Follow-up for breast cancer survivors consists of after care and surveillance. The benefits of routine surveillance visits remain debatable. In this study we compared the severity of locoregional recurrences (LRRs) and the subsequent risk of a distant metastasis (DM) between LRRs detected at routine and interval visits.Women diagnosed with early breast cancer between 2003 and 2008 in one of the 15 participating hospitals, and who developed a LRR as first event after primary treatment, were selected from the Netherlands Cancer Registry (Cohort A). Chi-squared tests were used to compare the severity of routine- and interval-detected local recurrences (LRs) and regional recurrences (RRs), using tumor size, tumor grade, and number of positive lymph nodes. Data on the development of a subsequent DM after a LRR were available for a subset of patients (Cohort B). Cohort B was used to estimate the association between way of LRR-detection and risk of a DM.Cohort A consisted of 109 routine- and 113 interval-LRR patients. The severity of routine-detected LRs or RRs and interval-detected LRs or RRs did not significantly differ. Cohort B consisted of 66 routine- and 61 interval-LRR patients. Sixteen routine- (24%) and 17 (28%) interval-LRR patients developed a DM. After adjustment, way of LRR-detection was not significantly associated with the risk of a DM .The current study showed that routine visits did not lead to less severe LRRs and did not decrease the risk of a subsequent DM.The online version contains supplementary material available at 10.1007/s10549-022-06757-3. Breast cancer is the most prevalent type of cancer among women worldwide . Due to Nevertheless, routine surveillance is not evidence based and the benefit of routine surveillance visits remains debatable. According to two systematic reviews, patients with asymptomatic- or mammography-detected LRRs or SPBCs have an improved survival compared to patients with symptomatic- or clinically detected LRRs or SPBCs , 7. HoweRoutine visits have disadvantages. They (1) can lead to false positive results, (2) can increase levels of psychological distress and anxiety –10, (3) As the benefit of routine surveillance visits is debatable and given the downsides of the visits, it is important to quantify the benefit and contribution of those visits to the early detection of LRRs and the prevention of distant metastases. Therefore, the current study aimed to compare the severity of the LRR  and the subsequent risk of a distant metastasis (DM) between LRRs detected at routine visits and interval visits.Data were obtained from the Netherlands Cancer Registry (NCR). The NCR is a nationwide population-based registry, hosted by the Netherlands Comprehensive Cancer Organisation (IKNL), that includes data on all newly diagnosed cancer patients since 1989. Reports of newly diagnosed malignancies are obtained from the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA). Subsequently, trained data managers record information on patient, tumor, and treatment characteristics from the patients’ files. In addition, all first recurrences, both LRRs or DMs, within 5 years following diagnoses were registered for women diagnosed in 2004 or between 2006 and 2008. All recurrences within 10 years following diagnoses were registered for women diagnosed in 2003 or 2005. Vital status was obtained by linkage to the Municipal Personal Records database, which was complete until February 1st, 2021. This study was approved by the Privacy Review Board of the NCR (reference number K20.057).Patients were eligible if they were diagnosed with invasive non-metastatic breast cancer between 2003 and 2008, were surgically treated in a Dutch hospital, and developed a LRR as first breast cancer-related event after primary treatment Fig. . PatientAdditional data for this study were collected of a subset of LRR patients treated in one of the 15 participating hospitals. These 15 hospitals included both academic, teaching and general hospitals, providing a good representation of the breast cancer care in the Netherlands. The additionally collected data included way of detection of the LRR  and characteristics of the LRR . Two cohorts were composed to answer our research questions. Cohort A was used to compare the severity of LRRs detected at routine visits and interval visits. This cohort consisted of all patients who met the in- and exclusion criteria and who were treated for their LRR in one of the 15 hospitals. Cohort B was used to investigate the association between way of detection of the LRR and risk of a subsequent DM. Patients from cohort A with a follow-up of 10 years were included in cohort B, as data on the development of recurrences after a LRR was previously collected for those patients.LRRs include both local recurrences (LRs) and regional recurrences (RRs). A LR was defined as the reappearance of invasive cancer in the ipsilateral breast or chest wall . A RR waThe development of a DM as first event after a LRR, was analyzed as the primary outcome. The development of a second LRR or a SPBC, or death, as first event after a LRR, were analyzed as secondary outcomes. If patients were diagnosed with multiple events within 30 days, the most severe event was registered as endpoint. DM was defined as most severe, followed by LRR and SPBC. Follow-up was calculated from the date of LRR diagnosis to date of event or the last date of observation.The socioeconomic status (SES) of a patient was based on their postal code at the time of diagnosis. Estrogen receptor (ER) and progesterone receptor (PR) status were combined into the variable hormone receptor (HR) status . Tumor size and stage were based on pathological assessment for the primary tumor and on pathological or clinical assessment for the LRR, depending on whether the pathological assessment was available. The disease-free interval (DFI) was defined as the number of years between date of diagnosis of the primary tumor and date of diagnosis of the LRR.Patient-, primary tumor-, LRR-, and treatment-related characteristics of cohort A and B were summarized and patients were compared according to way of LRR-detection using Chi-squared and Wilcoxon rank-sum tests.Tumor size, tumor grade, and number of positive lymph nodes of the LRR were regarded as proxies for the severity of the LRR. As those characteristics differ between LRs and RRs it was decided to stratify the baseline characteristics by type of LRR.As some variables were missing, multiple imputation by chained equations (MICE) was used to impute missing values . Missingp < 0.1). The following variables were selected as confounders: tumor size, tumor grade, lymph node status, and type of surgery of the primary tumor, and tumor grade, and lymph node status of the LRR. Log–log plots and scaled Schoenfeld residuals showed that all variables met the proportionality assumptions.Cause-specific cumulative incidence functions (CIFs) were calculated to estimate the probability of a DM, second LRR, SPBC, or death as first event after a LRR, specified by way of detection. CIFs calculate the crude risk of a certain event, while taking into account that a patient might have experienced one of the other events. Hence, they take the occurrence of competing events into account. Cox proportional hazard models were used to estimate cause-specific hazard ratios (cHRs) and 95% confidence intervals (CI) for the association between way of LRR-detection and risk of a subsequent DM, second LRR, SPBC, or death. cHRs take the occurrence of competing events into account. The development of a DM, second LRR, SPBC, or death was regarded as a competing event, when not regarded as the outcome of interest. Patients were censored at the occurrence of a competing event or at the last date of observation. The cHRs for the secondary outcomes, i.e., second LRR, SPBC, or death, were shown to give a complete overview of the association between way of LRR-detection and risk of a DM . The selA sensitivity analysis was performed without patients with a DFI of 5 years or longer, as surveillance consist of annual mammography for at least 5 years after diagnosis in the Netherlands. A second sensitivity analysis was performed to investigate whether the risk of a subsequent DM differed between asymptomatic and symptomatic patients. This analysis was performed to investigate whether early diagnosis was beneficial for patients most likely to benefit from it, i.e., asymptomatic patients.p-value < 0.05 was considered statistically significant. All data were analyzed with Stata version 16 software.A two sided Cohort A included 109 routine-LRR patients (49.1%) and 113 interval-LRR patients (50.9%). The median DFI was comparable between interval-LRR  and routine-LRR patients . Interval-LRR patients had more often a grade 3 primary tumor (41.6% vs. 30.3%) and received less often radiotherapy as treatment for their primary tumor (45.1% vs. 59.6%) compared to routine-LRR patients (Table p = 0.06) (Supplementary 1 and 2). Tumor grade did not differ between interval-detected LRs and routine-detected LRs (p = 0.84). Tumor grade and number of positive lymph nodes did not differ between interval-detected RRs and routine-detected RRs .Concerning the severity of the LRR, interval-LRs were more often smaller than 2 cm compared to routine-LRs (73.4% vs. 65.8%), although tumor size did not statistically differ between the two groups (Sixteen of the routine-LRR patients developed a DM 24.2%), 7 developed a second LRR (10.6%), 4 developed a SPBC (6.1%), 3 died (4.6%), and 36 were event free at the end of the 10 year follow-up period (54.6%). In comparison, 17 of the interval-LRR patients developed a DM (27.9%), 3 developed a second LRR (4.9%), 2 developed a SPBC (3.3%), 11 died (18.0%), and 28 were event free (45.9%). The CIF of developing a DM as first event within 5 years after diagnosis of a LRR was 27.5% (95% CI 16.6–39.5%) for routine-LRR patients and 32.9% (95% CI 20.4–46.0%) for interval-LRR patients , cannot be used to draw a conclusion about the protective effect of routine visits on the development of a secondary outcome, since these analyses were unadjusted. In addition, no conclusion can be drawn about the protective effect of routine visits on the risk of death, since patients were censored if they developed a DM, second LRR, or SPBC. The analyses showing the risk of developing one of the secondary outcomes were only performed to provide a complete overview of the risk of developing a DM.Interval-LRR patients received more often endocrine therapy for their primary tumor compared to routine-LRR patients. Patients receiving endocrine therapy regularly visit the medical oncologist. These visits are not regarded as routine surveillance visits, as they are rarely aimed at detecting recurrences. However, it is possible that patients experiencing symptoms might ask their medical oncologist for a physical examination during these visits or that the medical oncologist performs a physical examination as routine. Furthermore, routine-LRR patients received more often radiotherapy as treatment for their primary tumor compared to interval-LRR patients. A previous Dutch study showed that patients receiving radiotherapy received more routine surveillance visits compared to patients not receiving radiotherapy . Hence, In our study 24.2% of the routine- and 27.9% of the interval-LRR patients developed a DM within a median of 2.8 and 2.9 years of follow-up, respectively. Previous studies showed that approximately 21% of the lymph node negative patients with a LRR and 38% of the lymph node positive patients with a LRR developed a subsequent DM within 3 years of follow-up , 35. In The main sources of bias in studies investigating the beneficial effect of early detection are lead and length time bias. Lead time bias means that patients with a routine-LRR live longer than patients with a self-detected interval-LRR, even when earlier detection did not change the natural course of the disease. Length time bias means that the less aggressive, slowly growing, tumors are more likely to be detected at routine visits and the more aggressive, faster growing, tumors at interval visits. However, since the median DFI, the IQR of the DFI, and the tumor characteristics hardly differed between the group of patients with a routine- or interval-LRR, we believe that it is unlikely that lead or length time bias was present.Strengths of this study include the availability of data on a large number of variables, the relatively long follow-up and the use of cause-specific hazard ratios to take the occurrence of competing events into account . A limitTo conclude, our study showed that routine surveillance visits did not lead to less severe LRRs and did not decrease the risk of a subsequent DM. We therefore advise clinicians to plan surveillance visits in close consultation with the patient’s wishes and their risk on a LRR.Supplementary file1 (docx 62.9 KB)Below is the link to the electronic supplementary material.


The Chinese government has set long-term carbon neutrality and renewable energy (RE) development goals for the power sector. Despite a precipitous decline in the costs of RE technologies, the external costs of renewable intermittency and the massive investments in new RE capacities would increase electricity costs. Here, we develop a power system expansion model to comprehensively evaluate changes in the electricity supply costs over a 30-year transition to carbon neutrality. RE supply curves, operating security constraints, and the characteristics of various generation units are modelled in detail to assess the cost variations accurately. According to our results, approximately 5.8 TW of wind and solar photovoltaic capacity would be required to achieve carbon neutrality in the power system by 2050. The electricity supply costs would increase by 9.6 CNY¢/kWh. The major cost shift would result from the substantial investments in RE capacities, flexible generation resources, and network expansion. This study indicates that approximately 5.8 TW of wind and solar photovoltaic capacity would be required to achieve carbon neutrality in China’s power system by 2050. The electricity supply costs would increase by 19.9% or 9.6 CNY¢/kWh. The electricity sector in China consumes over 50% of the total coal supply nationally, leading to annual carbon dioxide (CO2) emissions of 4.5 billion tonnes2. Numerous studies have indicated that the power sector must take the lead among China’s sectors by achieving national carbon neutrality by 2050, in advance of the economy-wide target of 20603. In 2019, 62.1% of China’s electricity supply came from coal-fired units. Although China already features the world’s largest installed power generation capacity for renewable energy (RE), a profound transformation of the power system will still be required over the next 30 years to achieve carbon emission goals.During the 75th UN General Assembly, the Chinese government announced that China aims to reach peak carbon emissions before 2030 and achieve carbon neutrality by 2060 4. The VRE capital cost per kW has decreased significantly, with values for PV and wind power decreasing by 73.3% and 15.9%, respectively, since 2012 in China5. The declining trends in the cost of RE technology are clearly helping to decarbonize the power system7.The total unit capacity of the key technologies required for decarbonization, e.g., variable renewable energy , increased by a factor of ten in the past decade, due to massive feed-in tariffs and other policy support8. Such costs of the transition to carbon neutrality are affected by many factors, and the scale of these effects remains unclear. The costs of the power system transition are eventually paid by consumers through renewable surcharges in electricity bills. It is, therefore, crucial to evaluate the factors driving the electricity supply cost for carbon neutrality to inform effective energy policy design and implementation.However, the integration of VRE entails considerable external costs to guarantee secure and stable power system operations. The intermittency and uncertainty of VRE outputs require the coordinated dispatching of flexible generation resources, such as conventional units or energy storage systems. Moreover, the significant geographic mismatch between load centres and RE generation centres requires extensive grid interconnection. As the installed capacity increases, the completion of projects in areas with the best wind and solar resources will force later development into lower-quality regions, thus increasing the levelized cost of energy (LCOE). In addition, the need to achieve carbon neutrality in a short time frame of 30 years may force development that strains manufacturing capacities and induces massive investment in RE units before their cost decline has been fully realized11, focusing on the natural resource endowments. Based on these results, RE capital costs considering the external costs of RE integration are studied at a fine spatial resolution12. To assess the impacts of low-carbon goals on power systems, studies on the resulting power system morphology14, comparative development status across countries15 and associated environmental benefits17 have been conducted. However, few studies have focused on the costs of the electricity supply required to achieve carbon neutrality, including detailed consideration of the practical challenges associated with high renewable capacity shares. Simply estimating changes in electricity supply costs based on only the power balance will result in the underestimation of the comprehensive effects of critical factors. In particular, thorough cost accounting must consider the VRE supply curves, the operating security concerns caused by the high uncertainty and low inertia of VRE units, and the projection of interprovincial network developments.Previous studies have explored the wind and solar potential in China and other regions around the worldTo accomplish these tasks, we established a power system planning model with a high RE capacity share to evaluate the impacts on the electricity supply cost associated with China’s transition to carbon neutrality. The power system generation and transmission expansion is formulated, and the changes in power system morphology over time are simulated. The stability problems caused by high RE penetration, such as decreased inertia and lack of peak regulation reserves, are considered in detail via security constraints in the planning model. The supply curves of VRE based on high-resolution meteorological and geographic data are integrated into the planning model to comprehensively assess costs. Furthermore, a simulation of the long-term development of China’s power system at the province level is conducted. For the local grids including within-provincial transmission networks and distribution networks, we project expansion based on historical network data and simulation results. Finally, the modelling results provide an accurate estimation of the external costs of RE integration into the overall power system.The electricity supply cost changes caused by the carbon neutrality transformation are evaluated based on the power system expansion planning model considering the RE distribution characteristics and system security constraints under high renewable penetration. The model structure is presented in Fig. China’s wind and solar PV potential and supply curves in this paper are assessed using the Global Renewable-energy Exploitation Analysis (GREAN) database. The GREAN database contains detailed geographic information, RE resource endowment data, and human activity information for the quantitative evaluation of economic potential. Figure Although the total potential is high, the spatial distribution and corresponding LCOEs are not uniform and are highly mismatched with the electricity demand. RE-rich areas for both wind and PV are mainly located in northern and northwestern China, while the load centres are in coastal areas. The VRE supply curves for each province are detailed in Supplementary Figs. The VRE supply curves for each province are integrated into the GTEP model in a piecewise manner .The capacity mix and the interprovincial network topology of China’s power system as of 2020 are considered in the model. Other key parameters such as equipment costs and load demands are also updated to 2020. The parameters of the generation and transmission technologies are presented in Supplementary Tables Such detailed modelling of RE development costs, power system security constraints, and grid expansion investments ensure an accurate assessment of electricity supply costs with the carbon neutrality target. In addition, compared with models in the existing literature, our model provides advantages related to power equipment modelling, a high temporal resolution, and a long planning period duration. The details are discussed in Supplementary Note To precisely calculate the electricity supply costs of a power system with high renewable penetration, we have considered the following critical factors and new features in detail:Four carbon mitigation scenarios considering different government policies are designed to compare electricity supply costs with increased carbon mitigation. The specific descriptions for the scenarios are shown in Table Note that the “3060” target for China applies to the entire economy and society. Carbon neutrality is easier to achieve in the power sector than that in other sectors due to the relative maturity of low-carbon generation technologies. In addition, the power sector must achieve carbon neutrality in advance of the manufacturing sector, the transportation sector and other sectors, to provide zero-carbon power for the anticipated load increase from electrification. Hence, we regard the CN2050 scenario, which achieves carbon neutrality for the power sector in 2050, as a practical translation of the “3060” target to the power sector, and we set it as the base case for comparison and analysis. The electricity supply costs and the variation in the power system morphology, including the capacity mix and transmission network structure, are calculated for each scenario.2 capture.Figure 8, the investments required to expand the total installed capacity will increase the electricity supply costs.VRE units, specifically PV and wind, would be primarily deployed to offset the decline in coal power. For the BAU scenario, VRE would be developed only in later years, a result of its growing unsubsidized cost competitiveness, with a total capacity of 2485.9 GW in 2050, accounting for 32.8% of all power generation. For the CN2050 scenario, the VRE penetration reaches 72.8% in 2050 at 3579.0 GW of PV and 2247.1 GW of wind (ten times the VRE capacity in 2020). The installed capacities of VRE for the NDC and GM2.0 scenarios by 2050 are 4121.8 and 5247.7 GW, respectively. Figure Power system security and stability require auxiliary services from flexible generation resources to enable the integration of VRE. Currently, such services are mainly provided by coal-power units, although this cannot continue to be the case under carbon neutrality. With the forced withdrawal of coal power, auxiliary services must be provided by natural gas units because of their relatively low rate of carbon emissions and generation flexibility. The NDC scenario has a relatively low gas capacity in 2050 at 137.4 GW, because considerable coal-power capacity remains. Under the GM2.0 scenario, the gas capacity increases from 111 to 449.4 GW by 2050. The installed capacity in CN2050 is slightly smaller, at 389.1 GW. To achieve carbon neutrality in CN2050, the emissions of these gas units must be offset by negative emissions achieved by generation using biomass energy with CCS (BECCS). Due to its highly variable operating cost , biomass power is developed at a large scale only in the CN2050 scenario. Among the 187.5 GW of biomass power capacity, 43.4% is from BECCS units, contributing 182.7 million tonnes of negative carbon emissions per year. However, the supplies of natural gas and biomass fuel for power generation are relatively scarce in China, limiting the total corresponding capacities. These two types of units are mainly distributed in coastal and central China because the primary fuel resources in those provinces are relatively abundant. The high fuel costs of natural gas and biomass in China would also contribute to increasing the electricity supply costs.Energy storage systems (ESSs) play a critical role in accommodating high RE penetration and ensuring system security, as they are able to provide high-performance ancillary services including the contingency reserve, peak regulation, fast frequency regulation, and virtual inertia functions. Pumped hydro storage (PHS) and battery energy storage (BESS) are considered in the model. Due to the low VRE penetration and high battery capital costs in the early stages, BESSs are mainly deployed in the later stages of planning (after 2030). The total installed capacity of ESSs in GM2.0 and CN2050 is 967.7 and 1116.9 GW, respectively. The distribution of ESSs is roughly consistent with the distribution of VRE, primarily in areas with rich VRE resources and load centres. Concentrated solar power (CSP) plants with heat storage devices play a similar role as ESSs, while also providing RE generation resources. Because of limited solar irradiation conditions, CSPs are located mainly in northwest China and Inner Mongolia, where particularly high-quality solar energy resources exist. The investment in ESSs and CSP reflects the external costs that are needed for VRE integration, which would be a considerable part of the electricity supply costs to achieve carbon neutrality.High renewable penetration also increases the demand for long-distance transmission lines, and tighter interconnection is required to cope with the generation-load imbalance. Figure Dramatic changes in power system morphology will inevitably cause changes in the electricity supply cost. We observe considerable but arguably affordable costs associated with transforming China’s power system to support carbon neutrality. The total costs in the CN2050 and BAU scenarios over the 30-year period are 50.4 trillion CNY and 31.4 trillion CNY , respectively. The carbon neutrality goal results in an additional cost of 60.5% compared with that for BAU. The average annual additional costs are ~0.62% of China’s GDP in 2020. It is a large but manageable value, especially recognizing that it does not account for the considerable external cobenefits of fossil fuel substitution, such as reduced health and agricultural damages from air pollution.Figure By comparison, electricity supply costs would decrease 6.3% from 2020 to 2050 under BAU without carbon controls. Annual carbon emissions would reach 5.62 billion tonnes in 2035, and then begin to fall because the capital costs of RE would become sufficiently low, thus promoting competition with conventional units, even when considering their external costs. Specifically, declines in the per-kW VRE capital costs of 35% would incentivize the construction of many RE units even without carbon controls. The electricity supply costs in the other two scenarios exhibit slight decreases in the first five years and increases in the later years because of the more stringent emission requirements. The additional costs of emission reduction in 2050 for NDC and GW2.0 are 1.9 and 5.0 CNY¢/kWh, respectively, compared with emissions in the BAU case.18. As the emission reduction requirements become more stringent, the same amount of emission reduction will result in a rapid increase in emission reduction costs. These results reflect the trade-off between low-carbon targets and the total costs. Hence, the less aggressive carbon reduction goals are much less expensive to achieve not only in aggregate terms but also on a per tonne basis, and the per-tonne expenditure increases rapidly under ambitious targets, such as those of CN2050.The average carbon mitigation costs are the additional costs paid per tonne of carbon emissions between the two scenarios. The average carbon mitigation costs grow rapidly when strengthening the emission limits across the four scenarios as shown in Fig. The electricity supply cost structure shifts substantially over 30 years as changes are made to meet the carbon neutrality target. Figure Figure Wind and PV power are the main driving factors in decarbonization. Since the two have zero operating costs, their capacity growth does not cause increases in operating costs. However, carbon neutrality requires an RE installed capacity of over 5.8 TW, reflecting in part the low capacity factors of wind and PV. Therefore, the capital and maintenance costs per kWh increase, even as the VRE capital cost per kW rapidly decreases. Under the assumptions of the CN2050 scenario, the supply cost increments contributed by VRE total 15.5 CNY¢/kWh, although the capital costs per kW decline by ~60%. The fact that VRE capital costs account for a considerable fraction of the supply costs illustrates the necessity of considering the supply curves for each province in detail. Absent consideration of the spatial distribution of LCOE, the costs in 2050 will be underestimated by 2.2 CNY¢/kWh. Explanations of the calculations and method used for comparison are presented in Supplementary Note Security and stability constraints in a power system with a very high RE capacity share require flexible generation resources and regional network expansion. Adding gas power, biomass power, ESSs, CSP and transmission expansion results in an additional supply cost increases of 18.4 CNY¢/kWh. This increase is a result of the requirements for optimal development and operations considering the spatiotemporal imbalances and low-inertia characteristics of VRE.A sensitivity analysis is conducted on the CN2050 scenario to consider the impact of the uncertainties of five key factors: RE and BESS capital costs, the load growth rate, security requirements, the RE and BESS manufacturing capability, and transmission capacity limits. We vary the parameters within the given intervals for each factor to assess their influence on the electricity supply cost increase in each stage  result in the largest differences in the final electricity supply cost at approximately ±6.5%. The electricity supply costs in 2050 under carbon neutrality vary from 54.3 to 61.9 CNY¢/kWh as VRE capital cost increases from the low- to the high-penetration cases. Due to the accumulation of capital cost differences, such variation increases as the carbon emission limits tighten over the planning period. The results are expected considering the large contribution of RE capital costs to the total cost increases. However, the final supply cost would still rise by 6.0 CNY¢/kWh even in the case in which the cost decreases the fastest.The uncertainties of security and inertia requirements, including power reserve constraints, spinning reserve constraints, and minimum inertia requirements, result in a ±3.0% difference in final supply costs. The variation increases as the penetration of VRE increases since VRE provides neither a robust reserve capacity nor inertia to the power system and reduces system security and stability. Stricter system security requirements would require increased investment in ESSs, CSP, and transmission lines to accommodate VRE, leading to rapidly rising costs. Without the consideration of three security requirements in the GTEP model, the electricity supply cost of achieving carbon neutrality in 2050 drops by 2.4 CNY¢/kWh.The sensitivity of the transmission capacity limits exhibits a similar trend as that for the security requirements since transmission expansion is also an important means to ensure system security. Transmission capacity limits impact the electricity supply cost mainly after 2035, when the RE installed capacity grows rapidly and greater electricity delivery is needed. The uncertainties of transmission capacity limits result in a ±1.7% difference in the electricity supply cost in 2050.The impacts of the load growth rates and manufacturing capability exhibit different patterns, in which the influence on total costs is greater in the early to mid-term stages of system development. For load growth rates, the impact on cost decreases from a peak value of 4.1 CNY¢/kWh in 2035 to 1.3 CNY¢/kWh in 2050 because each additional kilowatt-hour of load demand in the initial stage would cause more carbon emissions and require more RE generation, which is relatively expensive at that time. Hence, the supply costs in the early stage are more sensitive to load demands than those in the completion stage.The RE and BESS manufacturing capabilities represent the maximum capacities that can be newly installed each year, which are driven by the production capacities of RE generation and BESS units and related policies. Their sensitivity is highest in 2040 since the carbon emission limits are reduced by 78.1% from 2030 to 2040, and the ability to install RE generation and BESS becomes critical. Such capabilities become redundant in the late stage, so their sensitivity to electricity supply costs diminishes. However, when the manufacturing capacity is too low to produce enough VRE units more than a certain threshold over the future 30 years (~5000 GW according to our results), considerable investment in coal-based CCS units would need to meet the emission reduction goals, leading to gross increases in costs  and various flexible generation resources serving as auxiliary service contributors. Moreover, the interprovincial grid interconnection must be gradually transformed from a simple power transmission channel to a platform supporting bidirectional energy sharing between regions with different generation resources.Sufficient manufacturing capabilities for RE and ESS units are important for achieving a tremendous transformation in such a short time. From 2030 to 2045, the installed capacities of wind, PV, and ESS would need to increase annually by at least 91.2 GW, 139.4  GW, and 54.0 GW on average, respectively. For comparison, the average annual growth in the capacities of wind and PV in the past 5 years was 30.0 GW and 41.8 GW respectively. In addition, the total installed capacity of ESSs in China was just 35.6  GW at the end of 2020. Therefore, the current manufacturing of RE and ESS units is still insufficient for supporting the needed energy transformation, although the growth rate in China is the highest in the world. Ensuring the integrity of the supply chain and implementing appropriate policy incentives are critical steps for achieving the decarbonization of the power sector by 2050.20 has not been changed since 2018, despite a 49.1% increase in VRE installed capacities. Hence, a spot market is required to obtain accurate and timely electricity prices. In addition, the electricity supply cost increases are derived from not only VRE capital costs but also from additional external costs to ensure system security due to the intermittency and low inertia of VRE. Flexible generation units, such as gas power, biomass power, CSP, and ESSs, are not major contributors to baseload power generation but support system security through their regulation ability and inertia. A capacity market and ancillary service market should be established to internalize the external costs of high RE penetration and to fairly allocate the benefits to these flexible units.Rapid capacity mix changes cause considerable electricity supply cost increases. A complete set of power market mechanisms including spot, capacity, and ancillary markets, is urgently needed to reflect such changes in the electricity prices and fairly allocate the corresponding benefits. As mentioned, the electricity supply costs would increase by 9.6 CNY¢/kWh (1.39 USD¢/kWh) in an attempt to achieve carbon neutrality in 2050 even though as costs of RE technologies decrease following the current global trends. The cost increases will ultimately be paid by consumers through the renewable surcharges in electricity bills under China’s current electricity pricing mechanism. The current administrative pricing method is rigid. For example, the RE surcharge of 1.9 CNY¢/kWhOur analysis indicates that achieving carbon neutrality in the power sector in China by 2050 is technically feasible but will increase the electricity supply costs significantly by 19.9%. Policy subsidies based on feed-in tariffs or renewable energy certificates should continuously be provided to newly emerging low-carbon generation and storage technologies, such as CSP and BESSs, in the early stages of their deployment. Historically, the Chinese government’s feed-in tariffs for the wind and PV industries in the past 10 years have resulted in considerable cost reductions thus providing the foundation for the carbon neutrality transition. Similar policy supports remain vital for technological innovation and scaling for the newly emerging technologies to ensure the transition is not only successful but also cost-effective. Determining how policy formulation impacts the techno-economic performance and how to formulate concrete policies are topics that must be considered in future work.To evaluate the factors that increase the electricity supply cost under high wind and solar penetration to achieve carbon neutrality in China’s power system, we apply the integrated analysis platform Global Renewable-energy Exploitation ANalysis (GREAN) established by the Global Energy Interconnection Development and Cooperation Organization (GEIDCO). The key points for accurately assessing the LCOE are twofold: (1) detailed databases of weather, geographic and related infrastructure information are needed and (2) comprehensive assessment models that consider the costs of construction of flexible generation and grid interconnection under high wind and solar penetration are required.21 include wind speed, wind direction, air density, temperature, and other parameters. The solar energy resource data provided by SolarGIS22 include global horizontal irradiance and direct normal irradiance. Combined with the characteristics of wind turbines and PV modules, these data are applied to evaluate provincial VRE capacity factors and generate the annual VRE power output. The geographic data include a global land cover map, the distribution of major water bodies, terrain elevation data, and other information. Human behaviour-related data include the distribution of major conservation areas, information on the power grid and transportation infrastructure, and other information. Geographic information and human behaviour data are used to evaluate the availability of RE sites, the challenges of construction, and grid-connection costs. The specific data categories and sources of the database used in the GREAN platform are detailed in Supplementary Fig. The GREAN platform includes data on worldwide VRE resources, geographic characteristics, and human activities. The global VRE (wind and solar) energy resource data have a resolution of 9 km × 9 km. In addition, the wind energy resource data provided by VortexComprehensive assessment models are established based on the databases above to conduct systematic calculations of theoretical, technical, and economic VRE potentials. The theoretical VRE potential refers to the maximum developable capacity for VRE units considering only the VRE resource data. The technical VRE potential is obtained by excluding areas that are unsuitable for development due to poor resource endowment, disadvantageous geographic conditions, or policy prohibitions, such as conservation area designations. Based on the technical VRE potential, the economic VRE potential is calculated considering the construction constraints and grid connection costs and is then used to calculate the overall electricity supply cost under carbon control and neutrality targets. To evaluate the economic VRE potential, the GREAN platform first calculates the year-round hourly power output of wind and solar units in each 500 m × 500 m area based on the generation characteristics of 2.5 MW wind turbines with hub heights of 100 m, which meet IEC standards, and 300 W monocrystalline PV modules with fixed-tilt systems. The capacity factors are then calculated for each province. The LCOE is finally calculated considering the grid connection cost and device transportation cost according to the distance between the construction location and related infrastructure. The supply curves of the VRE generation potential around China can be obtained based on the LCOE at a fine spatial resolution to reveal the different characteristics of the RE resources in different regions.23.With all the key factors considered, the GREAN platform provides LCOE and supply curve estimates that are as close as possible to the actual values based on the data available. The technical details of the platform are presented in the referencesA generation and transmission expansion planning (GTEP) model is formulated to simulate the long-term development roadmap of China’s power system under carbon emission reduction targets in the next 30 years. The multi-period planning model is structured at the provincial level with embedded operation simulation at an hourly scale. Hourly dispatch schedules based on our model in 2020 are presented in Supplementary Fig. 24. The per-kW capital cost projections for generation technologies in the GTEP model refer to the results of NREL’s annual technology baseline (ATB) model published in 20218, which includes three trajectories: conservative, moderate, and advanced. The per-kW capital costs in the NDC, GM2.0 and CN2050 scenarios follow moderate trajectories from 2020 to 2050. We conduct a sensitivity analysis of RE capital costs, where the NREL conservative projection is set as the high-cost case and the advanced projection represents the low-cost case  in 2050 under the CN2050 scenario.We collect annual installed transformer capacity and transmission line length data at each voltage level  in each province from 2008 to 201829. To consider such impacts of changes in the capacity mix on local power grids, we modify the local grid investment based on historical data according to the changes in the capacity factors of the local capacity mix. Assuming that the utilization rate of power grid equipment decreases by the same rate as the power supply capacity factor, the modification formula is as follows:r in planning stage y, respectively. cfr,0 and cfr,y denote the current capacity factors and capacity factors in planning stage y for the capacity mix in region r, respectively. The capital costs and the maintenance costs for the local grid network are calculated based on the modified network capacities.The capacity factors of the entire capacity mix will decrease as VRE penetration increases, which will further reduce the utilization rate of network equipmentFurther information on research design is available in the Supporting InformationPeer Review FileReporting Summary


The next-generation sequencing method was developed in the second half of the 2000s and marked the beginning of high-throughput sequencing (HTS) analyses of viral communities ,2. With With the HTS approach, a novel badnavirus, provisionally designated chinaberry tree badnavirus 1 (ChTBV1), was identified from chinaberry that has been cultivated in China for use in traditional medicines . ThereafChenopodium album via recombination; the study suggests that weeds serve as mixing vessels for begomoviruses, thereby facilitating recombination [Genetic recombination between viral isolates has been known to be one of the sources of speciation  and is abination .Moreover, the spatiotemporal spread of a plant virus, cherry leaf roll virus (CLRV), was studied using a Bayesian phylodynamics framework based on the coat protein gene sequences of 81 viral isolates collected from 5 different countries. It revealed that Germany played an important role in CLRV transmission, which is consistent with the trade of cherry .The genome sequence of several isolates of apple rubbery wood virus 2 (ARWV-2) and citrus virus A (CiVA), two negative-sense single-stranded RNA viruses, infecting pear trees grown in China was characterized by using the HTS approach combined with conventional RT-PCR assays. The results show that genome-wide nt sequence identities were above 93.6% among the ARWV-2 isolates and above 93% among CiVA isolates, and sequence diversity occurred in the 5′ untranslated region of the ARWV-2 genome and the intergenic region of the CiVA genome .This Special Issue informs us of up-to-date knowledge that genetic recombination, spatiotemporal spread, and host plants have largely shaped viral communities, including their speciation, evolution, ecology, and diversity of plant viruses. Altogether, this will help us better understand plant viruses and the development of a more efficient strategy for controlling plant viral diseases.


Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested  During mitosis, a cell’s replicated chromosomes are equally segregated to two daughter cells. In order for this process to happen accurately and efficiently, the microtubule cytoskeleton must completely remodel to form a bipolar spindle. Replicated chromosomes then attach to microtubules, emanating from opposite spindle poles, through a large dynamic chromosome-associated protein complex called the kinetochore. Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of cancer and birth defects . TherefoDuring establishment of biorientation, kinetochores have been observed to first attach to the lateral surface of a microtubule . Such kiEstablishment of end-on kinetochore attachments to microtubules, for chromosomes that are initially attached to spindle microtubules laterally, can be divided into two processes: (1) chromosome lateral movement along the microtubule surface and (2) conversion from lateral to end-on attachment. With regard to the kinetochore reaching the microtubule tip, CENP-E and chromokinesins are particularly important for chromosome movement to the spindle midzone in metazoans . HoweverDrosophila kinesin-8, Klp67A, shares many of the roles of other kinesin-8 proteins including stabilizing and destabilizing microtubules, but this kinesin also stabilizes kinetochore-microtubule attachments  of the gene that encodes a subunit of the anaphase-promoting complex, to arrest cells in metaphase at the non-permissive temperature  Cnn1, Okp1 (COMA complex), Mif2, Ctf3 (CBF3 complex), and Cse4 (a centromeric histone variant) cdc7-1) or anaphase (cdc15-2) arrested extracts  which do not physically bind to microtubules, are in fact able to bind to and translocate on microtubules in our assay provided evidence that these kinetochore proteins were not alone or in small sub-complexes, but, in fact, were part of complete or nearly complete kinetochores. To explore this possibility further, we turned to dual-color imaging of kinetochore protein pairs and colocalization analyses. Due to its well-documented and canonical microtubule binding abilities, Ndc80 was chosen as a fiducial marker to which the localization of other kinetochore proteins was compared . One posIn our assay, the mScarlet-I fluorophore used to tag proteins blinked on and off . Other rCryo-electron tomography (cryo-ET) is a powerful method for studying the structural organization of multi-protein complexes in their fully hydrated, native state . A challAfter establishing that the kinetochore protein signals observed in our reconstitution assay represented intact kinetochore complexes, we began to probe the mechanism of the plus-end directed kinetochore movement. We postulated that the movement was driven by a kinesin motor protein. In budding yeast, there are only 5 kinesins, Kip2, Kip3, Cin8, Kip1, and Kar3 . To testkip3Δ lysates. Moreover, those laterally bound kinetochores that did move in kip3Δ lysates spent significantly less time moving toward the microtubule plus end compared to wild-type, and their run lengths were about half as far as those in Kip3 +lysates  and each of the motors assayed in the motor deletion screen described above. We observed Cin8 and Kip1 coming on and off the microtubule lattice stochastically at a high frequency, but they were not motile in our assay . Kar3 wakip3Δ lysates, not in kip2Δ lysates , we observed kinetochores that are laterally attached to microtubules, but these kinetochores rarely became end-bound. This observation can be explained if fewer kinetochores make it to the end of the microtubule. Specifically, in kip3Δ lysates, the inability to establish end-on attachments is likely largely due to a combination of impaired kinetochore motility and due to the microtubule dynamics being shifted toward more elongation with fewer catastrophes, making it less likely that kinetochores will reach microtubule plus ends  it induces microtubule catastrophe to allow kinetochores to catch up with elongating microtubule plus ends and (2) it moves kinetochores to the microtubule plus ends. Kinetochores did not move as frequently, directionally, or as far along microtubules in the  lysates . Howeverlus ends . In contlus ends .kip3Δ cells metaphase kinetochores are declustered and aligned linearly along the spindle microtubules. This declustered kinetochore phenotype is also seen in kip3ΔT-LZ cells  in DMSO and 50 mM potassium phosphate buffer, pH 6.2, for 30 min before harvesting, 2.5 hr after the temperature shift. Cells were then harvested by serial centrifugation at 6000 x g in a Sorvall RC5B centrifuge using an SLA-3000 rotor for 10 min at 4°C. Cell pellets were then resuspended in cold ddH2O and pelleted in a Hermle Z446K centrifuge for 3 min at 3430 x g at 4°C. This wash procedure was repeated once more, with any remaining liquid removed from the pellet via aspiration. Using a stainless steel lab spatula , the cells were then scraped out as small clumps into a 50 mL conical filled with liquid nitrogen, snap-freezing them, for storage at -80°C.Cells were harvested as previously described in As described previously by As previously described in Both glass side preparation and passivation of coverslips followed the protocol previously described in 2O, the coverslips were submerged for 2 hr in 2% Hellmanex III solution . After this, they were again rinsed in ddH2O three times. Before proceeding to passivation, the coverslips were blown dry with nitrogen gas. For passivation, a solution containing a 0.1 mg/mL mixture of PLL(20)-g[3.5]-PEG(2):PEG(3.4)-biotin(50%) , at a ratio of 1:19, in 10 mM HEPES was prepared. Fifty µL drops were then placed on Parafilm in a humid chamber and coverslips were gently placed onto the drops. After 1 hr, the passivated coverslips were washed for 2 min in PBS and rinsed in ddH2O for 1 min. The coverslips were then air-dried with nitrogen gas and stored in an airtight container at 4°C. These passivated coverslips were stored for use only for three weeks.To prepare the coverslips for assembly into a flow chamber, cover glass  was first cleaned by sonication in acetone for 30 min. The coverslips were then soaked for 15 min in 100% ethanol. After 3 thorough, but short, rinses in ddHPreparation and assembly of the flow chamber for use in the TIRF based dynamics assay was exactly as described in Similarly to what is described in After clarified lysate supernatant was added to the prepared chamber, the slides were loaded onto a Nikon Ti2-E inverted microscope with an Oko Labs environmental chamber pre-warmed to 28°C. Images were acquired using a Nikon 60 X CFI Apo TIRF objective (NA 1.49) and an Orca Fusion Gen III sCMOS camera  at ×1.5 magnification using the Nikon NIS Elements software. Using a LUNF 4-line laser launch  and an iLas2 TIRF/FRAP module  total internal reflection fluorescence (TIRF) illuminated a single focal plane of the field and was imaged every 5 s for 30 min.Similar coverslip, slide, assembly of flow chamber, and lysate preparation were all followed as described above. However, instead of preparing one lysate for one genotype, two lysates were prepared side-by-side. To observe the initial behavior, 32 µL of the first lysate was flowed through the chamber and imaged for 10 min. While the slide was still mounted on the microscope, the movie was paused for 1 min while the second lysate was carefully flowed in, replacing the first. After replacing the lysate, the movie was restarted and the same field of view was captured for another 20 min.Cells were grown in rich medium (YPD) overnight to saturation at 25. The next day the cells were diluted into fresh YPD and grown for 4 hr (2 doublings) at 25°C, until the culture reached log phase (OD600=0.5). They were then spun down and washed with minimal Imaging Medium three times . Cells were then immobilized on coverslips coated with 0.2 mg/ml concanavalin A and were imaged in Imaging Medium. Using a Nikon Ti2-E inverted microscope with an Oko Labs environmental chamber pre-warmed to 25°C. Images were acquired with a Nikon 60 X CFI Apo TIRF objective (NA 1.49) and an Orca Fusion Gen III sCMOS camera (Hamamatsu) at 1.5 X magnification using the Nikon NIS Elements software. For imaging, a LUNF 4-line laser launch (Nikon) and an iLas2 TIRF/FRAP module (Gataca Systems) was used for HiLo total internal reflection fluorescence . Images For the microtubule dynamics assay, analysis was done as described previously in For the colocalization quantification, pair-wise kinetochore tracks (that moved as defined above) were pooled into three categories  based on the amount of time that the two proteins were colocalized. Because the red fluorophore blinked, colocalization was scored solely for the mScarlet tagged protein (either Ndc80 or Mtw1).For the live cell imaging analysis, images were analyzed using Fiji (NIH). Maximum intensity Z-projections were made and bleach corrections were applied using the ‘histogram matching’ macro. Metaphase cells were identified by the presence of a 2–3 µm spindle at the entrance to the bud neck. Cells were then counted as either ‘bilobed’ or ‘declustered’ based on the presence of two distinct kinetochore puncta. Line scans were done to assist in this binary classification. If two clear peaks were present, the cell was ‘bilobed’. Any other line scan shape, that is, 3+peaks or 1 long kinetochore signal that matched the spindle, were classified as ‘declustered’. Data are from two independent technical trials of one biological replicate. In each replicate, n=50 cells were counted. Graphs are of the mean and standard error of the mean (GraphPad Prism).Holey carbon electron microscopy grids  were washed in 99.5% acetone while agitating on a rocker for 15–30 min to remove potential residual plastic backing, then washed in H2O, and dried with filter paper. Cell lysate of DDY5818  strain was prepared as described above. After ultracentrifugation, supernatants were aliquoted into pre-chilled microcentrifuge tubes with a volume of 9.5 µL supernatant per aliquot and kept on ice. About 15 min before the lysate centrifugation was finished, far-red labeled GpCpp-stabilized MT seeds were polymerized in microcentrifuge tubes at 37°C for 10 min. Grids were coated with 10 nm BSA Gold Tracer (Electron Microscopy Sciences) by dripping a total of 10–20 µL of the BSA Gold Tracer solution on the grids and removing the drops with filter paper. Seeds were diluted 1/50 in 1 X PEM buffer. 0.5 µL of seeds were mixed with 9.5 µL cleared lysate. Three to 6 µL of the mix were pipetted on the pre-coated grids. For sample vitrification, either a Vitrobot Mark IV  or a Leica EM GP2  was used. Sample chamber conditions were set to 28°C and 75–90% humidity. Samples were incubated in the sample chamber for 10–15 min and then blotted from the backside and vitrified by plunging into liquid ethane. To set up the Vitrobot Mark IV for back-blotting, the filter paper facing the sample side was replaced with a custom-made Teflon sheet of the same size to blot only the fluid from the back of the sample. To ensure that suitable grids were produced for each imaging session, a range of plot forces between 0 and 10 (for Vitrobot Mark IV only), blotting times of 3–6 s, and a 1 s drain time, were used. Grids were fixed into AutoGrid carriers (Thermo Fisher) and stored in liquid nitrogen until they were imaged.Cryo-samples were imaged on a Leica EM Cryo CLEM system (Leica Microsystems). The system consists of a Leica DM6 FS widefield microscope that is equipped with a motorized Leica EM Cryo stage and a short working distance (<0.28 mm) 50 x Leica EM Cryo CLEM ceramic tipped objective . These specifications allow sample imaging at liquid nitrogen temperatures. A halogen lamp powered by a CTR6 halogen supply unit was used as a light source. We used GFP ET , RFP  and Y5  filter cubes for imaging. For cryo-correlative light and electron microscopy, a grid overview map was recorded using transmitted light and Y5 or GFP channels. The map was used to identify grid squares with good ice quality and intact carbon film. Z-stacks  of regions with clear fluorescence signals were recorded using the transmitted light, GFP (Spc105), RFP (Tub1), and Y5 (seeds) channels. These images were used to identify Spc105-GFP-positive regions of interest for subsequent cryo-electron tomography imaging. The same imaging conditions were used for grid squares on individual grids. Imaging conditions were varied between grids to obtain sufficient signal to allow correlation in later steps. Images for panel generation in Adobe Illustrator were prepared using Fiji and Adobe Photoshop. No nonlinear gamma correction was applied during image processing.2. Data were collected in 0.5 binning mode or without binning. Frame time was 0.2–0.25 s. Tilt series alignment and tomogram reconstruction were done using the freely available IMOD software package (Samples were imaged on a Titan Krios transmission electron microscope (FEI) equipped with an X-FEG electron source, a BioQuantum energy filter  and a K3 direct electron detecting device (Gatan) and operated at 300 kV. Samples were visually inspected for ice quality using the FEI flu cam. Overview grid maps were acquired at 0.2 µm pixel size. These grid maps were used to identify grid squares of interest from the cryo-fluorescence light microscopy overview maps. Grid square images and polygon maps of the regions of interest with an overlap of 20–25% between individual images were recorded. The polygon maps were used to pick tilt series acquisition points based on the fluorescent signal from cryo-fluorescence light microscopy. The hole pattern of the carbon film was used as a guide. Acquisition points were chosen with adequate distance between individual points to prevent electron dose exposure damage prior to data collection. SerialEM  in low-d package . Tilt se Kinetochores are large protein complexes that mediate faithful chromosome segregation in eukaryotes. The authors develop an in vitro approach to study interactions between kinetochores and microtubules in yeast cell extracts. They use this powerful lysate-based system to characterize a new role for the budding yeast kinesin-8, Kip3, in powering lateral kinetochore movement along microtubules. This paper should be of interest to researchers working in the field of mitosis, the cell cycle, and the cytoskeleton, and, more broadly, for those studying macromolecular complexes with reconstitution and in vitro imaging approaches. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Anna Akhmanova as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jason Stumpff (Reviewer #2); Stefan Westermann (Reviewer #3).Thank you for submitting your article "Reconstitution of Kinetochore and Microtubule Dynamics Reveals a Role for a Kinesin-8 in Establishing End-on Attachments" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. An interesting observation is that Kip3 doesn't consistently co-localize with the kinetochore particles, which raises a question regarding the mechanism of Kip3-mediated kinetochore movement. The authors suggest that weak transient interactions with Kip3 facilitate kinetochore movement or alternatively, that Kip3 is indeed stably associated with kinetochores, but low stoichiometry prevented detection in light of high background of free Kip3 bound to microtubules. To address this, the authors should consider titrating in recombinant Kip3 into Kip3 deficient extracts – this may allow for detection of a kinetochore-bound population of Kip3, and will reveal how much Kip3 is required to initiate kinetochore movement in this system.2. It is not clear if the authors are arguing for a model in which: (1) Kip3 travels to the plus end, where it causes depolymerization, which then allows for end-on conversion; or (2) Kip3 travels to the plus end, where it converts kinetochore attachments from lateral to end-on, which then causes depolymerization . To help clarify this, the authors should report the following for the WT, Kip3-depleted, and Kip3 mutant conditions: In what percentage of cases where kinetochores did arrive to the MT end, did this cause depolymerization ?3. In Figure 4e, the authors show images suggesting that adding exogenous Kip3 to Kip3-depleted lysates (in which kinetochores have assembled and bound to microtubules) promotes translocation of the kinetochore particles along the microtubules. Please provide quantification for this, so that it is clear if these are rare or common events.4. Please provide quantification for the data in Figure 2.5. The kinetochore densities in the tomographic slices shown in Figure 3b and 3d are not easy to see in the Figure, and are only a bit easier to see in the supplemental movies. It may be helpful to add another panel of the same images with the kinetochores outlined to aid with visualization.6. In Figure 1, is there an explanation for why the plus-end directed velocities of kinetochores with fluorescent labels on Mif2 and Mtw1 move significantly slower than those with other labels? The Mtw1-labeled tip-bound kinetochores also seem to spend more time in minus-end directed movement than the other conditions. Is this expected? Please address this in the text.7. On Line 174, the authors state "Interestingly, although less frequent and covering shorter distances, kinetochore movements in kip3∆ lysates had the same velocity as the more frequent and longer movements observed in Kip3+ lysates." However, in the discussion on Line 310, the authors state "Kinetochores did not move on microtubule in the kip3∆ lysate." The apparent contradiction between these statements should be addressed. It would also be interesting to know if the authors have thoughts on how kinetochores may be moving directionally in the absence of Kip3. Is there an intrinsic mechanism that promotes kinetochore movement towards the microtubule plus-end? If so, has this been observed in previous in vitro studies of kinetochores?8. The data in Figure 4b appear to indicate that loss of cin8 activity increases the percentage of time that tip-bound kinetochores spend in minus-end directed movement. Is this a significant increase and, if so, is there a logical explanation for this effect? Cin8 has been proposed to promote disassembly of kinetochore microtubule plus-ends (PMID: 19041752).9. Please provide in the discussion a comparison of this study to the Tanaka re-activated CEN system , which is still widely considered the gold standard in the field to directly observe different attachment configurations in yeast cells. In this system, no motor except Kar3 showed an effect. This should be addressed in the text.10. Please provide a brief discussion regarding conceptual limitations of these lysates: Kip3 has roles in nucleus and cytoplasm, yet this distinction is lost in these whole cell lysates. This is especially critical for budding yeast which undergoes a closed mitosis. What are the prospects of using nuclear extracts instead?Reviewer #1 (Recommendations for the authors):Specific Comments:(1) In figure 3D, the authors note a "cloud-like density" at the tips of microtubules. While I see this density in 3b, it is not obviously present in 3d. Is it possible to provide clearer images?(2) In Figure 4e, the authors show images suggesting that adding exogenous Kip3 to Kip3-depleted lysates (in which kinetochores have assembled and bound to microtubules) promotes translocation of the kinetochore particles along the microtubules. Providing quantification of this would help the reader understand if this a rare or common event.(3) On line 202, the authors state that only Kip3 and Kip2 moved processively on the microtubules, and the callout indicates "Figure 5," but I don't see the data.(4) In many instances it is unclear as to whether the authors are suggesting that Kip3 functions to decrease or increase microtubule dynamics. They state in several cases that Kip3 attenuates microtubule dynamics  and in other instances they mention that Kip3 functions to increase dynamics . For a reader not familiar with the details of the Kinesin-8 family of motors, this is somewhat confusing. It would be helpful to provide some clarity here.Reviewer #2 (Recommendations for the authors):Overall, I think this is a well-executed study with conclusions that are supported by high quality data. I do have a few minor suggestions for the authors to consider that might improve the manuscript.1. In Figure 1, is there an explanation for why the plus-end directed velocities of kinetochores with fluorescent labels on Mif2 and Mtw1 move significantly slower than those with other labels? The Mtw1-labeled tip-bound kinetochores also seem to spend more time in minus-end directed movement than the other conditions. Is this expected?2. On Line 174, the authors state "Interestingly, although less frequent and covering shorter distances, kinetochore movements in kip3∆ lysates had the same velocity as the more frequent and longer movements observed in Kip3+ lysates." However, in the discussion on Line 310, the authors state "Kinetochores did not move on microtubule in the kip3∆ lysate." The apparent contradiction between these statements should be addressed. It would also be interesting to know if the authors have thoughts on how kinetochores may be moving directionally in the absence of Kip3. Is there an intrinsic mechanism that promotes kinetochore movement towards the microtubule plus-end? If so, has this been observed in previous in vitro studies of kinetochores?3. The data in Figure 4b appear to indicate that loss of cin8 activity increases the percentage of time that tip-bound kinetochores spend in minus-end directed movement. Is this a significant increase and, if so, is there a logical explanation for this effect? Cin8 has been proposed to promote disassembly of kinetochore microtubule plus-ends (PMID: 19041752).4. The kinetochore densities in the tomographic slices shown in Figure 3b and d are not easy to see in the figure and are only a bit easier to see in the supplemental movies. It may be helpful to add another panel of the same images with the kinetochores outlined to aid with visualization.5. For the histograms shown in 4 and 5, changing the y-axis to percent of total may make it easier to compare the data sets displayed on each graph.Reviewer #3 (Recommendations for the authors):eLife.I think in principle this is a well-developed study and I hesitate to ask for many more experiments. I think in principle this is suitable for Given that the mechanism of Kip3-mediated kinetochore movement is still somewhat unclear, it would be interesting to titrate in recombinant Kip3 into the Kip3 deficient extracts, and not just perform the complementation with Kip3 containing extracts. This might facilitate the detection of a Kinetochore-bound population of Kip3 or determine how much Kip3 is required to initiate kinetochore movement in this system.To improve data presentation and discussion I would further suggest the following points:1) Some more quantification of data on key observations: Does the arrival of a kinetochore at a plus-end really switch that microtubule to depolymerization? In some of the presented kymographs it looks to me like the depolymerization initiates before the arrival . What is the fraction of arrival events that lead to depolymerization versus the frequency of spontaneous depolymerization without KT arrival? Also, in Figure 2 some more quantification on the co-localization would be helpful.2) The discussion should include two additional points:Comparison to the Tanaka re-activated CEN system . In this system no motor except Kar3 showed an effect.Also, a brief discussion regarding conceptual limitations of these lysates: Kip3 has roles in nucleus and cytoplasm, yet this distinction is lost in these whole cell lysates. This is especially critical for budding yeast which undergoes a closed mitosis. What are the prospects of using nuclear extracts instead? Essential revisions:1. An interesting observation is that Kip3 doesn't consistently co-localize with the kinetochore particles, which raises a question regarding the mechanism of Kip3-mediated kinetochore movement. The authors suggest that weak transient interactions with Kip3 facilitate kinetochore movement or alternatively, that Kip3 is indeed stably associated with kinetochores, but low stoichiometry prevented detection in light of high background of free Kip3 bound to microtubules. To address this, the authors should consider titrating in recombinant Kip3 into Kip3 deficient extracts – this may allow for detection of a kinetochore-bound population of Kip3, and will reveal how much Kip3 is required to initiate kinetochore movement in this system.To address the possibility that unbound Kip3 obscures the visibility of Kip3 associated with kinetochores, the reviewers state, “the authors should consider titrating in recombinant Kip3 into Kip3 deficient extracts”. We appreciate this suggestion, which we attempted to act on as described below, and which motivated us to add a new passage into the Discussion.Unfortunately, we are currently unable to purify functional Kip3 and add it to the extract assay without diluting the extract and completely losing all extract activity. However, in response to this reviewer’s comment, we performed two experiments that utilized the same principle in an attempt to address the suggestion of titrating Kip3 into the assay.While these two types of experiments (fully described below) failed to generate persistently colocalized tracks of moving kinetochores and Kip3, they do not rule out the possibility that Kip3 is present below the threshold of detection on motile kinetochores. However, these new negative results encouraged us to entertain alternative mechanisms for Kip3’s action at the end of the Discussion:“In the absence of the ability to consistently observe direct Kip3 stable association with motile kinetochores, we cannot rule out other mechanisms to explain the Kip3-dependent kinetochore motility that we discovered. Further investigation is required to determine the exact molecular mechanism by which Kip3 mediates plus end-directed kinetochore transport on the microtubule lattice. It is possible, for example, that Kip3 transports an unidentified factor to kinetochores, and that this hypothetical factor mediates kinetochore movement.”kip3∆ strain to control the amount of motor protein in our assay. At low concentrations of Kip3-GFP lysate, we did not observe a sufficient number of microtubule-associated kinetochores to analyze. However, increasing the amount of Kip3-GFP lysate in the mixture resulted in a dense population of motors on microtubules that obstructed any observation of colocalization with motile kinetochores, similar to what was observed in Figure 5c. Unfortunately, we were unable to achieve an optimal condition where we could have an unobstructed view of both motile side-bound kinetochores and individual Kip3 motors simultaneously.The goal of both experiments we tried was to “titrate” a labeled Kip3 protein into our assay, but in two different ways. In each experiment the goal was also to maintain extract assay microtubule functionality by avoiding dilution of overall cellular extract protein concentration. In the first experiment, we made a strain that expressed Kip3-HaloTag. By limiting the amount of dyeligand conjugate added to our lysates, we could control the total amount of fluorescent Kip3 visible on microtubules in the assay and could then look specifically for Kip3 tracks that colocalize with kinetochores. With the addition of JF646 ligand (Janelia Farms) to 25, 50, or 100 nM amounts, individual Kip3 tracks were easily distinguished. However, analysis of ~450 kymographs from lysates of metaphase-arrested cells did not reveal any labeled Kip3 colocalizing with slower moving Spc105-GFP (which marks kinetochores). A representative kymograph is shown to the right. One possible reason why this attempt might have failed is that unlabelled Kip3 might outcompete Kip3-Halo for binding to kinetochores, especially if the kinetochore was already assembled and had incorporated unlabeled Kip3 before the labeled Kip3 was added. Furthermore, in this experiment, limiting amounts of JF646 dye might not label Kip3-Halo already bound to kinetochores as efficiently as unbound Kip3-Halo, and using more dye to force binding would only increase the Kip3 background issue.In the second experiment, we titrated lysate from a Kip3-GFP strain with lysate from a 2. It is not clear if the authors are arguing for a model in which: (1) Kip3 travels to the plus end, where it causes depolymerization, which then allows for end-on conversion; or (2) Kip3 travels to the plus end, where it converts kinetochore attachments from lateral to end-on, which then causes depolymerization . To help clarify this, the authors should report the following for the WT, Kip3-depleted, and Kip3 mutant conditions: In what percentage of cases where kinetochores did arrive to the MT end, did this cause depolymerization ?We thank the reviewers for pointing out the alternative way our statements could be interpreted. In order to clear up any ambiguity in what we believe the data indicates, we made the following changes.Figure 5c text box: Changed “Percent of kinetochores that transition from side to end-bound” to “Percent of side-bound kinetochores that reached the microtubule plus end”.kip3∆T-LZ (12.4%) strain because this Kip3 mutant has impaired ability to induce catastrophe . However, this mutant retains the ability to move kinetochores along microtubules. Since few kinetochores reach the microtubule plus-end in kip3∆T-LZ or kip3∆ lysates, a comparison of the behaviors of kinetochore-associated microtubule ends between lysates of mutant and wild-type strains be of limited value because it would be very hard to achieve statistical significance due to low frequency of kinetochores associating with MT plus ends. Nevertheless, we did attempt to quantify the behaviors and found the following:Lines 234-240: Reworded several sentences to more clearly indicate support for model (1) and to avoid misinterpretation of our words as supporting model (2). We are proposing two specific roles for Kip3: (1) facilitating movement of kinetochores along the lateral surface of microtubules toward the plus-end and (2) induction of microtubule catastrophe triggered by Kip3, which decreases the distance required for laterally moving kinetochores to reach the microtubule tip. With respect to the two models put forth above by the reviewers, we propose that model (1) is correct because fewer side-bound kinetochores catch up to the microtubule end in a lysate made from a kip3 mutants immediately after they reach the microtubule tip is consistent with the possibility that Kip3 associated with end-bound kinetochores might induce depolymerization, we do not want to propose this idea in the manuscript because the observable sample size was too small and other possible explanations exist.While the decrease in the percentage of kinetochores that track depolymerizing microtubules in lysates from kip3 mutant strains made it difficult to study Kip3’s role directly. We are currently trying to design new strategies to monitor the transition of side-bound to end-bound kinetochores in vitro to test which proteins are essential for the process.With respect to model (2) proposed by the reviewers, while the Kip3 motor protein may facilitate the transition of kinetochores from a side-bound to end-bound orientation on microtubules, our current data neither supports nor refutes this possibility. The small number of events in which kinetochores reached the microtubule plus-end in lysates from 3. In Figure 4e, the authors show images suggesting that adding exogenous Kip3 to Kip3-depleted lysates (in which kinetochores have assembled and bound to microtubules) promotes translocation of the kinetochore particles along the microtubules. Please provide quantification for this, so that it is clear if these are rare or common events.kip3∆ lysates became motile  once Kip3+ lysate was added, best shown in the representative kymograph in Figure 4e.The text has been updated to indicate that 53.7% of stationary kinetochores in 4. Please provide quantification for the data in Figure 2.Quantification for this figure has been added and the figure legend has been updated along with the Materials and methods.5. The kinetochore densities in the tomographic slices shown in Figure 3b and 3d are not easy to see in the Figure, and are only a bit easier to see in the supplemental movies. It may be helpful to add another panel of the same images with the kinetochores outlined to aid with visualization.We have added another panel to Figures 3b and d with the densities colored in, as suggested.6. In Figure 1, is there an explanation for why the plus-end directed velocities of kinetochores with fluorescent labels on Mif2 and Mtw1 move significantly slower than those with other labels? The Mtw1-labeled tip-bound kinetochores also seem to spend more time in minus-end directed movement than the other conditions. Is this expected? Please address this in the text.cdc23-1 (doi: 10.1016/j.addr.2012.09.039). Despite the difference in measured lateral movement rates when these two kinetochore proteins were tagged, we believe that Figure 2 conclusively shows that Mif2- and Cse4- colocalize and move together with other kinetochore proteins.We appreciate this comment and the opportunity to provide the following explanations and to modify our manuscript. In Figure 1, Mif2- and Cse4-labeled kinetochores move more slowly than kinetochores in which other proteins were tagged with fluorescent proteins. This observation is now addressed in the Results. These observations are consistent with the possibility that c-terminal GFP tags on Mif2 and Cse4 affect protein function and therefore dynamics on the microtubule. Both of the corresponding strains have slow growth rates. In addition, we had to make the linker for the GFP tag on Cse4 longer or else it was synthetic lethal with With respect to Mtw1, while the speed was not significantly different from what was observed when kinetochore proteins other than Mif2 or Cse4 were tagged, kinetochores with tagged Mtw1 spend more time in minus-end directed movement when bound to the end of a microtubule than kinetochores in which other subunits were tagged. Whether this reflects a modification of Mtw1 function due to the fluorescent tag or some other factor whose nature is currently a mystery is unclear. However, the colocalization experiment in Figure 2 rules out the possibility that Mtw1 has a localization independent of other kinetochore proteins.Taken together, our results indicate that the kinetics of microtubule-bound kinetochores in lysates is sensitive to perturbations in protein function and activity, but also that functional kinetochores all exhibit the same baseline characteristics of binding and plus-end directed movement on the lateral surface of microtubules followed by end-binding and tracking the depolymerizing ends of microtubules. We have modified the manuscript text to highlight these issues.7. On Line 174, the authors state "Interestingly, although less frequent and covering shorter distances, kinetochore movements in kip3∆ lysates had the same velocity as the more frequent and longer movements observed in Kip3+ lysates." However, in the discussion on Line 310, the authors state "Kinetochores did not move on microtubule in the kip3∆ lysate." The apparent contradiction between these statements should be addressed. It would also be interesting to know if the authors have thoughts on how kinetochores may be moving directionally in the absence of Kip3. Is there an intrinsic mechanism that promotes kinetochore movement towards the microtubule plus-end? If so, has this been observed in previous in vitro studies of kinetochores?cdc23-1 (doi: 10.1016/j.addr.2012.09.039). Despite the difference in measured lateral movement rates when these two kinetochore proteins were tagged, we believe that Figure 2 conclusively shows that Mif2- and Cse4- colocalize and move together with other kinetochore proteins.We appreciate this comment and the opportunity to provide the following explanations and to modify our manuscript. In Figure 1, Mif2- and Cse4-labeled kinetochores move more slowly than kinetochores in which other proteins were tagged with fluorescent proteins. This observation is now addressed in the Results. These observations are consistent with the possibility that c-terminal GFP tags on Mif2 and Cse4 affect protein function and therefore dynamics on the microtubule. Both of the corresponding strains have slow growth rates. In addition, we had to make the linker for the GFP tag on Cse4 longer or else it was synthetic lethal with With respect to Mtw1, while the speed was not significantly different from what was observed when kinetochore proteins other than Mif2 or Cse4 were tagged, kinetochores with tagged Mtw1 spend more time in minus-end directed movement when bound to the end of a microtubule than kinetochores in which other subunits were tagged. Whether this reflects a modification of Mtw1 function due to the fluorescent tag or some other factor whose nature is currently a mystery is unclear. However, the colocalization experiment in Figure 2 rules out the possibility that Mtw1 has a localization independent of other kinetochore proteins.Taken together, our results indicate that the kinetics of microtubule-bound kinetochores in lysates is sensitive to perturbations in protein function and activity, but also that functional kinetochores all exhibit the same baseline characteristics of binding and plus-end directed movement on the lateral surface of microtubules followed by end-binding and tracking the depolymerizing ends of microtubules. We have modified the manuscript text to highlight these issues.8. The data in Figure 4b appear to indicate that loss of cin8 activity increases the percentage of time that tip-bound kinetochores spend in minus-end directed movement. Is this a significant increase and, if so, is there a logical explanation for this effect? Cin8 has been proposed to promote disassembly of kinetochore microtubule plus-ends (PMID: 19041752).We appreciate this comment. The data do indicate that in the absence of Cin8, end-bound kinetochores spend more time in minus-end directed movement. As highlighted in , Cin8 works with Kip1 as a microtubule crosslinker important for spindle integrity. Furthermore, their model for Cin8 promoting microtubule disassembly predicts a graded effect within the symmetry of the bipolar mitotic spindle. In contrast, our assay utilizes single microtubules grown from widely spaced seeds in order to achieve better resolution of individual molecular dynamics. Thus, our assay provides clearer observations of effects on single microtubules while sacrificing some of physiological geometry relevant to Cin8 function. Possible reasons why microtubules with end-bound kinetochores spend more time depolymerizing in Cin8 mutants include a change in protein composition at the kinetochore upon the loss of Cin8 or a more direct effect of Cin8 on kinetochore-microtubule dynamics. We prefer not to speculate on the biological implications of this observation in the absence of more data to form the basis for a model.9. Please provide in the discussion a comparison of this study to the Tanaka re-activated CEN system , which is still widely considered the gold standard in the field to directly observe different attachment configurations in yeast cells. In this system, no motor except Kar3 showed an effect. This should be addressed in the text.We appreciate the opportunity to make this comparison. A new paragraph was added in the Discussion to frame our observations in the context of the Tanaka observations.. We believe that both mechanisms may work together to facilitate accurate chromosome congression and biorientation, making chromosome segregation robust to different cellular stresses.10. Please provide a brief discussion regarding conceptual limitations of these lysates: Kip3 has roles in nucleus and cytoplasm, yet this distinction is lost in these whole cell lysates. This is especially critical for budding yeast which undergoes a closed mitosis. What are the prospects of using nuclear extracts instead?We appreciate this suggestion and have added a discussion of the nuclear vs cytoplasmic compartmentalization to the Discussion section. The idea of using nuclear extracts or cytoplasmic extracts is very appealing to us. We have however not yet seen that achieving a clean separation of these fractions is possible for yeast, particularly for studies like ours that depend on minimizing lysate dilution.