Poor ovarian response (POR) to controlled ovarian stimulation (OS) presents a major challenge in assisted reproduction. The Bologna criteria represented the first serious attempt to set clear criteria for the definition of POR. However, the Bologna criteria were questioned because of the persistent heterogeneity among POR patients and the inability to provide management strategies. Based on these facts, a more recent classification, the POSEIDON  classification, was developed to provide a homogeneous and refined definition of POR that significantly reduces the heterogeneity of the Bologna criteria definition of POR and helps in the clinical handling and counseling of patients. In this review, we discuss the impact of the POSEIDON classification on the clinical management of patients with POR. The ovarian response to ovarian stimulation (OS) reflected by the number of oocytes retrieved is a keystone in in vitro fertilization (IVF) cycles and an independent factor in the success of treatment ,2. AlthoIn 2016, a new classification by a panel of fertility experts known as the POSEIDON  group  was intrThe success of ART treatment is increasingly being measured by the live birth rate (LBR) per transfer or per OS cycle, the so-called cumulative live birth rate (CLBR). As mentioned, The POSEIDON Group introduced a new measure of success, which is the number of oocytes needed to achieve at least one euploid blastocyst for transfer in the individual patient . The avaThe prediction of an ovarian response to OS is crucial for the optimal and individualized management of patients undergoing ART. Both AFC and AMH have been successfully used to predict both poor and hyper responses ). In conhttp://www.members.groupposeidon.com/Calculator.As a pretreatment predictive model, the ART calculator  has beenPOSEIDON groups 1 and 2 include women who had a poor (<4) or suboptimal (4–9) number of oocytes retrieved after OS despite the presence of an adequate ovarian reserve defined as AFC > 5 and/or AMH > 1.2 ng/mL. These patients are classified as group 1 if younger than 35 years or group 2 if ≥ 35 years of age. In practical terms, POSEIDON groups 1 and 2 are hyporesponders who unexpectedly show a low ovarian response or resistance to OS with exogenous gonadotropins. Both groups are likely to have a lower CLBR than normal or high responder patients . ApproxiThe pathophysiological mechanism of the hyporesponse noted in POSEIDON groups 1 and 2 is not fully understood; however, it seems that the decreased sensitivity of follicles to exogenous follicle-stimulating hormone (FSH) may be the most plausible explanation. In line with this some specific genetic polymorphisms, affecting gonadotropins and their receptors have been suggested. Thus, a common LH beta subunit variant was associated with increased FSH consumption during stimulation ,26,27,28Environmental contaminants might also affect ovarian response during OS ,30,31. TSince the new marker of success in ART according to the POSEIDON group is to retrieve the number of oocytes needed to have at least one euploid blastocyst for transfer, the goal of the management of POSEIDON groups 1 and 2 is to find a way to increase oocyte yield and subsequently the reproductive outcome. According to the available literature, five main strategies should be considered for use alone or in combination, namely: (i) use of recombinant FSH (rFSH) in preference over urinary gonadotropins, (ii) an FSH dosage increase, (iii) the use of recombinant LH (rLH), (iv) dehydroepiandrosterone (DHEA) supplementation before OS, and (v) a double-stimulation (DuoStim) protocol. The suggested management of POSEIDON groups 1 and 2 is illustrated in Multiple RCTs and meta-analyses have shown that the use rFSH is associated with significantly more oocytes retrieved as compared to urinary FSH formulations, both in long gonadotropin releasing hormone (GnRH) agonist (GnRHa) and GnRH antagonist cycles ,38,39. Tp < 0.001) and good quality embryos  was retrieved compared to the previous cycle. According to the authors, an increase of 50 IU of the initial rFSH dose would lead to the retrieval of one more oocyte. Even patients with FSHR polymorphism seem to benefit from increases in rFSH dosage. Thus, Behre et al. randomized Ser680/Ser680 carriers to receive a daily rFSH of either 150 IU or 225 IU. The 225 IU/day dose was able to restore estradiol levels of Ser680/Ser680 carriers similar to those of women with the wild-type genotype at the end of stimulation [Increasing the FSH dose in patients with a history of suboptimal response is a commonly used strategy. This strategy might be used to rescue an ongoing OS in women with initial slow response to gonadotropins or in a subsequent stimulation cycle in women with a history of suboptimal response. In a retrospective study by Drakopoulos et al., a total of 160 women with a normal ovarian reserve and a history of suboptimal response (4–9 oocytes retrieved) were recruited to receive an increase in rFSH in the subsequent fixed GnRH antagonist co-treated cycle . A signimulation . In concp < 0.01). In addition, the implantation rate  and ongoing pregnancy rates  were similar to those observed in the control group, consisting of normal responders. These results were recently corroborated in an RCT by Yilmaz et al. [p < 0.02).Several studies evaluated the addition of rLH to the OS protocol in women with ovarian hyporesponse ,46,47,48z et al. , in whicz et al. . Patientp < 0.05) [As for the rLH dosage, 150 IU rLH was previously proven to be superior to 75 IU rLH when a long GnRHa downregulation protocol was used  were ran < 0.05) ,51 repor < 0.05) . ImportaThe most recent systematic review and a further meta-analysis have shown that adding rLH to the OS protocol is beneficial for two subgroups of patients, (i) women with an adequate ovarian reserve having an unexpected hyporesponse to rFSH monotherapy, i.e., POSEIDON groups 1 and 2, and (ii) patients 36–39 years of age ,52. Therp < 0.05). In another RCT, Moawad et al. [p < 0.05) in patients who received DHEA pretreatment.Pretreatment with DHEA before OS has been shown to significantly improve the birth rate in POR and patients of advanced age, according to the latest Cochrane meta-analysis . As a prd et al.  reportedDouble stimulation or so-called DuoStim combines conventional follicular phase stimulation with luteal phase stimulation during the same cycle. The aim of DuoStim is to maximize the number of oocytes obtained per cycle. A large multicenter study has shown comparable fertilization, blastocyst, euploidy, and pregnancy rates from follicular phase stimulation and luteal phase stimulation . MoreovePOSEIDON groups 1 and 2 respond unexpectedly poorly to OS despite a good ovarian reserve. The main difference between these two groups of patients is their age and consequently a difference in oocyte euploidy and thus LBR. Group 1 is considered to have a better prognosis due to an age-related lower oocyte aneuploidy rate. Patients in group 2, although older and with an age-related higher oocyte aneuploidy, still have a good chance of reaching the estimated number of oocytes needed for one euploid blastocyst if properly stimulated. For both groups, this might be achieved by increasing the dosage of rFSH, rLH supplementation, and/or using a DuoStim protocol.The subdivision achieved in the POSEIDON classification can help us better understand the specifics of each homogeneous subgroup of patients undergoing ART. Future clinical trials should study each group separately aiming to find the optimal clinical management for each group. This will lead to the development of personalized treatments and tailored stimulation protocols. Research should evaluate the benefit of screening patients for specific FSH and LH receptor polymorphisms as well as the presence of variant LH-β to explore specific treatments for each trait. The role of practical indices such as FOI and the ART calculator in the management of POSEIDON groups 1 and 2 should also be further evaluated. Finally, research on the use of antioxidants to alleviate the possible negative effects of ROS on ovarian resistance to OS is warranted.POSEIDON Groups 3 and 4 are patients who are expected to have a poor response to OS due to their low ovarian reserve in terms of a total AFC < 5, and/or AMH < 1.2 ng/mL. Group 3 includes patients < 35 years of age and group 4 ≥ 35 years of age. The latter group has a high risk of having poor oocyte quality in addition to a reduced quantity due to the age-related increase in oocyte aneuploidy . InteresPreviously, it was suggested that natural cycle IVF or mild stimulation would be more optimal for the Poseidon group 3 and 4 patients when compared to conventional OS ,60, as oAs for the choice of the stimulation protocol, Sunkara et al. reported a non-significant increase in the number of mature oocytes by one oocyte, and a significant decrease in cancellation rates when a long GnRH agonist protocol was used in patients with POR compared to a GnRH antagonist protocol . This inRecombinant FSH is the preferred choice of gonadotropins in POR patients, and there is ample high-quality evidence showing that rFSH is superior to urinary FSH and human menopausal gonadotropin (hMG) as a means to increase the oocyte yield ,39,72,73Finally, two systematic reviews suggested that rLH supplementation during OS is beneficial in women with hyporesponse to exogenous FSH-only stimulation and specifically in women aged 36–39 years ,52. HumaIn addition to the individualized OS, an individualized ovulation trigger (OT) strategy may also be used to increase the mature oocyte yield. One of the reasons for a low FOI could be mutations of the LH receptor ,20,78, wTwo new OT strategies, a dual trigger and double trigger, have been introduced for the management of a low FOI or a low mature follicle/mature oocyte rate. These two strategies have been suggested to improve IVF outcomes by overcoming impairment in follicular function, oocyte meiotic maturation, and cumulus expansion . In dualclinicaltrial.gov identifier NCT02418572) currently evaluates a daily dose of 5.5 mg transdermal testosterone as a pretreatment for 60 days, and the results of this trial might provide further evidence for the possible clinical use of androgens in POSEIDON group 3 and 4 patients.Pretreatment with androgens could be considered in patients with expected POR. The biological evidence has shown that androgens induce FSH receptors on granulosa cells , which sThe rationale for the use of growth hormone (GH) in ART is the fact that GH itself and through IGF-1 has a synergistic effect with FSH on follicular development ,95. AdjuThe hypothesis behind using coenzyme Q 10 (CoQ 10) is the reduction in mitochondrial oxidative stress improving oocyte competence. A recent RCT assessed the effectiveness of CoQ 10 pretreatment for 60 days prior to OS in POSEIDON group 3 patients , reportiDue to the limited ovarian reserve, POSEIDON group 3 and 4 patients have a low reproductive prognosis and pose a clinical challenge. The current best practice to manage these patients is to individualize OS in order to increase the oocyte yield, which is essential to optimize the LBR. Even increasing the oocyte yield from 2 to 3 results in a 25% relative increase in the LBR across all age groups. Ongoing pregnancy rates above 20% per cycle of DuoStim in these difficult groups could be considered a successful achievement; however, more research is needed to suggest firm clinical recommendations.Randomized controlled trials (RCTs) taking into consideration the novel patient stratification suggested by the POSEIDON Group are warranted. Thus, future trials investigating the most optimal OS strategy for groups 3 and 4 are needed. This might be achieved through studies comparing the long GnRH agonist downregulation protocol to estrogen or contraceptive pill primed GnRH antagonist protocols and the DuoStim protocol. The outcome of these studies should include the cumulative LBR and the ability to obtain the number of oocytes needed in each induced patient to obtain at least one euploid blastocyst for transfer. More studies are also needed in the area of pretreatment with adjuvants, including androgens, GH, and antioxidants that explore the timing, duration, and dosages of the treatment. On a more experimental basis, the future management of the expected POR might include in vitro follicle activation , autologThe POR patient constitutes a major challenge in reproductive medicine. The recently proposed POSEIDON criteria for POR stratifies patients into four homogeneous groups, leading to a better understanding of the different subgroups of POR, better development of individualized treatment regimens, and better research design. Groups 1 (<35 years) and 2 (≥35 years) are the unexpected POR patients with a good ovarian reserve, and groups 3 (<35 years) and 4 (≥35 years) are those who are expected to have POR due to a low ovarian reserve. The primary goal of the management of the POSEIDON POR patient is to maximize the oocyte yield so as to increase the likelihood of having at least one euploid blastocyst for transfer. This demands an individualized approach at all steps of the ART treatment, including a targeted pretreatment evaluation, the choice of GnRH analogues, gonadotropin type, and doses, ovulation trigger, and the possible use of adjuvant therapies. Tools such as FOI and the ART calculator can help develop individualized treatment regimens and most importantly help counsel patients about their reproductive prognosis. With this approach, even the very poor prognosis patients in POSEIDON group 4 may achieve an ongoing pregnancy rate of more than 20% per cycle (DuoStim). Future well-designed RCTs are warranted to evaluate the stratifications and the recommendations set forward in this review. In this aspect, POSEIDON groups 1 and 2 need to be studied separately from POSEIDON groups 3 and 4.


Leaders around the world have embraced the idea of a “15-minute city”. This urban planning concept proposes a city where residents can meet their essential needs within a short walking or cycling trip from their home. Local access to grocery stores is a necessary component for cities to achieve the 15-minute city. This study aims to evaluate local accessibility to grocery stores by walking and cycling in the City of Vancouver. We used a cu-mulative opportunity measure to count the number of grocery stores available within a 15-minute walk and cycle from people’s homes. To evaluate accessibility from the perspective of younger and older travellers, we considered different travel speeds. Our results show there is good accessibility to grocery stores when cycling, with less than 1% of the city’s population not having a grocery store within a 15-minute cycle. When assuming a walking speed of an older pedestrian, around one-fifth of the population did not have access to a grocery store in their local area. The neighbourhoods that did not have a store within a 15-minute walk had higher proportions of children, older adults, and visible minorities, and lower rates of employment and post-secondary education attainment. In seeking to improve accessibility via walking and cycling, cities should prioritize grocery store locations and investments in pedestrian and cycling infrastructure to underserved neighbourhoods and populations. The 15-minute city proposes a city where all residents can meet their daily needs within a short walking or cycling trip from their home . This coThe concept has garnered considerable interest from cities and international organizations, however, some question whether this concept translates from a European to a North American context . Many EuIn this paper, we explored whether the 15-minute city is within reach in a Canadian context by evaluating local accessibility to grocery stores by walking and cycling in the City of Vancouver. A secondary aim was to explore social equity in access to grocery stores by comparing the sociodemographic characteristics of the population who had access to at least 1 grocery store within a 15-minute walk compared to those who did not. We focused exclusively on grocery stores because this amenity is an essential component of the 15-minute city and a destination that almost all households need to access on a regular basis. We selected Vancouver as our case study because it has the highest population density of any Canadian city – making it one of the more likely candidates to achieve the 15-minute city. Vancouver is also already working towards its towards its own vision of the 15-minute city as part of its Climate Emergency Response. The City of Vancouver has set target that 90% of residents live within an easy walk or roll of their daily needs by 2030 .We used the cumulative opportunity measure to count the number of grocery and produce stores available within a 15-minute walk and cycle from people’s homes. The cumulative opportunity measure counts the number of opportunities reached within a given travel time or cost threshold, which makes it well suited to evaluate the 15-minute city concept.We considered different travel speeds in the analysis to compare accessibility from the perspective of younger and older travellers.2, Vancouver has the highest population density of any Canadian city .We used dissemination block centroids to represent the origins for all residents in a dissemination block. Dissemination blocks are geographical areas equivalent to one city block. They are the smallest geographical unit provided by Statistics Canada for which population counts are available , and theWe aimed to include food stores that provided access to healthy food options, which we considered to be supermarkets, grocery stores, and produce stores. We excluded convenience stores. We acquired the locations of Vancouver’s supermarkets, grocery stores, and produce stores from the Business Licenses dataset in the City of Vancouver’s Open data catalogue (downloaded March 2021) . Since tImpedance between origins and destinations was operationalized by measuring travel time along the street network. We used Open-StreetMaps (OSM) data for the street network. Since the distance covered along the street network in 15-minutes will depend on the traveller’s speed, which may be impacted by age or topography, we used different values for walking and cycling speeds of younger and older travellers. For pedestrians, we used speeds provided in the Highway Capacity Manual: 4.8 km/hr for younger pedestrians (<65 years) and 3.6 km/hr for older pedestrians (≥ 65 years) . For cycTo generate travel time matrices between dissemination block centroids and grocery stores, we used the R package R5R . The traThe R5 routing engine makes use of OSM’s tags to determine what streets are suitable for walking and cycling. Tags are used to describe specific features in OSM. For example, the R5 routing engine removes all road segments with the tag “highway = motorway” when determining routes for pedestrians and cyclists. For cycling, a maximum LTS can be specified to further filter the network to only include routes where most cyclists would feel comfortable cycling. LTS is a rating system for classifying streets and intersections based on tolerance for traffic stress . The scoWe generated four travel time matrices for the following scenarios: (1) walking: younger traveller (speed 4.8 km/hr); (2) walking: older traveller (3.6 km/hr); (3) cycling: younger traveller (16.2 km/hr); and (4) cycling: older traveller (13.9 km/hr). The travel time matrices were used to count the number of grocery stores within a 15-minute travel time of each dissemination block in the four scenarios. We calculated descriptive statistics to summarize the number of grocery stores within 15-minutes.To examine social equity in access to grocery stores, we compared the average sociodemographic characteristics between two groups: those who did not have a grocery store within a 15-minute walk to those who had at least one store within a 15-minute walk . Since population-level sociodemographic characteristics are not available at the dissemination block level, we calculated the average number of grocery stores accessible in each dissemination area (DA). For both groups , we calculated the average proportion for seven sociodemographic variables: % aged 0–14 years, % aged 65 and over, employment rate, % with post-secondary education, % low income, % visible minority, % of recent immigrants. We used the after-tax low-income measure calculated by Statistics Canada . VisibleA total of 169 grocery stores were identified in the study area (n = 131 in Vancouver and n = 38 in Burnaby). The majority of dissemination blocks in the City of Vancouver were within a 15-minute walk of at least one grocery store and almost all dissemination blocks were within a 15-minute cycle . HoweverThe dissemination blocks with the greatest accessibility to grocery stores by walking are in the downtown core and surrounding neigh-bourhoods. Dissemination blocks with poor accessibility to grocery stores were concentrated in the south area of city.Assuming a walk speed of a younger pedestrian, dissemination blocks had access to 4 grocery stores within 15 minutes, on average. Only 11% of dissemination blocks did not have a grocery store within 15 minutes, which corresponds to roughly 9% of the city’s population. An additional quarter of the city’s population only had one or two stores within 15 minutes at a walking speed of a younger pedestrian.Accessibility to grocery stores by walking was reduced considerably when a walking speed of an older pedestrian was assumed. Dissemination blocks had an average of 2 grocery stores within 15 minutes. Over a quarter of dissemination blocks (27%) had no grocery store within 15 minutes, which corresponds to about one fifth (21%) of the city’s population. An additional third (34%) of the population only had one or two stores within 15 minutes at a walking speed of an older pedestrian.There is good accessibility by cycling to grocery stores across the city. The majority of dissemination blocks had access to more than 10 grocery stores within a 15-minute cycle. Dissemination blocks in the corners of the city – particularly the south-west neighbourhoods – have lower accessibility.Assuming a cycling speed of a younger cyclist, dissemination blocks had an average of 20 grocery stores within a 15-minute cycle. The majority of dissemination blocks (80%) had access to 11 or more stores, which corresponds to 85% of the city’s population. There are no dissemination blocks without access to a grocery store within 15 minutes.For cycling speeds of older travellers, dissemination blocks had an average of 16 stores within a 15-minute cycle. Nearly three-quarters of the population (72%) had access to 11 or more grocery stores, and only 5 dissemination blocks did not have access to any grocery store within 15 minutes.Our findings suggest that the 15-minute city is within reach in the City of Vancouver in terms of access to grocery stores. There is particularly good accessibility by cycling, with over three-quarters of the population having over 10 grocery stores within a 15-minute cycle. This highlights the important role that cycling can play in achieving the 15-minute city in the North American context. In terms of accessibility by walking, most dissemination blocks had at least one grocery store within 15-minutes, however in many areas the options were limited to one or two stores. We found that neighbourhoods that did not have access to a grocery within 15-minutes had higher proportions of children, older adults, visible minorities, and had lower employment rates and levels of post-secondary attainment.The findings from this study highlight the importance of considering different perspectives in working towards the 15-minute city. We considered travel speed since this affects the number of amenities available within a 15-minute walk or cycle. At a walk speed typical of adults younger than 65 years, only 9% of the population did not have a grocery store within a 15-minute walk. At a walk speed typical of adults older than 65 years, the proportion that did not have a grocery store within 15-minutes more than doubled to 21% of the population. Applying more conservative estimates of walk-speed in evaluating local access to amenities will ensure that the 15-minute city is accessible to more people. Another perspective that could be considered in a future analysis is accessibility by cycling from the perspective of children or parents travelling with children. We specified a maximum LTS of 2 for routing cyclists along the street network, which includes routes with a level of traffic stress that is tolerable by most adults. To consider accessibility from the perspective of children, a maximum LTS of 1 could be specified, which would include only the lowest traffic stress routes.Future work should also study perceptions of accessibility to consider whether the local opportunities available are meeting the needs of residents in the area. The accessibility analysis in this paper provides a starting point for identifying areas where there are high and low levels of accessibility to grocery stores by walking and cycling. A complementary body of work assesses people perceptions of their local neighbourhood, including the perceived safety and comfort of walking and cycling environments, the amount of time that people are willing to walk or cycle to amenities, and whether the local opportunities serve resident’s needs . We can A second area for future research to explore is the role of online shopping in the 15-minute city. Widespread use of digital technology is changing the way we access services and opportunities . AccordiWe used a cumulative opportunity measure to quantify the number of grocery stores within a 15-minute threshold. The advantage of this measure is that it provides an output that is absolute, easy to interpret, and comparable across modes and contexts . On the There are a few limitations relating to the data used in this analysis. The grocery stores identified from the business licenses dataset may not capture all stores. A validation study of the 2015 Business Licenses data in the City of Vancouver found that the dataset undercounted the number of grocery stores compared to ground-truthed observational data . HoweverThe 15-minute city envisions a city where all residents can access their daily needs within a short walking or cycling trip. This study evaluated whether the 15-minute city was within reach for the City of Vancouver in terms of access to grocery stores. Overall, our findings showed that the 15-minute city is within reach in the Vancouver context. Four fifths (79%) of Vancouver’s population had access to a grocery store within a 15-minute walk or cycle, assuming a travel speed of an older traveller. We found there was some inequities in access to grocery stores, with lower access in areas that had higher proportions of children, older adults, visible minorities, and lower employment and post-secondary education attainment rates. To help guide the development of a 15-minute city that is inclusive of all ages and abilities we recommend that researchers and city planners use conservative estimates of walk speed and level of traffic stress for cycling, while prioritizing investments that ensure more equitable access across neighbourhoods and populations.


Saccharomyces cerevisiae is amenable to studying membrane traffic by live-cell fluorescence microscopy. We used this system to explore two aspects of cargo protein traffic through prevacuolar endosome (PVE) compartments to the vacuole. First, at what point during Golgi maturation does a biosynthetic vacuolar cargo depart from the maturing cisternae? To address this question, we modified a regulatable fluorescent secretory cargo by adding a vacuolar targeting signal. Traffic of the vacuolar cargo requires the GGA clathrin adaptors, which arrive during the early-to-late Golgi transition. Accordingly, the vacuolar cargo begins to exit the Golgi near the midpoint of maturation, significantly before exit of a secretory cargo. Second, how are cargoes delivered from PVE compartments to the vacuole? To address this question, we tracked biosynthetic and endocytic cargoes after they had accumulated in PVE compartments. The results suggest that stable PVE compartments repeatedly deliver material to the vacuole by a kiss-and-run mechanism. Saccharomyces cerevisiae have identified many conserved components of the biosynthetic and endocytic machineries  compartments, which are multivesicular bodies reminiscent of mammalian late endosomes , plays an additional role as an early and recycling endosome  versions of the secretory and vacuolar cargoes. The vacuolar membrane was visualized with Vph1-GFP. Compared to the non-aggregating secretory cargo, which accumulated at low levels in the vacuole , the nonVPS10 wild-type strain and a vps10∆ mutant strain expressed the vacuolar cargo together with the vacuole marker Vph1-GFP. After SLF was added to initiate cargo transport, the cells were imaged by 4D confocal microscopy for 60 min  prior to imaging, a fraction of the fluorescent cargo molecules are in the vacuole or other non-PVE compartments, and (2) a typical cell contains several PVE compartments that can undergo fission and homotypic fusion , making Traffic of the biosynthetic vacuolar cargo from PVE compartments to the vacuole was visualized by generating a yeast strain expressing the vacuolar cargo together with the PVE marker Vps8-GFP and the vacuole marker Vph1-HaloTag. At 10–15 min after SLF addition, the majority of the cargo molecules had passed through the Golgi and accumulated in PVE compartments see  above. 4We wondered whether the putative kiss-and-run events could also deliver transmembrane cargoes that are encapsulated in intraluminal vesicles within PVE compartments . This exFor both the vacuolar cargo and Mup1, quantification revealed that gradual transfer to the vacuole was punctuated, about once every 5 min on average, by bursts in which > 15% of the remaining cargo moved to the vacuole. The average amount of remaining cargo transferred during a burst was ~35–40%. Individual PVE compartments differed greatly in the total fraction of the cargo that was transferred in bursts, but on average this number was ~75%. These results favor a PVE-to-vacuole traffic mechanism involving repeated kiss-and-run events of varying size.Live-cell fluorescence microscopy complements other methods by providing unique insights into membrane traffic . RecentlThe convenience of working with an artificial vacuolar cargo is accompanied by some caveats. Compared to CPY, which is monomeric , our vacUsing the vacuolar cargo, we asked a basic question: at what point during cisternal maturation does the vacuolar cargo depart from the Golgi? Unlike the regulatable fluorescent secretory cargo, which persists in Golgi cisternae until they are terminally mature , the vacSuperficially, these results are at odds with the established view that various types of biosynthetic cargoes travel through the entire Golgi until being packaged into distinct carriers at the TGN . While tAfter the vacuolar cargo reaches PVE compartments, how does it move to the vacuole? Previously, based on evidence that PVE compartments are long-lived organelles that rarely if ever fuse completely with the vacuole, we proposed that transfer of material from PVE compartments to the vacuole might involve kiss-and-run fusion events . This moOur inference about the kiss-and-run mechanism is based on fluorescence microscopy and will benefit from validation by other techniques. Notably, an electron tomographic reconstruction carried out by the Odorizzi group , reprintIf kiss-and-run transfer occurs between PVE compartments and the vacuole, the transient continuities between these organelles might permit exchange of freely diffusing transmembrane proteins. For example, the transmembrane precursor to the vacuolar hydrolase carboxypeptidase S is normally internalized into intraluminal vesicles of PVE compartments, but a mutant carboxypeptidase S precursor that cannot be internalized moves from the limiting membranes of PVE compartments to the vacuole membrane . AlthougCargo transfer from PVE compartments to the vacuole is slow relative to other membrane traffic steps in budding yeast. Historically, kinetics of traffic to the vacuole have been measured by pulse-chase experiments that rely on the maturation of CPY by vacuolar proteases , but tholeu2-3,112 ura3-52 rme1 trp1 his4)  . Yeast wp1 his4)  or in riPDR1, PDR3, and GGA1 genes was accomplished by replacement with a G418, nourseothricin, or hygromycin resistance cassette from pFA6a-kanMX6, pAG25, or pAG32, respectively . Newly generated plasmids have been archived with Addgene .Yeast proteins were tagged by gene replacement using the pop-in/pop-out method to maintain endogenous expression levels . Secretorminator . To ensuFor live-cell fluorescence imaging, yeast strains were grown in NSD (pH ∼5.5) at 23°C. Where indicated, SLF was diluted from a 100 mM stock solution in ethanol  to a final concentration of 100 µM, and cycloheximide was added from a 100 mg/mL stock solution in DMSO. Cells were attached to a concanavalin A-coated coverglass-bottom dish containing NSD  for imagStatic images and 4D movies were processed as follows. Static images were converted to 16-bit and average projected , then raFor photobleaching prior to 4D imaging of PVE compartments, a region of interest was drawn to include all fluorescent structures within the cell except the PVE compartments. The regulatable vacuolar cargo and Mup1-mScarlet were bleached by maximum-intensity illumination with a 561 nm laser for 40 s or 5 s, respectively. These bleaching durations were chosen by determining the minimal times needed to bleach the fluorescence signals completely.To quantify the frequency at which bursts of cargo moved from PVE compartments to the vacuole, each 10 min video was examined at 10 s intervals. Bursts were scored as intervals in which more than 15% of the remaining cargo transferred to the vacuole. Six movies for the vacuolar cargo and seven movies for Mup1 were analyzed. The two cargoes displayed similar frequencies of large bursts as well as similar amounts of cargo transferred per burst, so the data were combined to obtain the averaged numbers stated in the text.646 ligand  for 2 min in a microcentrifuge. The culture medium supernatant was transferred to a fresh microcentrifuge tube on ice, and the cells were washed once with deionized water. Then the cells were resuspended in 100 µL 4% trichloroacetic acid. Glass beads  were added to bring the total volume to ∼200 µL, and the sample was vortexed three times for 1 min each separated by 1 min intervals on ice. Finally, 800 µL of PBS was added, the solution was mixed, and 800 µL of the cell lysate was transferred to a fresh microcentrifuge tube. Supernatant protein samples were precipitated with 4% trichloroacetic acid on ice for 20 min. Precipitated proteins were centrifuged at maximum speed in a microcentrifuge for 15 min at 4°C. Finally, each protein pellet was resuspended in 50 µL SDS-PAGE sample buffer.Immunoblotting was performed as follows Treatment with endoglycosidase H was performed as described by the manufacturer . Briefly, Glycoprotein Denaturing Buffer was added to the protein sample, which was boiled for 5–10 min, followed by addition of GlycoBuffer 3 and endoglycosidase H. The reaction was performed at 37°C for at least 1 hr.For immunoblotting, 9 µL of each cell lysate and 14 µL of each secreted protein sample were run on a 4–20% Tris-glycine gel  together with the Precision Plus Protein Dual Color Standards molecular weight marker . The separated proteins were transferred to a PVDF membrane  using the Trans-Blot Turbo system (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in TBST  with shaking at room temperature for 1 hr, and then incubated with 1:1000 polyclonal rabbit anti-FKBP12 antibody  in 5% milk/TBST with shaking overnight at 4°C. After four 5 min washes in TBST, the membrane was incubated with a 1:1000 dilution of goat anti-rabbit secondary antibody conjugated to Alexa Fluor 647  for 1 hr at room temperature. The membrane was then washed twice with TBST and twice with 50 mM Tris-HCl at pH 7.6, 150 mM NaCl. Analysis was performed with a LI-COR Odyssey CLx imaging system. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study provides compelling evidence that a yeast vacuolar precursor protein is sorted from an earlier Golgi compartment than previously thought, and secondly, that transfer of vacuolar cargo from the pre-vacuolar endosome to the vacuole is gradual and does not 'consume' the endosome. The results suggest that cargo transfer between the pre-vacuolar endosome and the vacuole occurs via a 'kiss and run' mechanism.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, including Christopher G Burd as the Reviewing Editor and reviewer #1, and the evaluation has been overseen by Vivek Malhotra as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: David Teis (Reviewer #2).Thank you for submitting your article "A microscopy-based kinetic analysis of yeast vacuolar protein sorting" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Summary:The reviewers found your manuscript to clearly present a well-executed study of biosynthetic and endocytic protein trafficking to the yeast vacuole. Regarding the two major conclusions of the manuscript, all three reviewers considered the finding that a fluorescent vacuolar enzyme reporter is exported from the Golgi at the early-to-late cisterna transition interesting and well supported by the analyses. The reviewers found the analysis of content transfer between the pre-vacuolar endosome and the vacuole interesting and potentially important. However, each reviewer considered the conclusion that content is transferred by a kiss and run fusion mechanism to be poorly supported. Their consensus opinion is that substantial additional experimentation is necessary to rigorously prove this point. The core points that the reviewers suggest need to be addressed are the following.1) The use of Vps8 as an accurate marker of the PVE needs to be more firmly established. It is a component of the CORVET complex that tethers clusters of late endosomes with Vps21, hence, it is a reasonable possibility that individual late endosomes from the cluster fuse with the vacuole. The reviewers also point several concerns with tagged/inappropriately expressed Vps8 that need to be addressed.2) An ultra-structural analysis of the PVE and vacuole is required to clarify the nature of the tethered PVE (single vs. multiple compartments) and to define the structural relationship between the PVE and vacuole (fused?). The single reprinted micrograph showing MVBs apparently fused to the vacuole membrane via a stalk underscores the importance of the approach, which seems suitable for rigorously characterizing the PVE-vacuole junctions.Revisions for this paper:Given the impact of the COVID-19 pandemic to the research community, the reviewers considered the strength of the study as it currently stands. From this perspective, the reviewers agreed that the results presented in the study would provide a substantial and healthy advance to the field if the following considerations were addressed by additional analysis of data already in hand and modifications to the presentation, as follows:1) The presentation of transfer by kiss and run fusion should be consistent throughout the manuscript. For example, the Abstract states that the data "imply" kiss and run. The last sentence of the Introduction states that transfer during kiss and run fusion has been "demonstrate(d)". The reviewers agree that the results can be explained by a kiss and run fusion mechanism, as indicated in the Abstract, but not that cargo transfer via kiss and run fusion has been demonstrated. The text should accurately and consistently indicate that kiss and run fusion remains to be demonstrated.2) In Figures 7B/C and 8B/C, the two distinct types of PVE-to-vacuole transfer events  should be quantified so that it can be determined which occurrence, if either, is most prevalent.Revisions expected in follow-up work:An ultra-structural analysis of the PVE and vacuole is required to clarify the nature of the tethered PVE (single vs. multiple compartments) and to define the spatial relationship between the PVE and vacuole. Summary:The reviewers found your manuscript to clearly present a well-executed study of biosynthetic and endocytic protein trafficking to the yeast vacuole. […] The core points that the reviewers suggest need to be addressed are the following.1) The use of Vps8 as an accurate marker of the PVE needs to be more firmly established. It is a component of the CORVET complex that tethers clusters of late endosomes with Vps21, hence, it is a reasonable possibility that individual late endosomes from the cluster fuse with the vacuole. The reviewers also point several concerns with tagged/inappropriately expressed Vps8 that need to be addressed.If we understand correctly, the concern is that Vps8 might specifically mark clusters of PVE compartments and not label individual PVE compartments. We have enhanced the text to allay this concern. In our previous work , we showed that Vps8GFP colocalizes with a variety of other PVE markers, and that PVE compartments showed similar dynamics when labeled with Vps8-GFP or with other PVE markers. There was no hint that any PVE compartments might fail to label with Vps8.The use of Vps8-GFP as a PVE marker is based on a paper from the Ungermann lab , who have studied Vps8 and related topics extensively.2) An ultra-structural analysis of the PVE and vacuole is required to clarify the nature of the tethered PVE (single vs. multiple compartments) and to define the structural relationship between the PVE and vacuole (fused?). The single reprinted micrograph showing MVBs apparently fused to the vacuole membrane via a stalk underscores the importance of the approach, which seems suitable for rigorously characterizing the PVE-vacuole junctions.We agree that ultrastructural analysis would be needed for a definitive conclusion about the mechanism of cargo transfer from PVE compartments to the vacuole. The reprinted micrograph is included here because it suggests that our interpretation is plausible. We have modified the text to be appropriately cautious.Revisions for this paper:Given the impact of the COVID-19 pandemic to the research community, the reviewers considered the strength of the study as it currently stands. From this perspective, the reviewers agreed that the results presented in the study would provide a substantial and healthy advance to the field if the following considerations were addressed by additional analysis of data already in hand and modifications to the presentation, as follows:1) The presentation of transfer by kiss and run fusion should be consistent throughout the manuscript. For example, the Abstract states that the data "imply" kiss and run. The last sentence of the Introduction states that transfer during kiss and run fusion has been "demonstrate(d)". The reviewers agree that the results can be explained by a kiss and run fusion mechanism, as indicated in the Abstract, but not that cargo transfer via kiss and run fusion has been demonstrated. The text should accurately and consistently indicate that kiss and run fusion remains to be demonstrated.We fully agree. The text has been modified to emphasize that kiss-and-run is merely a hypothesized mechanism that can potentially explain the data, and that this hypothesis needs to be tested by future analysis.2) In Figures 7B/C and 8B/C, the two distinct types of PVE-to-vacuole transfer events  should be quantified so that it can be determined which occurrence, if either, is most prevalent.This quantification is now included in the text at the end of the Results section. The procedure is described in a new paragraph in the Materials and methods section.Revisions expected in follow-up work:An ultra-structural analysis of the PVE and vacuole is required to clarify the nature of the tethered PVE (single vs. multiple compartments) and to define the spatial relationship between the PVE and vacuole.We acknowledge the limitations of the current data, but further thought will be needed for a suitable follow-up analysis. An ideal experiment would reveal ultrastructural data of the same PVE compartments before and after bursts of cargo transfer to the vacuole, but such an experiment is not feasible. We are more optimistic about the prospect of distinguishing single from clustered PVE compartments by fluorescence microscopy, perhaps with some form of super-resolution imaging. However, such a method remains to be developed.


Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a ‘bind and discard’ mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of  Notch (N) haploinsufficiency, which was discovered in Drosophila, causes a variety of tissue-specific defects including wing notching and extra sensory bristle formation that can vary greatly in penetrance and expressivity /Lag1 (CSL) transcription factor (TF) and the adaptor protein Mastermind (Mam), and induces gene expression via two types of DNA binding sites: independent CSL sites that bind monomeric NICD/CSL/Mam (NCM) complexes, and Su(H) paired sites (SPS) that are oriented in a head-to-head manner to promote cooperative binding between two NCM complexes  from the membrane . NICD suomplexes . Once boNotch1 mutations that remove an NICD degron sequence have been associated with increased NICD levels and the development of T-cell Acute Lymphoblastic Leukemia (T-ALL) in mice and humans  affect both short and long duration processes. Collectively, these findings provide new insights into how distinct Notch-dependent cellular processes can be differentially impacted by both enhancer architecture and signal duration to induce tissue-specific Notch defects within a complex animal.In this study, we use Notch signals in Drosophila, we designed synthetic enhancers with comparable numbers of either CSL monomer or SPS dimer sites (Drosophila (Su(H), NICD, and Mam) proteins. For this experiment, an equal amount of differentially labeled 2xCSL  and 1xSPS  probe was added into the same binding reaction containing different concentrations of the RBPJ/Su(H) TF with and without the species matched NICD/Mastermind co-activators . Surprisingly, flies homozygous for 6xSPS-3xGBE-lacZ (6SG-lacZ) developed a notched-wing phenotype that mimics a classic Notch haploinsufficiency , 6xSPS alone (6S-lacZ), or mutated SPSs (6SmutG-lacZ)  The 6SG-lacZ caused notched wings when inserted in another locus and regardless of the order of GBE and SPS, although with differences in penetrance and severity (Notch haploinsufficiency phenotype in the wing.We next generated transgenic fly lines containing reporter genes with either CSL or SPS binding sites. Since prior studies found that including sites for the Grainyhead (Grh) pioneer TF enhanced Notch reporter activity  and induficiency . In conttG-lacZ)  insertedtG-lacZ) . To defiseverity ; ii) Theseverity ; and iiiseverity . In tota6SG-lacZ induced wing phenotype could be modified by genetic changes in Notch pathway components, we analyzed flies carrying different gene copy numbers of either N or the Hairless (H) co-repressor that antagonizes Notch-mediated gene activation  or removing one allele of H significantly suppressed the notched wing phenotype induced by two copies of 6SG-lacZ  that associates with the Mediator complex (cdk8 (cycC (kto (Med12) (skd (Med13)  (#28813), H[1] (#515), kto[T241] (#63126), kto[T631] (#63125), skd[T13] (#63123) and skd[T413] (#63124). cdk8[K185] and cycC[Y5] alleles were gifts from Professor The following alleles were obtained from the Bloomington To analyze the wing notching and macrochaetae phenotypes, flies of the appropriate genotypes were mated in cornmeal-containing vials and transferred to fresh food every day. During our studies, we observed that changes in food quality and overcrowding could change the severity/penetrance of wing phenotypes, introducing variation. Hence, all experiments quantifying wing phenotypes within each Figure panel contained control flies that were grown on the same batch of food and with a similar animal density. Offspring of the listed genotypes were selected and the number of nicks on each wing was recorded and/or the number of dorsocentral and scutellar macrochaetae was counted. A Fisher’s exact test was used to determine significance between samples when penetrance was being assessed (i.e. no phenotype versus a phenotype), whereas a proportional odds model was used to determine significance when the analysis included phenotype severity.For L5 wing vein length, fly wings of the proper genotypes were dissected, mounted on glass slides and imaged using a Nikon NiE upright widefield microscope. The total length of the presumptive L5 vein and the vein-missing gap were measured using Imaris software. Student’s t-test was used to determine significance.22A6xSPS-GFP and either 3xGBE-lacZ, 3xGBE-6xSPS-lacZ, 2-lacZ(3xGBE-6xSPS), or 3-lacZ(3xGBE-6xSPS) were mated to either yw (wild type) or skd[T413]/TM6B males. Imaginal discs from male non-TM6B wandering 3rd instar larvae (skd[T413] heterozygotes) were dissected and fixed in 4% formaldehyde for 15 min. Samples were subsequently washed 4 times with PBX (0.3% Triton X-100 in PBS) and incubated with an antibody that recognizes the Cut antigen  followed by a fluorescent-conjugated secondary antibody . For quantitative purposes, at least eight imaginal discs were analyzed for each genetic condition tested and the entire wild type imaginal disc series was harvested, fixed and imaged at the same time using a Nikon A1R inverted confocal microscope (40x objective). For the skd heterozygote series, a set of wild type imaginal discs with 3xGBE-lacZ was performed simultaneously to normalize the responses between series. All imaging was performed with constant settings for GFP levels, and GFP pixel intensity in wing margin cells was determined from Z-stack images using Imaris software. Two-sided Student’s t-test was used to determine significance between samples.To systematically assess Notch transcription responses in larval wing imaginal discs, animals homozygous for For recombinant protein purification, constructs that correspond to mouse RBPJ (aa 53–474), mouse N1ICD (aa 1744–2113), human MAML1 (aa 1–280), fly Su(H) (aa 98–523), fly NICD (aa 1763–2412) and fly Mastermind (aa 87–307) were expressed and purified from bacteria using a combination of affinity (Ni-NTA or Glutathione), ion exchange, and size exclusion chromatography as previously described . PurifieStability of mammalian N1ICD was analyzed using the previously described split-luc HEK293T cells . These cTo confirm the activity of Actinomycin D, mK4 cells with a stably integrated a 6xSPS-NanoLuc reporter or a 0xSPS-NanoLuc construct as control were used. Cells were cultured for 8 hr with 50 nM Actinomycin D or 0.1% DMSO and activated for 10 min with 0.05% Trypsin-EDTA (Gibco). Nano-Luc activity was measured after 3 hr using the Nano-Glo Luciferase Assay (Promega) and imaged with the IVIS Lumina LT system.Since the decay curves in Figure 3 did not exhibit simple exponential decay, we calculated the half-life values by fitting the luciferase activity values to a decreasing Hill function, 2, 1 mM DTT) and incubated with Calf intestinal phosphatase  for 60 min at 37°C. Equal amounts were loaded on 6% Acrylamide gels for SDS-PAGE and blotted on Nitrocellulose membranes . Membranes were blocked in 5% dry milk powder in PBS with 0.1% Tween and incubated with anti-N1ICD , anti-Notch1  and anti-β-actin  overnight at 4°C. After incubation with HRP-conjugated secondary antibodies , signals were detected using SuperSignal Femto West Chemoluminescent Substrate (Thermo Fisher Scientific) and imaged with the BioRad ChemiDoc system.Mouse Kidney 4 (mK4) cells, which were developed by and a kind gift from Steve Potter’s lab , were roMouse embryonic fibroblast cells deficient for RBP-JK (OT-11)  or wild-We generated mK4 cells deficient for all mastermind-like proteins through CRISPR Cas9 mediated deletion of exon 1 of Mastermind-like 1, 2, and 3. Briefly, parental mK4 cells were simultaneously transfected with PX458 and PX459 containing guide RNAs flanking exon1 for each of the Mastermind-like genes and subjected to selection with puromycin for 2 days. Surviving clones were picked and authenticated the following week using cloning disks and screened by PCR analysis and Western blot to identify clones that lacked expression of Mastermind-like 1, 2, and 3. The sequences of guide RNAs and genotyping PCR primers are in Key Resources Table. All cell lines used throughout these studies were tested negative for mycoplasma contamination and no cell lines were used from the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee.The mathematical model describes the concentrations of the possible states of NICD. These include unphosphorylated unbound NICD, Here, The total nuclear NICD concentration, The total phosphorylated and unphosphorylated NICD are the sum of the bound (index b) and unbound (index ub) fractions:Combining echanics . The con=[X]/kdx . The parThe partition function is the normalization factor by which the probabilities of the different states of the system are calculated so that the probability for state j is:For our model, the statistical weights of the states of bound unphosphorylated and phosphorylated NICD are:The total number of SPS sites, GBE-SPS transgenes.Note, Combining This statistical mechanics approach is based on three main assumptions: (1) The binding dynamics (on and off rates) are much faster than the dynamics determining the level of NICD in the nucleus. This is clearly valid as the DNA binding time scales are of the order of seconds  and degrWe also consider the situation where the nuclear NICD concentration is much higher than the dissociation rate: We use Since phosphorylation of NICD by Cdk8 occurs only for bound NICD, it can be assumed that for a low number of SPS sites The total concentration of NICD is then:We now assume the phosphorylation rate of This analysis predicts that the slope in the linear regime isskd heterozygotes .All materials used in this study will be made freely available. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Brian Gebelein (https://github.com/OhadGolan/NICD-concentration-in-the-nucleus-as-by-binding-site-coupled-NICD-degradation.git (https://github.com/elifesciences-publications/NICD-concentration-in-the-nucleus-as-by-binding-site-coupled-NICD-degradation/tree/master).All simulation codes are available in the GitHub repository at  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Your work details a novel mechanism of Notch haploinsufficient phenotypes that is interesting and likely important to a broad range of biological systems given that it is involved in many biological processes.Decision letter after peer review:eLife. Your article has been reviewed by Aleksandra Walczak as the Senior Editor, a Reviewing Editor (Hugo Bellen), and two reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Enhancer architecture sensitizes cell-specific responses to Notch gene dose via a bind and discard mechanism" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Drosophila genetics, mammalian cell culture and mathematical modeling to show that a specific enhancer configuration can impact Notch signaling in a tissue-specific manner, likely by regulating the level of the Notch intracellular domain. They have found, unexpectedly, that inserting enhancers containing as few as 12 SPS binding sites together with binding sites for a pioneer transcription factor in the genome of Notch[+/+] flies can generate a wing margin phenotype reminiscent of the Notch haploinsufficient wing margin phenotype. This phenotype is quite sensitive to the gene dosages of Notch, a negative regulator of Notch called Hairless, and multiple components of the CDK8 kinase module (CKM) of the Mediator complex. Furthermore, experiments in mammalian cell lines provided evidence that CDK8 plays a key role in regulating Notch1 intracellular domain (N1ICD) turnover. Active transcription is not required for the observed effect in flies and mammalian cells, but formation of the Notch/CSL/Mastermind is. The authors also used quantitative analysis and mathematical modeling to conclude that changes in NICD degradation rate preferentially impact long duration Notch-dependent processes, whereas genetic changes in NICD production rate (consequence of Notch haploinsufficiency) affect both short and long duration processes. Finally, they show that transgenes harboring two copies of an endogenous SPS site behave similarly to the synthetic sites used in the study, and that presence of a pioneer factor binding site is critical for the resulting reduction in Notch signaling in a Notch[+/+] context.In this manuscript, the authors have used Overall, this is an interesting manuscript with novel and important results. A number of Notch pathway components show significant haploinsufficient phenotypes in animal models and in human patients. However, the basis for tissue-specific phenotypes observed in these diseases and their phenotypic variability is not fully understood. The current work provides a new potential mechanism that might contribute to some aspect of phenotypic variability in these diseases.Essential revisions:Some sections of the manuscript are not well explained, and some conclusions do not seem to be fully supported by the data. Moreover, although the idea that duration of signaling determines which cell types are sensitive to SPS sites is appealing, it is only one of the possibilities.1) Please describe the EMSA data shown in Figure 1 and Figure 1—figure supplement 1 and provide your conclusion. Currently, the EMSA data is not discussed in the Results section. The cooperative binding to SPS seems to be clear for the mammalian complex but not as much for the fly complex. Is it known whether the complex binds the 1x SPS better/stronger that 2x CSL? In other words, have people done competition assays with cold 2x CSL probe in the 1x SPS EMSA and vice versa? Experiments along this line or referring to literature about relative affinity of the RBPJ/Notch/Mam complex for tandem single versus SPS sites might provide clues for the observed difference between single and SPS sites.Notch phenotype”, "The relative order of GBE and SPS did not matter ": The data shown in this figure do not support this conclusion. Moreover, no statistics is provided. On the other hand, comparison of Figures 1J and 1K supports this conclusion. Are the quantifications shown in Figure 1—figure supplement 2H accurate? 2) Subsection “Enhancers with specific TF binding sites can induce a tissue-specific +/-N animals, unlike other components of the CKM complex.3) How specific is Senexin-A for CDK8? It seems that the only direct evidence used to link mammalian NICD and CDK8 in the manuscript is this compound. If Senexin-A has not been shown to be highly specific for CDK8, knock-down or knockout of CDK8 in a mammalian cell line should be considered to make the link stronger. This is important for two reasons: (a) No direct evidence has been provided for decreased stability of the fly NICD in animals harboring 6SG transgenes; (b) Removing one copy of Cdk8 did not significantly change the wing margin phenotype of 4) In subsection “Cdk8 induces Notch turnover independent from transcription activation” the authors state that "…we found that NICD was stabilized in both mammalian OT11 cells deficient for RPBJ and mK4 cells deficient for three mastermind-like proteins. While the data for the OT11 cells is convincing , the data for mK4 cells in Figure 3—figure supplement 2 lack a control comparison. In subsection “Cdk8 induces Notch turnover independent from transcription activation”, the authors suggest that the upper band represents NICD with a posttranslational modification. Can the authors show that there is decreased ubiquitination of NICD in Rbpj knockout cells or in cells treated with Senexin-A? Alternatively, since phosphorylation is a key aspect of the mathematical modeling in the manuscript, it might be worth testing whether treating the cell lysate from control cells with a phosphatase increases the mobility of NICD in the gel. Also, please mention what the open and filled arrowheads represent in Figure 3F (perhaps in the legend).1/+H genetic background, the removal of one copy of cycC seems to significantly decrease macrochaetae (at p = 8.7X10-3), yet the authors make no mention of this data, stating only that "removing and allele of skd or cdk8 did not significantly alter macrochaetae formation". Although the data supports their conclusion that macrochaetae are relatively unaffected by these genetic manipulations, one wonders if there is any biological significance to the cycC data in this context?5) In Figure 4J, in the cis-inhibitory interactions in the Notch haploinsufficient phenotypes in the wing, depending on the relative ligand-to-Notch levels in each context, this number might be lower or higher than one half. In fact, the reason Notch haploinsufficient phenotypes are sensitive to the presence of SPS in some contexts but not others might be the relative contribution of cis-inhibition in each context (and/or the relative levels of ligands to Notch in each context). A simplistic example will be to have 10 Notch molecules and 4 ligand molecules in a given cell. Normally, 6 Notch molecules will be competent to generate NICD in this cell. Upon removal of one copy of Notch, only 1 Notch molecule might be available to generate NICD in this cell.6) Subsection “Altered NICD degradation sensitizes tissues requiring long duration signals”, "We assume that N heterozygotes lower NICD production by one half without impacting NICD.": This assumption is likely to be too simplistic and cell type dependent. Given the importance of 7) Discussion section, paragraphs 1-2: Is there an estimate for the number of SPS sites in the fly genome? The authors mention that Severson et al., suggested that SPS sites contribute to the regulation of about a third of direct Notch1 targets in T-cells. If there are so many SPS sites scattered in the genome, why adding 12 SPS sites can have such a dramatic effect? Does this suggest that the endogenous SPS sites are rarely close to the binding sites of pioneer factors? The mathematical modeling predicted that the contribution of endogenous SPS sites is equivalent to ~5 active synthetic SPS sites. It is worth adding some sentences to the Discussion section to better explain the significance of their findings in the regulation of endogenous Notch signaling, in a wild-type background and in contexts of haploinsufficiency for Notch or its ligands.8) Discussion section, paragraph 3: In my opinion, linking Notch pathway duration to the observed differences is rather speculative. Although wing margin development takes longer than lateral inhibition, it is not a simple sending-receiving situation that is extended for two days. The direction of signaling and the identity and level of ligands used during wing margin development evolve over time. Moreover, there are other differences between wing margin formation and macrochaetae lateral inhibition. For example, both Delta and Serrate play key and non-redundant roles in wing margin formation, and Serrate loss-of-function phenotype is more severe in the wing margin than the Delta loss-of-function phenotype . However, loss of Serrate by itself does not have any macrochaetae phenotype, and Serrate only contributes redundantly to macrochaetae lateral inhibition . Similarly, Serrate only plays a redundant role during wing vein development , and based on Figure 4H, even 4 copies of 6SG do not recapitulate the wing vein expansion phenotype of Notch[+/-] animals. The authors should consider modifying this paragraph to acknowledge that Notch signaling duration is just one possibility.yw background. Was the X chromosome crossed out and replaced with a different genetic background when establishing the stocks?9) Subsection “Cdk8 induces Notch turnover independent from transcription activation”, about the conclusion that removing an allele of each gene of the Cdk8-kinase module significantly suppressed the penetrance and severity of the 6SG-induced wing nicking: Although the data shown in Figure 3 support this conclusion, there is a dramatic variation in the penetrance of wing nicking phenotypes caused by 2 copies of 6SG-lacZ among the figures . The integration sites used in this study seem to be on a 10) Figure 4D-G: Although a subset cells show loss of Cut-positive cells, there seems to be a broadening of Cut expression across the dorso-ventral boundary in all three genotypes with (G6S)n-lacZ. Are these images full projection views? Do the authors have an explanation for this observation (assuming the images are representative for the Cut expression in these genotypes)?11) Subsection “Quantitative analysis of Enhancer binding site induced Notch turnover”, "We found that GFP levels decreased as a function of added GBE-SPS sites": Potentially related to the above note on Figure 4D-G, although addition of two or three cassettes of G6S clearly results in decreased GFP expression from S-GFP, it appears that the GFP expression domain is broader, with somewhat brighter signal upon addition of one G6S site. Please make sure representative images are shown that match the quantification in 4H. Whether the wing margin region becomes broader in all of these transgenes or not remains an open question. It is worth noting that decreasing CKM gene dosage also affected macrochaetae numbers in the opposite direction of what was expected. Is any other component of the Notch pathway known to be regulated by the CKM complex? For example, if Delta is also a target, a change in the level of Delta might explain both phenotypes .12) The mathematical model used to describe the dynamics of NICD in the cells assumes that dephosphorylation of NICD is absent. While there is no evidence that dephosphorylation of NICD occurs and even if it does there would either be little effect on the model (if dephosphorylation rates were low) or add an additional complexity unnecessary to the model and beyond the scope of the present study, it is an inherent assumption of the model and should be stated as such. Essential revisions:Some sections of the manuscript are not well explained, and some conclusions do not seem to be fully supported by the data. Moreover, although the idea that duration of signaling determines which cell types are sensitive to SPS sites is appealing, it is only one of the possibilities.We revised the text throughout the manuscript to better explain our experimental results and conclusions with each change highlighted under the relevant specific points raised below. Moreover, we acknowledge that while our data is consistent with the duration model, other factors may contribute to the different sensitivities observed in the wing versus macrochaetae tissues. Hence, we added several sentences in the Discussion section highlighting that additional tissue-specific factors besides signal duration could contribute to the differences in phenotypes observed between the wing and macrochaetae.1) Please describe the EMSA data shown in Figure 1 and Figure 1—figure supplement 1 and provide your conclusion. Currently, the EMSA data is not discussed in the Results section. The cooperative binding to SPS seems to be clear for the mammalian complex but not as much for the fly complex. Is it known whether the complex binds the 1x SPS better/stronger that 2x CSL? In other words, have people done competition assays with cold 2x CSL probe in the 1x SPS EMSA and vice versa? Experiments along this line or referring to literature about relative affinity of the RBPJ/Notch/Mam complex for tandem single versus SPS sites might provide clues for the observed difference between single and SPS sites.Notch phenotype” of the revised submission).We apologize for this omission. We added several sentences to the Results section to describe the EMSA results and conclusions derived from this line of experimentation . Hence, direct comparisons can be made in regard to the binding characteristics of the Su(H)/RBPJ transcription factors and the NICD/MAM co-activators to these two probes. These data show that in the absence of co-activators, the 2xCSL probe is bound slightly better by the Su(H)/RBPJ transcription factors than the 1xSPS probe. However, in the presence of NICD/Mam, the 1xSPS is preferentially bound by both the fly and mammalian proteins over the 2xCSL. We describe these results in the modified Results section and included the fly protein EMSAs in Figure 1C rather than in the figure supplement.We also agree that the mouse proteins appear to be more cooperative than the fly proteins in our EMSAs and we describe and show this observation in Figure 1—figure supplement 1. We decided not to include it in the main text for two reasons: First, our main goal was to test if the fly proteins, which to our knowledge were never tested on SPS sites by EMSAs, generally behave like the mouse proteins. Our data clearly show that the fly proteins do indeed bind cooperatively to the 1xSPS site, but not to the 2xCSL sites. Second, while the main driver of NICD-induced cooperativity is a dimer interface in the Notch ANK domains, which is found in both species (see the data in https://doi.org/10.1101/2020.02.13.948224) slight differences in the polypeptides used in EMSA may impact the degree of cooperativity. Moreover, these differences may reflect the challenge in purification of the NICD and Mam proteins. Thus, we cannot be certain that the differences in cooperativity observed between the mouse and fly proteins in this assay are biologically meaningful.2) Subsection “Enhancers with specific TF binding sites can induce a tissue-specific Notch phenotype”, "The relative order of GBE and SPS did not matter ": The data shown in this figure do not support this conclusion. Moreover, no statistics is provided. On the other hand, comparison of 1J and 1K supports this conclusion. Are the quantifications shown in Figure 1—figure supplement 2H accurate? 6SG-lacZ caused notched wings when inserted in another locus and regardless of the order of GBE and SPS, although with differences in penetrance and severity” (subsection “Enhancers with specific TF binding sites can induce a tissue-specific Notch phenotype”). We also added statistical tests to Figure 1—figure supplement 2.We agree with the reviewers that we overstated this conclusion and reworded the sentence as follows: “i) The vs (2 copies in 86Fb) vs (1 copy in 51C and 1 copy in 86Fb) vary in penetrance/severity. These data are shown and described in Figure 1—figure supplement 2 and statistical tests are now included in this figure to account for the differences in penetrance/severity. Second, we found that the severity/penetrance can vary depending upon environmental changes . To deal with this source of variability, all experiments assessing wing notching within each Figure panel were performed with appropriate controls using food made from the same batch and similar fly densities to avoid environmental confounders. We included this information in the Materials and methods section of the paper to better explain our experimental protocol.The reviewers are also correct that differences in penetrance and severity are sometimes observed between experiments and these differences arise from two sources: First, some differences in penetrance and severity can be attributed to the insertion location: flies with (2 copies in 51C) +/- animals, unlike other components of the CKM complex.3) How specific is Senexin-A for CDK8? It seems that the only direct evidence used to link mammalian NICD and CDK8 in the manuscript is this compound. If Senexin-A has not been shown to be highly specific for CDK8, knock-down or knockout of CDK8 in a mammalian cell line should be considered to make the link stronger. This is important for two reasons: (a) No direct evidence has been provided for decreased stability of the fly NICD in animals harboring 6SG transgenes; (b) Removing one copy of Cdk8 did not significantly change the wing margin phenotype of NSenexin A was first described as an optimized inhibitor for CDK8/CDK19 paralogs and the authors claimed that it showed “striking selectivity” . Senexin A has since been used by multiple other research groups . To further address this concern, we performed the same half-life measurement with SEL120-34A, another CDK8/CDK19 inhibitor, which was shown to be highly selective and efficient for CDK8/CDK19 . Since Senexin A and SEL120-34A are structurally distinct, they are unlikely to share the same targets other than CDK8/CDK19. Importantly, we observed a similar result with SEL120-34A as to that we observed with Senexin A – inhibitor treatment prolonged NICD half-life. These new findings are described in the Results section and the data are now included in Figure 3—figure supplement 2B and 2D.+/-N animals, we would like to point out that catalytic components of multi-subunit enzymes are often not rate limiting factors. In fact, a previous research study found that enzymes, in general, were depleted from the list of haploinsufficiency genes, whereas structural/regulatory proteins that form macromolecular complexes were enriched in haploinsufficiency genes . Hence, it is not surprising that CDK8 is not the rate limiting factor in this genetic assay. Instead, the wing phenotype is far more sensitive to the gene dose of the structural/regulatory proteins encoded by the med12 (kto), med13 (skd) and the cycC genes, which collectively form the CDK8-kinase module with Cdk8. Moreover, this finding is in agreement with prior reports that cycC (CCNC) has been reported to be a haploinsufficient tumor suppressor . We make these points more clearly in the revised Results section and Discussion section.For the finding that removing one copy of Cdk8 did not significantly change the wing margin phenotype of 4) In subsection “Cdk8 induces Notch turnover independent from transcription activation” the authors state that "…we found that NICD was stabilized in both mammalian OT11 cells deficient for RPBJ and mK4 cells deficient for three mastermind-like proteins. While the data for the OT11 cells is convincing , the data for mK4 cells in Figure 3—figure supplement 2 lack a control comparison. In subsection “Cdk8 induces Notch turnover independent from transcription activation”, the authors suggest that the upper band represents NICD with a posttranslational modification. Can the authors show that there is decreased ubiquitination of NICD in Rbpj knockout cells or in cells treated with Senexin-A? Alternatively, since phosphorylation is a key aspect of the mathematical modeling in the manuscript, it might be worth testing whether treating the cell lysate from control cells with a phosphatase increases the mobility of NICD in the gel. Also, please mention what the open and filled arrowheads represent in Figure 3F (perhaps in the legend).To address these concerns, we did the following: First, we added the control Western blot for the MAML knockout cells . Second, we performed a Western blot using mK4 cells treated with SEL120-34A and found that this CDK8/CDK19 inhibitor resulted in both increased NICD stability and mobility, consistent with reduced NICD phosphorylation . Third, we treated the protein lysates from mK4 cells with Calf intestinal phosphatase (CIP) and confirmed that the slower migrating (upper) band of NICD is phosphorylation-dependent . Fourth, we added the following description for the arrows in the figure legends of Figure 3 and Figure 3—figure supplement 2: “Open arrow denotes post-translationally modified NICD and closed arrow denotes unmodified NICD.”1/+ genetic background, the removal of one copy of cycC seems to significantly decrease macrochaetae (at p = 8.7X10-3), yet the authors make no mention of this data, stating only that "removing and allele of skd or cdk8 did not significantly alter macrochaetae formation". Although the data supports their conclusion that macrochaetae are relatively unaffected by these genetic manipulations, one wonders if there is any biological significance to the cycC data in this context?5) In Figure 4J, in the HNotch or Hairless allele with mutations in the CDK8 kinase module , and skd (MED13)). In particular, we added the following statements in the Results section:To address this concern, we provided a more thorough description of the macrochaetae results generated in conjunction with the compound heterozygotes containing either a “However, we did find that removing an allele of cycC had a small, but significant impact on macrochaetae formation in H;cycC compound heterozygotes. […] Nevertheless, these data suggest that macrochaetae development in general is relatively insensitive to changes in CKM gene dose in comparison to Notch dependent wing margin phenotypes.”In addition, we also changed the Discussion section to include that the CDK8 kinase module is likely to have additional targets outside of the Notch signaling pathway. Hence, we cannot exclude that additional tissue-specific CDK8 module targets may contribute to the observed phenotypes.6) Subsection “Altered NICD degradation sensitizes tissues requiring long duration signals”, "We assume that N heterozygotes lower NICD production by one half without impacting NICD.": This assumption is likely to be too simplistic and cell type dependent. Given the importance of cis-inhibitory interactions in the Notch haploinsufficient phenotypes in the wing, depending on the relative ligand-to-Notch levels in each context, this number might be lower or higher than one half. In fact, the reason Notch haploinsufficient phenotypes are sensitive to the presence of SPS in some contexts but not others might be the relative contribution of cis-inhibition in each context (and/or the relative levels of ligands to Notch in each context). A simplistic example will be to have 10 Notch molecules and 4 ligand molecules in a given cell. Normally, 6 Notch molecules will be competent to generate NICD in this cell. Upon removal of one copy of Notch, only 1 Notch molecule might be available to generate NICD in this cell.Notch heterozygous animals. The main point was to show that the reduction in production affects both long duration and short duration processes. As the reviewer pointed out, cis-inhibition is expected to reduce NICD levels in the Notch heterozygotes even more, and if such a mechanism impacts one tissue to a higher degree than another, it would potentially make that tissue more sensitive to changes in Notch gene dose. We now added several sentences addressing the potential effect of cis-inhibition, and how it, and other potential tissue-specific factors, may impact the underlying parameters that contribute to the duration model (Discussion section).As pointed out by the reviewer, we have indeed used the simplified assumption that NICD production is halved for 7) Discussion section, paragraphs 1-2: Is there an estimate for the number of SPS sites in the fly genome? The authors mention that Severson et al., suggested that SPS sites contribute to the regulation of about a third of direct Notch1 targets in T-cells. If there are so many SPS sites scattered in the genome, why adding 12 SPS sites can have such a dramatic effect? Does this suggest that the endogenous SPS sites are rarely close to the binding sites of pioneer factors? The mathematical modeling predicted that the contribution of endogenous SPS sites is equivalent to ~5 active synthetic SPS sites. It is worth adding some sentences to the Discussion section to better explain the significance of their findings in the regulation of endogenous Notch signaling, in a wild-type background and in contexts of haploinsufficiency for Notch or its ligands.Drosophila genome has not been accurately determined, we do have estimates from mammalian cells. Moreover, we also have some estimates for the number of Su(H) binding loci in a Drosophila wing cell line, although this study didn’t differentiate CSL vs. SPS sites. In addition to incorporating this information into the discussion, we also discussed the possibility that concentrating the SPS sites at a specific locus may have an “avidity” impact such that any NICD molecule recruited to the locus has a very high probability of being marked for degradation. By integrating these data into the manuscript, we believe it puts our finding that transgenes with as few as 12 SPS sites can induce phenotypes into a better context relative to the entire endogenous genome.To address this concern, we have added a paragraph in the Discussion section describing the current literature in regards to numbers of SPS sites in the genome. In this paragraph, we discuss that while the number of functional SPS sites in the 8) Discussion section, paragraph 3: In my opinion, linking Notch pathway duration to the observed differences is rather speculative. Although wing margin development takes longer than lateral inhibition, it is not a simple sending-receiving situation that is extended for two days. The direction of signaling and the identity and level of ligands used during wing margin development evolve over time. Moreover, there are other differences between wing margin formation and macrochaetae lateral inhibition. For example, both Delta and Serrate play key and non-redundant roles in wing margin formation, and Serrate loss-of-function phenotype is more severe in the wing margin than the Delta loss-of-function phenotype . However, loss of Serrate by itself does not have any macrochaetae phenotype, and Serrate only contributes redundantly to macrochaetae lateral inhibition . Similarly, Serrate only plays a redundant role during wing vein development , and based on Figure 4H, even 4 copies of 6SG do not recapitulate the wing vein expansion phenotype of Notch[+/-] animals. The authors should consider modifying this paragraph to acknowledge that Notch signaling duration is just one possibility.We agree that the proposed duration model is correlative by nature and it is difficult to definitively test the model  in the wing margin and macrochaetae. Hence, we expanded the Discussion section to point out that while our data is consistent with the duration model, other tissue-specific factors and parameters may also contribute to the observed differences in sensitivity between the wing margin and macrochaetae.9) Subsection “Cdk8 induces Notch turnover independent from transcription activation”, about the conclusion that removing an allele of each gene of the Cdk8-kinase module significantly suppressed the penetrance and severity of the 6SG-induced wing nicking: Although the data shown in Figure 3 support this conclusion, there is a dramatic variation in the penetrance of wing nicking phenotypes caused by 2 copies of 6SG-lacZ among the figures . The integration sites used in this study seem to be on a yw background. Was the X chromosome crossed out and replaced with a different genetic background when establishing the stocks?6SG-lacZ transgenes induce wing notching phenotypes regardless of chromosomal integration site, the insertion location can impact the penetrance and severity of the phenotype. Thus, the data in Figure 2F (6SG-lacZ-51C/6SG-lacZ-51C) and Figure 3A  are not comparable since the two alleles of 6SG-lacZ are on different chromosomes, and we found that flies with 6SG-lacZ-51C/+; 6SG-lacZ-86Fb/+ have a higher wing-phenotype penetrance than flies with either 6SG-lacZ-51C/6SG-lacZ-51C or 6SG-lacZ-86Fb/6SG-lacZ-86Fb . Second, food quality and fly density can impact the penetrance and severity of the wing nicking phenotype observed, and we described both this result and how we addressed it in point #2 above. As for the X chromosome, all of our transgenes, including the GBE-CSL reporters, the GBE alone reporters, and the SPS alone reporters that do not induce phenotypes, were created and tested in an identical manner as the transgenes containing the SPS sites. Moreover, we always used yw as our control, even when crossing with the Hairless and Notch heterozygous animals. Given that the yw X chromosome never showed phenotypes on its own, nor with any of the other reporters except those with GBE-SPS or when crossed to Notch heterozygous animals, we think that it is highly unlikely to contribute in any meaningful way to the observed phenotypes.As for the variability between experiments, please see our response to point #2 above regarding two sources of phenotype variability. First, while the 10) Figure 4D-G: Although a subset cells show loss of Cut-positive cells, there seems to be a broadening of Cut expression across the dorso-ventral boundary in all three genotypes with (G6S)n-lacZ. Are these images full projection views? Do the authors have an explanation for this observation (assuming the images are representative for the Cut expression in these genotypes)?The images in Figure 4D-G were taken as Z-stacks before being max-projected. To address this concern, we carefully analyzed all of the wing disc data and found that while the width of the Cut-expressing cells does vary from wing disc to wing disc, we observe similar variation in all genotypes. Also, see our response to the related point 11 below.11) Subsection “Quantitative analysis of Enhancer binding site induced Notch turnover”, "We found that GFP levels decreased as a function of added GBE-SPS sites": Potentially related to the above note on Figure 4D-G, although addition of two or three cassettes of G6S clearly results in decreased GFP expression from S-GFP, it appears that the GFP expression domain is broader, with somewhat brighter signal upon addition of one G6S site. Please make sure representative images are shown that match the quantification in 4H. Whether the wing margin region becomes broader in all of these transgenes or not remains an open question. It is worth noting that decreasing CKM gene dosage also affected macrochaetae numbers in the opposite direction of what was expected. Is any other component of the Notch pathway known to be regulated by the CKM complex? For example, if Delta is also a target, a change in the level of Delta might explain both phenotypes .GBE-SPS sites , the variation was similar between genotypes. Moreover, it is important to point out that all of the data collected from each imaginal disc was included in the quantified data shown in Figure 4H, as each dot on the graph is the average GFP intensity in the Cut-positive cells from a single wing imaginal disc and none of the discs were excluded from this analysis.To address the concern regarding the GFP levels and pattern in the wing discs shown in Figure 4D-G, we reanalyzed the data and selected new images that are representative of GFP levels, pattern, and the width of the Cut expression domain. While looking at all of the discs analyzed in this experiment, we did notice variation in the width of Cut expression domain between imaginal discs. However, besides observing gaps in the Cut expression domain for those flies carrying 6, 12, or 18 The reviewer also raised a second point: Is any other component of the Notch pathway known to be regulated by the CKM complex? To our knowledge, CKM activity is restricted to the nucleus and does not modify the Delta ligand or the unprocessed Notch receptor. However, CKM activity is known to phosphorylate other transcriptional regulators including Mastermind, CycC, Med12, and Med13. Taking these potential targets into account, removing an allele of CKM components would still be predicted to increase Notch signal strength, which is consistent with our current model. In addition, to these points, we also included a statement that we cannot exclude the possibility that CKM activity alters additional transcriptional regulatory proteins, and that they could contribute to the observed phenotypes. We make these points clearer in the modified Discussion section.12) The mathematical model used to describe the dynamics of NICD in the cells assumes that dephosphorylation of NICD is absent. While there is no evidence that dephosphorylation of NICD occurs and even if it does there would either be little effect on the model (if dephosphorylation rates were low) or add an additional complexity unnecessary to the model and beyond the scope of the present study, it is an inherent assumption of the model and should be stated as such.We agree with the reviewer that adding additional complexity to the model is beyond the scope of the present study. We have specified in the new version that we assume no dephosphorylation of NICD is considered in the model in the Results section.