Gait disorders can reduce the quality of life for people with neuromuscular impairments. Therefore, walking recovery is one of the main priorities for counteracting sedentary lifestyle, reducing secondary health conditions and restoring legged mobility. At present, wearable powered lower-limb exoskeletons are emerging as a revolutionary technology for robotic gait rehabilitation. This systematic review provides a comprehensive overview on wearable lower-limb exoskeletons for people with neuromuscular impairments, addressing the following three questions: (1) what is the current technological status of wearable lower-limb exoskeletons for gait rehabilitation?, (2) what is the methodology used in the clinical validations of wearable lower-limb exoskeletons?, and (3) what are the benefits and current evidence on clinical efficacy of wearable lower-limb exoskeletons? We analyzed 87 clinical studies focusing on both device technology  and clinical aspects , and make available the database with all the compiled information. The results of the literature survey reveal that wearable exoskeletons have potential for a number of applications including early rehabilitation, promoting physical exercise, and carrying out daily living activities both at home and the community. Likewise, wearable exoskeletons may improve mobility and independence in non-ambulatory people, and may reduce secondary health conditions related to sedentariness, with all the advantages that this entails. However, the use of this technology is still limited by heavy and bulky devices, which require supervision and the use of walking aids. In addition, evidence supporting their benefits is still limited to short-intervention trials with few participants and diversity among their clinical protocols. Wearable lower-limb exoskeletons for gait rehabilitation are still in their early stages of development and randomized control trials are needed to demonstrate their clinical efficacy. Gait disorders affect approximately 60% of patients with neuromuscular disorders  and geneRobotic gait rehabilitation appeared 25 years ago as an alternative to conventional manual gait training. Compared with conventional therapy, robotic gait rehabilitation can deliver highly controlled, repetitive and intensive training in an engaging environment , reduce Wearable exoskeletons are emerging as revolutionary devices for gait rehabilitation due to both the active participation required from the user, which promotes physical activity , and theThere have been several reviews surveying the field of wearable exoskeletons for gait rehabilitation. Some of these reviews have focused on reviewing the technological aspects of exoskeletons from a general perspective , 30, whiThis review provides a comprehensive overview on wearable lower-limb powered exoskeletons for over ground training, without body weight support, that are intended for use with people who have gait disorders due to neuromuscular impairments. In comparison with other reviews, we analyse a wide range of aspects of wearable exoskeletons, from their technology to their clinical evidence, for different types of pathologies. This systematic review was carried out to address the following questions: (1) what is the current technological status of wearable lower-limb exoskeletons for gait rehabilitation?, (2) what are the benefits and risks for exoskeleton users?, and (3) what is the current evidence on clinical efficacy for wearable exoskeletons? AND (robot* OR power* OR active) AND  AND ((leg OR lower) AND (limb OR extremity)) AND  NOT (“body weight support” OR BWS OR treadmill OR upper OR hand OR arm). This literature search resulted in 855 publications, 57 of which were added in a second search for commercially available exoskeletons: 175 in PubMed, 348 in Web of Science, 296 in Scopus, 36 in IEEE Xplore. Additionally,  29 studies from exoskeleton websites were added.We searched for scientific publications in four online databases from 2000 until 18th March 2019 using the following search terms: After removing duplicates, 777 publications were screened first by their title and secondly by their abstract. 127 publications were full-text assessed for eligibility. The identification, screening and eligibility check of the studies were all done by the same author . In case of uncertainty during the screening or the classification process, a decision was reached in agreement with the three authors of the manuscript. Finally, 87 studies were included in this review  studies had to use a wearable and powered lower-limb exoskeleton, (2) report overground outcome measures, and (3) participants had to have a neuromuscular impairment. There were no limitations regarding the participants’ age or gender. Note that we considered as wearable exoskeletons those that present a rigid external structure and therefore, soft exoskeletons or exosuits were not included in the present survey. Studies that used body weight support or a treadmill were excluded with the purpose of focusing only on studies that solely investigated the effect of wearable exoskeleton technology. Note that for the analysis, only data from patients who used the robotic devices were included, i.e., patients in the intervention group.The information of each study was classified according to technical aspects of the exoskeleton and clinical aspects. The technical aspects included: (1) exoskeleton design and structure, (2) control methods, and (3) type of actuators. The clinical aspects included: (4) patient demographics, (5) patient impairments, (6) training protocol, (7) outcome measures, (8) the walking aids used during training, and (9) the training environment.The neuromuscular impairments of the patients were classified into three groups: spinal cord injury (SCI), stroke, and other pathologies. This classification was used to analyse the technical and clinical aspects of the 87 studies. Due to the large number of studies involving SCI patients, we carried out a specific analysis on the level of injury (LOI) building upon the previous analysis carried out by Contreras-Vidal et al. .Ambulation assessments, which includes measures to assess locomotor ability based on time or distance measures; (2) balance and level of assistance/independence, which evaluates the stability and the dependency on walking aids; (3) physiological improvements, which considers effects related to pain, skin, bowel/bladder function and spasticity; (4) energy expenditure, which quantifies the effort and metabolic energy consumption needed when using the device; (5) usability and comfort, which evaluates the ergonomics and the subjective feedback of the user; and (6) biomechanics, which contains the kinematic and kinetic metrics.The classification of primary and secondary outcome measures were grouped using the five categories proposed by Contreras-Vidal et al.  and a siSelected studies were grouped in four categories according to their study design: experimental validation , pilot study, observational study  and experimental study .This review identified 25 exoskeletons  in wearable exoskeletons ranges from one to three per leg in the sagittal plane  and the most frequent number of DOF is two Fig. . Joints Exoskeletons with two active joints were tested by 76.4% of the total number of patients reported in the included studies, and focused mostly on SCI patients Fig. a. In conIn agreement with the trend previously detected by Young and Ferris  and VealWearable exoskeletons are still heavy and bulky devices due to their rigid structures, actuators and batteries. For example, the average weight of hip-knee exoskeletons is 14.28 kg (7.14 kg/leg), which approximately corresponds to more than half the weight of an average adult human leg . Note tExoskeletons for SCI patients have the highest mean weight  have recently arisen to mitigate some of the limitations of conventional, rigid wearable exoskeletons mentioned above. Soft exoskeletons stand out for doing away with rigid frames presented in wearable exoskeletons. Standard soft exoskeletons are characterized for being textile devices actuating on user’s joints through Bowden cable-based transmissions , 70. TheWearable exoskeletons started implementing rigid control methods based on predefined trajectories . NeverthRegardless of the type of control, there are two elements that are crucial for the operation of the exoskeleton: the algorithms for gait phase detection and step initiation . From these RCTs, 5 of them were studies with post-stroke patients , 95–97 aWe found that the total number of sessions shows a large variability (range: 1–120), being the range from 1 to 5 sessions the most common 33%). Concerning the number of sessions per week, 3 sessions was the most common frequency (46%) followed by 5 sessions per week , open doors, or elevator use Fig. . We founAdditional file ambulation assessments category were the most used (44%), followed by biomechanics measures (17%), energy expenditure (14%), balance and level of assistance (13%), physiological improvements (8%), and metrics related to usability and comfort  was the most common outcome measure of balance and level of assistance category, used mainly for stroke patients, although the main outcome measure in stroke studies was the Fugl-Meyer Assessment (FM). Spasticity and pain were the most frequent outcomes in physiological improvement category. Moreover, this category, together with energy expenditure and usability and comfort categories, was mainly focused on people with SCI. In contrast, outcome measures related to biomechanics were widely studied independently of the pathology, with knee and hip angles being the most interesting biomechanical outcome measures assessed.We found that outcome measures belonging to the 3%; Fig. d. We fouAs previously mentioned, outcome measures varied across studies and were mainly focused on aspects related to functional mobility, instead of focusing on analyzing physiological and psychological effects. Only a few studies assessed the improvement related to SHCs. For example, Baunsgaard et al.  and JuszThis section analyses the benefits and risks of using wearable exoskeletons and summarizes the most relevant clinical evidence of this technology. In this section we show the most remarkable information and detailed information can be found in Additional files Additional file In contrast to studies with patients with SCI, studies with post-stroke patients assessed the gait performance, without wearing exoskeleton, after training with the exoskeleton and compared the results with the baseline measurements. In general, we found that the degree of mobility improvement was not as substantial as with the studies focusing on SCI patients: 12 out of 16 studies that analyzed gait speed reported an improvement , 112–116To asses the clinical evidence of wearable exoskeletons as a therapy for walking rehabilitation in people with neuromuscular impairments, we analyzed the results obtained in the aforementioned studies that conducted a RCT see "" sectionAs we have just seen in the previous section, studies focused on people with SCI showed promising results. However, these studies were mainly observational and pilot studies, which implies a questionable evidence. This finding has already been detected in previous works , 120. InRegarding studies with post-stroke patients, we identified 5 RCTs that involved a total of 183 patients. Buesing et al.  and JayaLastly, we identified one randomized cross over trial in which the authors evaluated the effects of the Keeogo exoskeleton on the physical performance of people with multiple sclerosis, both in a clinical setting and in a home setting . Note thFrom the 87 studies screened in this review, only 36 provided information on adverse effects derived from the use of wearable exoskeletons. We found only one study  reportinDespite the fact that, in general, studies show that wearable exoskeletons are safe devices, these results may not be fully representative. According to He et al. , studiesIn this review, we did not use delimiters related to study design nor assessed the study quality. The lack of delimiters could produce some bias, especially for the conclusions related to clinical effectiveness of wearable lower-limb exoskeletons. However, we tried to mitigate this bias by focusing only on experimental studies  when discussing the clinical evidence of wearable exoskeletons. Nonetheless, the main aim of this review was to provide a comprehensive overview of wearable lower-limb exoskeletons for clinical applications, so we considered that displaying all the literature without limiting by study design would provide a broader view of the topic.In this paper we reviewed the design and clinical evaluation of wearable lower-limb exoskeletons intended to support walking in people with neuromuscular impairments. Since its nascence around 20 years ago, the field of wearable exoskeletons has shown significant progress at supporting the walking function for individuals with neuromuscular impairments. However, it is still challenged by its small evidence base, slow acceptability, complex technical problems and inordinate costs for purchasing. We conclude this review paper by summarizing the main conclusions for each of the proposed research questions.Wearable exoskeletons are still heavy and bulky devices that in general require supervision  and the use of walking aids, which hinders mobility and independence. All the reviewed exoskeletons use deterministic gait phase detection algorithms following button press or a threshold-based approach. For the latter, foot-ground contact force measurement through insole sensors is the most common metric used. The most frequent type of measurement in wearable exoskeletons is joint angle, since the vast majority of actuators are used together with encoders or potentiometers to provide position feedback. Regarding actuation, the most frequent actuators are electric motors, probably due to the fact that they are easy to control and exhibit great precision with high specific power . ControlWearable exoskeletons need to progress towards modular systems capable of adapting to the user’s motor capabilities and limitations. In the same way, control methods should be based on Assist-As-Needed algorithms to conveniently adapt actuation to the user needs according to the rehabilitation process. Moreover, neuronal technology may have an important role for the next generation of wearable exoskeletons. Brain machine interfaces (BMI) allow direct and voluntary control of the devices irrespective of the user capabilities  which coClinical validation studies of wearable exoskeletons are currently in their early stages, thus evidence is still limited to short intervention trials with few participants, as it was concluded in a previous study by Mekki et al. . Study dProtocol design and outcome measures vary across studies, which hinders their comparison. Outcome measures, despite presenting encouraging results, are mainly focused on ambulation assessments  rather than being centered on physiological and psychological changes to improve or avoid SHCs. Since prevention of SHCs is a primary aim, especially in SCI , studiesRobotic therapy is progressing toward wearable exoskeletons since they offer the advantages of grounded exoskeletons, as well as providing more active participation of the user. Wearable exoskeletons offer the opportunity to socialize more easily with the environment, increasing quality of life and decreasing depression rate , 135. LiDespite the previous benefits, the optimal type of rehabilitation robot for a specific patient’s needs still remains unclear –140. LitIn any case, overground wearable exoskeletons stand out for providing more movement freedom during gait, the opportunity of independent training at home, and the possibility to carry out more activities of daily living such as sitting, turning and climbing stairs. These advantages activate mechanisms of neural plasticity and connectivity re-modulation , 141; whIn conclusion, efforts should be invested in developing lightweight and easy-to-use exoskeletons, which should be validated through well-defined protocols to provide the best patient-specific rehabilitation training and offer the possibility of benchmarking.Size and weight of wearable exoskeletons should be reduced, and structures should be simplified to allow independent donning/doffing and transportability, while increasing user acceptance.Balance capabilities of wearable exoskeletons should be improved to reduce the use of supportive devices.Control methods should focus on Assist-As-Needed control algorithms to conveniently adapt assistance to the user needs, increase active participation and promote neural plasticity.Studies need standard clinical guidelines that define protocols for clinical validation, and regulations to have a better understanding of the adverse events and risks of using wearable exoskeletons.Research studies should focus more on outcome measures related to SHCs, since prevention of secondary health problems is a primary aim in rehabilitation.Randomized control trials are needed to demonstrate clinical efficacy of wearable exoskeletons when comparing with conventional gait therapy and/or other types of robotic gait therapy, since most of the literature is based on observational and pilot studies.Additional file 1. Clinical trial identification assessment.Additional file 2. Wearable lower-limb exoskeletons.Additional file 3. Clinical evidence of wearable lower-limb exoskeletons.Additional file 4. Outcomes measures.


It is therefore important that we understand the biophysical, molecular composition, activation pathways, and physiological significance of SOCs in VSMCs as these maybe future therapeutic targets for conditions such as hypertension and atherosclerosis. Archetypal SOCs called calcium release-activated channels (CRACs) are composed of Orai1 proteins and are stimulated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) following store depletion. In contrast, this review focuses on proposals that canonical transient receptor potential (TRPC) channels composed of a heteromeric TRPC1/C5 molecular template, with TRPC1 conferring activation by store depletion, mediate SOCs in native contractile VSMCs. In particular, it summarizes our recent findings which describe a novel activation pathway of these TRPC1-based SOCs, in which protein kinase C (PKC)-dependent TRPC1 phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) are obligatory for channel opening. This PKC- and PIP2-mediated gating mechanism is regulated by the PIP2-binding protein myristoylated alanine-rich C kinase (MARCKS) and is coupled to store depletion by TRPC1-STIM1 interactions which induce Gq/PLCβ1 activity. Interestingly, the biophysical properties and activation mechanisms of TRPC1-based SOCs in native contractile VSMCs are unlikely to involve Orai1.In vascular smooth muscle cells (VMSCs), the stimulation of store-operated channels (SOCs) mediate Ca This rise in Ca2+i is important for initiating vasoconstriction, which determines vascular resistance and blood pressure and ultimately regulates blood flow to our tissues and organs. In addition, an excessive increase in Ca2+i is linked to cell proliferation, migration and growth, phenotypes which are involved in the development of vascular disease. Therefore, understanding cellular pathways which mediate increases in Ca2+i may help to identify future therapeutic targets for conditions such as hypertension and atherosclerosis.In vascular smooth muscle cells (VSMCs), neurotransmitters and hormones such as noradrenaline, adrenaline, angiotensin II (Ang II), and endothelin 1 (ET-1) produce an increase in cytosolic Ca+i)  activity, phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, and inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) generation [3 mediates an increase in Ca2+i by causing the release of Ca2+ from sarcoplasmic reticulum (SR) Ca2+ stores, and this subsequent rise in Ca2+i and DAG-mediated pathways induce Ca2+ influx from the extracellular medium. A component of this Ca2+ influx occurs through activation of voltage-gated Ca2+ channels (VGCCs), but there is also a significant contribution from voltage-independent Ca2+-permeable non-selective cation channels. Stimulation of Ca2+-permeable cation channels are thought to mediate Ca2+ entry pathways through direct Ca2+ influx, and through Na+ influx which leads to depolarization and activation of voltage-gated Ca2+ channels and stimulation of the Na+/Ca2+ exchanger in reverse mode.Vasoconstrictors induce a rise in Casphate IP and diacneration ,7. IP3 m2+-permeable non-selective cation channels classified as receptor-operated (ROCs) and store-operated (SOCs) are expressed in VSMCs [3-mediated depletion of SR Ca2+ stores, whereas SOCs are activated by pathways coupled to depletion of SR Ca2+ stores but not by the subsequent increase in Ca2+i. These definitions infer that stimulation of Gq-protein-coupled receptors will activate both ROCs and SOCs. In addition, it is proposed that SOCs may be activated by Gq-protein-coupled receptor stimulation independently of store depletion, hence SOCs may also function as ROCs [2+ signal recordings but possible using single channel recordings [2+ stores in the absence of receptor stimulation using SR Ca2+-ATPase inhibitors  and thapsigargin), and high ethane-N,N,N’,N’-tetraacetic acid (BAPTA)) and low affinity -1,2-ethnediamine (TPEN)) Ca2+ chelators to passively deplete Ca2+i and SR Ca2+ levels respectively.There is substantial evidence that Cain VSMCs ,2. ROCs  as ROCs . Togethecordings ,10,11. M2+ ATPase inhibitors or Ca2+ chelators to prevent complications from activation of ROCs, although in the final section we discuss the potential physiological significance of this store-operated activation pathway in vasoconstrictor-induced TRPC1-based SOCs.This article reviews our current understanding of the biophysical properties, molecular composition, and activation mechanisms of SOCs in VSMCs, in particular, it focuses on recent studies from our laboratory which have described a novel activation pathway of SOCs composed of canonical transient receptor potential 1 (TRPC1) proteins in native contractile VSMCs. In the majority of these studies SOCs were activated by SR Ca2+ permeabilities (PCa2+:PNa+ > 1000:1), pronounced inward rectification with reversal potentials (Erev) greater than +50 mV, unitary conductances in the fS range, and are composed of Orai1 proteins , are characterized by high Cains  [2 which do not require Orai1.However, it is apparent that many cell types express SOCs with very different characteristics to Orai1-based CRACs such as much lower Caces (see  for reviRPC1–C7) . These TRPC1–C7) . There iRPC1–C7) ,14. For RPC1–C7) ,16,17,18RPC1–C7)  for revi2+ permeabilities (PCa2+:PNa+ of 1–50), and unitary conductances of 2–3 pS and 2–8 pS in 1.5 mM and 0 mM external Ca2+ respectively [−/− mice, molecular knockdown , and pharmacology  [2+ permeability and unitary conductance. For example, inclusion of TRPC6 within the TRPC1/C5 template produces biophysical properties which indicate a decreased Ca2+ permeability [3 on TRPC1-based SOCs [−/− VSMCs indicate that TRPC1 is essential for conferring activation by store depletion, and therefore these TRPC1/C5 channel templates are termed TRPC1-based SOCs [−/− mice [The molecular composition and activation mechanisms of SOCs in VSMCs reflects the controversies highlighted above as there is significant evidence to indicate that both Orai1-based CRACs and TRPC-based SOCs are present in VSMCs, but that these channels are differentially expressed according to cell phenotype. In freshly isolated VSMCs or primary cultured VSMCs maintained in low serum, conditions in which VSMCs retain their native contractile phenotype, evidence suggests that SOCs have biophysical properties which resemble TRPC-based SOCs and not Orai1-based CRACs with relatively linear, S-shaped, or slightly outward rectification, low Caectively ,27,28,29Pico145) ,31 indicPico145) ,33. As seability , whereassed SOCs ,34. Studsed SOCs . These fsed SOCs . Severalsed SOCs ,36 and ssed SOCs ,38 for r2+ entry with similar properties to Orai1-based CRACs such as pronounced inward rectification, inhibition by a Orai1 selective inhibitor, and inhibition by knockdown of Orai1 and STIM1 protein levels but not by knockdown of TRPC1, TRPC4 or TRPC6 proteins [2+ entry are unaffected in cultured VSMCs maintained in high serum conditions from TRPC1−/− mice [In contrast to native contractile VSMCs, long-term cultured VSMCs maintained in high serum, conditions that induce a non-contractile synthetic phenotype with proliferative, migrative and growth characteristics, express whole-cell SOCs and store-operated Caproteins ,40,41,42−/− mice  further −/− mice ,38.These complexities mean that when studying SOCs in VSMCs it is essential to clearly define cell phenotype of VSMCs. In this regard, when investigating SOCs in native contractile VSMCs it is advantageous to use freshly isolated cells whenever possible, and when required only use primary culture cells maintained in low serum conditions for as short a time as possible.2) regulated by myristoylated alanine-rich C kinase (MARCKS) are obligatory gating partners, with these mechanisms coupled to store depletion by a novel STIM1-mediated Gq-PLCβ1 pathway.The following sections discuss our recent findings that TRPC1-based SOCs in freshly isolated and primary cultured VSMCs with a native contractile phenotype exhibit a complex activation mechanism, in which interactions between PKC-dependent phosphorylation of TRPC1 and phosphatidylinositol 4,5-bisphosphate (PIP2 are important in activating TRPC1-based SOCs. The stimulation of whole-cell and single channel TRPC1-based SOCs is significantly reduced by PKC inhibitors and anti-PIP2 antibodies [2 (a water-soluble form of PIP2) stimulate TRPC1-based SOCs in WT but not in TRPC1−/− VSMCs [2 antibodies and pharmacological agents such as wortmannin to deplete PIP2 levels, whereas diC8-PIP2-activated TRPC1-based SOCs are reduced by PKC inhibitors [2 and TRPC1 [2 have obligatory roles in activation of TRPC1-based SOCs; PKC cannot activate TRPC1-based SOCs without PIP2, and vice versa. This supports the hypothesis that PIP2 is the activating ligand of TRPC1-based SOCs and that store-operated PKC-dependent phosphorylation of TRPC1 is required for this opening mechanism to occur.There are several lines of evidence which indicate that PKC activity and PIPtibodies ,25,44. I/− VSMCs ,25,44. Ind TRPC1 ,27,28,442 on TRPC1-based SOCs are different to their roles in the activation of TRPC3/C6/C7-based ROCs in native contractile VSMCs  require both Ca2+ and diacylglycerol (DAG), novel PKC isoforms  require DAG but are Ca2+-insensitive, and atypical PKC isoforms  are activated by lipid mediators such as phosphatidylserine and do not require Ca2+ or DAG (see [2+ chelators BAPTA and BAPTA-AM), or produce little change in Ca2+i . These findings suggest that the PKC isoform involved is likely to require DAG but is Ca2+-insensitive, which are characteristics of the novel group of PKC isoforms. Our preliminary data indicate that PKCδ is the most highly expressed novel PKC isoform in VSMCs, and that selective PKCδ inhibitory peptides and knockdown of PKCδ using morpholino sequences prevent activation of TRPC1-based SOCs [Important omissions from our understanding are what PKC isoform(s) is involved, which amino acid(s) within TRPC1 protein structure is phosphorylated, and how a PKC-dependent phosphorylation process alters interactions between TRPC1 and PIPDAG , protein kinase G (PKG), and calmodulin kinase II (CaMKII) have inhibitory actions on TRPC1-based SOCs in VSMCs [2 interactions by lowering PIP2 affinity. Similar roles for kinase activities in modulating lipid-protein interactions are well-established in the regulation of different K+ channel subtypes [Interestingly, the predication of PKCδ-dependent phosphorylation sites within the TRPC1 sequence using GPS 3.0 reveals five intracellular serine residues, with Ser619 and Ser752 at the C-terminal domain of potential significance as both these sites are close to a known PIPg domain . It is tin VSMCs ,50,51, a2 gating of TRPC1-based SOCs when physiological activators of SOCs involve stimulation of Gq-coupled receptors which drive PLC activity and PIP2 hydrolysis. To provide answers to this question, we investigated the role of MARCKS in activation of TRPC1-based SOCs. MARCKS is a membrane-bound PIP2-binding protein that acts a PIP2 buffer, which can provide a distinct pool of PIP2 at the plasma membrane that is protected from breakdown by PLC thus enabling this phospholipid to be released in a coordinated manner into the local environment [2-binding dot-blot assays, we showed that in unstimulated conditions MARCKS is bound to TRPC1, with PIP2 predominately associated with MARCKS and not TRPC1 [2 being released and associated with TRPC1 to cause channel opening. Importantly, both dissociation of MARCKS from TRPC1 and redistribution of PIP2 from MARCKS to TRPC1 are stimulated by phorbol esters and prevented by PKC inhibitors indicating that PKC activity is central to these mechanisms. It will be important to ascertain how receptor stimulation and store-depleting agents couple to these actions of MARCKS. A potential idea is that stimulation of CaM is involved, as this Ca2+-binding protein is known to bind to MARCKS and to cause dissociation from the plasma membrane and PIP2 release [An important question is how PKC-dependent phosphorylation regulates PIPironment ,54. Usinot TRPC1 . Upon st release ,55. Furt release .2? A crucial finding was the discovery that store depletion activates a Gq-PLCβ1 pathway which drives the PKC-dependent phosphorylation of TRPC1 [2 and IP3 . In the future, it will be important to investigate structural interactions between STIM1 and TRPC1 and discover where Gq subunits binds. Initial work might focus on the CRAC-activating (CAD) and polybasic domains which have been linked to binding and activation of TRPC1 by protein-protein interactions and electrostatic interactions respectively ,16,17,18Interestingly, differential blocking effects of N-terminal and C-terminal anti-STIM1 antibodies on TRPC1-based SOCs using whole-cell and inside-out patch clamp recordings may suggest that store-operated STIM1/TRPC1 interactions lead to STIM1 acting as a transmembrane protein at the plasma membrane . In this2+ entry pathways in VSMCs with a synthetic phenotype, although it is unclear whether Orai1 and TRPC1 interact together or form separate STIM1-mediated ionic mechanisms (see earlier). In addition, it is unclear whether Orai1 is required for activation of TRPC1-based SOCs in native contractile VSMCs. In our recent study, we showed that the properties of store-operated whole-cell and single channel currents in freshly isolated and primary cultured native contractile WT and Orai1−/− VSMCs were similar to previously described TRPC1-based SOCs, and that store-operated STIM1-mediated PLCβ1 activity and STIM1-TRPC1 interactions were unaffected in Orai1−/− cells [−/− or TRPC1−/− preparations [There is substantial evidence that TRPC1, Orai1 and STIM1 have important roles in mediating store-operated Ca/− cells . These f/− cells ,36,37,38/− cells , and thearations ,29.2+-independent phospholipase A2 (iPLA2) in store-operated conductances in native and primary cultured contractile VSMCs [2-mediated mechanism involving production of lysophospholipids (see [2+ channels ([−/− mice [2 has been inferred in many studies using the proposed inhibitor bromoenol lactone (BEL), which has also been shown to block heteromeric TRPC1/C5, TRPC5, and TRPC6 channels and VGCCs [It should be noted that other studies using pharmacological and molecular techniques have implicated Orai1 and Cale VSMCs . These sids  instead of receptor stimulation to study SOCs (see Introduction). However, it is important to examine whether proposed store-operated mechanisms are involved in activation of SOCs by physiological receptor stimulation. This is particularly important for TRPC1-based SOCs in native contractile VSMCs as these channels also behave as ROCs, and so may be activated by distinct store-independent and -dependent pathways [B receptor stimulation induce TRPC1-based SOCs via a PLC-mediated pathway that requires PKC activity and PIP2 for channel opening in portal vein, mesenteric artery and coronary artery VSMCs respectively [2 interactions similar to those produced by store depleting agents [3-mediated depletion of SR Ca2+ stores and activation of TRPC1-based SOCs by store depletion.To avoid complications from receptor-operated pathways, most studies use agents that selectively deplete SR Caectively ,11,23,72ectively ,28. Moreg agents . These fA receptor stimulation induces TRPC1-based SOCs through a Gβγ-protein-phosphoinositol 3-kinase (PI3K)-mediated pathway, with PIP3 formation thought to both induce PKC activity and act as the channel activating ligand; a pathway which is unlikely to involve IP3-mediated depletion of SR Ca2+ stores [A and ETB receptor stimulation through separate store-independent and -dependent pathways respectively, both requiring obligatory roles of PKC activity and phospholipids [It is likely that certain receptors only activate TRPC1-based SOCs via store-independent pathways. In native contractile coronary artery VSMCs, ET-1-activated ET+ stores ,72. Therholipids ,66.3-mediated depletion of SR Ca2+ stores are likely to activate two different Gq-PLC pathways mediated by distinct PLC isoforms. To test this hypothesis, it would be important to identify the PLC isoform linked to α1-adrenoceptor stimulation. The critical role of MARCKS in stimulation of TRPC1-based SOCs is further highlighted when considering the physiological significance of our proposed activation pathway. Stimulation of Gq-protein-coupled receptors and IP3-mediated depletion of SR Ca2+ stores will both induce Gq-PLC activities leading to PIP2 hydrolysis. Therefore, MARCKS provides the essential role of a buffering a separate pool of PIP2, which is protected from PLC-mediated hydrolysis enabling the phospholipid to be available for channel opening.Interestingly, the knockdown of PLCβ1 and STIM1 had little effect on noradrenaline-stimulated PLC activity measured using the GFP-PLCδ-PH biosensor, although it was inhibited by U73122 a general PLC isoform inhibitor ,28. Thes2+ store depletion by receptor-induced IP3-mediated generation or store depleting agents such as CPA and BAPTA stimulate STIM1 to translocate to the plasma membrane where it interacts with TRPC1 subunits to form a receptor for Gq G-protein subunits which induces PLCβ1 activity, production of DAG, and PKC activity. PKC-dependent phosphorylation of TRPC1 increases PIP2 binding and channel opening. The interactions between TRPC1 and PIP2 are regulated by MARCKS, a plasma membrane PIP2-binding protein bound to TRPC1 at rest, which upon store depletion dissociates from TRPC1 and releases PIP2 into the local environment where the phospholipid acts as the gating ligand. This review highlights recent findings that SOCs in native contractile VSMCs are mediated by TRPC1-based SOCs, which have very different biophysical properties to the well-characterized channels formed by Orai proteins, including archetypal Orai1-based CRACs. Moreover,


New drugs are needed for treating acute myeloid leukemia (AML). We analyzed data from genome-edited leukemia cells to identify druggable targets. These targets were necessary for AML cell survival and had favorable binding sites for drug development. Two lists of genes are provided for target validation, drug discovery, and drug development. The deKO list contains gene-targets with existing compounds in development. The disKO list contains gene-targets without existing compounds yet and represent novel targets for drug discovery.Refractory disease is a major challenge in treating patients with acute myeloid leukemia (AML). Whereas the armamentarium has expanded in the past few years for treating AML, long-term survival outcomes have yet to be proven. To further expand the arsenal for treating AML, we searched for druggable gene targets in AML by analyzing screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library and gene knockout (KO) dependency scores in 15 AML cell lines . Ninety-four gene KOs met the criteria of (A) specifically essential to AML cell survival, (B) non-essential in non-AML cells, and (C) druggable according to three-dimensional (3D) modeling or ligand-based druggability scoring. Forty-four of 94 gene-KOs (47%) had an already-approved drug match and comprised a drug development list termed “deKO.” Fifty of 94 gene-KOs (53%) had no drug in development and comprised a drug discovery list termed “disKO.” STRING analysis and gene ontology categorization of the disKO targets preferentially cluster in the metabolic processes of UMP biosynthesis, IMP biosynthesis, dihydrofolate metabolism, pyrimidine nucleobase biosynthesis, vitellogenesis, and regulation of T cell differentiation and hematopoiesis. Results from this study serve as a testable compendium of AML drug targets that, after validation, may be translated into new therapeutics. Acute myeloid leukemia (AML) is challenging to treat due to its refractory nature. Despite achieving initial morphologic remissions in 50–70% of patients, the 10-year disease free survival rate is 16.6% in younger AML patients (<60 years old) and 2.4% in older AML patients (≥60 years old) [Although AML can be subdivided into subgroups defined by cytogenetic abnormalities and/or genetic mutations, we hypothesized that AML in aggregate depend on a common core of functions. For example, in the clinic we already use pyrimidine analogs as backbone drug agents for the treatment of all AML patients, based on all AML’s essential need for DNA synthesis. We speculated that there may be additional common functions in AML that have yet to be fully investigated. However, searching for leukemia-essential functions has been challenging. In contrast, the scientific strategy of biological reductionism—for example, focusing on FLT3 mutant AML—is generally easier to understand and requires fewer resources than investigating for common essential functions. Major challenges in finding common AML functions is lack of a variety of specimens and culling leukemia-essential from cell-essential. Long-term, inhibiting leukemia-essential functions may be applicable to a greater number of AML patients compared to targeted therapies.Loss-of-function screens in well annotated cell lines represent a feasible experimental approach to interrogate gene function and cancer cell dependency . The BroWe hypothesized that a safe and effective drug target in AML is necessary for AML cell survival , non-essential to non-AML cells (specific), and can be inhibited by drugs (druggable) A. Thus, The DepMap Avana 19Q2 CRISPR screening library contained CRISPR-Cas9 dropout data for 17,634 genes in 15 AML cell lines  within a library of 1714 cell lines , as the n = 1585, 8.988%) were assigned null scores and excluded from screening. 3D measurements included number of successful binding models and percentage of models successful at binding for both “druggability” and “tractability”, qualities defined by CanSar but are herein discussed synonymously as druggability. Ligand-based measurements included standardized scores and percentiles, which are directly related. As a result, they were not compared against each other and only the standardized ligand-based score was used.CanSAR Black was queried on 20 June 2020 to obtain druggability measures for each gene product identified above, drawing from experimentally derived ligand-based drug interactions and X-ray crystallography derived computational 3D protein-drug binding in the Protein Data Bank (PDB) . UnmatchUtilizing the Drug Gene Interaction Database (DGIdb) accessed on 20 June 2020, we constructed a list of all known and predicted drug-gene interactions for the genes in Avana 19Q2 . This liCutoff values for criteria measurements of essentiality, specificity, and druggability, discussed in results, were set at an estimated specificity of 0.90 based on receiver–operator curve (ROC) analysis of the three drug categories in IBM SPSS. The genes meeting both criteria  but not currently targeted by a US FDA-approved or experimental drug therapy formed a drug discovery knock-out list termed “AML disKO.” Genes targeted by existing drugs formed a drug development knockout list termed “AML deKO”. An analysis of functional interactions was conducted using StringDB , on the A stepwise method was used to identify essential, specific, and druggable genes for the treatment of AML B. First,p < 0.001) for these existing leukemia chemotherapeutic drug targets (AML Targets) as compared to a set of all other genes from the Avana 19Q2 dataset (Non-AML Targets) (p < 0.001) for common essential genes  A,B, withTargets) . These ral genes C,D, withal genes . TogetheTo identify AML-specific drug targets with reduced systemic effects, we developed a novel metric, the AML Z-score. The AML Z-score for each gene represents the standardized difference between mean dependency in 15 AML cell lines and mean dependency of all cell lines in the Avana 19Q2 data set. AML-specific Z-score distributions were approximately normal and positive-skewed C. The pot-tests comparing AML-specific Z-scores were significant (p < 0.01) only for genes targeted by common essential genes and AML chemotherapy but not for FDA drug targets or other chemotherapy targets  compared to other gene products  were not targeted by an existing or experimental drug and were annotated in a list called AML discovery gene knockout set (AML disKO)  which reWe note that while all gene products in disKO and deKO had a high Z-score indicating greater mean dependency in AML cells than other cells, 56/94 (60%) are previously identified common essential genes (red) while 38/94 (40%) are not common essential genes (blue). Targeting of the disKO common essential genes , red maySTRING analysis identified clustering around distinct protein interaction clusters . FurtherPathway analysis conducted through STRING highlighted proteins among our top AML essential genes, with enrichment in MAPK signaling A, JAK-STMLL translocations, which likely skewed their results toward MLL-driven AML [AURKB, MAP2K1, MAP2K2, IGF1R, and HDAC3. Although none of these targets passed our screening criteria, related genes such as AURKA, HDAC7, RUNX1, and KMT2A were identified as targets with existing inhibitors  contained iven AML . The prihibitors . We noteated AML ,15. In tThe genes identified in our deKO list  are targOther deKO genes with inhibitors designed for other diseases may also represent drugs for repurposing. We found LDLR as a highly essential gene in AML cell lines, indicating that cholesterol metabolism is essential in AML. Pravastatin in phase 2 Southwest Oncology Group (SWOG) clinical trials has shown improved morphologic remission in combination with Idarubicin and Cytarabine .Additionally, we identified novel targets for AML drug discovery in our disKO list . These tOur results highlight the importance of AML metabolism and opportunities for therapeutic intervention. Uridine and inosine biosynthesis are known essentials for AML cell proliferation and differentiation ,27. ReceWhen interpreting our results, several considerations need to be made. First, the disKO list was constructed from a genome-wide data analysis. Before embarking on any drug development work, target validation testing is needed. Second, Avana 19Q2 included only 15 cell lines, which may not fully represent the heterogeneity of AML. Follow-up tests using primary AML cells would validate the AML cell-line results in this study and add clinical relevance. Third, in vivo experimental data is also needed before clinical testing.In conclusion, we used an agnostic, genome-wide approach to identify potential new therapeutic targets for AML and provide hit lists for target validation, drug discovery, and drug development.Data analysis of an agnostic, genome-wide CRISPR screen led to the identification of 94 druggable genes specific and essential for AML survival. These genes represent potential drug targets after validation testing.


NTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CA The structural polyprotein Gag is conserved among all retroviruses and mediates virus assembly via oligomerization into incomplete lattices that are stabilized by dimeric, trimeric and hexameric contacts. Despite a high degree of conservation at the secondary and tertiary structure level, the quaternary interactions between the CA domains of retroviral Gag vary. Recently, the small cellular molecule IP6 was identified as an assembly co-factor of the lentivirus HIV-1. To better understand the structural variability of retroviruses and to determine if IP6 is an assembly cofactor of other lentiviruses we determined the structure of the HIV-1 related retrovirus EIAV. Using cryo-electron tomography and subtomogram averaging, in vitro assembly, mutation analysis, and molecular dynamics simulations, we determined and characterized the structure of the EIAV immature lattice. Furthermore, we found that IP6 is an assembly cofactor of EIAV, and other lentiviruses. NTD and CACTD), rearrange to create a new lattice of hexamers, but with an entirely new set of interactions We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.Sincerely,Félix A. ReyAssociate EditorPLOS PathogensMichael MalimSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: In this study, the authors examine the role of IP6 in the assembly of equine infectious anemia virus (EIAV) and several other lentiviruses. By using a variety of approaches, they observe that IP6 markedly stimulates the assembly of EIAV and the other lentiviruses. EIAV Gag is observed to assemble in vitro into several morphologically distinct tubes and sphere; interestingly, the Gag lattice in all these structures is in the immature conformation. Both EIAV and HIV-1 maintain similar immature inferfaces, and both lentiviral Gag proteins bind IP6 via conserved Lys residues in the CA C-terminal domain and adjacent spacer peptide.In general, the study provides some interesting insights into lentiviral assembly and the role of IP6 in assembly. Some of the results might have been predicted based on earlier work with HIV-1, whereas other findings were unexpected. One major issue, related to how particle production is measured in cell-based assays, needs to be addressed. In their previous work, Dick and colleagues (Nature 2018) measured virus particle production by quantifying the amount of infectious virus released into the supernatant. This approach has a serious limitation: it does not distinguish between effects on particle assembly/release and effects on particle maturation/infectivity. Thus it is not clear from the previous work whether KO of IPPK, a key enzyme in the IP6 biosynthetic pathway, impairs particle assembly/release, maturation/infectivity, or both. This information is vital to understanding the role of IP6 in lentivirus replication. In the current study, the authors use the same approach to evaluate the effect of IPPK KO on virus particle production.Reviewer #2: In this manuscript, Dick et al. characterise the immature Gag lattice of the lentivirus EIAV. They first recombinantly express different Gag constructs and establish the conditions for their assembly. They do this in the presence or absence of IP6, and find that, while IP6 promotes EIAV Gag assembly, the dependence of EIAV on IP6 is lower than that for HIV. They then structurally characterise some of the assembled lattices by cryo-electron tomography and subtomogram averaging, which allows the authors to produce atomic models for different rearrangements of the capsid domains. They use these atomic models for: (1) studying the key interactions to maintain the lattice; (2) comparing the capsid proteins of EIAV and HIV; and (3) producing mutants to confirm some of their findings. Finally, the authors show that other lentiviruses also require IP6.Overall, this is a very complete piece of work, with a state-of-the-art subtomogram averaging results, complemented by mutagenesis experiments that confirm the authors conclusions.Reviewer #3: This paper studied the role of IP6 in the immature particle assembly of EIAV through in vitro and in viro assembly assays, cryo-ET reconstruction and molecular modeling methods. The major results and conclusions of the papers are: (1) IP6 promotes in vitro assembly of EIAV Gag proteins into immature particle structures via conserved lysine residues within the CA-CTD and SP. (2) This function of IP6 is conserved for all lentivirus Gag proteins, although at varied magnitudes. (3) EIAV Gag lattice was resolved from in vitro assembled tubular and spherical structures, at sub-4Å resolution using cryo-ET and subtomogram averaging techniques. (4) When comparing that with HIV-1, the molecular contacts involved with CA-CTD organization are conserved, however the CA-NTD arrangement are not conserved.Comments, concerns or questions:1) Although stimulated by IP6, in vitro assembly of EIAV Gag can happen without presence of IP6 (line 331-335). The authors think this is because that in EIAV most of the interactions of the six-helix bundle are hydrophobic in nature, which strengthen the six-helix bundle structure. In contrast to HIV-1, “MD simulations the EIAV 6HB was stable both with and without IP6 likely due to additional side chain contacts between helices in the six-helix bundle” (line 404-405). Can you perform a sequence alignment of this region of all lentiviruses studied in the paper and address if this property is unique for EIAV?2) Line 264-269: EIAV CA was found to have a shorter helix 1, and therefore in EIAV Gag lattice, the putative dimer interface between neighboring Gag hexamers (found in HIV-1 lattice) is missing. According to HIV-1 mature CA lattice, helix 1 is important in intra-hexamer interactions. Could this be one of the reasons that in the in vitro assembly systems, EIAV Gag always form immature lattice, not mature lattice? Is this property unique for EIAV?3) The authors found that the EIAV CA-NTD organization is not the same as that in HIV-1. Several molecular contacts in HIV-1 Gag lattice are missing in EIAV which include: (a) there is no intra-hexamer interactions among CA-NTD (line 238-239) (b) there is no dimeric inter-hexamer interaction formed by helix 1 (c) there is no strong contact between CA-NTD and CA-CTD (line 263-264). The studied EIAV assemblies are rather small: narrow tube 35nm, wide tube 70nm and spherical particles are 100nm. In general, retroviruses have a larger dimension. Is it possible that these missing contacts actually present in the authentic particles?4) Line 109-110: The authors suggests that “the retroviral CA-NTD and CA-CTD are independent structural entities that act autonomously in regulating immature virus particle diameter and curvature”. This idea was repeated at line 363-364: “The spatial separation of EIAV CA-NTD and CA-CTD, and the lack of strong interactions between them, allow these two halves of CA to behave almost as independent entities, as suggested previously for other retroviruses”. This statement seems overly strong. In HIV-1, helix 11 and helix 8 interact with MHR and helix 7. This interaction would limit the orientation of NTD relative to CTD and the role of NTD in deciding the curvature of the Gag lattice.5) Line 292-295: Line 292 lists the SP sequence as (LAKAL) in which the residue 359 is L. However in line 295, the last residue is listed as T395. Please clarify.6) Supplement figure 4, the electron density looks low resolution. A better figure is desired.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1. Supp Fig. 3. The authors measure the effect of IPPK KO on EIAV and HIV-1 particle production by measuring the infectivity of virus produced from KO cells. As mentioned above, this approach does not differentiate between effects on particle production and effects on infectivity. The authors need to perform virus assembly assays in which levels of HIV-1 and EIAV Gag in the cell and virus fractions are quantified so that virus release efficiency can be determined, a infectivity values need to be normalized based on viral input (measured by RT or capsid protein).2. P. 16. The authors speculate about the role of IP6, or potentially other positively charged molecules, in EIAV assembly in vivo. They suggest that, in the cells used, EIAV infectivity is significantly lower than that of HIV-1. Again, this issue needs to be addressed by measuring the efficiency of virus particle production and determining the relative infectivity  of the EIAV stocks. These crucial experiments will resolve this issue.3. Line 425. The authors discuss how future work will evaluate the effect of IP6 depletion on assembly, budding and infectivity. This work will need to be performed by measuring virus release efficiency and specific particle infectivity as described above.4. Supp Fig. 7. Have the authors measured the efficiency of virus particle production of the Q350A mutant in IPPK KO cells? This result would be interesting.Reviewer #2: My only major concern is related to the assignment of the density for IP6. In order to be certain that this density corresponds indeed to IP6, the authors should obtain an immature lattice average without it (i.e. a negative control), for example using the EIAV GAGdeltaMA preparation with 0µM IP. This is specially required as IP6 sits at the symmetry axis, and non-relevant densities can appear there due to symmetrisation.Reviewer #3: Comments, concerns or questions:1) Although stimulated by IP6, in vitro assembly of EIAV Gag can happen without presence of IP6 (line 331-335). The authors think this is because that in EIAV most of the interactions of the six-helix bundle are hydrophobic in nature, which strengthen the six-helix bundle structure. In contrast to HIV-1, “MD simulations the EIAV 6HB was stable both with and without IP6 likely due to additional side chain contacts between helices in the six-helix bundle” (line 404-405). Can you perform a sequence alignment of this region of all lentiviruses studied in the paper and address if this property is unique for EIAV?**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Minor: Fig. 6A. The sequence of SP1 is incorrect, as is the placement of the CA-SP1 cleavage site. The correct sequence is KARVL/AEAMSQ, where / denotes the cleavage site.Reviewer #2: - Can the authors estimate the occupancy of IP6 in their averages?- It would be good if the authors could provide a list/table of the conditions attempted to produce a mature EIAV lattice. Related to this, there is a reference missing in line 473Reviewer #3: Comments, concerns or questions:2) Line 264-269: EIAV CA was found to have a shorter helix 1, and therefore in EIAV Gag lattice, the putative dimer interface between neighboring Gag hexamers (found in HIV-1 lattice) is missing. According to HIV-1 mature CA lattice, helix 1 is important in intra-hexamer interactions. Could this be one of the reasons that in the in vitro assembly systems, EIAV Gag always form immature lattice, not mature lattice? Is this property unique for EIAV?3) The authors found that the EIAV CA-NTD organization is not the same as that in HIV-1. Several molecular contacts in HIV-1 Gag lattice are missing in EIAV which include: (a) there is no intra-hexamer interactions among CA-NTD (line 238-239) (b) there is no dimeric inter-hexamer interaction formed by helix 1 (c) there is no strong contact between CA-NTD and CA-CTD (line 263-264). The studied EIAV assemblies are rather small: narrow tube 35nm, wide tube 70nm and spherical particles are 100nm. In general, retroviruses have a larger dimension. Is it possible that these missing contacts actually present in the authentic particles?4) Line 109-110: The authors suggests that “the retroviral CA-NTD and CA-CTD are independent structural entities that act autonomously in regulating immature virus particle diameter and curvature”. This idea was repeated at line 363-364: “The spatial separation of EIAV CA-NTD and CA-CTD, and the lack of strong interactions between them, allow these two halves of CA to behave almost as independent entities, as suggested previously for other retroviruses”. This statement seems overly strong. In HIV-1, helix 11 and helix 8 interact with MHR and helix 7. This interaction would limit the orientation of NTD relative to CTD and the role of NTD in deciding the curvature of the Gag lattice.5) Line 292-295: Line 292 lists the SP sequence as (LAKAL) in which the residue 359 is L. However in line 295, the last residue is listed as T395. Please clarify.6) Supplement figure 4, the electron density looks low resolution. A better figure is desired.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level, and was found to be improved with respect to the original submission. There are two minor issues that we would like you to consider before formal acceptance, as you will see listed below. In addition, when you are ready to resubmit, please be prepared to provide the following:(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, We hope to receive your revised manuscript within 60 days or less. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.[LINK]If you have any questions or concerns while you make these revisions, please let us know.Sincerely,Félix A. ReyAssociate EditorPLOS PathogensMichael MalimSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************The revised manuscript by Dick et al is significantly improved with respect to the original version as the authors have now quantified the amount of released EIAV particles from wild type and IPPK KO cells, showing that there is only a minor decrease in particle release which correlates with the drop in infectiousness, contrary to HIV-1 infected cells for which there is apparently no particle released into the supernatant in the IPPK KO cells. This was one of the main issues raised by reviewer 1 of the original version. The authors have also adequately answered the other questions, except for the request by reviewer 2, who asked to provide a control reconstruction of immature assemblies in the absence of IP6. We had editorially considered that answering to such a request was not a requirement for acceptance, given the amount of work involved and that the observed density for IP6 conformed to the expected shape for this molecule, that it was as strong as the surrounding protein density (including in regions where only 2-fold averaging instead of 6-fold was done). The new version is now acceptable for publication, provided that the authors pay attention to two minor issues:1. In the discussion, I suggest that the authors add a sentence considering a potential basal immature assembly in the absence of IP6, which would be similar for all lentiviruses but that is detected here in the case of EIAV because the cells used are poorly infectable by this virus. As the same cells are much more susceptible to HIV1 infection, the difference in particle release in the case of EIAV is not very different to basal, whereas it would be more than two logs in the case of HIV1. Depending on the infectivity assays and on the reactivity of the antibodies used in the western blot and on the, this basal assembly in the case of HIV-1 could be missed. In other words, it could be that the striking difference in the requirement for IP6 suggested by Fig. S3 is due to the cell line used for the experiment and does not reflect an intrinsic difference between the two viruses.2. In Figure 1, I suggest that the authors take a look at the “representative images” that they provide in relation with the quantification provided. For instance, panel A suggests that there are predominantly spheres at 0uM IP and at 10 uM IP5, whereas the quantification in panel B indicates that there are mainly tubes under these conditions. Similarly, panel C  suggests that there are essentially tubes at 10 uM IP5, whereas the quantification in panel D indicates more spheres than tubes. This can be confusing to some readers, so if possible, please provide more representative micrographs in each case  11 Dec 2019Dear Dr. Schur,We are pleased to inform that your manuscript, "Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly", has been editorially accepted for publication at PLOS Pathogens. Before your manuscript can be formally accepted and sent to production, you will need to complete our formatting changes, which you will receive by email within a week. Please note that your manuscript will not be scheduled for publication until you have made the required changes.IMPORTANT NOTESplospathogens@plos.org.(1) Please note, once your paper is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. 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Go to http://journals.plos.org/plospathogens/s/latex in the meantime.(8) LaTeX users only: Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines (9) If you have associated protocols in protocols.io, please ensure that you make them public before publication to guarantee immediate access to the methodological details.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Félix A. ReyAssociate EditorPLOS PathogensMichael MalimSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XGrant McFaddenEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-2556-3526***********************************************************The authors have adequately addressed the minor editorial suggestions made in the previous round of revision, and the manuscript is now acceptable for publication. This is a very high quality paper with data obtained using state-of-the-art electron cryo-tomography and subtomogram averaging methodologies that the authors have strongly contributed to develop. It will certainly be of interest the broad readership of PLoS Pathogens.Reviewer Comments : 16 Jan 2020Dear Dr. Schur,We are delighted to inform you that your manuscript, "Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. 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Tumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure. 53BP1 is a crucial factor involved in double strand break repair which blocks DNA end resection affecting DNA repair pathway choice. Here the authors reveal by live cell nuclear architecture analysis the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response. To ensure cellular homoeostasis, it is critical that this type of genomic lesion is detected with high fidelity and resolved accurately. To do just this, a cellular surveillance system termed the DNA damage response (DDR) has evolved and it resolves DSBs by one of two main DNA repair pathways—homologous recombination (HR) or non-homologous end joining (NHEJ)5. A key factor in a DSB’s DDR is TP53-binding protein 1 (53BP1)4. 53BP1 is a large  DNA repair factor that is recruited to DSBs to regulate DNA repair pathway choice by blocking DNA end resection (part of HR) and promoting DSB repair by NHEJ10. Biochemically, it has been demonstrated that DSBs elicit a local histone modification cascade that triggers 53BP1 recruitment and binding of 53BP1 dimers to chromatin flanking a DSB12. However, the spatiotemporal dynamics that underpin this mechanism of 53BP1 dimer recruitment to a DNA DSB within the nuclear architecture of a living cell remains unknown.DNA double-strand breaks (DSBs) represent a serious threat to genomic integrity. A single DSB left unrepaired can lead to mutations that promote chromosomal rearrangements, loss of an entire chromosome arm and ultimately cell death or oncogenic transformation16 and a series of histone ubiquitination events performed by RING finger 8 (RNF8) and RNF168, which includes lysine 15 of core histone 2A (H2AK15ub)22. Although the biophysical mechanism by which 53BP1 molecules arrive at a chromatin-signaled DSB has not been directly observed within an intact cell nucleus, it is known from cryogenic electron microscopy that dimers of 53BP1 directly bind H2AK15ub-containing nucleosomes via the 53BP1 ubiquitylation-dependent recruitment motif and this interaction requires simultaneous engagement of the 53BP1 tandem tudor domain with dimethylated lysine 20 of histone 4 (H4K20me2)—a mark present throughout the genome23. It has also been shown by quantitative chemical proteomics that in addition to H2AK15Ub and H4K20me2, 53BP1 directly interacts with γH2AX via the 53BP1 BRCT domain in specific contexts such as late repairing foci24.53BP1 recognition of a DNA DSB starts with Ataxia-telangiectasia-mutated (ATM) kinase phosphorylating serine 139 of histone variant 2AX (γH2AX) within proximal chromatin25, which has been shown to promote 53BP1 recruitment to DSBs27 and self-assembly of 53BP1 into phase-separated condensates28. Although it is known from co-immunoprecipitation experiments that 53BP1 dimers exist in the nucleoplasm independent of DNA damage signaling25, it has never been shown in a living cell when or where 53BP1 dimers are formed during the DNA DSB recruitment phase. Furthermore, upon arrival at a DNA DSB, it is unknown how the different components of the DSB histone code  regulate 53BP1 dimer retention or oligomer formation. Thus, here we set out to quantify the spatiotemporal dynamics of 53BP1 self-association during a DSB DDR and test whether the DSB histone code differentially regulates 53BP1 dimer recruitment, retention, and oligomer formation in the context of live-cell nuclear architecture. To do so we couple fluorescence fluctuation spectroscopy (FFS)30 of fluorescently tagged constructs of 53BP1 with the DSB inducible via AsiSI cell system (DIvA)32.53BP1 dimerization and self-association into higher-order oligomers is mediated by the 53BP1 oligomerization domain30. DIvA cells are a stable cell line generated in the human U2OS background harbouring a 4-hydroxytamoxifen (4OHT)-inducible AsiSI restriction enzyme, which allows for induction of approximately 100 site-specific DSBs in the genome upon 4OHT treatment32. Using the DIvA system, we designed two different types of FFS-based experiments aimed at characterizing 53BP1 dimer localization and transport dynamics during DSB repair. The first experiment is based on Number and Brightness (NB) analysis35, an FFS method that can extract the amplitude spectrum of eGFP-53BP1 fluorescence fluctuations recorded in a frame scan acquisition and convert this into a spatial map of the oligomeric state of 53BP1 throughout the nucleoplasm. The second experiment is based on cross-pair correlation function (pCF) analysis38, an FFS method that can compare the temporal spectrum of eGFP-53BP1 fluorescence fluctuations with spatially distinct mKate2-53BP1 fluorescence fluctuations along a line scan and quantify 53BP1 dimer mobility in or across different chromatin environments.FFS describes a family of methods that statistically analyse fluctuations in fluorescence intensity within live-cell microscopy data to extract single molecule dynamics41, we find the DSB histone code to regulate a spatiotemporal redistribution in 53BP1 oligomerization. Specifically, upon DSB induction a population of 53BP1 dimer that is present independent of DDR signaling is recruited to DSB sites. Upon arrival at these nuclear locations, H2AK15Ub mediates efficient 53BP1 dimer loading onto a DSB, while consecutive engagement with H4K20me2 enables this population of 53BP1 dimer to be retained. Collectively, it is this stepwise interaction that leads to the assembly of 53BP1 oligomers and mature 53BP1 foci.From application of NB and cross-pCF to DIvA cells expressing different fluorescent constructs of 53BP1, and independent validation of this FFS-based strategy by fluorescence anisotropy imaging microscopy (FAIM) of homo-Förster resonance energy transfer (FRET)YY1258,1259AA) or are constitutively oligomeric (eGFP-GCA-53BP1)26. An NB FFS experiment is a time series of confocal frames  tagged to wild-type 53BP1 (eGFP-53BP1) vs. 53BP1 mutants that either have a diminished capacity to dimerize  and eGFP-GCA-53BP1 (positive control)  Fig. , we findol) Fig.  to accurol) Fig.  and spatol) Fig.  53BP1 diol) Fig. . This reol) Fig. . Thus, o32. Immunofluorescence (IF) for γH2AX in fixed DIvA cells following 4OHT treatment confirmed (1) multiple DSB foci to form across the genome within 1 h of addition that were localized outside of HP1α foci   eGFP-53BP1 to co-localize at newly formed DSB foci  at which we spatially cross correlate the two different colored 53BP1 fluorescence fluctuations. Thus, in co-transfected DIvA cells treated with 4OHT for 60 min , as well as over three different spatial scales   amin Fig.  and (2) ls) Fig.  to estabδr = 0 (pCF0), and comparison of this analysis with the cross pCF0 profile of 53BP1 dimers only, we find the fraction of 53BP1 dimer present to be 31 ± 4% , we find the spatial evolution of this transport to be effectively captured with cross-pCF4 to pCF7 (30–50% efficiency) and not beyond this distance range at cross pCF12 (0% efficiency)  in the nucleoplasm at  4% Fig.  after co 4% Fig. . From crcy) Fig. . Given tcy) Fig. , we proccy) Fig. . This ancy) Fig.  and, (2)cy) Fig. . Thus, cD1521R)12, H2AK15ub (eGFP-53BP1L1619A)23, and γH2AX (eGFP-53BP1K1814M)24. In the literature, it is reported that H4K20me2 and H2AK15ub are critical for DSB recruitment of 53BP112, whereas γH2AX plays a secondary role in 53BP1 DSB foci formation24. Thus, in DIvA cells transfected with eGFP-53BP1 vs. eGFP-53BP1D1521R, eGFP-53BP1L1619A, or eGFP-53BP1K1814M, we first performed γH2AX IF 60 min after 4OHT treatment  ent Fig. .Fig. 4ThD1521R and eGFP-53BP1L1619A in the DIvA cell system is significantly less than eGFP-53BP1 or GFP-53BP1K1814M  dimer accumulation in the absence of H2AK15ub recognition  was digested with AgeI-HF and BamI-HF (NEB) followed by Antarctic Phosphatase treatment (NEB: M0289). The vector fragment was then gel extracted (NEB Monarch gel extraction kit). Overlapping PCR primers containing the desired mutant codons paired with common PCR primers with ~30 bp overhangs for assembly into the digested eGFP-53BP1 vector were used in two separate PCR reactions (NEB Q5 polymerase). PCR products were then gel extracted and assembled with the vector (~1 ng/100 bp of DNA), transformed into chemically competent Escherichia coli DH5α cells (Thermo Fisher 18265017), and plated onto selective Lysogeny broth agar media. The 53BP1 mutant K1814M and small interfering RNA (siRNA)-resistant 53BP1 constructs on the other hand, were generated using gBlocks (IDT) flanking the desired changed base pairs. The digested vector and gBlock were assembled by Gibson Assembly with a 1 : 3 molar ratio of vector to gBlock. eGFP in the eGFP-53BP1 vectors was replaced with mKate2 using Gibson Assembly with a PCR product , which had compatible overhangs and cloning it into Acc651/AscI digested 53BP1 vector. All constructs and mutants were confirmed by Sanger sequencing .53BP1 mutants YY1258, 1259AA, D1521R, and L1619A were generated using mutagenesis via Gibson Assembly2. For live-cell microscopy experiments, the DIvA cells were plated 24 h before experiments onto 35 mm glass bottom dishes and transiently transfected or co-transfected with the following plasmids via use of Lipofectamine 3000 according to the manufacturer’s protocol: (1) eGFP , eGFP-53BP1 , eGFP-53BP1YY1257,1258AA, eGFP-GCA-53BP1 , eGFP-53BP1D1521R, eGFP-53BP1L1619A, eGFP-53BP1K1814M, mKate2-53BP1, mKate2-53BP1D1521R, mKate2-53BP1L1619A, and mKate2-53BP1K1814M. For IF against γH2A.X (S139)  and 53BP1 , cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 1 mg/ml Triton X-100 for 15 min at room temperature, and blocked with 1% bovine serum albumin, each in a phosphate-buffered saline buffer. Primary antibody (1 : 200 dilution) was incubated overnight at 4 °C. Secondary antibody  was incubated for 1 h at room temperature. For DNA staining, cells were incubated with 1 μM Hoechst 33342 for 10 min at room temperature.DIvA cells  were grown in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% bovine growth serum (Gibco), 1× Pen-Strep (Lonza), and 1 µg/ml puromycin (Thermo Fisher Scientific) at 37 °C in 5% CO49. RNF8 and RNF168 siRNA were as follows, respectively: RNF8 5′-ATGGTAAACTGTACGCTA-3′, 5′-TTCAGAATAGCGTACAGT-3′; RNF168 5′-GAAAGCTAAGCTAAGCATTGATA-3′, 5′-TTGTTAATATCAATGCTT-3′ (from IDT). The siRNA-resistant eGFP-53BP1 construct was transiently transfected via use of Lipofectamine 3000 according to the manufacturer’s protocol at 24 h post siRNA transfection. Microscopy experiments were conducted at 48 h after DIvA cells being transfected with siRNA duplexes and IF was used to test 53BP1 knockdown efficiency at the same time.All siRNA-mediated knockdown of RNF8, RNF168 and 53BP1 was achieved using Lipofectamine RNAiMAX (Thermo Fisher) mediated transfection according to the manufacturer’s protocol: 25 pmol of siRNA duplexes per 0.25 million cells. 53BP1 siRNA sequence was as follows 5′-AGAACGAGGAGACGGUAAUAGUGGG-3′. To generate siRNA-resistant 53BP1 expression constructs, A231G, A234G, and A237C silent point mutations were introduced into the 53BP1 cDNA2. For single channel NB FFS measurements the eGFP-tagged plasmids were excited by a solid-state laser diode operating at 488 nm and the resulting fluorescence signal directed through a 405/488/561 dichroic mirror to a photomultiplier detector (H7422P-40 of Hamamatsu) fitted with an eGFP 500/25 nm bandwidth filter. For the dual channel cross-pCF FFS measurements the eGFP and mKate2 plasmids were excited by solid-state laser diodes operating at 488 nm and 561 nm, respectively, and the resulting signal was directed through a 405/488/561 dichroic mirror to two internal GaAsP photomultiplier detectors set to collect 500–540 nm and 600–700 nm, respectively. For the FAIM measurements of homo-FRET the eGFP-tagged plasmids were excited by a solid-state laser diode operating at 488 nm and the resulting fluorescence signal directed through a 405/488/561 dichroic mirror as well as a thin film polarizer cube, which split the parallel and perpendicular signal with respect to the excitation light into two photomultiplier detectors (H7422P-40 of Hamamatsu). For the IF measurements Hoechst 33342 and Alexa Fluorophore 647 (AF647) were excited by solid-state laser diodes operating at 405 nm and 640 nm, respectively, and the resulting signal was directed through a 405/488/561/640 dichroic mirror to two internal GaAsP photomultiplier detectors set to collect 430–470 nm and 650–750 nm, respectively.All microscopy measurements were performed on an Olympus FV3000 laser scanning microscope coupled to an ISS A320 Fast FLIM box for fluorescence fluctuation data acquisition. A ×60 water-immersion objective 1.2 NA was used for all experiments and the cells were imaged at 37° in 5% CO30 and then selecting a 10.6 μm region of interest (ROI) within that DIvA cell’s nucleus  was then acquired with the pixel dwell time set to 12.5 µs, which resulted in a line time of 4.313 ms and a frame time of 1.108 s. Cross-pCF FFS measurements of the different eGFP and mKate2 tagged 53BP1 constructs involved selecting a DIvA cell exhibiting low and comparable co-expression of the two fluorescent constructs and then selecting a 5.3 μm line across the middle of a 4OHT-induced DSB foci  was then acquired with the pixel dwell time set to 8 μs, which resulted in a line time of 1.624 ms (our sampling frequency). FAIM of the different eGFP-tagged 53BP1 constructs employed the same scan settings as NB , which 35. In brief, within each pixel of a frame scan we have an intensity fluctuation (F(t)) that has an average intensity 〈F(t)〉 (first moment) and a variance (F(t) − 〈F(t)〉)2 (second moment). As defined in Eq. (B) of the molecules that give rise to the intensity fluctuation.The brightness of a fluorescently tagged protein is a readout of that protein’s oligomeric state that can be extracted by a moment-based NB analysis of an FFS frame scan acquisitiond in Eq. , the ratɛ) of the molecules is related to the measured apparent brightness (B) by B = ε + 1, where 1 is the brightness contribution of our photon counting detector. Calibration of the apparent brightness of monomeric eGFP (Bmonomer = 1.15) enabled extrapolation of the expected apparent brightness of eGFP-53BP1 dimers (Bdimer = 1.30) and oligomers (Boligomer > 1.60)  were used as parameters to quantify the degree of 53BP1 self-association detected across multiple cells .The true molecular brightness (60) Fig. , which i60) Fig.  and spat60) Fig.  the fraclls Fig. . An inte41. In brief, homo-FRET induces a depolarization to a fluorescence emission that can be detected as a reduction in fluorescence anisotropy, and as defined by Eq. (r) is the intensity-corrected difference between the emission parallel (I||) and emission perpendicular  that can be monitored by FAIMd by Eq. , anisotrg factor accounts for any systemic differences introduced by the optics or detectors into the parallel vs. perpendicular channel and this was calibrated in our system by measurement of fluorescein, a fast-rotating molecule whose emission polarization should be isotropic. Depolarization effects caused by use of a high numerical aperture objective were taken-into-account by incorporation of an additional correction factor into r that was derived from referencing eGFP anisotropy values acquired with a 60X water objective to eGFP anisotropy values acquired with a ×10 air objective50. After application of this correction factor to FAIM measurements acquired in live DiVA cells expressing free eGFP we find that in our system the fluorescence anisotropy value of eGFP in the absence of homo-FRET (reGFP) is 0.32. In the presence of homo-FRET due to eGFP-53BP1 dimer or oligomer formation, the expectation is that reGFP will be reduced. Thus, we express eGFP-53BP1 homo-FRET in terms of delta r, which is equal to the difference between reGFP and reGFP-53BP1. An intensity-based mask was used to quantify these parameters in the nucleoplasm vs. within foci. All anisotropy and homo-FRET analysis of FAIM data acquisitions were carried out in a custom code written in Matlab.The 38. In brief, in each pixel of a two-channel line scan we have an intensity fluctuation (F(t)), which we can format into two intensity carpets, where the x-coordinate in each carpet corresponds to the point along the line (pixel) in that channel and the y-coordinate corresponds to the time of acquisition. The carpet data format enables temporal cross correlation of pairs of intensity fluctuations separated by a set distance (δr) for every possible delay time (τ) either (1) within a single channel, by application of the pCF function (defined in Eq. 51 or (2) between the two channels, by application of the cross-pCF function  it takes a population of molecules  to translocate a set distance (δr), and because the measurement is exquisitely local to a pair of points, the translocation time into or out of different environments along the line scan can be extracted with a spatial resolution of ~260 nm ). Thus, given that the two-channel FFS line scan measurements presented here were acquired with a pixel size of ~80 nm, the spatial evolution of 53BP1 transport was tracked by performing pCF and cross-pCF analysis at a distance of δr = 0, 4, 7, and 12 . These four pCF distances  enabled quantification of the fraction of 53BP1 dimer present in the nucleoplasm, the time it takes this fraction to diffuse onto a DSB vs. off this structure and the efficiency of these two transits at a selected optimal distance.The pCF and cross-pCF profiles that result from cross correlation of intensity fluctuations within or between channels, respectively, reports the time (G0CH1) and mKate2-53BP1 mobility (G0CH2) were compared with the amplitude of the cross pCF0 profile derived for eGFP-53BP1 in complex with mKate2 mobility (G0CC) and the ratio was taken with the limiting channel  52 that was derived from simulation of the experimentally measured percentage of eGFP signal detected in the mKate channel .To quantify the fraction of 53BP1 dimer present in the nucleoplasm the amplitude of the pCF0 profile derived for eGFP-53BP1 mobility (For 53BP1 knockdown quantification, DIvA cell nuclei were identified by thresholding the corresponding Hoechst 33342 signal in dual channel IF confocal images by ImageJ. The coordinates of the identified nuclei were saved in the ImageJ ROI manager and then the mean 53BP1 intensity in the identified ROIs was calculated via use of the ImageJ ‘measurement’ function. The number and intensity of γH2AX, 53BP1 WT, and 53BP1 mutant foci were quantified in ImageJ. Confocal images were smoothed twice with the smooth function and foci were thresholded out by selecting the top ~5% pixels. Each identified ROI was counted as a foci and foci intensity was calculated by the sum intensity of all foci divided by sum intensity of the whole nucleus.Statistical analysis was performed by using GraphPad Prism software. Figures were prepared by use of Adobe Illustrator, MATLAB, and SimFCS.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary