phase that occurs in the ex situ population, whether this represents failure to conceive or early embryo loss.The reproductive biology of the cheetah has been studied for decades, with great advances in the understanding of this species both  species , 20. Becdefenses , low fecdefenses , 23. Whidefenses , 25, indTemporal tracking of IgJ abundance over the first 9 weeks post-breeding provided insight into the intrauterine events that result in the success or failure to establish pregnancy in the cheetah. Females in our study that were bred with an intact male demonstrated an elevation of IgJ early post-breeding compared to exogenously stimulated females with no exposure to a male. This increase in IgJ could be the result of multiple factors. Exposure to a new male, as well as the act of copulation can be a stressful event for the female. While this may provoke a stress response, it is unclear what effect this stress has on secretory immunity or IgJ expression. Intromission may also result in the introduction of bacteria or other foreign substances into the reproductive tract of the female, increasing the expression of IgJ and provoking an immune response at the mucosal surface. Another possible explanation is that a secretory immune response was stimulated by the presence of seminal plasma in the reproductive tract which, as a foreign substance interacting with a mucosal surface, could induce an upregulation in IgJ. An immune response to semen has been documented previously in mice, as lymphocyte synthesis and cytokine activation is triggered in response to the constituents of seminal plasma . The immThe timing of early intrauterine events, including implantation and the development of the placenta, is unknown in wild felids. In the domestic cat, fertilization takes place in the oviduct up to 48 h after ovulation, and implantation occurs at day 13–14 post-breeding . These f2ɑ (PGF2ɑ) by the placenta during week 8 post-breeding, as levels of a fecal PGF2ɑ metabolite (PGFM) were found to be elevated in the pregnant cheetah beginning at day 48 post-breeding and increased through parturition [2ɑ is known to have a strong luteolytic effect in many species [Pregnancy in the cheetah first becomes distinguishable from a non-pregnant luteal phase using progestogen metabolite monitoring at ~day 55 post-breeding, as progestogen levels in fecal samples drop to pre-ovulatory levels in non-pregnant individuals. It can be theorized that around this time in non-pregnant individuals a regression of the corpora lutea (CL) occurs which results in a subsequent decrease in progesterone production, followed by a plateau at lower basal levels. It is unknown whether luteal regression occurs in pregnant females as well, or if the CL is maintained until parturition. Recent studies have suggested that the placenta is a source of progesterone production in the domestic cat , 30, andturition . While P species , suggestIt is possible that the presence of external immune stressors, which potentially could alter the production of systemic IgJ, could influence the results and interpretation of our experiment. IgJ is a protein that is upregulated in response to an activation of the secretory immune system, which is present in all mucosal surfaces of the body in order to protect from foreign pathogens. An immune challenge to a mucosal surface that is independent of pregnancy could increase expression of IgJ, resulting in high IgJ values in our assay that do not correspond to intrauterine events and could affect the accuracy of the assay. Inter-individual variability in IgJ expression was controlled for here by assessing relative changes in concentration from pre-breeding levels within each individual. Because the assay in this study utilizes fecal material, the IgJ that is being measured is likely excreted by the cells of the intestinal mucosa. Immune stressors, whether related to pregnancy or not, likely increase IgJ expression across all mucosal surfaces, allowing for excreted IgJ abundance to be a useful indicator of pregnancy related events. A future goal is to extend the Western analysis to a reliable benchtop enzyme-linked immunosorbent assay (ELISA) for measuring fecal IgJ levels after breeding, allowing for the confirmation of pregnancy at week 4 post-breeding. Daily quantification using this method would improve the accuracy and efficiency of IgJ monitoring and may be able to reveal moments of immune challenge that impact establishment of pregnancy with greater precision. The occurrence of a non-pregnant luteal phase is common in other species, indicating the potential for a felid-wide or carnivore-wide assay for determining pregnancy using IgJ monitoring.ex situ management of the species, and further research will continue to advance the understanding of cheetah reproductive physiology following breeding, aiding future conservation efforts for the species.In summary, this study detailed IgJ levels throughout the first 9 weeks of pregnancy and non-pregnant luteal phase in the cheetah. Females that were bred naturally with intact males tended to have a higher immediate IgJ response than females that were exogenously stimulated to ovulate, indicating a potential immune response to the constituents of seminal plasma or other foreign substances introduced by the act of breeding that may have the effect of promoting maternal tolerance of fetal tissue upon implantation. There was a tendency towards increased IgJ abundance at week 4 post-breeding in pregnant cheetahs compared to non-pregnant cheetahs, indicating an activation of the secretory immune system in response to implantation and the invasion of the maternal endometrium by the fetal trophoblast. A significant increase in IgJ abundance was also found in week 8 post-breeding in pregnant cheetahs compared to non-pregnant cheetahs. Taken together, these data support the suggestion that the window of implantation in the cheetah is between 19–21 days post-breeding, and that the placenta is a source of extragonadal progesterone during the third trimester. Further refinement of this assay will allow for fecal IgJ monitoring to become a reliable non-invasive tool for determining pregnancy in the cheetah at week 4 post-breeding, much earlier than current methods. These findings will help to improve S1 Fig(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a ‘handover’ of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions. Caenorhabditis elegans, symmetry breaking of the plasma membrane is caused by PAR proteins that sort into distinct anterior and posterior cortical domains and generate cell polarity  and GTPase activating proteins (GAPs) . Most smIt has been proposed that small GTPase patterning can arise from the coupling of GEF activity and effector binding . In thisRabex5/Rabaptin5 is one of the best characterized GEF/effector complexes in eukaryotes. Rabex5 is a 57 kDa Vps9 domain containing GEF for Rab5 . RabaptiElucidating the precise mechanisms of self-organization of peripheral membrane proteins is critical to understanding endomembrane identity and functionality. We hypothesize that, similar to what has been observed for Cdc42 in silico, Rab5, Rabex5/Rabaptin5 and GDI comprise a minimal system that is capable of spatially organizing Rab5. We made use of in vitro reconstitution to test this hypothesis and elucidate the contributions of individual components to membrane association and organization. Our biochemical reconstitution system allowed for in-depth study of the biochemical interactions underlying the self-organization of Rab5 and its interacting molecules on the membrane.To directly test the positive feedback loop model, we investigated the structural rearrangements occurring in Rab5 and Rabex5/Rabaptin5 in the course of nucleotide exchange by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS). Rabex5/Rabaptin5 was first premixed with Rab5:GDP and the resulting ternary complex diluted into deuterated buffer in the absence  top or p3:MeOH (1:2), as described by To reconstitute Rab5 membrane recruitment and organization, we developed an in vitro system consisting of recombinant proteins and synthetic membranes. The lipid composition of the synthetic membrane was chosen based on the lipid composition of an enriched early endosomal fraction determined in a previous study . BrieflyEE-MCBs incubated with 10 nM GFP-Rab5/GDI showed membrane recruitment of GFP-Rab5 with a random distribution see . The addIn order to understand how these domains form, we monitored EE-MCBs over time See . DomainsIn order to understand the mechanisms by which Rab5 domains form, we dissected the contribution of each component of our reconstituted system. GDI delivers and extracts Rab5, as seen in Next, we verified that the Rabex5/Rabaptin5 complex indeed localizes to the Rab5 domain. For this, we used a fluorescent Rabex5/Rabaptin5-RFP complex and observed both enrichment of Rabaptin5-RFP signal inside the domain See  and coloWe next wanted to investigate whether the full Rabex5/Rabaptin5 complex was necessary for domain formation . In the Finally, we quantified the domain size distribution as a function of concentration of the components in the reaction. Interestingly, neither the domain diameter nor the area differed significantly when decreasing Rabex5/Rabaptin5 concentration, but the mean intensity of domains decreased with decreasing concentration of Rabex5/Rabaptin5 See .In addition to protein-protein interactions, protein-lipid and lipid-lipid interactions also play a role in Rab5 domain formation. The above experiments  and 3 weIn order to determine whether these interactions have an effect on domain formation, the same MCBs were incubated with Rab5/GDI, Rabex5/Rabaptin5, GDI and GTP. Strikingly, domain formation was most efficient on EE-MCBs with 1 mol% PI(3)P, less efficient on EE-MCBs with 0 mol% PI(3)P and completely abolished on PC/PS membranes regardless of PI(3)P content see . DomainsO) domains (data not shown).To investigate further which components of the EE lipid composition contribute to domain formation, the simple PC/PS lipid composition was sequentially modified to include the three most abundant lipids in the EE lipid composition: cholesterol (32.3 mol%), sphingomyelin  and ethanolamine plasmalogen  See ; Table 5The observation that Rab5 can be recruited efficiently to PC/PS/PI(3)P membranes but cannot be organized into domains in the presence of Rabex5/Rabaptin5, excess GDI, and GTP suggest that Rab5 interacts differently with the complex EE membrane that with a simple PC/PS membrane. Our results demonstrate that PI(3)P enhances recruitment of Rab5 to MCBs and the presence of lipids that contribute to acyl chain packing  together with coupling of GEF and effector activities (via Rabex5/Rabaptin5) are sufficient to reconstitute domain organization of Rab5 in vitro. Geranylgeranylated Rab5 was observed to be recruited to EE-like membranes from the Rab5/GDI complex. Whereas in the absence of other factors Rab5 was randomly distributed in the plane of the membrane, upon the addition of GDI, Rabex5/Rabaptin5 and GTP, it reorganized into discrete domains in a GTP-dependent manner. Key to Rab5 domain formation was the ‘handover’ of Rab5 from Rabex5 to Rabaptin5 and the lipid composition of early endosomes, suggesting a hitherto unknown cooperativity between lipids and Rab-dependent membrane self-organization.Self-organizing systems that form spatial patterns on membranes often depend on non-linear dynamics . In our The membrane diffusion of multiple small GTPases has been shown to be integral in their capacity to self-organize . An impoO domains. Rab5 domain formation therefore requires the synergy between the Rab5 minimal machinery and lateral lipid packing. We suggest that the EE lipid composition supports Rab5 domain formation in our in vitro system through a combination of 1) direct interactions between Rab5 and PI(3)P enhancing recruitment to the membrane, 2) cholesterol stabilizing the geranylgeranyl anchor insertions to support a nucleotide-dependent orientation of Rab5 relative to the membrane, and 3) the presence of saturated lipids allowing for dense packing of geranylgeranyl chains, contributing to domain stabilization and growth.Similar to Rab5 recruitment, domain formation was lipid composition dependent and most strongly observed on membranes containing the full EE lipid mixture. The observation that simple, highly diffusive, PC/PS membranes do not support domain formation suggests that the EE lipid composition facilitates lateral lipid packing and protein-lipid interactions that are necessary for domain formation. 2 on the PM . The non-linearity of the nucleotide cycle coupled to specific lipid interactions make small GTPases widespread regulators of membrane self-organization. K-Ras for example, has long been known to cluster and alter the local lipid environment, e.g. by forming nanoclusters of PIPn the PM . HoweverHerein, we reconstituted a minimal system for the formation of Rab5 GTPase domains in vitro and demonstrated that both GEF/effector coupling and lipid interactions contribute to the self-organization of Rab5 on the membrane, where the lipid composition plays an important role beyond that of a solvent for lipidated proteins. This appears to be a universal system deploying small GTPases to pattern membranes from mono-cellular to multi-cellular organisms.GSAGSAAGSGAAA; C-terminal: linker: GAPGSAGSAAGSG). As the addition of a fluorescent tag to a protein always carries the risk of altering protein behavior by interfering with protein folding, fluorescent proteins were compared to non-fluorescent constructs known to fold properly by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) and discarded if they showed any aberrant dynamics. The following constructs were used in this study: 6xHis-GFP-Rab5, GST-GDI, GST-Rabaptin5, Rabex5-6xHis, RFP-Rabaptin5, 6xHis-RabexCAT.Rab5, Rabex5, Rabaptin5, and GDI were cloned into pOEM series vectors (Oxford Expression Technologies), modified to contain a Human Rhino Virus (HRV) 3C cleavable tag at either the Nor C-terminus, followed by a protease cleavage site  for insect (SF9) cell expression. Cleavable tags consisted of either 6x-Histidine (6xHis), for Rab5 and Rabex5, or Gluthathione S-Transferase (GST), for GDI and Rabaptin5. In order to monitor membrane association and organization, fluorescent Rab5 and Rabaptin5 constructs were created. The proteins were cloned into SF9 expression vectors containing either an N or C-terminal fluorescent tag (GFP or RFP) attached to the protein by a 13 amino acid flexible linker  and co-transfected with linearised viral genome and expression plasmid. P1 and P2 virus was generated per manufacturers protocol and yield was optimised by expression screens and infection time course experiments. The P2 virus was used to infect SF9 cells (grown to a density of 1 million cells/ml) at 1% (v/v). Rabex5/Rabaptin5 and geranylgeranylatedRab5/GDI complexes were produced by co-infection. Cells were harvested after 30–40 hr by spinning in a tabletop centrifuge at 500 g for 10 min. Cell pellets were resuspended in Standard Buffer  supplemented with DNAse one and protease inhibitor cocktail . Pellets were flash frozen and stored at −80◦C. All subsequent steps performed at 4◦C or on ice. Cells were thawed on ice and lysed by sonication (previously frozen SF9 cell pellets were not sonicated as freeze-thawing is sufficient for lysis). Cell lysates were spun with a JA 25.50 rotor at 22500 rpm for 20 min at 4◦C. Histidine-tagged proteins were bound to Ni-NTA Agarose resin (1L of culture = 1 mL resin) in the presence of 20 mM Imidazole. Resin was washed with STD buffer supplemented with 20 mM Imidazole. Proteins were eluted using 200 mM Imidazole only followed by Histidine-tag cleavage during overnight dialysis with 3C protease. GST tagged proteins were bound to Glutathione Sepharose resin  for 2 hr at 4◦C, washed with Standard Buffer and cleaved from resin overnight with a GST-3C protease. Rabex5/Rabaptin5 and Rab5/GDI complexes were purified by both His- and GST-tag affinity purification to obtain pure complex. Size Exclusion Chromatography was performed in STD on a Superdex200 Increase 10/30. Concentrations were determined by a bicinchoninic acid protein Assay  and purity was assessed by SDS-PAGE followed by colloidal Coomassie staining. Proteins were aliquoted, flash frozen in liquid nitrogen and stored at −80◦C.The lipids listed below were purchased and resuspended in either CHCl3, CHCl3:MeOH (2:1 for GM3) or CHCl3:MeOH:H2O (1:2:0.8 for PI(3)P) as per manufacturer’s instructions and stored at −20◦C. To form liposomes, lipids were mixed together and the solvent was evaporated under a stream of nitrogen. Residual solvent was removed by drying under vacuum overnight in a desiccator. Lipids were rehydrated for at 37◦C in SLB Buffer  and vortexed to form a stock solution of 1 mM lipid. Small unilamellar vesicles (SUV) were prepared by freeze-thaw cycles (10x snap freezing and thawing at 37◦C). Vesicles were stored at −20◦C and sized by sonication before each application. Size distribution of liposome preparations was assessed by Dynamic Light Scattering using a Zetasizer Nano ZSP Malvern.DOPC:DOPS:DOPE:Sphingomyelin:Cholesterol:ethanolamine plasmalogen (1-(1Z-octadecenyl)−2-oleoyl-sn-glycero-3-phosphoethanolamine):choline plasmalogen (1-(1Z-octadecenyl)−2-oleoyl-sn-glycero-3-phosphocholine):GM3: PI(3P) (diC16 Phosphatidylinositol 3-phosphate): DiD  (13.8:6.1:6.8:12.6:32.3:12.9:3.6:9:1:0.1) See .DOPC: DOPS: Phosphatidylinositol 3-phosphate (PI(3P)diC16): DiD  (83.95:15:1:0.1) See .Silica beads (10 μm standard microspheres for microscopy) were coated with a supported lipid bilayer as described  with minMicroscopy experiments were performed on either Nikon TiE  or Cell Voyager 7000S (CV7000S) (automated imaging for time lapse experiments). For manual imaging in Nikon TiE, reactions were prepared in an 8-well NuncTM Lab-TekTM Chamber Slide for imaging. Images were acquired with a Nikon TiE equipped with a 100x/1.45NA Plan Apochromat, DIC oil immersion objective, Yokogawa CSU-X1 scan head and Andor DU-897 back-illuminated CCD. Images were acquired with 80 ms exposure at λ 488, 561 and 660 with the following laser intensities: 15% 488; 5% 561; and 2% 660. For automated imaging, reactions were prepared in a Greiner Square bottom 384 well plate. Images were acquired with Cell Voyager 7000S (CV7000S) equipped with a 60x/1.2NA water immersion objective at 30% 488 and 660 laser. Color and illumination corrections were applied though CV7000S software. Imaging support by M. Stöter .Intensity quantifications at MCB equators were performed manually in FIJI . Beads wTo quantify size, intensity, and number of domains on MCBs, a novel image analysis pipeline was developed. The pipeline consists of the following steps, illustrated in The membrane surface wass extracted by selecting all pixels in the upper 0.5% intensity percentile for the membrane channel. The sphere center and radius were fitted using a linear least-squares solver with the normal residuals as cost function. Next, particles were distributed on the fitted sphere on a latitude-longitude mesh with  schemes . FinallyUneven background in the tangent space of beads was corrected using a ‘rolling ball’ algorithm, with a radius of 2 μm .Rab5 domains were segmented using a globally optimal model-based method Squassh . The segA marching cubes algorithm  was usednMDP) as a measure of correlation between the corresponding pair of particles with intensities according to the formula:Correlation maps were created by computing the normalized mean deviation product  for 30 ms at Q of 0.25. HCD fragmentation NCE was 35 eV. Peptides were identified using Mascot (Matrix Science) and manually verified to remove ambiguous peptides. For measurement of deuterium uptake, 10 uM protein is diluted 1:9 in Rab5 buffer prepared with deuterated solvent. Samples were incubated for varying times at 22 °C followed by the aforementioned digestion, desalting, separation and mass spectrometry steps. The intensity weighted average m/z value of a peptide’s isotopic envelope is compared plus and minus deuteration using the HDX workbench software platform. Individual peptides are verified by manual inspection. Data are visualized using Pymol. Deuterium uptake is normalized for back-exchange when necessary by comparing deuterium uptake to a sample incubated in 6M urea in deuterated buffer for 12–18 hr at room temperature and processed as indicated above.HDX-MS was performed essentially as previously described . Protein In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Rab GTPases are master regulators of membrane trafficking that template the organization and directionality of the secretory and endocytic pathway. Using a reconstitution approach, the authors describe how a molecular feedback loop is created to regulate Rab function in coordination with specific lipids, effector proteins and guanine nucleotide exchange factors. Understanding feedback loops such as that generated in this system is of broad interest and much remains to be learned about how proteins are organized on intracellular membrane surfaces.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Suzanne Pfeffer as the Senior and Reviewing Editor. The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Thank you for submitting your article "A non-linear system patterns Rab5 GTPase on the membrane" for consideration by This is a high-quality study in which the authors reconstitute prenylated Rab5 GTPase on bead bound liposomes and reveal macromolecular clustering that depends on a Rab5 GEF and GEF-binding effector. Understanding feedback loops such as that generated in this system is of broad interest and much remains to be learned about how proteins are organized on intracellular membrane surfaces. The surprising result here is that Rab5-GTP alone does not organize but in the presence of the GEF and effector, it does, and the PI3P composition of the membrane is also important.All the reviewers were highly supportive of the manuscript but suggested various approaches to enhance the significance of the story without too much additional work. We leave it to you to decide how to accomplish this, thus we have included all the reviews--but it would make sense to characterize more fully how PI3P works and add some data more related to lipid composition.Reviewer #1:This is a high-quality study in which the authors reconstitute prenylated Rab5 GTPase on bead bound liposomes and reveal macromolecular clustering that depends on a Rab5 GEF and GEF-binding effector. Understanding feedback loops such as that generated in this system is of broad interest and much remains to be learned about how proteins are organized on intracellular membrane surfaces. The surprising result here is that Rab5-GTP alone does not organize but in the presence of the GEF and effector, it does, and the PI3P composition of the membrane is also important. Although there is a large amount of work presented herein, additional experiments would add greatly to the overall impact of the current story. For example, although others proposed in MD simulations a role for an arginine residue in the interaction between Rab5 and PI3P, the authors could easily test this in their system. Not explored is the issue of a catalytic GEF linked to a Rabaptin-5 stoichiometric binding site, suggesting that in this simple system, the GEF may continue to act on a single Rab. This should be discussed more fully and the authors should use standard fluorescent proteins to try to determine the number of molecules in their liposome-bound complexes. What fraction of Rabex is Rabaptin bound in cells, and is it in equilibrium with a soluble pool? If they dimerize the Rabaptin with GFP, do they get twice as large a cluster? And why is there such a great difference between 500nM and 1µM GDI? Is it there to rescue some prenylated proteins that crash out of the system if not able to jump into the membrane? Is there an effect of PRA1 that is so essential for their in vitro endosome fusion reaction? Finally, the authors were not able to remove cholesterol from their system but they could use cholesterol binding protein toxins to segregate cholesterol and look at the effect?In summary, a slightly deeper consideration of each of the take home messages will add much to this interesting and high-quality study.Reviewer #2:In this paper, the authors have used the purified Rabex5/Rabaptin5 complex and supported lipid bilayers to study the association of Rab5 with the bilayer. They report that Rab5 forms domains at the surface of the membrane, that Rabex5 hands Rab5 over to Rabaptin5 upon nucleotide exchange, and that a minimal system consisting of Rab5, RabGDI, and Rabex5/Rabaptin5 is necessary to pattern Rab5 into membrane domains. They also report that early endosomal lipids were required for Rab5 pattern formation. The authors conclude that the prevalence of GEF/effector coupling in nature suggests the existence of a universal system for small GTPase patterning.The paper is very interesting, because the notion that Rab5, or other small GTPases, forms functional effector domains has broad physiological implications, and was proposed a long time ago by the authors. This is the first, direct in vitro evidence of GTPase domain formation because of a dynamic activation loop controlled by the GEF-GAP-GDI. The study is globally very convincing, but I have a few comments that the authors may wish to address1) The patterns observed by the authors are dynamically stable. While in a dynamic Turing model of patterning, some area of the phase diagram form stable patterns, it is more likely to obtain travelling waves/spirals in such systems. Did the author observe any propagation or dynamic change of patterns through time? Otherwise, can the author justify using the known time constants or proper feedback loops in their system why it is expected to have a stable pattern rather than travelling waves?2) The formation of such patterns should strongly depend on the diffusion coefficient of lipids (and proteins), which, I guess is pretty limited onto the supported bilayers used in this study. Did the author try to used GUVs instead of the beads? I would expect that the domains do not form, or that they transform into travelling waves. It would be an important point to make about the role of diffusion in the formation of domains. Diffusion of membrane components on the beads and on the GUVs could also be measured by FRAP, to have an idea of the diffusion change.3) To reconstitute Rab5 domain formation in vitro, the authors used the lipid composition of enriched early endosomes previously determined by mass spectrometry in Perini (2012). I find it surprising that PC corresponds only to 14 mol% of EE lipids . As far as I can remember, PC accounts for 45-55 mol% of all cellular membranes in higher eukaryotes. Also, Perini (2012) is a thesis from the MPI-CBG in Dresden and is not readily available. Sufficient information on fractionation and analysis should be made available in the paper so that readers can evaluate the data. Similarly, the pseudocylindrical projection used to segment and visualized EE-membrane-coated beads is described in a submitted paper .4) Along the same lines, and due to the high content in sphingolipids and cholesterol in the EE lipid composition used by the authors, how can the authors rule out that Rab5 domains are not formed onto pre-existing lipid domains, in particular because they see domains forming only with the EE composition?5) I find it a bit difficult to evaluate the projections in Figure 5 A-B, after recruitment of GG-GFP-Rab5 to EE and PC/PS bilayers (without Rabex5/Rabaptin5). I agree with the authors that GFP-Rab5 distribution without Rabex5/Rabaptin5 looks different from the patterns with domains observed in the presence of Rabex5/Rabaptin5 . Yet, GFP-Rab5 without Rabex5/Rabaptin5 does not seem evenly or randomly distributed on the surface with PI3P (both EE or PC/PS): GFP-Rab5 seems to form numerous, smaller domains. Is this so? Are these domains more or less dynamic by FRAP? Also, GFP-Rab5 fluorescence , but not DiD fluorescence , seems to be restricted to the projection above the equator.Reviewer #3:The manuscript by Cezanne et al. constitutes a significant breakthrough not only for the narrow field of small GTPases, or even for the cell biology as a whole, but also for the broadest interdisciplinary community interested in biological systems from the systems point of view. Cellular pattern formation and morphogenesis is currently of great interest and is very timely because both the experimental tools and the theoretical methods have reached the needed maturity. The senior author was probably the first to propose, already back in 1997, that the interaction between the GTPase effectors and GEFs can generate positive feedback loops capable of breaking spatial symmetry and generating membrane clusters, the simplest structures to form a prepattern for a great variety of much more complex structures. Zerial and colleagues are again ahead of the competition. Formation of clusters of activated GTPases had been theoretically predicted and computationally modeled for over a decade, but the in vitro reconstitution of this process remained out of reach. Recent tremendous success in reconstituting the MinD system in vitro showed how much can be learned from a reliable, reproducible and controllable in vitro system about cellular morphogenesis. Several groups had been able to reconstitute various Rho GTPases in vitro, but nobody attacked the pattern formation problem. Cezanne et al. do exactly this, they convincingly show that all theoretical predictions were correct, a minimal system consisting of a small GTPase, its GDI and the positive feedback molecule (the effector-GEF complex) is fully competent to break spatial symmetry and generate clusters of activated GTPase.eLife, very timely and novel and, thus, should be published by eLife upon some, mostly minor, improvements.Therefore, I believe the paper is extremely interesting to a broad readership of 1) The discovery that only the native endocytic lipid mixture (EE), but not the PC/PS artificial membrane, supports the pattern formation is striking but remains unexplained in the manuscript. It is even more surprising given the result that addition of 1% of PI3P rescues the Rab5 recruitment. Although the authors discuss this result at length, the reader is left feeling somehow unsatisfied… I realize that the authors could not remove lipids from the EE without jeopardizing the integrity of the membrane. What about instead modifying/doping the PC/PS mix? One potential explanation of this result is that some specific lipid/ electric charge is required to make a functional membrane. The authors could "add back" some of the natively found lipids  or increase the negative charge by adding more PS and see if this rescues pattern formation. There is also another potential explanation, which is more interesting from the theoretical point of view and the authors could confirm or reject this possibility with the tools/methods they already have in place! Modeling work predicted and some experiments  confirmed that pattern formation is very sensitive to the diffusion coefficient of the GTPase on the membrane. The manuscript shows that the authors used oleic acid derivatives for both PC and PS. While this lipid tail is long, it is also unsaturated, which likely supports liquid disordered lipid phase. To change the diffusion coefficient without affecting the chemistry of lipid head groups, the authors could use longer and fully saturated fatty acid derivatives. Addition of cholesterol to this mix should even further increase the thickness of the membrane bilayer and reduce the diffusion coefficient of Rab5. These experiments are doable without any new protein constructs and could significantly increase the impact of the paper.2) Another suggestion organically follows from the previous one. The authors site a number of modeling studies but, surprisingly, offer no computational model of their dramatic pattern-forming system themselves! A model could increase the impact of the work and assist in understanding the role of molecular transport (GDI) of Rab5, membrane diffusion coefficient, etc. All the reviewers were highly supportive of the manuscript, but suggested various approaches to enhance the significance of the story without too much additional work. We leave it to you to decide how to accomplish this, thus we have included all the reviews--but it would make sense to characterize more fully how PI3P works and add some data more related to lipid composition.Reviewer #1:This is a high-quality study in which the authors reconstitute prenylated Rab5 GTPase on bead bound liposomes and reveal macromolecular clustering that depends on a Rab5 GEF and GEF-binding effector. Understanding feedback loops such as that generated in this system is of broad interest and much remains to be learned about how proteins are organized on intracellular membrane surfaces. The surprising result here is that Rab5-GTP alone does not organize but in the presence of the GEF and effector, it does, and the PI3P composition of the membrane is also important. Although there is a large amount of work presented herein, additional experiments would add greatly to the overall impact of the current story. For example, although others proposed in MD simulations a role for an arginine residue in the interaction between Rab5 and PI3P, the authors could easily test this in their system.This is an interesting suggestion, unfortunately, due to the Covid-19 crisis which forced us to close the institute, we were unable to complete these experiments for inclusion in this manuscript. We will pursue this as a part of our follow-up work further dissecting the mechanisms controlling Rab5 domain formation.Not explored is the issue of a catalytic GEF linked to a Rabaptin-5 stoichiometric binding site, suggesting that in this simple system, the GEF may continue to act on a single Rab. This should be discussed more fully and the authors should use standard fluorescent proteins to try to determine the number of molecules in their liposome-bound complexes. What fraction of Rabex is Rabaptin bound in cells, and is it in equilibrium with a soluble pool? If they dimerize the Rabaptin with GFP, do they get twice as large a cluster?We improved the description of the stoichiometry of the Rabex5/Rabaptin5 complex to make it clearer that it is a heterotetramer composed of two Rabex5 and two Rabaptin5 molecules (Introduction). Rabex5 and Rabaptin5 form a constitutive complex that is stable to gel filtration and we do not observe exchange between a soluble pool of subunits. The precise interaction between these molecules has been investigated in a previous study . We also amended the Discussion to include an estimate of the number of Rab5 molecules in our reconstituted domains (paragraph four).And why is there such a great difference between 500nM and 1µM GDI?In preliminary experiments we found that 1µM GDI was required to fully remove 10nM GFP-Rab5 from EE-MCBs.Is it there to rescue some prenylated proteins that crash out of the system if not able to jump into the membrane? Is there an effect of PRA1 that is so essential for their in vitro endosome fusion reaction?Pra1 was previously included by Ohya et al., 2009, to stimulate the delivery of Rab5 to proteoliposomes and render them fusogenic. We did not look at the addition of Pra1 because we found that a minimal system consisting of GDI and the Rabex5/Rabaptin5 complex was sufficient to create Rab5 domains.Finally, the authors were not able to remove cholesterol from their system but they could use cholesterol binding protein toxins to segregate cholesterol and look at the effect?Pra1 was previously included by Ohya et al., 2009, to stimulate the delivery of Rab5 to proteoliposomes and render them fusogenic. We did not look at the addition of Pra1 because we focused on creating a minimal system consisting of GDI and the positive feedback loop provided by the Rabex5/Rabaptin5 complex that was sufficient to create Rab5 domains. In such minimal system the requirement for Pra1 can be bypassed.Reviewer #2:In this paper, authors have used the purified Rabex5/Rabaptin5 complex and supported lipid bilayers to study the association of Rab5 with the bilayer. They report that Rab5 forms domains at the surface of the membrane, that Rabex5 hands Rab5 over to Rabaptin5 upon nucleotide exchange, and that a minimal system consisting of Rab5, RabGDI, and Rabex5/Rabaptin5 is necessary to pattern Rab5 into membrane domains. They also report that early endosomal lipids were required for Rab5 pattern formation. The authors conclude that the prevalence of GEF/effector coupling in nature suggests the existence of a universal system for small GTPase patterning.The paper is very interesting, because the notion that Rab5, or other small GTPases, forms functional effector domains has broad physiological implications, and was proposed a long time ago by the authors. This is the first, direct in vitro evidence of GTPase domain formation because of a dynamic activation loop controlled by the GEF-GAP-GDI. The study is globally very convincing, but I have a few comments that the authors may wish to address1) The patterns observed by the authors are dynamically stable. While in a dynamic Turing model of patterning, some area of the phase diagram form stable patterns, it is more likely to obtain travelling waves/spirals in such systems. Did the author observe any propagation or dynamic change of patterns through time? Otherwise, can the author justify using the known time constants or proper feedback loops in their system why it is expected to have a stable pattern rather than travelling waves?We investigated this possibility but did not observe wave-like or other macroscopic dynamic behaviors in our system. However, we cannot exclude the possibility that our system lacks sufficient resolution to detect waves. We amended the Discussion to include a brief discussion of Turing type pattern formation and how stable patterns rather than travelling waves might be formed/observed in our set-up (paragraph five). We are in the process of working on an in-depth computational model to investigate the mechanisms of pattern formation in this system and possible dynamic behaviors.2) The formation of such patterns should strongly depend on the diffusion coefficient of lipids (and proteins), which, I guess is pretty limited onto the supported bilayers used in this study. Did the author try to used GUVs instead of the beads? I would expect that the domains do not form, or that they transform into travelling waves. It would be an important point to make about the role of diffusion in the formation of domains. Diffusion of membrane components on the beads and on the GUVs could also be measured by FRAP, to have an idea of the diffusion change.We have not yet tried to use free-standing membranes as this would necessitate another optimization of the system. However, this is something we would like to investigate in future. A consideration here is that the incorporation of 10 lipid components into GUVs is technically challenging. We chose another approach to achieve the same goal. We instead increased the membrane rigidity of the simple PC/PS lipid composition by adding cholesterol, sphingomyelin and ethanolamine plasmalogen (PlasmPE). Interestingly, we observed that it is not a global change in membrane diffusivity that allows for pattern formation, but most likely local changes induced by increased acyl chain packing and accommodation of the geranylgeranyl anchor.3) To reconstitute Rab5 domain formation in vitro, the authors used the lipid composition of enriched early endosomes previously determined by mass spectrometry in Perini (2012). I find it surprising that PC corresponds only to 14 mol% of EE lipids . As far as I can remember, PC accounts for 45-55 mol% of all cellular membranes in higher eukaryotes. Also, Perini (2012) is a thesis from the MPI-CBG in Dresden and is not readily available. Sufficient information on fractionation and analysis should be made available in the paper so that readers can evaluate the data. Similarly, the pseudocylindrical projection used to segment and visualized EE-membrane-coated beads is described in a submitted paper .We added a description of our fractionation protocol and our image analysis pipeline  to make this information accessible. The image analysis pipeline is further detailed in a separate submitted publication that we hope will be available soon.While the total phospholipid content of cells is indeed estimated to be composed of 45-55mol% PC, there are significant variations in the PC content between different internal membrane structures. In our EE lipid composition PC and PlasmPC combined make up around 20% of the total phospholipid. This is in agreement with studies reporting the lipid composition of the plasma membrane which is known to be very similar to that of the early endosome and we now comment on this in the Results (subsection “Reconstituting Rab5 domain formation in vitro”). Other endo-membranes, e.g. later steps in the endocytic pathway or the ER, on the other hand have been shown to contain a higher percentage of PC .4) Along the same lines, and due to the high content in sphingolipids and cholesterol in the EE lipid composition used by the authors, how can the authors rule out that Rab5 domains are not formed onto pre-existing lipid domains, in particular because they see domains forming only with the EE composition?This is a plausible interpretation, especially in light of our new findings that cholesterol and sphingomyelin appear to be necessary for Rab5 domain formation. In order to test this, we stained EE-MCBs with C-laurdan. However, we could not detect pre-existing phases of lipid order.5) I find it a bit difficult to evaluate the projections in Figure 5 A-B, after recruitment of GG-GFP-Rab5 to EE and PC/PS bilayers (without Rabex5/Rabaptin5). I agree with the authors that GFP-Rab5 distribution without Rabex5/Rabaptin5 looks different from the patterns with domains observed in the presence of Rabex5/Rabaptin5 . Yet, GFP-Rab5 without Rabex5/Rabaptin5 does not seem evenly or randomly distributed on the surface with PI3P (both EE or PC/PS): GFP-Rab5 seems to form numerous, smaller domains. Is this so? Are these domains more or less dynamic by FRAP? Also, GFP-Rab5 fluorescence , but not DiD fluorescence , seems to be restricted to the projection above the equator.We agree that the images originally chosen highlighted inhomogeneity in GFP-Rab5 distribution that might be misleading. To remove a reliance on visual inspection to detect domains, we use automated image analysis methods to detect the presence of domains. We substituted the images in question with other images from the same condition, which are in better agreement with the results from the automated detection algorithm.Reviewer #3:The manuscript by Cezanne et al. constitutes a significant breakthrough not only for the narrow field of small GTPases, or even for the cell biology as a whole, but also for the broadest interdisciplinary community interested in biological systems from the systems point of view. Cellular pattern formation and morphogenesis is currently of great interest and is very timely because both the experimental tools and the theoretical methods have reached the needed maturity. The senior author was probably the first to propose, already back in 1997, that the interaction between the GTPase effectors and GEFs can generate positive feedback loops capable of breaking spatial symmetry and generating membrane clusters, the simplest structures to form a prepattern for a great variety of much more complex structures. Zerial and colleagues are again ahead of the competition. Formation of clusters of activated GTPases had been theoretically predicted and computationally modeled for over a decade, but the in vitro reconstitution of this process remained out of reach. Recent tremendous success in reconstituting the MinD system in vitro showed how much can be learned from a reliable, reproducible and controllable in vitro system about cellular morphogenesis. Several groups had been able to reconstitute various Rho GTPases in vitro, but nobody attacked the pattern formation problem. Cezanne et al. do exactly this, they convincingly show that all theoretical predictions were correct, a minimal system consisting of a small GTPase, its GDI and the positive feedback molecule (the effector-GEF complex) is fully competent to break spatial symmetry and generate clusters of activated GTPase.Therefore, I believe the paper is extremely interesting to a broad readership of eLife, very timely and novel and, thus, should be published by eLife upon some, mostly minor, improvements.1) The discovery that only the native endocytic lipid mixture (EE), but not the PC/PS artificial membrane, supports the pattern formation is striking but remains unexplained in the manuscript. It is even more surprising given the result that addition of 1% of PI3P rescues the Rab5 recruitment. Although the authors discuss this result at length, the reader is left feeling somehow unsatisfied… I realize that the authors could not remove lipids from the EE without jeopardizing the integrity of the membrane. What about instead modifying/doping the PC/PS mix? One potential explanation of this result is that some specific lipid/ electric charge is required to make a functional membrane. The authors could "add back" some of the natively found lipids  or increase the negative charge by adding more PS and see if this rescues pattern formation.As the simple PC/PS lipid composition already includes 15mol% DOPS which is a significant amount of negative charge, we modified the PC/PS lipid composition by sequentially adding PI(3)P, cholesterol and either ethanolamine plasmalogen (PlasmPE) or sphingomyelin (the next most abundant lipids in the EE lipid composition which both influence membrane rigidity). We show that the presence of sphingomyelin, but not PlasmPE, is necessary for Rab5 domain formation . We conclude that lipids that are known to mediate acyl chain packing  are vital for formation and stability of the Rab5 domain. These points have been expanded in the discussion as well.There is also another potential explanation, which is more interesting from the theoretical point of view and the authors could confirm or reject this possibility with the tools/methods they already have in place! Modeling work predicted and some experiments  confirmed that pattern formation is very sensitive to the diffusion coefficient of the GTPase on the membrane.The papers mentioned are indeed very interesting and we now cite these papers in the Discussion.The manuscript shows that the authors used oleic acid derivatives for both PC and PS. While this lipid tail is long, it is also unsaturated, which likely supports liquid disordered lipid phase. To change the diffusion coefficient without affecting the chemistry of lipid head groups, the authors could use longer and fully saturated fatty acid derivatives. Addition of cholesterol to this mix should even further increase the thickness of the membrane bilayer and reduce the diffusion coefficient of Rab5. These experiments are doable without any new protein constructs and could significantly increase the impact of the paper.m of DSPC (55°C) and DSPS (68°C). Instead, we addressed the question of Rab5 diffusion by modifying the simple DOPC/DOPS lipid composition to include cholesterol and other components of the EE lipid composition that influence membrane rigidity, as described above.We tried to, either fully or partially, replace DOPC(18:1) and DOPS(18:1) with the saturated DSPC(18:0) and DSPS(18:0) species, either fully or partially. Unfortunately, the addition of saturated DSPC and DSPS prevented the formation of stable MCBs in our set up as we conduct experiments at 25°C, well below the TIn the future, we aim to explore the effect of altering lateral diffusion of Rab5 in the membrane in the framework of the computational model described below.2) Another suggestion organically follows from the previous one. The authors site a number of modeling studies but, surprisingly, offer no computational model of their dramatic pattern-forming system themselves! A model could increase the impact of the work and assist in understanding the role of molecular transport (GDI) of Rab5, membrane diffusion coefficient, etc.We have been working on an in-depth computational model of this system. This will be submitted as a separate manuscript as it is a full study on its own. The model will delve into the roles of protein-protein, protein-lipid and lipid-lipid interactions highlighted in this study.


The coronavirus disease 2019 pandemic has necessitated almost exclusive National Health Service focus on emergency work and cancer care. There are concerns that increased hospital and community pressures will lead to decreased referrals and worse outcomes for head and neck cancer patients.This is a retrospective review of all cases referred for suspected head and neck cancer to our institution in January and April 2020.vs 98.33 per cent for April). Referrals for laryngopharyngeal reflux rose from 27.5 per cent to 41.9 per cent. Referrals for those aged over 60 years fell from 42 per cent to 26 per cent.There was a 55 per cent decrease in referrals but diagnostic yield rose from 2.9 per cent in January to 8.06 per cent in April. In both months, 100 per cent of patients met the 31- and 62-day targets, with similar 14-day wait time success (97.83 per cent for January It is suggested that further research be conducted into the reasons why fewer patients were referred, particularly elderly patients, and why laryngopharyngeal reflux is so prevalent in fast-track referrals. The cause was identified as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a novel coronavirus strain that causes coronavirus disease 2019 (Covid-19) and is suspected to be of zoonotic origin.3 In the community, general practice had to rapidly adapt to a sudden change from face-to-face patient reviews to remote consultations via telephone and video. Patients aged over 70 years or those with underlying health conditions were advised to shield at home, to decrease the risk of becoming infected with Covid-19, as the mortality rate for this group is 15 per cent.4In order to cope with the expected influx of patients with Covid-19, the National Health Service (NHS) had to fundamentally change the way it functions so as to narrow its focus on emergency care, including Covid-19 and cancer care. In order to achieve this, NHS hospitals across the UK were instructed to cancel elective work across all specialties, thereby freeing bed space and allowing the redeployment of staff to areas of need.The changes to the NHS and the increased anxiety surrounding patients being exposed to SARS-CoV-2 in healthcare settings are barriers to patients seeking medical care. There is concern that an increase in barriers to healthcare will be reflected in decreased referrals to cancer services and potentially missed diagnoses. In addition to decreased referrals, there are further concerns that cancer services themselves may be affected, with decreased staff availability because of self-isolation, Covid-19 infection or shielding. Further compounding this problem is staff redeployment to different areas of the hospital, limiting clinic numbers and the availability of radiological investigations.This audit of a busy tertiary centre head and neck fast-track referral service assessed how these multiple factors affected head and neck cancer referrals by comparing practice in January 2020 to April 2020. The study also assessed whether the fast-track service at our institution maintained function in line with UK head and neck cancer multidisciplinary team (MDT) guidelines during the Covid-19 pandemic.We conducted a retrospective review of all cases referred for suspected head and neck cancer to the ENT fast-track clinic at our institution during the months of January and April 2020. Referral details were obtained from our electronic referrals system. Data on demographics, investigations and management of patients were collected from our electronic patient record system. Practice was compared against the UK head and neck cancer MDT guidelines.vs 7.29 days). The proportion of patients being seen within 14 days from general practitioner referral, as per MDT guidelines, remained high in both months (97.83 per cent vs 98.33 per cent).There was a 55 per cent decrease in referrals between January and April 2020 , with thvs 8.06 per cent in April).At first review, a significant proportion of patients were discharged without any further investigation for malignancy in both months, with 63.04 per cent of cases in January, rising to 75.8 per cent in April. Whilst there was a decrease in the proportion of patients referred for suspected malignancy, there was a rise in the proportion of patients who had a malignancy diagnosed . In addition, the proportion of patients with no pathology on review remained similar, with 11 per cent in January compared to 7 per cent in April.The most common diagnosis for patients who were seen was laryngopharyngeal reflux. In January, this accounted for 27.5 per cent of total referrals, rising to 41.9 per cent in April . The proAnalysis of the demographics showed that the change in referral pattern during April was not evenly split by sex and age. In January, males accounted for 47 per cent of referrals, whereas this figure dropped to 40 per cent in April. There was a more marked variation in referrals when analysed by age group; the largest decrease was noted in the late-middle-aged to elderly population, with referrals for patients aged 60 years or more falling from 42 per cent of total referrals in January to 26 per cent in April .Fig. 2.vs 5 referrals). Ultrasound scanning remained the most common investigation; 91 per cent of patients referred in January had an ultrasound scan, with an average wait for the ultrasound scan from first review of 5.7 days. This compared to 60 per cent of referrals in April receiving an ultrasound scan, with an average wait time of 5.3 days. There was a rise in cross-sectional imaging, with 9 per cent of patients in January undergoing magnetic resonance imaging (MRI) compared to 20 per cent of patients in April, with an average wait time of 4 days in January and 6 days in April. Diagnostic yield for malignancy increased from 18 per cent of patients referred with a suspicious neck lump in January to 40 per cent in April.Of those patients referred for a suspicious neck lump, there was a reduction of 55 per cent in total referrals  in January, falling to 50 per cent in April. Fine needle aspiration for cytology was performed in 12 per cent of patients in January compared to 50 per cent in April, with a diagnostic yield for malignancy rising from 5.88 per cent to 50 per cent of referrals from January to April.Our results showed that the expected drop in referrals to our head and neck fast-track service amounted to 55 per cent. This reduction in referrals could be the result of a wide range of factors. For instance, it is likely that patients did not seek medical attention in order to avoid healthcare settings where the risk of Covid-19 infection is likely to be higher, or because they were shielding. It is also possible that the increased use of remote reviews only by general practitioners decreased the likelihood of patients wishing to contact them, which removed the potential for incidental findings on clinical examination.5Analysis of referrals by age group showed that the biggest drop in referrals was seen in the over-60 years age group, who are the most likely patients to be shielding because of age and co-morbidities. This is particularly concerning given the peak incidence for head and neck cancers in the 70–74 years age group.5 Further research is needed to determine which factors were most prominent in causing a decrease in referrals.Breakdown of referrals by sex showed a small decrease in male referrals, from 47 to 40 per cent. This could be reflective of less health-seeking behaviour in males in general, which may be exacerbated during a pandemic. However, males are almost three times as likely to develop head and neck cancer.6It was suspected that a decrease in referrals would lead to an increased yield of diagnosed malignancies associated with a decrease in ‘worried well’ patients presenting. This is reflected in the overall malignancy rate, which rose from 2.9 per cent to 8.1 per cent. However, there was also an unexpected increase in benign disease being diagnosed at first review. Analysis of outcomes after the first review showed a decreased proportion of patients being investigated for malignancy, falling from 38 per cent to 24 per cent. This may reflect a higher threshold for investigating patients in order to prevent those with a very low risk of malignancy being unnecessarily exposed to potential Covid-19 in hospital. This practice is likely to have been partly supported by the ENT UK Head and Neck Cancer Telephone Triage service tool, which risk stratifies patients into high and low risk groups.The increase in benign diagnosis at first review is particularly marked for the diagnosis of laryngopharyngeal reflux, with 42 per cent of patients being diagnosed with laryngopharyngeal reflux in April 2020 compared to 27.5 per cent in January 2020. Possible causes for this include: anxiety and stress caused by the pandemic, exacerbating reflux symptoms; or patients being assessed remotely prior to referral and consequently being more likely to be referred than if they had attended a face-to-face consultation. Laryngopharyngeal reflux continues to be a large burden on head and neck cancer services, and further research is required to assess why this is.vs 5.3 days), which may reflect the cancellation of routine imaging decreasing the wait time. The decrease in ultrasound scans performed, coupled with an increase in MRI from 9 per cent to 20 per cent, could be the result of more widespread use of telephone consultations, which may have increased diagnostic uncertainty and therefore increased requests for cross-sectional imaging.•There has been a large decrease in fast-track referrals during the coronavirus disease 2019 pandemic, particularly in the elderly population•Head and neck cancer services at our institution continue to meet 14-, 31- and 62-day target times, despite changes in services•There has been an increased diagnostic yield of malignancies•There has been a large rise in referrals for laryngopharyngeal reflux disease•More research is required to establish why there has been a large decrease in referral of elderly patientsThere was a 55 per cent decrease in the number of patients referred with suspicious neck lumps. This decrease is greater than expected given that neck lumps are a ‘red flag’ presentation of possible head and neck cancer, and they are recognised by most patients as a finding that needs to be reviewed by a medical practitioner. This likely represents a decrease in health-seeking behaviour for the reasons outlined above regarding attempts to reduce the risk of potential Covid-19 exposure. There was also a decrease in patients receiving an ultrasound scan for their suspicious neck lump, falling from 91 per cent to 60 per cent. The average wait for ultrasound scanning remained similar  for 65 per cent of scans in January and 50 per cent in April. The most common reason for not reporting a U grade was that normal thyroid tissue was not documented as ‘U1’.The decrease in referrals and increase in proportion of patients with red flag symptoms led to an increase in yield for diagnosing thyroid malignancy, rising from 5.88 per cent to 50 per cent. This suggests that if British Thyroid Association guidelines are followed, there would potentially be fewer patients referred with benign or no disease.The results of our audit show that despite the Covid-19 pandemic, the fast-track head and neck service at our institution met the 14-day, 31-day and 62-day target times in the month of April 2020, when compared to pre-pandemic data. There has not been a drop in the overall number of malignancies diagnosed, as may have been expected despite the significant fall in referrals, particularly among the elderly.Future audits will be required to see if there is a rise in malignant diagnoses in the coming months or whether those patients not referred represent the ‘worried well’. With an easing of lockdown restrictions across the UK, it is expected that health-seeking behaviours will begin to normalise and this may cause a spike in referrals; with hospitals still readjusting following Covid-19, this may make it difficult for the two-week wait guidance to be adhered to in the near future.7 We recommend that further studies investigate why these patients are being referred so commonly to head and neck cancer services, in order to prevent unnecessary patient anxiety and to allow more time for patients with malignancy to be reviewed.A final key finding of this audit is the markedly low yield of diagnosed malignancies, both before and during the pandemic, with huge numbers of laryngopharyngeal reflux cases. This mirrors other studies in the literature.