When three SARS-CoV-2 vaccines came to market in Europe and North America in the winter of 2020–2021, distribution networks were in a race against a major epidemiological wave of SARS-CoV-2 that began in autumn 2020. Rapid and optimized vaccine allocation was critical during this time. With 95% efficacy reported for two of the vaccines, near-term public health needs likely require that distribution is prioritized to the elderly, health care workers, teachers, essential workers, and individuals with comorbidities putting them at risk of severe clinical progression.We evaluate various age-based vaccine distributions using a validated mathematical model based on current epidemic trends in Rhode Island and Massachusetts. We allow for varying waning efficacy of vaccine-induced immunity, as this has not yet been measured. We account for the fact that known COVID-positive cases may not have been included in the first round of vaccination. And, we account for age-specific immune patterns in both states at the time of the start of the vaccination program. Our analysis assumes that health systems during winter 2020–2021 had equal staffing and capacity to previous phases of the SARS-CoV-2 epidemic; we do not consider the effects of understaffed hospitals or unvaccinated medical staff.%) of vaccine supply to individuals over the age of 70 is optimal in terms of reducing total cumulative deaths through mid-2021. This result is robust to different profiles of waning vaccine efficacy and several different assumptions on age mixing during and after lockdown periods. As we do not explicitly model other high-mortality groups, our results on vaccine allocation apply to all groups at high risk of mortality if infected. A median of 327 to 340 deaths can be avoided in Rhode Island (3444 to 3647 in Massachusetts) by optimizing vaccine allocation and vaccinating the elderly first. The vaccination campaigns are expected to save a median of 639 to 664 lives in Rhode Island and 6278 to 6618 lives in Massachusetts in the first half of 2021 when compared to a scenario with no vaccine. A policy of vaccinating only seronegative individuals avoids redundancy in vaccine use on individuals that may already be immune, and would result in 0.5% to 1% reductions in cumulative hospitalizations and deaths by mid-2021.We find that allocating a substantial proportion (>75%) and no major changes in distancing, masking, gathering size, hygiene guidelines, and virus transmissibility between 1 January 2021 and 1 July 2021 a combination of vaccination and population immunity may lead to low or near-zero transmission levels by the second quarter of 2021.Assuming high vaccination coverage . The international effort to bring a SARS-CoV-2 vaccine to market began in January 2020 with the release of the viral genome sequence . DevelopIn vaccination campaigns, prioritization of certain population groups over others can influence the success of the campaign. The classic trade-off in vaccine distribution programs is between vaccinating high-contact versus high-risk individuals –15, and Vaccinating populations with real-time information on seroprevalence or attack rate—used interchangeably here as the seroprevalence is simply a 3-week delayed version of the attack rate—allows a public health system to introduce efficiencies into vaccine allocation. Here, we evaluate different age-distributions for vaccine rollout given current age-stratified attack rate estimates in Rhode Island and Massachusetts . While wUsing a mathematical model whose fit to Rhode Island and Massachusetts COVID-19 data was described in Wikle et al. , we cons% (95% CredInt: 17.3–24.1%) in Rhode Island and 12.5% (95% CredInt: 11.5–13.5%) in Massachusetts [We use a mathematical model developed by Wikle et al.  and fit chusetts . We upgrchusetts . The eigchusetts  were trachusetts . Since tAll simulation results are presented as medians. We draw ten sets of parameters from posterior distributions of the new model fit, and we present simulation results as the median value of these ten simulations. The new model’s median 30 November 2020 attack rates ares 19.6% for RI and 12.9% for MA.Z1 to Z24 in Fig. x% efficacy at n weeks post-vaccination. Clearly, 24 vaccine efficacy estimates for different time points have not been published, but we use this 24-compartment chain to model the expected gradual changes in vaccine efficacy from the early and measured stages in clinical trials  to stages 12 or 18 months later when the vaccine is assumed to have an exponentially waned efficacy . Even for early-stage efficacy, these data are not yet available because we do not know the average enrollment duration of patients in the two key trials. Nevertheless, we make several reasonable choices for shapes of this efficacy function based on the enrollment dates of each trial [s/(HLs+Ts), where HL is the efficacy half-life, s is the slope, and T is the time post-vaccination. To translate this individual efficacy into a population-level model, we define the vaccine efficacy (VEj) in the vaccinated class Zj as To model vaccination with waning vaccine efficacy, we add a 24-stage vaccinated state . We assume an FOI of 0.001 per day, and for tj=60 or tj=90 days we assume 95% efficacy and solve for aj. For other values of tj, the vaccine efficacy is assumed to follow one of the seven patterns in Fig. aj values (relative risk parameters for a fixed time point after vaccination) are solved for. We assume the force of infection from 1 December 2021 onward is constant and we explore three transmission scenarios. In the low-transmission scenario, transmission levels in Rhode Island and Massachusetts revert to their late spring and summer levels. Under medium transmission, transmission levels are set to their September through November mean value. Under high transmission, wintertime transmission levels continue through spring 2021; see Fig. where ρ in Wikle et al. [E or the asymptomatic class A can be chosen for vaccination but we assume that they progress through their normal course of infection with no effect of the vaccine, as vaccination will have occurred for these individuals at a time when their immune system has already been exposed to whole live virus SARS-CoV-2.Individuals can be vaccinated if they are in the susceptible class. Individuals who experienced asymptomatic infection and are in the recovered class can also be vaccinated, according to whether they were likely to have been asymptomatic or not  and confe et al. ). IndiviS and R are moved into the vaccinated class Z1. This means that we assume all vaccinated individuals become seropositive after vaccination. These individuals can then be infected, despite their seropositivity, at the relative rate aj in the class Zj, solved for in equation (1). Individuals progress from Z1 through to Z24 and back to S over the course of 540 days. Likewise, individuals entering the recovered class R are all assumed to be seropositive, with naturally immunity waning after a mean 540 days.Vaccinated individuals from % or 28.3% of the population. We call these the low and high supply scenarios: 50,000 or 300,000 vaccinations available in RI, and 300,000 or 1.8 million vaccinations available in MA. Distribution lasts from 14 December 2020 through 13 January 2021 (4.7% vaccine coverage) or 14 December 2020 to 4 March 2021 (28.3% vaccine coverage).Vaccination strategies are considered where doses are made available for 4.7The following age-based strategies are considered: (1) random, where any individual ≥16 in the population can be chosen for vaccination on a particular day; (2) 16–29 age group only; (3) 30–59 age group only; (4) 60-and-above age group only; vaccine supply is allocated to the 20–39 and 60+ age groups in proportions of (5) 75/25, (6) 50/50, (7) 25/75; vaccine supply is allocated to the 20–49 and 70+ age groups in proportions of (8) 75/25, (9) 50/50, and (10) 25/75. If there is sufficient supply to cover an entire age group, the remaining vaccines are allocated to the second age group. If both age groups have been covered completely, the remaining vaccines are distributed at random in the population to all individuals over the age of 16.9−1=511 possible combinations of age bands in the vaccination strategy. We take this approach to see if including or excluding a particular age band has a large effect on the results, after marginalizing over the inclusion/exclusion of the other age groups. As evaluation criteria, we consider the median total cumulative number of cases, hospitalizations, and deaths through to 30 June 2021, where the median is taken over ten forward simulations using ten parameter samples from the posterior of the model fit. Our model does not evaluate the benefits of vaccinating health care workers. We assume that health care and front-line medical staff are vaccinated first, and that hospital capacity and staffing during the winter wave are not affected by absenteeism or a surge of COVID-19 cases.In addition, using the nine 10-year age bands in our model, we consider all 2As the duration of immunity from the two current high-efficacy vaccines (Pfizer/BioNTech and Moderna) to have completed phase 3 clinical trials is not known, we build several vaccine profiles that have 95% vaccine efficacy at 60 to 90 days post-vaccination, with four different slopes of waning vaccine efficacy Fig. . In the The most straightforward approach to maximizing public health utility out of every vaccine dose is to vaccinate either high-contact and high-risk individuals. It is known that contact rates vary across the age groups, and that for SARS-CoV-2 risk and clinical severity increase monotonically with increasing age. To determine the average sizes of the effects of including or excluding certain age groups for SARS-CoV-2 vaccination in Rhode Island and Massachusetts, we considered all possible strategies  defined by inclusion/exclusion based on 10-year age band. We assumed that transmission from 1 December 2020 onwards would persist at the mean level observed in September - November 2020 (the “medium transmission” scenario). The violin plots in Fig. % coverage) during the initial rollout, allocating 25% of vaccines to the 20–49 age group and the remaining 75% to the 70+ age group is optimal  in terms of minimizing deaths and hospitalizations; see left three columns, Fig. In a low-supply scenario with 50,000 vaccinations available in Rhode Island (4.7% population coverage)—strategies focused on the elderly can reduce cumulative death numbers by as much as 10% when compared to a random distribution strategy  among those examined for Massachusetts. Under a scenario of low vaccine supply, deaths are reduced by approximately 8% when comparing to a strategy of random vaccine allocation; under high vaccine supply, deaths are reduced by approximately 13% when comparing to a random strategy. As in Rhode Island, prioritization of the 70+ group  leads to optimal outcomes .% coverage), a strategy that is targeted 25/75 at the 20–49 and 70+ age groups will results in 335–353 (from the least to the most optimistic vaccine efficacy profile) fewer hospitalizations and 327–340 fewer deaths by 30 June 2021 than a random allocation strategy. Compared to the unmitigated scenario (no vaccine), this vaccination strategy will save 639–664 more lives and reduce hospitalization numbers by 1563–1633 by the end of June 2021. In Massachusetts, with an ample supply of vaccines (1.8M vaccines distributed by March), this same approach translated to 1647–1766 fewer hospitalizations and 3444–3647 fewer deaths compared to a random strategy, and a total of 11,716–12,424 fewer hospitalizations and 6278–6618 fewer deaths compared to a scenario with no vaccine. Our current seroprevalence estimates indicate that when the distribution campaign is finished on 4 March 2021, the population-level immunity  in Rhode Island will be 71.3% and in Massachusetts will be 61.1%.For Rhode Island, assuming a total of 300,000 vaccinations  current seroprevalence levels in Rhode Island and Massachusetts and (2) the waning effect of vaccine efficacy. End-of-2020 attack rate was estimated to be 25.9% (95% CredInt: 24.5–27.2%) for Rhode Island and 20.6% (95% CredInt: 19.0–22.8%) for Massachusetts , 30. TheThe basic outcome observed in our evaluations is that high-mortality groups need to be vaccinated first in order to minimize death counts. In our model, groups that are at high risk of death if infected are the older age groups, but our analysis implies that any high-risk group—whether the risk factor is age, obesity, diabetes, past lung disease, lack of health care access, or anything else—should have equally high priority to vaccination. The obvious second-order implication is that individuals who have high-contact rates with risk groups or cohabit with someone at elevated risk of death should similarly be prioritized for vaccination. Although the highly specific context in which we have been managing COVID-19 risk over the past year allows us to identify such individuals on a case by case basis, there does not seem to be a systematic way to define this second-order group who have either frequent contacts or cohabitation with high-risk individuals; the clear exceptions are health care workers and employees of long-term care facilities.Vaccination of high-contact groups alone is not an optimal approach, primarily due to the large mortality differences between the youngest and oldest age groups. This conclusion is identical to the one reached in Bubar et al.  and MoorOne efficiency that is important to evaluate in the early part of a vaccination campaign—especially when supply is moderate—is the deprioritization of vaccination for individuals with past confirmed COVID-19 infections or recent confirmed COVID-19 infections. Although this would normally be voluntary, public health communication around this topic could ask individuals with known past infection to offer up their place in the vaccination queue to those that are still fully susceptible. In Rhode Island and Massachusetts, more than 75% of symptomatic COVID-19 cases were likely identified through testing during 2020, meaning that a large majority of individuals would be aware that they had a past infection. Asymptomatically infected individuals would not know that they were COVID-positive at some point in 2020, unless they were included in a random screening campaign focused on nursing home staff/residents or essential workers. Most asymptomatic infections during 2020 would have occurred in the <20 or <30 age groups, individuals who would not be prioritized for vaccination, thus reducing the effect that serostatus would have on vaccine wastage in the early part of the campaign. Despite the good intentions of such a policy, our analysis shows that serological testing prior to vaccination would not provide a meaningful benefit to the campaign overall, and it would likely add to delays unless rapid high-specificity point-of-care IgG tests were used. Our model currently assumes that individuals with past known infection are not included in the winter/spring vaccination campaign; if these individuals are included, the hospitalization and mortality benefits of the vaccination campaign are predicted to be lower by about 0.5% to 1%.The major benefit in focusing on state-level analyses of vaccine rollout is that cumulative seroprevalence and vaccination numbers can be tracked on a month by month basis. This is critical for public communication in the initial months of a vaccination campaign as the public will be eager to know whether total infection and vaccination counts are in the range of “30+10” or “40+20”, allowing the public health system to describe risk in terms of the fraction of individuals that are still not immune. Assuming our medium transmission scenario through spring and improved vaccination trends at approximately 0.5% of the population per day, both Massachusetts and Rhode Island were predicted  to crossOur modeling approach has several limitations. First, although age stratification allows for a straightforward strategy design focused on protecting the elderly, our model does not include any variables on race, comorbidities, health care workers, or other essential workers. This means that some key high-contact and high-risk groups are omitted from the modeling, and it is unlikely that an age-group proxy would be a suitable substitute for any of them. For example, health care workers would likely fall into the high-contact category, but they may preferentially be in contact with non-susceptibles  making them more a high-exposure group than a high-contact group. In addition, the benefits of vaccinating HCWs and essential workers is that certain essential services  can continue functioning, a benefit not captured in traditional epidemiological models.Second, phase 3 efficacy trials of the Pfizer/BioNTech and Moderna vaccines were not designed to evaluate reductions in transmission. Thus it is not possible to state whether the vaccines’ high efficacy could be compromised by asymptomatic or sub-clinical infections occurring in vaccinated individuals and allowing for the continuation of transmission. Asymptomatic individuals do have lower viral loads and fewer opportunities to transmit via large droplets projected out through coughs, sneezes, speech, or breathing. Therefore, inadequate vaccine prevention of potentially-transmissible asymptomatic infections may reduce the indirect benefits of vaccination, but this effect size is currently unknown . Asymptomatic and pre-symptomatic infection does occur for SARS-CoV-2, but the naturally observed infectivities in household and contact tracing contexts are likely to be different than the infectivity of a vaccinated infected asymptomatic individual. Preliminary reports from a Moderna FDA filing  were intThird, results on long-term efficacy and efficacy by age are currently unknown. The combination of these two is critical as older individuals may be protected for a shorter time than younger healthier individuals. If vaccine-induced immunity were to wane quickly in older individuals, the vaccine would be least effective in the group that needs it the most. All vaccination campaigns would then need to be restructured as routine/repeat campaigns that focus on the most vulnerable individuals and account for the fact that re-vaccination may need to occur often to realize the vaccination campaign’s intended mortality benefits.Real-time knowledge of seroprevalence can guide decisions on vaccine allocation. Knowing which population groups have experienced the most infection and how far seroprevalence has advanced population-wide can help determine the total vaccine supply needed as well as its distribution priorities. This highlights the value of (1) real-time attack-rate estimation, (2) completeness of population surveillance, and (3) widespread testing. Comprehensive surveillance and widespread testing ensure that individuals are as informed as possible about their serostatus to SARS-CoV-2 and allows for the health system to communicate to seropositive individuals that they should forego vaccination in the first few rounds of allocation while immunologically naive individuals are vaccinated first. Given the 1 January 2021 seroprevalence levels in Rhode Island and Massachusetts, older age groups and vulnerable populations with substantial risk of hospitalization or death resulting from SARS-CoV-2 infection should be the priority groups for vaccination, after the completion of vaccine rollout for front-line health and medical workers.Additional file 1 Supplementary figures.


Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6β and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of  Correct organization of the microtubule cytoskeleton is essential for many cellular processes such as the establishment of cell shape, organelle positioning, or intracellular transport . In prolDrosophila tracheal cells. It was shown that the transcription factor trachealess, which specifies tracheal fate, is required for the spastin-mediated release of centrosomal components from the centrosome and their subsequent Piopio-mediated anchoring to the apical membrane  . Among tNE-MTOC formation involves the localization of different MTOC proteins to the nuclear envelope. These include the PCM components pericentrin (PCNT), CDK5RAP2, and AKAP9  as well as γ-tubulin, key component of γ-tubulin ring complexes (γTuRCs) . At the Akap6 and Syne1 that encodes nesprin-1α.Based on loss- and gain-of-function experiments, quantitative analysis utilizing different cell types, and promoter studies, we show here that myogenin is required and sufficient for the formation of an NE-MTOC by controlling the expression of muscle- and NE-MTOC-specific isoforms of To gain insight into the regulation of NE-MTOC establishment in skeletal muscle cells, we correlated the expression of MyoD and myogenin during mouse C2C12 myoblast differentiation with two key steps of NE-MTOC formation: (1) the expression of nesprin-1α, the nuclear envelope anchor for MTOC proteins , and (2)Prominent MyoD expression was detected in all cells that had upregulated nesprin-1α and recruited PCM1 to the nuclear envelope , suggestα+ nuclei was not significantly affected by myogenin depletion  important for recruitment of other proteins to the centrosome  and (2) Collectively, our data suggest that the myogenin-independent early myogenic differentiation is sufficient to induce nesprin-1α expression, whereas myogenin regulates the nuclear envelope targeting of AKAP9 and PCM-1, which in turn recruits PCNT.All MRFs are able to ‘transdifferentiate’ permissive non-muscle cells with varying efficiency. They induce skeletal muscle markers such as the expression of contractile proteins or cell fusion into myotubes . HoweverMyog levels, Myod1 levels were not detectably affected in myogenin-depleted cultures. Taken together, these results further argue that myogenin is required for the localization of MTOC proteins to the nuclear envelope.The results obtained upon MyoD-GFP expression are potentially explained by the fact that MyoD can activate myogenin transcription . AnalyziIn order to elucidate how myogenin – whose expression did not correlate with nesprin-1α in C2C12 cells  – induceSyne1 is upregulated upon myogenin induction  and myoblasts of healthy donors were differentiated into myotubes and AKAP6 localization was compared  PCM-1 enables the recruitment of the dynein regulator p150glued and other motor proteins to the nuclear envelope and promotes alignment of nuclei , and (2)Next, we aimed to confirm that the observed nuclei mispositioning in AKAP6-depleted cells is due to aberrant microtubule nucleation and motor protein recruitment at the nuclear envelope. Immunofluorescence analysis showed that 2 days post siRNA transfection the microtubule network organization was similar in AKAP6-depleted and control myotubes showing the typical organization of microtubules in longitudinal arrays . SimilarSyne1 and Akap6 in the precipitated DNA. For this, we performed qPCR using primer pairs targeting myogenin consensus binding sites  in regions predicted by ENCODE data (Syne1 α-isoform transcript (nesprin-1α2) was 4.5-fold higher than the promoter region of the long Syne1 isoform (nesprin-1-giant)  . Similaranscript . This inAkap6 as well as with promoter regions of the giant- or α-isoform of Syne1 located directly upstream of a luciferase coding sequence. These promoter constructs were then co-transfected into human HEK293T cells together with GFP or myogenin-GFP. Co-transfection of myogenin with the Akap6 β-isoform promoter construct increased luciferase activity 10.9-fold, while co-transfection with the Akap6 α-isoform promoter construct did not show a significant increase compared to GFP-transfected control  required for the NE-MTOC in differentiated skeletal muscle cells and (2) sufficient to recruit MTOC proteins to the nuclear envelope in cells with a centrosomal MTOC.We conclude that the myogenic transcription factor myogenin controls NE-MTOC formation and that myogenin-induced AKAP6β expression is one of the central molecular components required for NE-MTOC formation . This coAkap6 β-isoform as well as Syne1 α-isoform promoters and regulate the switch from centrosomal to NE-MTOC in these cell types. For this purpose, promising cardiomyocyte and osteoclast ‘transdifferentiation’ tools using multiple transcription factors are available  are needed to activate the NE-MTOC after MTOC proteins have been recruited.SYNE1 have been described in EDMD patients, pointing towards a potential role of NE-MTOC defects in this pathology  contributes to a better understanding of the striated muscle NE-MTOC, (2) presents a mechanistic framework that may be applicable to ncMTOC formation in other cell types and tissues, and (3) provides a cellular system to elucidate further molecular mechanisms inducing the switch from centrosomal to ncMTOCs.Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Felix Engel (felix.engel@uk-erlangen.de).Cell types were authenticated as follows: human myoblasts and C2C12, myotube formation; NIH3T3, morphology; HEK293, efficiency in protein production. Note that the identity of NIH3T3 and HEK293T cells is not essential for this study. All cell lines were mycoplasma-free (tested every 12 months).2. Growth medium for C2C12, NIH3T3, Hela, and Hct116 consisted of high glucose DMEM supplemented with GlutaMAX containing 10% FBS, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. C2C12 cells were maintained at 50% confluence to preserve differentiation capacity. For differentiation, cells were cultured to 90% confluence and subsequently changed to differentiation medium high glucose DMEM with GlutaMAX containing 0.5% FBS and insulin, transferrin, selenium, sodium pyruvate solution (1:1000 of 100× ITS-A).Reagents used for cell culture are listed in the Key resources table. All cells used in this study were cultured at 37°C in a humidified atmosphere containing 5% COSYNE1 gene (nucleotide 23560 G>T) were immortalized by Kamel Mamchaoui and Vincent Mouly  as previously described via transduction with retrovirus vectors expressing hTERT and Cdk4  for ~2 min at room temperature. Detachment of myotubes was constantly monitored by phase contrast microscopy. After sufficient myotube detachment was observed, Trypsin/EDTA solution was carefully aspirated and a myotube-enriched suspension was collected by rinsing the plates five times with normal growth medium. Enriched myotubes were then plated on glass coverslips coated with 25 µg/ml fibronectin in PBS for >45 min at 37°C. After 24 hr incubation at 37°C, myotube cultures were subjected to siRNA treatment and/or microtubule regrowth assays.Myod1 and Myog coding sequences were obtained by PCR using cDNA from C2C12 cells differentiated for 2 days. The cDNAs were then cloned into the peGFP-N1 backbone by Cold Fusion Cloning  following the manufacturer’s instruction. Positive clones were identified by restriction digest and Sanger sequencing.Syne1 and Akap6 genes were identified from ENCODE data accessed through the SCREEN web interface (https://screen.wenglab.org/). Potential promoter regions were amplified from genomic DNA obtained from NIH3T3 cells using the primers listed in Renilla luciferase ORF (hRluc) into the psiCHECK-2 vector using NEBuilder HiFi DNA Assembly Master Mix  according to the manufacturer’s instructions.Candidate regulatory elements associated with myogenin binding in Plasmid transfection into NIH3T3 cells was carried out with 500 ng DNA per well of a 24-well plate using 1 µl Lipofectamine LTX  according to the manufacturer’s instructions. Transfection complexes were formed by incubating DNA with Lipofectamine LTX in Opti-MEM for 20 min at room temperature.For transfection of luciferase constructs in HEK293T cells, 250 ng luciferase vector and 250 ng myogenin-eGFP or eGFP control plasmid were used per well of a 24-well plate. Transfection complexes were assembled by incubating DNA with PEI MAX  in a 1:3 ratio in Opti-MEM for 20 min at room temperature.Cells were transfected using 2 µl Lipofectamine RNAiMAX reagent  and 40 nM final siRNA concentration per well of a 24-well plate. Transfection complexes were formed by incubating siRNA with RNAiMAX in Opti-MEM for 20 min at room temperature. C2C12 cells were transfected 24 hr prior to induction of differentiation (~50% confluence) and enriched C2C12 myotubes were transfected 24 hr after re-plating. NIH3T3 were transfected with siRNA 48 hr after plasmid transfection.3 LB 960 96‐well plate reader luminometer  using the Dual‐Luciferase Reporter Assay System according to the manufacturer’s instructions. In brief, HEK293T cells were harvested 48 hr after transfection in passive lysis buffer and stored at –80°C until measurement. Activities of firefly luciferase  and Renilla luciferase (promoter activation) were measured sequentially for each sample. Values of Renilla luciferase activity were normalized to those of firefly luciferase for each measurement.Luciferase activity was measured in Centro XShttp://n2t.net/addgene:12260; RRID: Addgene_12260; pMD2.G: Addgene plasmid #12259; http://n2t.net/addgene:12259; RRID: Addgene_12259). The transfer plasmids pLenti CMVtight Blast DEST (w762-1) and pLenti CMV rtTA3 Hygro (w785-1) used for creating tetracycline-inducible cell lines were gifts from Eric Campeau . The coding sequences of human myogenin  and mScarlet  were cloned into w762-1 by NEBuilder HiFi DNA Assembly according to the manufacturer’s instructions using the primers indicated in The lentiviral packaging plasmid psPAX2 and the VSV-G envelope plasmid pMD2.G were gifts from Didier Trono . Cells were transduced overnight in 6-well plates using 1 ml of diluted lentiviral vector. The following morning, medium was refreshed and cells were selected for transgene integration 72 hr after transduction. We first generated cells expressing a reverse tetracycline activator (rtTA3), which were subsequently transduced with lentiviral vectors carrying the desired transgene to express mScarlet or MYOG-2A-mScarlet under control of a tetracycline-responsive element (TRE). After selection, Tet-ON cell lines were used for 20 passages.RNA was isolated using a column-based RNA purification kit. For production of cDNA, 1 µg of RNA was reverse transcribed using Oligo (dT) 12-18mer primers and M-MLV Reverse Transcriptase (Sigma-Aldrich #M1302) according to the manufacturer’s instructions. For PCR, ~20 ng of cDNA were used with Redtaq master mix (Genaxxon #M3029) and products were analyzed using agarose gel electrophoresis.Cells were harvested in lysis buffer containing 1% NP-40, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 20 mM Tris-HCl (pH 7), and EDTA-free protease inhibitor cocktail . After 30 min incubation on ice, samples were sonicated and lysates were cleared by centrifugation at 16,000×g for 16 min at 4°C. For immunoprecipitation, 0.5 µg anti-AKAP6 antibody/mg of total protein were added to the lysate and incubated overnight at 4°C on a rotor. Subsequently, antibody complexes were purified by incubation of lysate with Protein A Sepharose beads  for 3 hr rotating at 4°C. Beads were washed three times with cold lysis buffer for 5 min and proteins were eluted from beads by incubation in 2× NuPAGE LDS sample buffer at 95°C for 5 min. Lysates and immunoprecipitated samples were analyzed by SDS-PAGE (4–12% NuPAGE Novex Bis-Tris gels) under reducing conditions and transferred to a nitrocellulose membrane by wet transfer at 350 mA and <60 V for 1.5 hr in 1× transfer buffer . The membrane was then blocked with 5% BSA in TBS-T  and incubated with primary antibodies against AKAP6 or PCM-1.3). Eluates were digested with proteinase K and RNAse A and DNA fragments were purified using a PCR purification kit .Protein and DNA were cross-linked by fixing cells for 10 min at 37°C using 1% formaldehyde in culture medium. Cross-linking was quenched by adding 125 mM glycine and gently agitating the cells for 5 min at room temperature. Then, cells were harvested in ice-cold PBS, centrifuged at 1000×g for 5 min at 4°C, and the resulting pellet was lysed in ChIP lysis buffer  for 30 min on ice. After lysis, samples were sonicated for 30 cycles consisting of 20 s sonication and 30 s pause inside an ice bath. Sonicated samples were centrifuged at 4°C for 30 min at 10,000×g. A small aliquot of the supernatant was saved as input control and the remaining supernatant was subjected to immunoprecipitation. Samples were diluted 1:5 in RIPA buffer and incubated overnight at 4°C with 1 µg/ml myogenin antibody  or IgG1 isotype control . Protein G agarose beads (Roche #11719416001) pre-blocked with salmon sperm  were used to precipitate antibody complexes from diluted samples. Beads were sequentially washed at 4°C with low-salt buffer , high-salt buffer , LiCl buffer , and TE buffer . Antibody complexes were eluted by incubating beads for 15 min at 30°C in elution buffer  with Airyscan image processing.Primary antibodies used in this study are listed in the Key resources table. Note that the MANNES1E antibody detects different nesprin-1 isoforms . Howeverhttp://fiji.sc, RRID:SCR_002285). For quantification of nuclear envelope coverage, confocal images were transformed into binary images by setting a manual intensity threshold. Regions of interests (ROIs) were obtained by detecting nuclei outline via DAPI staining and subsequent transformation of these outlines into 1-µm-wide bands. Coverage was quantified as the percentage of positive pixels inside bands in the binary images. ROIs for measuring intensities at centrosomes were generated by detecting local signal maxima in γ-tubulin channels and subsequent generation of circular ROIs with 1 µm diameter using the maxima as centers.All image analyses were carried out using the Fiji software package  for 30 s at room temperature prior to fixation.C2C12 cells were treated with 5 µM nocodazole  in culture media for 3 hr at 37°C to depolymerize microtubules. To observe microtubule regrowth, nocodazole-containing medium was removed, cells were rinsed three times with cold medium, and either fixed (0 min time point) with 4% formaldehyde in PBS for 10 min or immediately transferred to 37°C pre-warmed culture media for the desired length of time followed by formaldehyde fixation. Myotubes were extracted with 1% Triton X-100 in PHEM buffer  and Bioconductor . Myogenin ChIP-Seq data (GEO accession number: GSE36024) produced within the ENCODE project (https://genome.ucsc.edu/index.html (–5) was localized at a maximum distance of 1 kb from the annotated transcriptional start site.Data was analyzed with R  were obtained from the Gene Expression Omnibus (GEO) repository . Three sGene expression microarray data for rat heart development were obtained as described previously using the Affymetrix GeneChip RAT 230 Expression Set . Genes whttp://geneontology.org/ . Biological replicate means that cells were freshly plated, treated, fixed and stained, and then analyzed. For each biological replicate, two technical replicates were performed in the sense that two individual wells were processed at the same time. When analyzed, the two technical replicates were scored as one sample.For quantification in C2C12 cells, >500 nuclei were analyzed per condition and biological replicate. For MRF-GFP experiments, >50 GFP+ cells were analyzed per condition and biological replicate. For nuclear coverage and intensity quantifications of mScarlet and MYOG-mScarlet cells, >100 nuclei or centrosomes were analyzed per condition and pooled from three biological replicates to display distribution in violin plots.SCR_002798). Differences between groups were considered statistically significant when the p-value ≤ 0.05. The 95% confidence interval (CI) for the differences between compared groups are reported in the figure legends. Statistical significance of differences between groups was tested using the following:Statistical analysis was carried out using GraphPad Prism 5.02 or Prism 8.2.1  function from centrioles to the nuclear envelope during muscle cell differentiation. Assembling the non-centrosomal MTOC on the surface of nuclei and inactivating the centrosome is linked to the different cytoplasmic organization of differentiated, multi-nucleated myotubes and is required for proper alignment of nuclei along the elongated myotubes. The demonstration that myogenin expression is sufficient to induce MTOC re-distribution and the identification of the crucial role of AKAP6 in assembling the nuclear envelope MTOC are important novel findings and advance our understanding of this process. In myotubes AKAP6 depleted nuclei do not align properly, as shown previously for myotubes lacking MTOC function at the nuclear envelope. Finally, while myogenin expression is not able to induce MTOC redistribution in cancer cells, this process can be observed upon fusion with myogenin-expressing fibroblasts. The authors claim that this may serve as strategy to attenuate centrosomal MTOC activity, which has been linked to cancer development and progression.Overall, the study presents interesting novel findings, but lacks analytical depth regarding the underlying molecular mechanism. Also, several findings require more solid quantitative analysis. In particular, the part investigating attenuation of cancer centrosomes is quite weak in this regard.1) I am surprised that the authors seem to observe only nuclei that are either positive or negative for MTOC markers. Is this process (redistribution of centrosome proteins) not gradual? I would suggest a different way of quantifying this process, to account for the temporal aspect and include intermediate stages.2) Related to point 1: after myogenin expression, is the centrosomal MTOC inactivated first, after, or simultaneously with the appearance of the nuclear envelope MTOC? Are centrosomes in 2G active or inactive? Do 100% of cells look like the two examples in 2G?3) The attenuation of the centrosomal MTOC should be quantified upon myogenin expression should be quantified. PCM1 is considered a centriolar satellite marker. The authors should use additionally centrosomal markers such as pericentrin and γ-tubulin to evaluate the state of the centrosomal MTOC.4) 3D: the spatial distribution is not very clear from the image. A larger area of the nuclear envelope should be shown and some type of quantification (e.g. intensity profiles) that reveals the difference between nesprin and AKAP6. Are the locations of antibody epitopes (N-term. vs C-term.) consistent with the authors' interpretation?5) 4C: a different centrosomal marker that is not affected by myogenin is needed, to identify the presence of centrosomes/centrioles in all cells. Also, PCM1 is a satellite marker. The disappearance of satellites may or may not indicate changes in centrosome staining. Moreover, as the assembly of the ncMTOC, the disassembly of the centrosomal MTOC needs to be quantified.6) Figure 6: as before, other centrosome markers (including one that is unaffected) are needed to evaluate the state of the centrosomal MTOC. Higher magnifications are also required in particular of the centrosome region.7) The experiments in figure 6 are not convincing for claiming that centrosome attenuation can be induced in cancer cells. First, the quality of the provided data is simply not enough. Is the amount of PCM reduced? Is the activity reduced? Second, after fusion they are technically not cancer cells anymore.8) An important experiment would be to test whether nesprin-1alpha and AKAP6 ectopic expression can induce MTOC assembly at the nuclear envelope. If so, this may allow more detailed mechanistic studies (expression of mutants etc), to provide more depth to the study.Reviewer #2:Microtubule organization varies by cell type and function. Dividing cells, cancer cells, and some specialized cell types such as fibroblasts use centrosomes as microtubule organizing centers (MTOCs), but most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to obtain more complicated microtubule patterns during the process of differentiation. Despite decades worth of work on the centrosome, comparatively little is known about ncMTOC composition or how the cell regulates the switch in MTOC function from the centrosome to non-centrosomal sites during differentiation. Here Becker et al., use differentiated muscle cells as a model to understand how the well-defined nuclear ncMTOC, which is a hallmark of skeletal muscle, is established. Previous studies found that PCM proteins such as AKAP450, Pericentrin, and PCM1 localize to the nuclear envelope of muscle cells, that Nesprin1a recruits these proteins, and that Nesprin and AKA450 are required for microtubule nucleation at this site. Here Becker et al., appear to uncouple the expression of Nesprin and PCM; Nesprin and PCM1 are both expressed in cells positive for expression of the transcription factor MyoD, but Nesprin expression precedes expression of the transcription factor myogenin, and PCM1 but not Nesprin expression is controlled by myogenin. These results suggested that activation of MTOC function at the nucleus is downstream of Nesprin localizing there and requires expression of myogenin targets. Consistently, myogenin expression in naïve cells is sufficient to induce nuclear MTOC activation. By mining ChIP-seq, RNAseq and microarray data, the authors identify AKAP6 as this potential target, finding that AKAP6 localizes to the cytoplasmic face of the nucleus, fails to localize to the nucleus in SYNE1/Nesprin mutant patient derived myoblasts, and is required to localize PCM1, AKAP450, and Pericentrin to the nuclear envelope. AKAP6 also is required for microtubule regrowth from the nucleus of myotubes and for nuclear positioning in these cells, a MT controlled process. Finally, the authors find that fusion of a Myogenin induce muscle cell can convert a nive NIH3T# cells with a centrosome MTOC to a nuclear MTOC state. While I love a cell fusion experiment, this result is not surprising given that fusion of a myoblast to a U2OS cell can similar convert the non-muscle cell from a centrosome to a nuclear MTOC .eLife.The experiments appear well preformed and presented. However, although these studies appear to identify a novel ncMTOC factor at the nucleus of muscle cells, they do little in the way of uncovering mechanistic aspects of how AKAP6 functions there or on assembly of the nuclear ncMTOC in these cells. Is AKAP6 merely a bridge between Nesprin and AKAP450? How is its localization regulated to allow for the timely activation of the nucleus as an MTOC? Is AKAP6 sufficient to induce MTOC function? In the absence of more mechanistic insight on the role of AKAP6, I am not sure that this paper will be of broad enough interest for the readership of The findings here do little in the way of uncovering how AKAP6 functions at the nucleus in ncMTOC formation. This could be addressed in many ways including:– Does AKAP6 bind to Nesprin and AKAP450 (or other MTOC components) to create a bridge? If so, how is this association regulated? Limited by expression of AKAP6?– Is AKAP6 localization sufficient to promote MTOC activity? This could be addressed by artificially tethering AKAP6 to the plasma or mitochondrial membrane and seeing if MTOC proteins and microtubules are recruited. If this worked, the ability of such a structure to compete with the centrosome could also be directly addressed.The authors repeatedly claim that 'Myogenin is required for centrosomal protein recruitment to the nuclear envelope', but they really only look at PCM1 localization in most cases. Why not look at AKAP450? This is especially important when arguing that Myogenein controls MTOC formation downstream of Nesprin localization to the nucleus.If Nesprin expression is not controlled by myogenin, in naïve cells in which myogenin overexpressed, how does Nesprin become expressed and localized to the nucleus? The authors briefly comment on this, but I find their explanation unsatisfying especially given what they are arguing in Figure 1.The fusion experiment is cool and suggests that the nuclear MTOC is dominant to a centrosomal MTOC in interphase cells. However, almost the exact experiment has been done but is not cited  Similarly, although less directly relevant, dominance was tested between a mitotic centrosome MTOC and a membrane ncMTOC .– Line 49: PCM needs to be defined as material that is specific to the centrosome. PCM proteins might localize to other locations, but 'pericentriolar' refers to the material surrounding centrioles.– The authors interchange usage of expression and localization.Reviewer #3:The manuscript by Becker et al. focusses on the regulation of non-centrosomal microtubule organizing centers (ncMTOCs), specifically MTOC activity at the surface of the nucleus. The authors show that during muscle development, the MTOC activity transitions from the centrosome to the nucleus, and that this transition depends on the transcriptional activation of AKAP6 by myogenin, a TF that drives muscle differentiation. While it seems that several results can be found in the literature , the authors assemble them together well in a single manuscript to better link muscle differentiation to ncMTOC formation. Overall I found the study very interesting and most of the conclusions/model supported by the data, although we seem to disagree on direct vs indirect links with APAP6. Here are my comments and suggestions.A) Figure 1 is critical for setting up the order of events during differentiation. While the final conclusion of this section is correct, following the data in this section was extremely difficult. I had to read this section 4 times as I knew the data was in there somewhere, I just had to find it. I recommend replacing C and E with 4 graphs that show: 1) the % of the MyoD+ nuclei that are positive for PCM1, Myog, nesprin; 2) the % of PCM1+ nuclei that are positive for Myog, MoyD, nesprin; 3) the % of Myog+ nuclei that are positive for PCM1, MyoD, nesprin; 4) the % of nesprin+ nuclei that are positive for PCM1, MyoD, Myog. Then the description in the text should match these graphs.B) Related to the use of PCM-1 as the main PCM marker and an indicator of MTOC activity. I always thought of PCM-1 as a centrosome satellite component, which has little direct function on centrosome MTOC activity. In fact, in the original paper describing PCM-1  it is shown to leave centrosomes in the lead up to, and throughout mitosis, when the centrosome is at its highest MTOC activity. PCM-1's role in ncMTOC activity might be better understood, but ultimately γ-tubulin (g-tub) must be recruited to the site of nucleation. Thus, it would be nice to see g-tub and MTs in several experiments such as 1D, 2A-F, 3E, 4F.C) The model presented is that myognin binds AKAP6 promoter, increases expression, which drives AKAP6 binding to nesprin and triggers downstream PCM recruitment to the nuclear surface. While the order of events is nicely shown, a direct link upstream and downstream of AKAP6 is not. Many additional experiments must be performed to claim a direct role as shown in Figure 7. For example: mutating AKAP6 promoter, bypassing myog by overexpressing AKPA6, showing nuclear MTOCs when nesprin and AKAP6 are coexpressed in fibroblasts, bypassing nesprin by artificially tethering AKAP6 to nuclear envelope, mapping AKAP6 binding to PCM proteins and mutating that interaction site, and so on. If the reviewers and editor(s) feel this is beyond the scope of the manuscript, I suggest that the authors explicitly state, and show in the model, that indirect paths are possible/likely.D) It is not clear, given the similarity in steady state MTs between control and si-AKAP6, why nuclear positioning is different. The manuscript would be strengthened if it ended with the role of ncMTOCs in nuclear spacing instead of the current figure 6, which I think is not necessary and should be removed. Can the authors follow MT nucleation and nuclear positioning during the development of myotubes in control and si-AKAP6? Are the MTs more dynamic or more stable with or without nuclear MTOCs? What are the difference between myotubes with aligned nuclei and overlapping nuclei in the si-AKAP6 populations? Are the ones that end up overlapping start out too close to one another and therefore require ncMTOC activity to push the nuclei apart? Can the authors provide any further insight here?eLife. Your article has been reviewed by 3 peer reviewers, including Jens Lüders as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor.Thank you for submitting your article "Myogenin controls via AKAP6 non-centrosomal microtubule organizing center formation at the nuclear envelope" 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) The authors should clarify the relative regulation of MTOC activities. Centrosome inactivation may be due to factors that remove microtubule organizing capacity. Alternatively, the nuclear envelope MTOC could outcompete the centrosome by recruiting MTOC components. In cases where the nuclear envelope MTOC is perturbed, does the centrosome remain active? Does the centrosomal pool of PCM-1 remain prominent upon myogenin knockdown? After SYNE1 or AKAP6 knockdown, what is the state of the centrosome?2) Since they are at the core of the regulation mechanism, the IP/CHIP experiments need extra controls. The authors should probe for a protein that is not specifically retained by the precipitating antibody.3) What are the relative contributions of myoD versus myogenin in the regulation of MTOC assembly at the nuclear envelope? Experiments should be conducted to address: is myoD expression affected in the absence of myogenin? Does myoD expression also induce nesprin and AKAP6?4) Please provide a zero time point in regrowth experiments and check nucleation activity in the experiment in Figure 7G. This is needed to argue that an MTOC is present.5) The introduction and discussion should be improved by discussing:– The relevance of the presented findings for muscle function and disease.– The roles of the particular centrosomal proteins that are recruited to the nuclear envelope in generating and organizing microtubules.– The term "centrosomal proteins". As they shuttle from centrosome to the nuclear envelope, the term "MTOC proteins" or similar may be more appropriate.Reviewer #1:The study "Myogenin controls via AKAP6 non-centrosomal microtubule organizing center formation at the nuclear envelope" by Becker et al. identifies the transcriptional regulation and the recruitment factors required for assembly of the MTOC at the nuclear envelope in skeletal muscle cells. Assembly of the non-centrosomal MTOC is still poorly understood, but is important for the distribution of nuclei in multi-nucleated myotubes, which in turn is linked to muscle function and disease. The authors show that the transcription factor myogenin drives this process and that AKAP6beta and nesprin-1alpha are crucial transcriptional targets. Overexpression of these two proteins is sufficient to drive nuclear envelope MTOC formation even in the absence of myogenin expression.The manuscript extends previously published work by the same group on the role of AKAP6 in non-centrosomal MTOC assembly at the nuclear envelope. The current study elucidates the upstream factors that regulate this process and the provided data is of high quality.The strength of this study is the identification of transcription factors and MTOC assembly factors that are sufficient to induce nuclear envelope MTOC assembly, including in non-muscle cells. This provides the first framework for a mechanistic understanding of this process and for further analysis in future studies.Weaknesses are that experiments identifying transcriptional targets require additional controls and that the implications of the findings for muscle function and related diseases have not been described very well.The authors have addressed my concerns raised during review of a previous version. Regarding this new manuscript, which has a different focus, there are some remaining issues that need to be addressed:1) All IP/ChIP experiments lack controls . These are integral to the main claims of the paper and the authors should show control proteins that are detected in the input, but are not specifically retained by the precipitating antibody.2) For the non-expert reader, the introduction and discussion need to be improved in terms of the relevance of the presented findings for muscle function and disease. What is the role of the nuclear envelope MTOC? How does it affect nuclei positioning, why is this important?Reviewer #2:Here, Becker et al. set out to identify the factors that control MTOC formation at the nuclear envelope of skeletal muscle cells. The nuclear envelope has long been appreciate for its ability to grow and localize microtubules, however the mechanisms that induce this state and simultaneously inactivate the centrosome as an MTOC have remained elusive. The authors identify the transcription factor myogenin as both necessary and sufficient to induce MTOC formation at the nuclear envelope. By mining the literature and ChipSeq and RNAseq data, they identify Nespin and AKAP6 as downstream targets of myogenin, that together are sufficient to induce a nuclear envelope MTOC in fibroblasts, a cell type that normally maintains microtubules at the centrosome and Golgi. Furthermore, myogenin is required to specifically induce cell-type specific isoforms, of these proteins. Loss of AKAP6 and results perturbs the recruitment of MTOC proteins to the nuclear envelope and leads to nuclear positioning defects in myotubes. Together, these data establish a module within the differentiation program of muscle cells for establishing the nuclear envelope as an MTOC. This is a resubmission of a former manuscript and in general, the authors have greatly expanded the work and added significant mechanistic insight.1) I am left a bit confused about what the authors think are the relative contributions of myoD versus myogenin. It seems that the authors are setting up a linear pathway where MyoD induces expression of myogenin which in turn induces ncMTOC formation, especially since it is known that myoD activates myogenin transcription . If this is indeed the pathway the authors are testing, they should explicitly state that at the beginning of this section rather than at the end. Additionally, appropriate experiments should be conducted, i.e. is myoD expression affected in the absence of myogenin?; does myoD expression also induce nesprin and AKAP6 expression?2) The switch in MTOC function from the centrosome to non-centrosomal sites in differentiated cell is a general and interesting phenomenon. The authors propose that centrosome inactivation must be due to the expression of factors that remove microtubule organizing capacity from the centrosome. Alternatively, the nuclear envelope MTOC activity could outcompete the centrosome, stealing their shared components. Given that the authors have numerous cases where the nuclear envelope MTOC is perturbed, can they comment on whether the centrosome remains active? For example, what appears to be the centrosomal pool of PCM-1 remains prominent upon myogenin KD. Is this the case? Similarly, in the SYNE1 or AKAP6 knock down, what is the state of the centrosome?3) Regrowth experiments need a zero timepoint to be able to assess how well microtubules were removed upon depolymerization treatments. As presented, these experiments are hard to interpret. While it is unquestionable that microtubules are associated with the nuclear envelope in these cases, it is important to distinguish whether these are new microtubules or persistent anchored, stabilized ones.4) Does nesprin directly bind to AKAP6? The authors indicate that the interaction is direct.5) Figure 7G is very nice, but it would be much nicer to see that microtubules also grow from the nuclear envelope in this case in addition to the fact that PCM-1 is recruited.eLife. The introduction and/or discussion could be more extensive. For example, there is no discussion of why recruitment of these particular centrosomal proteins to the nuclear envelope would be important to generate microtubules. I also think it is important to explain what "centrosomal proteins" are as they clearly do not stay at the centrosome and instead shuttle to the nuclear envelope. Perhaps the authors could use "MTOC proteins" or something similar.6) I do not think the problem and players are set up well enough for the broad readership of Reviewer #3:eLife submission that focusses on the transition from centrosomal MTOCs to non-centrosomal MTOCs, specifically the surface of the nucleus. This submission more clearly focuses on triggering ncMTOC transition in developing skeletal muscles via the function of MyoD and Myogenin, members of the transcription factors family termed myogenic regulatory factors (MRFs). The final model shown in Figure 8 is well supported by the presented data, which I believe is much improved over the previous manuscript. Reading the recently published work from the same group (Vergarajauregui 2020), which appears to have been in review at the same time as the original submission of this manuscript, helped clarify some of the interaction and functional claims downstream of AKAP6. The authors have done a great job addressing all my previous concerns, they should be commended for this and my comments below are mostly small changes that do not move the goal line for the authors.The manuscript by Becker et al. is a new submission of previously 1) In several cases in the beginning of the manuscript (ex. First paragraph pf the results) it was not clear that ncMTOC specifically referred to nuclear envelope MTOCs. I would recommend explicitly stating in this first result section that throughout the manuscript, ncMTOCs refers to the nuclear envelope in this study. Alternatively, maybe the authors can use NE-MTOC to distinguish it from other ncMTOCs.2) On line ~181 the authors state that "both MRFs are sufficient individually to induce a switch from centrosomal MTOC to a nuclear envelope MTOC." At this point in the manuscript the authors had not yet shown the elimination of centrosomes, that comes in figure 3. I suggest removing a reference to a switch at this point.3) In several location in the manuscript the author state that myogenin is required to recruit centrosomal proteins. This can be found in lines 193 and 570 and other places. It is more accurate to say that myogenin is require for the localization of centrosome proteins to the nuclear envelope. Recruitment implies myogenin is the recruiting protein, which it is not, it is simply upstream in the ncMTOC formation pathway.4) The figures overall are much improved and the quantification is suitable. However, It is important to report the Ns in each of the experiments. For example in figure 3, how many nuclei were measured? How many experimental repeats were performed.5) Presentation of the analysis leading up to AKAP6 was great6) How reliable is the specificity of the E-Box PCR amplification? Have the authors confirmed specificity of the ChIP experiment by PCR amplifying other regions of the AKAP and nesprin promotors? Although, it does appear to be specific enough to distinguish the different isoforms.7) Direct protein interaction cannot be determined by IPs. The authors should change the conclusion that "AKAP6 localizes to the nuclear envelope in differentiated skeletal muscle cells via interaction with nesprin-1α" to AKAP6 localization depends on nesprin-1a. An interaction can only be determine using purified proteins or Y2H as the authors did with AKAP6 and PACT in the (Vergarajauregui 2020). If there is direct protein interaction data, the authors should present it, otherwise the current results should be presented in terms of being upstream or downstream.Another example of this is "AKAP6 is required for the localization of centrosomal proteins to the nuclear envelope, in part by interacting with PCM-1." If there is no direct protein interaction data available (here or in the literature), then the word interacting should be replaced by recruiting.eLife. Your article has been reviewed by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor.Thank you for submitting your article "Myogenin controls via AKAP6 non-centrosomal microtubule organizing center formation at the nuclear envelope" for consideration by The Reviewing Editor has drafted this to help you prepare a revised submission.Suggested revisions:The paper is very much improved and you have addressed all the remaining points raised by the reviewers. I only have a couple of suggestions before formal acceptance.eLife in the introduction and the result, where you describe identification of AKAP6. I would include statements referring to this work.1) It seems odd to not mention your recent work on AKAP6 published in 2) You showed that recombinant nesprin and AKAP6 expression is sufficient to recruit MTOC protein to the NE, but your new data now show that this is not sufficient to generate an active MTOC. In my opinion this finding is very important and deserves a discussion. It suggests that myogenin induces another factor that is crucial for MTOC activity. While you correctly state "recruitment of MTOC proteins" rather than "MTOC formation" in the paper, non-expert readers may assume that this is synonymous. Without clarification/discussion this could be misleading. Overall, the manuscript presents interesting novel findings regarding the regulation of ncMTOC assembly at the nuclear envelope and most experiments appear well performed and presented. However, although the study identifies AKAP6 as a novel ncMTOC factor, it does not provide sufficient mechanistic insight. Is AKAP6 merely a bridge between Nesprin and AKAP450? What does it interact with, what other centrosome proteins are involved? How is its localization regulated to allow for the timely activation of the nucleus as an MTOC? Is AKAP6 sufficient to induce MTOC function? Considering these and other issues raised by all three reviewers, we feel that the manuscript does not reach the level of insight and impact that we seek for eLife at this stage. However, if the authors are able to address all of the raised issues, we would happily reconsider a new manuscript and review it in relation to these reviewer comments.As discussed with the editor, several of the raised issues have been addressed in the companion paper, which has recently been accepted for publication.While the companion paper focussed on the elucidation of the role of AKAP6 as a critical player for ncMTOC formation in cardiomyocytes and osteoclasts, the focus of this manuscript is to elucidate the initiation of ncMTOC formation in vertebrate cells.eLife.In summary, the present manuscript together with the companion paper identify AKAP6 as a critical player for ncMTOC formation in the three vertebrate cell types known so far to have a nuclear envelope MTOC: skeletal muscle cells, cardiomyocytes, and osteoclasts. We illuminate the upstream regulation of ncMTOC formation, showing that a single transcription factor can induce the attenuation of the centrosomal MTOC and formation of the ncMTOC at the nuclear envelope. We further give mechanistic insight into how AKAP6 functions as a critical adaptor between nesprin-1 and centrosomal proteins to create a ncMTOC and to localize the Golgi in close vicinity to the nuclear envelope. Furthermore, both manuscripts show the functional significance of AKAP6 for microtubule- or Golgi-dependent processes in skeletal muscle cells, cardiomyocytes, and osteoclasts. Due to the different experimental approaches and model systems as well as the large variety of addressed points/questions, we strongly believe that both manuscripts deserve publication in Reviewer #1:[…] Overall, the study presents interesting novel findings, but lacks analytical depth regarding the underlying molecular mechanism. Also, several findings require more solid quantitative analysis. In particular, the part investigating attenuation of cancer centrosomes is quite weak in this regard.1) I am surprised that the authors seem to observe only nuclei that are either positive or negative for MTOC markers. Is this process (redistribution of centrosome proteins) not gradual? I would suggest a different way of quantifying this process, to account for the temporal aspect and include intermediate stages.We thank the reviewer for indicating this point. In our C2C12 experiments, <2% of total PCM1-positive nuclei showed clear intermediate stages and have therefore been included in the total percentage of PCM-1-positive nuclei. To clarify this issue, we included this information in the text and provide a Supplementary Figure with intermediate stages of PCM-1 redistribution . For pericentrin and AKAP9 in C2C12 we have manually distinguished between partially and fully positive nuclei, as an automated analysis was not possible due to high cell density required for C2C12 differentiation. However, an automated analysis was performed for ectopic myogenin expression experiments in NIH3T3 cells, which could be grown at lower density. In this case nuclear envelope coverage by centrosomal proteins was quantified to account for the temporal aspect of recruitment .2) Related to point 1: after myogenin expression, is the centrosomal MTOC inactivated first, after, or simultaneously with the appearance of the nuclear envelope MTOC?Our new data provided in Figure 2 and 3 indicate that attenuation of the centrosomal MTOC and activation of the ncMTOC occur simultaneously. In order to clarify this issue, we created a cell line with tetracyclineinducible myogenin, which yields a high number of cells that form an ncMTOC and therefore allows reliable quantification. We quantified ncMTOC formation  and centrosome attenuation  upon myogenin expression. Furthermore, microtubule regrowth experiments showed that, while centrosomes are attenuated, microtubules still regrow from centrioles alongside growth from the nuclear envelope . In addition, -tubulin signal intensity at centrioles was quantified.Are centrosomes in 2G active or inactive?As described under point 2a, our analysis indicates that centrosomes are attenuated upon myogenin expression but retain some microtubule nucleating activity.Do 100% of cells look like the two examples in 2G?To address this issue, we have quantified the nuclear coverage by centrosomal proteins as well as their intensity at the centrosome upon myogenin expression and displayed the results as violin plots to visualize frequency distributions in Figure 2 and Figure 3.3) The attenuation of the centrosomal MTOC should be quantified upon myogenin expression should be quantified. PCM1 is considered a centriolar satellite marker. The authors should use additionally centrosomal markers such as pericentrin and γ-tubulin to evaluate the state of the centrosomal MTOC.We thank the reviewer for these suggestions and have now quantified centrosome attenuation in Figure 3 based on pericentrin and -tubulin.4) 3D: the spatial distribution is not very clear from the image. A larger area of the nuclear envelope should be shown and some type of quantification (e.g. intensity profiles) that reveals the difference between nesprin and AKAP6. Are the locations of antibody epitopes (N-term. vs C-term.) consistent with the authors' interpretation?As requested, we provide quantifications and better images of a larger area of the nuclear envelope . The antibody epitopes do not allow a conclusion in regards to our interpretation. The nesprin-1 antibody was raised against the full-length nesprin-1 and a few amino acids of nesprin-1 . However, its epitope could not be mapped so far .Locations of the antibody epitopes:st spectrin domain nesprin-1 : epitope has not been mappedAKAP6 : 752-891 of 2319 aa total; 15) 4C: a different centrosomal marker that is not affected by myogenin is needed, to identify the presence of centrosomes/centrioles in all cells. Also, PCM1 is a satellite marker. The disappearance of satellites may or may not indicate changes in centrosome staining. Moreover, as the assembly of the ncMTOC, the disassembly of the centrosomal MTOC needs to be quantified.To address this issue, we now included -tubulin in all figures regarding centrosome activity. While -tubulin levels at centrosomes were affected by myogenin expression, the signal-to-noise ratio of -tubulin was still better than those of centriole markers like Cep135 and Centrin in our system and therefore allowed a reliable identification of centrosomes.6) Figure 6: as before, other centrosome markers (including one that is unaffected) are needed to evaluate the state of the centrosomal MTOC. Higher magnifications are also required in particular of the centrosome region.7) The experiments in figure 6 are not convincing for claiming that centrosome attenuation can be induced in cancer cells. First, the quality of the provided data is simply not enough. Is the amount of PCM reduced? Is the activity reduced? Second, after fusion they are technically not cancer cells anymore.Due to the extended critique on Figure 6 from all three reviewers, we will omit the current Figure 6. Instead we focused on providing more data on the initiation of ncMTOC formation.8) An important experiment would be to test whether nesprin-1alpha and AKAP6 ectopic expression can induce MTOC assembly at the nuclear envelope. If so, this may allow more detailed mechanistic studies (expression of mutants etc), to provide more depth to the study.Previously, it has been reported that overexpression of nesprin-1 is sufficient in myoblasts to recruit the PACT domain and small amounts of endogenous PCM-1 . However, this recruitment was inefficient.To address the raised issue, we ectopically expressed nesprin-1 and AKAP6 in undifferentiated C2C12 myoblast and observed that nesprin-1 and AKAP6 are sufficient to recruit endogenous PCM-1 to the nuclear envelope. A more detailed study of AKAP6 domains and mutants is part of our recently accepted manuscript. The focus of the present manuscript was the upstream regulation of ncMTOC induction, where we show that myogenin preferentially activates the promoters of nesprin-1 and AKAP6 which in turn recruit centrosomal proteins and enable microtubule nucleation from the nuclear envelope.Reviewer #2:Microtubule organization varies by cell type and function. Dividing cells, cancer cells, and some specialized cell types such as fibroblasts use centrosomes as microtubule organizing centers (MTOCs), but most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to obtain more complicated microtubule patterns during the process of differentiation. Despite decades worth of work on the centrosome, comparatively little is known about ncMTOC composition or how the cell regulates the switch in MTOC function from the centrosome to non-centrosomal sites during differentiation. Here Becker et al., use differentiated muscle cells as a model to understand how the well-defined nuclear ncMTOC, which is a hallmark of skeletal muscle, is established. Previous studies found that PCM proteins such as AKAP450, Pericentrin, and PCM1 localize to the nuclear envelope of muscle cells, that Nesprin1a recruits these proteins, and that Nesprin and AKA450 are required for microtubule nucleation at this site. Here Becker et al., appear to uncouple the expression of Nesprin and PCM; Nesprin and PCM1 are both expressed in cells positive for expression of the transcription factor MyoD, but Nesprin expression precedes expression of the transcription factor myogenin, and PCM1 but not Nesprin expression is controlled by myogenin. These results suggested that activation of MTOC function at the nucleus is downstream of Nesprin localizing there and requires expression of myogenin targets. Consistently, myogenin expression in naïve cells is sufficient to induce nuclear MTOC activation. By mining ChIP-seq, RNAseq and microarray data, the authors identify AKAP6 as this potential target, finding that AKAP6 localizes to the cytoplasmic face of the nucleus, fails to localize to the nucleus in SYNE1/Nesprin mutant patient derived myoblasts, and is required to localize PCM1, AKAP450, and Pericentrin to the nuclear envelope. AKAP6 also is required for microtubule regrowth from the nucleus of myotubes and for nuclear positioning in these cells, a MT controlled process. Finally, the authors find that fusion of a Myogenin induce muscle cell can convert a nive NIH3T# cells with a centrosome MTOC to a nuclear MTOC state. While I love a cell fusion experiment, this result is not surprising given that fusion of a myoblast to a U2OS cell can similar convert the non-muscle cell from a centrosome to a nuclear MTOC .The experiments appear well preformed and presented. However, although these studies appear to identify a novel ncMTOC factor at the nucleus of muscle cells, they do little in the way of uncovering mechanistic aspects of how AKAP6 functions there or on assembly of the nuclear ncMTOC in these cells. Is AKAP6 merely a bridge between Nesprin and AKAP450? How is its localization regulated to allow for the timely activation of the nucleus as an MTOC? Is AKAP6 sufficient to induce MTOC function? In the absence of more mechanistic insight on the role of AKAP6, I am not sure that this paper will be of broad enough interest for the readership of eLife.Drosophila identifying the transcription factor trachealess to be required for ncMTOC formation . The major finding of our study is the identification of the first transcription factor sufficient for induction of an ncMTOC and centrosomal MTOC attenuation.We thank the reviewer for this feedback. Please note, that mechanistic details on the role of AKAP6 at the ncMTOC has been elucidated in the companion paper, which has just been published. The focus of this manuscript is to elucidate the initiation of ncMTOC formation in vertebrate cells, as mechanisms that initiate the switch from centrosomal to non-centrosomal MTOCs during differentiation of vertebrate cells are unknown. The only mechanistic insight into ncMTOC induction has been gained by studying The findings here do little in the way of uncovering how AKAP6 functions at the nucleus in ncMTOC formation. This could be addressed in many ways including:– Does AKAP6 bind to Nesprin and AKAP450 (or other MTOC components) to create a bridge?This is not the scope of this manuscript and has been addressed in detail in the published companion manuscript.If so, how is this association regulated? Limited by expression of AKAP6?In this manuscript, we demonstrate by several experiments that AKAP6 expression is required for ncMTOC formation. In addition, we show that myogenin directly regulates AKAP6 expression. Finally, we also demonstrate that overexpression of AKAP6 and nesprin-1 is sufficient to recuirt endogenous centrosomal proteins.– Is AKAP6 localization sufficient to promote MTOC activity? This could be addressed by artificially tethering AKAP6 to the plasma or mitochondrial membrane and seeing if MTOC proteins and microtubules are recruited. If this worked, the ability of such a structure to compete with the centrosome could also be directly addressed.This is not the scope of this manuscript and has been addressed in detail in the published companion manuscript. Here, we show that overexpression of AKAP6 and nesprin-1 is sufficient to recuirt endogenous centrosomal proteins. Yet, the more important finding is that myogenin expression in fibroblasts was sufficient to initiate the formation of a functional ncMTOC.The authors repeatedly claim that 'Myogenin is required for centrosomal protein recruitment to the nuclear envelope', but they really only look at PCM1 localization in most cases. Why not look at AKAP450? This is especially important when arguing that Myogenein controls MTOC formation downstream of Nesprin localization to the nucleus.We thank the reviewer for this suggestion. In order to substantiate our conclusions, we expanded our analysis by quantifying pericentrin, AKAP9, and/or microtubule growth in myogenin-depleted C2C12 cells as well as myogenin overexpression cells.If Nesprin expression is not controlled by myogenin, in naïve cells in which myogenin overexpressed, how does Nesprin become expressed and localized to the nucleus? The authors briefly comment on this, but I find their explanation unsatisfying especially given what they are arguing in Figure 1.To clarify this issue, we performed chromatin immunoprecipitation and luciferase experiments, which demonstrate that myogenin binds and activates the nesprin-1 promoter in NIH3T3 fibroblasts.The fusion experiment is cool and suggests that the nuclear MTOC is dominant to a centrosomal MTOC in interphase cells. However, almost the exact experiment has been done but is not cited  Similarly, although less directly relevant, dominance was tested between a mitotic centrosome MTOC and a membrane ncMTOC .We thank the reviewer for pointing out the missing references to existing literature and apologize for it. Due to the extended critique on Figure 6 from all three reviewers, we will omit the current Figure 6. Instead we focused on providing more data on the initiation of ncMTOC formation.– Line 49: PCM needs to be defined as material that is specific to the centrosome. PCM proteins might localize to other locations, but 'pericentriolar' refers to the material surrounding centrioles.The text will be changed as requested.– The authors interchange usage of expression and localization.The text will be adjusted.Reviewer #3:The manuscript by Becker et al. focusses on the regulation of non-centrosomal microtubule organizing centers (ncMTOCs), specifically MTOC activity at the surface of the nucleus. The authors show that during muscle development, the MTOC activity transitions from the centrosome to the nucleus, and that this transition depends on the transcriptional activation of AKAP6 by myogenin, a TF that drives muscle differentiation. While it seems that several results can be found in the literature , the authors assemble them together well in a single manuscript to better link muscle differentiation to ncMTOC formation. Overall I found the study very interesting and most of the conclusions/model supported by the data, although we seem to disagree on direct vs indirect links with APAP6. Here are my comments and suggestions.A) Figure 1 is critical for setting up the order of events during differentiation. While the final conclusion of this section is correct, following the data in this section was extremely difficult. I had to read this section 4 times as I knew the data was in there somewhere, I just had to find it. I recommend replacing C and E with 4 graphs that show: 1) the % of the MyoD+ nuclei that are positive for PCM1, Myog, nesprin; 2) the % of PCM1+ nuclei that are positive for Myog, MoyD, nesprin; 3) the % of Myog+ nuclei that are positive for PCM1, MyoD, nesprin; 4) the % of nesprin+ nuclei that are positive for PCM1, MyoD, Myog. Then the description in the text should match these graphs.We changed Figure 1 and the corresponding section in the manuscript to clarify this issue. While we did not include all four suggested graphs, the new Figure 1 shows the percentage of C2C12 nuclei positive for MyoD, myogenin, nesprin-1, and PCM-1. In addition, the percentage of nesprin1-positive nuclei that are positive for MyoD and myogenin is shown and the text has been changed to clarify that always 100% of PCM-1-positive nuclei were postivie for MyoD, myogenin, and nesprin-1.B) Related to the use of PCM-1 as the main PCM marker and an indicator of MTOC activity. I always thought of PCM-1 as a centrosome satellite component, which has little direct function on centrosome MTOC activity. In fact, in the original paper describing PCM-1  it is shown to leave centrosomes in the lead up to, and throughout mitosis, when the centrosome is at its highest MTOC activity. PCM-1's role in ncMTOC activity might be better understood, but ultimately γ-tubulin (g-tub) must be recruited to the site of nucleation. Thus, it would be nice to see g-tub and MTs in several experiments such as 1D, 2A-F, 3E, 4F.The reviewer is right in the point that the amount of PCM-1 at the centrosome also changes during the normal cell cycle. The analysis of PCM-1 was chosen, as it is an integral part of centriolar satellites that play an important role in the transport of centrosomal proteins and centrosome assembly . For example, it has been shown that PCM-1 depletion impairs recruitment of ninein and Pcnt to the centrosome and disturbs microtubule organization .To address this issue, we included the information regarding PCM-1 in the text and expanded our analysis by analyzing microtubule regrowth as well as the centrosomal proteins pericentrin and AKAP9, the latter of which has been shown to be essential for microtubule nucleation in muscle cells .C) The model presented is that myognin binds AKAP6 promoter, increases expression, which drives AKAP6 binding to nesprin and triggers downstream PCM recruitment to the nuclear surface. While the order of events is nicely shown, a direct link upstream and downstream of AKAP6 is not. Many additional experiments must be performed to claim a direct role as shown in Figure 7. For example: mutating AKAP6 promoter/Using chromatin immunoprecipitation and luciferase assays we added data to show that myogenin preferentially binds and activates the promoter of the muscle-specific -isoform of AKAP6.Bypassing myog by overexpressing AKPA6, showing nuclear MTOCs when nesprin and AKAP6 are coexpressed in fibroblasts.To address the raised issue, we ectopically expressed nesprin-1 and AKAP6 in undifferentiated C2C12 myoblast and observed that nesprin-1 and AKAP6 are sufficient to recruit endogenous PCM-1 to the nuclear envelope. Additionally, we show that PCM-1 co-immunoprecipiates with AKAP6. Together with the finding that depletion of AKAP6 results in a loss of centrosomal proteins from the nuclear envelope while nesprin-1 remains unaffected, this indicates that AKAP6 acts as an adapter between the nuclear envelope anchor nesprin-1 and centrosomal proteins.Bypassing nesprin by artificially tethering AKAP6 to nuclear envelope, mapping AKAP6 binding to PCM proteins and mutating that interaction site, and so on.This issues are beyond the scope of this manuscript and have been addressed in the published companion manuscript.If the reviewers and editor(s) feel this is beyond the scope of the manuscript, I suggest that the authors explicitly state, and show in the model, that indirect paths are possible/likely.We adjusted Figure 8  to fit our new findings.D) It is not clear, given the similarity in steady state MTs between control and si-AKAP6, why nuclear positioning is different. The manuscript would be strengthened if it ended with the role of ncMTOCs in nuclear spacing instead of the current figure 6, which I think is not necessary and should be removed.As suggested, we removed the former Figure 6.The focus of the manuscript is the initiation of ncMTOC. Therefore, the manuscript end now on demonstrating that myogenin binds and activates the promoters of AKAP6 and nesprin-1a and that overexpresiion of AKAP6 and nesprin-1a is sufficient to induce the recruitment of endogenous centrosomal proteins to the nuclear envelope. Nuclear positioning was mainly utilized to validate the importance of AKAP6 for ncMTOC function. In order to address the raised questions regarding nuclear positioning, live cell imaging needs to be established which is beyond the scope of this study. Yet, we have added several data to provide further insides into the role of AKAP6 in nuclear positioning (see answers below).Can the authors follow MT nucleation and nuclear positioning during the development of myotubes in control and si-AKAP6?To understand the role of AKAP6 in nuclear positioning, we have depleted AKAP6 in myotubes and performed microtubule regrowth assay. Our data show, that AKAP6 depletion abolished microtubule growth from the nuclear envelope and resulted consequently in defects in nuclear positioning.Are the MTs more dynamic or more stable with or without nuclear MTOCs?To address this issue we have assessed the intensity of detyronisated microtubules a marker for stable microtubules. Our data reveal that AKAP depletion resulted in a reduction of detyronisated microtubules.What are the difference between myotubes with aligned nuclei and overlapping nuclei in the si-AKAP6 populations? Are the ones that end up overlapping start out too close to one another and therefore require ncMTOC activity to push the nuclei apart? Can the authors provide any further insight here?Early nucleus positioning in myotubes after fusion can be divided in two sub-processes. First, nuclei that cluster in the center of the newly formed myotube are pushed apart to spread along the length of the myotube.This process is dependent on AKAP9-mediated microtubule nucleation . Second, nuclei are aligned on the long axis of the cell. This process is mediated by microtubule-associated motors that are at least in part recruited to the nuclear envelope via PCM1 . AKAP6 is potentially required for both processes by recruiting AKAP9 as well as PCM1 to the nuclear envelope. We quantified defects in nuclei spreading (i.e. overlapping nuclei) and nuclei alignment separately to assess the effect of AKAP6 depletion on each process.To clarify this issue, we included a more detailed description in the text and a model of how AKAP6 depletion affects nuclear positioning in Figure 6. In addition, we confirmed that AKAP6 depletion impaired nuclear envelope localization of the dynein activator p150glued to the nuclear envelope in myotubes.Essential revisions:1) The authors should clarify the relative regulation of MTOC activities. Centrosome inactivation may be due to factors that remove microtubule organizing capacity. Alternatively, the nuclear envelope MTOC could outcompete the centrosome by recruiting MTOC components. In cases where the nuclear envelope MTOC is perturbed, does the centrosome remain active?Thank you very much for raising this important point. As detailed below, we have performed the suggested experiments to clarify the regulation of MTOC activity at the nuclear envelope and the centrosome and added a paragraph to the discussion (starting at line 553).Does the centrosomal pool of PCM-1 remain prominent upon myogenin knockdown?To address this issue, we have analyzed PCM-1 staining patterns in myogenin-depleted differentiating C2C12. The results are now described in lines 146-155 and representative images are shown in Figure 1—figure supplement 2. Importantly, in myogenin-depleted cultures, PCM-1 remains localized in a centrosomal pattern in cells with highly nesprin-1-positive nuclei. This suggests that endogenous myogenin contributes to the attenuation of the centrosome during NE-MTOC formation, which is in agreement with the observation that ectopic myogenin expression in fibroblast is sufficient to attenuate the centrosome.After SYNE1 or AKAP6 knockdown, what is the state of the centrosome?In order to clarify whether myogenin induces loss of MTOC proteins from the centrosome by establishing an NE-MTOC that outcompetes the centrosome or induces separate factors to attenuate the centrosome, we have depleted nesprin-1 or Akap6 in MYOG-mScarlet cells and analyzed pericentrin as well as γ-tubulin intensities at the centrosome. The analysis is now described in lines 393-399 and shown in Figure 5—figure supplement 2, revealing that levels of MTOC proteins at the centrosome are still reduced upon MYOG expression in nesprin-1- or AKAP6-depleted cells, meaning disturbing of the NE-MTOC does not prevent centrosome attenuation. These results indicate that competition between centrosome and NE-MTOC is not the mechanism underlying the attenuation of the centrosome during NE-MTOC formation.2) Since they are at the core of the regulation mechanism, the IP/CHIP experiments need extra controls. The authors should probe for a protein that is not specifically retained by the precipitating antibody.Syne1 or Akap6. To address this issue, we have included an intronic region of Syne1 or Akap6 as negative control as well as a promoter region of Desmin as positive control for the specificity of the myogenin antibody in the ChIP experiments. Additionally, we have probed lysates of Tet-ON mScarlet cells as well as non-induced Tet-ON MYOG-mScarlet cells. The results of these new experiments, presented in Figure 2 – supplemental figure 2 and Figure 4 – supplemental figure 1 and described in lines 242-251 and lines 345-349, further show that myogenin binds the promoter regions of Syne1 and Akap6.We are not sure what experiment the reviewer suggests. We assume that the reviewer would like additional controls regarding the specificity of the anti-myogenin antibody or other data that substantiate our conclusion that myogenin binds to the promoter regions of 3) What are the relative contributions of myoD versus myogenin in the regulation of MTOC assembly at the nuclear envelope? Experiments should be conducted to address: is myoD expression affected in the absence of myogenin?Myod RNA levels. As positive control, we show that depletion of MyoD also reduces myogenin levels, which is consistent with literature as well as our findings that MyoD induces myogenin expression in fibroblasts. These results have been added as panel C to Figure 2 – supplemental figure 1 and described in lines 196-202.To clarify this point, we have analyzed MyoD expression in myogenin-depleted C2C12 cells using RT-PCR and found that myogenin depletion does not affect Does myoD expression also induce nesprin and AKAP6?Analysis of MyoD-GFP-transfected NIH3T3 fibroblasts by immunostaining revealed that nesprin-1α and AKAP6 expression is induced . This is in accordance with our previous finding that overexpression of MyoD in this cell line induces myogenin expression as well as PCM-1 recruitment to the nuclear envelope . In order to address the relevance of myogenin in this system, we have repeated the experiments in the presence of siRNA targeting myogenin. Our results show that MyoD can induce AKAP6 expression via myogenin (see lines 409-422).In addition to adding new data to the manuscript, we have included a paragraph in the Discussion regarding the relative contributions of MyoD versus myogenin in the regulation of MTOC assembly at the nuclear envelope (lines 544-552). Furthermore, we adapted the model in Figure 8.4) Please provide a zero time point in regrowth experiments and check nucleation activity in the experiment in Figure 7G. This is needed to argue that an MTOC is present.Please note, we did not claim that an MTOC is present in Figure 7G but that nesprin-1α and AKAP6β are sufficient to recruit endogenous PCM-1 in the absence of myogenin. However, based on this question, we have now performed regrowth assays in myogenin-negative C2C12 cells after nesprin-1α and AKAP6β co-expression. Our results indicate that there is no significant NE-MTOC activity induced in this setting, whereas MTOC activity was observed at centrosomes. This is in agreement with the fact that myogenin appears to induce centrosome attenuation independent of the presence of nesprin-1α and AKAP6β at the nuclear envelope (see Point 1). The new data are shown in Figure 7 – supplement 1 and described in the text starting at line 497. For regrowth experiments, we have added “zero time points” in Figure 2 – supplemental figure 3.5) The introduction and discussion should be improved by discussing:– The relevance of the presented findings for muscle function and disease.As requested, we have added information in regards to the relevance of the NE-MTOC and our data in muscle function and disease to the Introduction (lines 68-77) and Discussion (lines 580-596).– The roles of the particular centrosomal proteins that are recruited to the nuclear envelope in generating and organizing microtubules.Information regarding the roles of the different MTOC proteins has been added to the Introduction (lines 104-117).– The term "centrosomal proteins". As they shuttle from centrosome to the nuclear envelope, the term "MTOC proteins" or similar may be more appropriate.As suggested, the term “centrosomal proteins” has been replaced with “MTOC proteins” throughout the manuscript.Reviewer #3:The manuscript by Becker et al. is a new submission of previously eLife submission that focusses on the transition from centrosomal MTOCs to non-centrosomal MTOCs, specifically the surface of the nucleus. This submission more clearly focuses on triggering ncMTOC transition in developing skeletal muscles via the function of MyoD and Myogenin, members of the transcription factors family termed myogenic regulatory factors (MRFs). The final model shown in Figure 8 is well supported by the presented data, which I believe is much improved over the previous manuscript. Reading the recently published work from the same group (Vergarajauregui 2020), which appears to have been in review at the same time as the original submission of this manuscript, helped clarify some of the interaction and functional claims downstream of AKAP6. The authors have done a great job addressing all my previous concerns, they should be commended for this and my comments below are mostly small changes that do not move the goal line for the authors.1) In several cases in the beginning of the manuscript (ex. First paragraph pf the results) it was not clear that ncMTOC specifically referred to nuclear envelope MTOCs. I would recommend explicitly stating in this first result section that throughout the manuscript, ncMTOCs refers to the nuclear envelope in this study. Alternatively, maybe the authors can use NE-MTOC to distinguish it from other ncMTOCs.Thank you for this recommendation. We have replaced as suggested “ncMTOC” with “NE-MTOC” in the manuscript where appropriate.2) On line ~181 the authors state that "both MRFs are sufficient individually to induce a switch from centrosomal MTOC to a nuclear envelope MTOC." At this point in the manuscript the authors had not yet shown the elimination of centrosomes, that comes in figure 3. I suggest removing a reference to a switch at this point.The text has been changed accordingly.3) In several location in the manuscript the author state that myogenin is required to recruit centrosomal proteins. This can be found in lines 193 and 570 and other places. It is more accurate to say that myogenin is require for the localization of centrosome proteins to the nuclear envelope. Recruitment implies myogenin is the recruiting protein, which it is not, it is simply upstream in the ncMTOC formation pathway.Thank you for pointing out this issue. The text has been changed accordingly.4) The figures overall are much improved and the quantification is suitable. However, It is important to report the Ns in each of the experiments. For example in figure 3, how many nuclei were measured? How many experimental repeats were performed.The requested information has been added.5) Presentation of the analysis leading up to AKAP6 was greatThank you.6) How reliable is the specificity of the E-Box PCR amplification? Have the authors confirmed specificity of the ChIP experiment by PCR amplifying other regions of the AKAP and nesprin promotors? Although, it does appear to be specific enough to distinguish the different isoforms.To assess the specificity of the ChIP experiments, we have performed additional experiments. For details, see response to essential revisions, point 3.7) Direct protein interaction cannot be determined by IPs. The authors should change the conclusion that "AKAP6 localizes to the nuclear envelope in differentiated skeletal muscle cells via interaction with nesprin-1α" to AKAP6 localization depends on nesprin-1a. An interaction can only be determine using purified proteins or Y2H as the authors did with AKAP6 and PACT in the (Vergarajauregui 2020). If there is direct protein interaction data, the authors should present it, otherwise the current results should be presented in terms of being upstream or downstream.We agree with the reviewer and have changed the text accordingly.Another example of this is "AKAP6 is required for the localization of centrosomal proteins to the nuclear envelope, in part by interacting with PCM-1." If there is no direct protein interaction data available (here or in the literature), then the word interacting should be replaced by recruiting.The text has been modified accordingly.Suggested revisions:The paper is very much improved and you have addressed all the remaining points raised by the reviewers. I only have a couple of suggestions before formal acceptance.1) It seems odd to not mention your recent work on AKAP6 published in eLife in the introduction and the result, where you describe identification of AKAP6. I would include statements referring to this work.eLife paper on AKAP6 in the Introduction and Results (lines 117-122 and 342-344).We included information about our recent 2) You showed that recombinant nesprin and AKAP6 expression is sufficient to recruit MTOC protein to the NE, but your new data now show that this is not sufficient to generate an active MTOC. In my opinion this finding is very important and deserves a discussion. It suggests that myogenin induces another factor that is crucial for MTOC activity. While you correctly state "recruitment of MTOC proteins" rather than "MTOC formation" in the paper, non-expert readers may assume that this is synonymous. Without clarification/discussion this could be misleading.We added a paragraph to the Discussion to clarify the results of the AKAP6β/nesprin‑1α co-expression experiment (lines 586-597).