Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72 Microtubules (MTs) are cylindrical polymers composed of tubulin, a heterodimer of α- and β-tubulin, with β-tubulin facing the more dynamic MT plus end and α-tubulin the less dynamic MT minus end . One facKnop and Schiebel, 1997Stu2, as well as XMAP215/ZYG-9/Dis1/Alp14/Msps/ch, belongs to a family of proteins that contain TOG (Tumour Overexpressed Gene) domains  and function as MT polymerases. They localize to both the plus and the minus end of MTs . The twoCaenorhabditis elegans SPD-5 or the budding yeast γ-TuSC receptor protein Spc72 target TOG domain proteins to sites of MT nucleation at the centrosome and the SPB . These proteins together concentrate α/β-tubulin, leading to MT nucleation in form of asters, without the action of γ-tubulin (Proteins such as the human ‘Transforming Acidic Coiled Coil’ (TACC) protein CDK5RAP2, the  the SPB . In vitr the SPB . Just re the SPB . Further-tubulin . The eme-tubulin ?Saccharomyces cerevisiae SPB provides an ideal system to address these questions because it has spatially distinct MT nucleation sites at the SPB. The SPB is embedded in the nuclear envelope and only the cytoplasmic side recruits Stu2 through binding to the γ-TuSC receptor protein Spc72  in vivo. Furthermore, in vitro reconstitution of MT assembly indicated that the purified Stu2–Spc72 complex was already sufficient to nucleate MT asters. Addition of recombinant γ-TuSC to the Spc72–Stu2 complex promoted γ-TuSC oligomerization into a functional MT nucleation template independent of the TOG domains of Stu2. However, efficient MT nucleation from these Spc72–Stu2–γ-TuSC complexes required the action of the TOG domains. The reason could be that the TOG domains of Stu2 facilitate binding of the first α/β-tubulin subunits to the γ-tubulin template. Complementary in vivo studies supported the role of Stu2 in MT nucleation. The depletion of γ-tubulin Tub4 did not inhibit cytoplasmic MT (cMT) nucleation, but these MTs assembled in the cytoplasm away from the SPB and were strictly dependent on Stu2. Together, this study illustrates specific roles for Stu2 and γ-TuSC in MT nucleation and in the maintenance of the γ-TuSC oligomer at the cMT minus end.Drosophila CNN and human CDK5RAP2   . spc72CMM1 motif .Candida albicans Spc110 or human CDK5RAP2 impair binding to the γ-tubulin complexes  and a Stu2-binding deficient mutant . In pulldown experiments, γ-TuSC showed reduced binding to Spc72NCM1 in comparison to Spc72N or Spc72N∆Stu2 (d) of the Spc72N–γ-TuSC interaction with the high value of 1.1 µM, which reflects a relatively low binding affinity  showed reduced binding affinity (Amino acid substitutions in CM1 of omplexes . To testaffinity . The Spcis assay . Importais assay . Also inaffinity . In concd) of Spc72 for γ-TuSC raises the question of how this receptor recruits the γ-TuSC to the SPB efficiently. Stu2, which interacts with Spc72, could mediate the binding of γ-TuSC to Spc72. To test this notion, we quantified the interactions between Spc72 and Stu2, as well as the Spc72–Stu2 complex and the interaction with γ-TuSC. The requirement for the last 33 aa of Stu2 (d of the Spc72N and Stu2 interaction as 115 nM (∆Stu2 showed 23-fold reduced binding to Stu2 (∆C) did not interact with Spc72N . As expected, α/β-tubulin bound efficiently to the Stu2TOG construct  and theonstruct  but suchCM1  shifted a larger portion of the γ-TuSC into the Dextran blue marked void volume fractions than did Spc72N alone . Thus, the recombinant Stu2–Spc72WT complex predominately assembles MTs in the form of asters.Purified Spc72N did not have MT nucleation activity as it behaved similarly to the α/β-tubulin control , but the was low . The SpcWT alone , leadingWT alone . The astTOGAA protein failed to bind α/β-tubulin in gel filtration and pulldown assays  experiments. Spc72–yeGFP at detached cMTs showed no recovery after bleaching. By contrast, 58% of Stu2–yeGFP recovered with a half time of 3.3 s, indicating a dynamic and stable Stu2 pool . In spc7 kar1∆15 . In concSPC72 kar1∆15 cells  that was even further increased to 3.27 in ∆Stu2 kar1∆15spc72 cells -tag -tag . AdditioKar9–AID  and decrKar9–AID . By contKar9–AID . These died Tub4 . The norreported . This reKAR9–AID in combination with the ∆Stu2spc72 mutation in order to reduce Stu2 specifically at the minus end of cMTs. kar1∆15 cells were incubated with α-factor for 60 min simultaneously with the addition of IAA or the solvent control. ∆Stu2 KAR9–AID kar1∆15spc72 cells that were exposed to IAA showed a reduction in cMT numbers  in comparison to the SPC72 KAR9–AID kar1∆15 cells  to 25% within 60 min after the addition of IAA  failed to assemble cMTs  Stu2 is important for stabilization of the γ-TuSC oligomer at the minus end of detached cMTs . (2) StuThe functions of TOG domain proteins at the MT minus end could be structural or enzymatic, of general importance or context-specific. The ability to measure the binding affinities and MT nucleation activity of purified Spc72, Stu2 and γ-TuSC in vitro, in combination with the analysis of the function of these proteins in vivo, has now shed light on the role of Stu2 and its cooperation with the γ-TuSC during cMT nucleation.∆CC complex lacked MT nucleation activity , γ-TuSC dissociated from the minus end of detached cMTs  and are listed in 1 arrest, cells were treated with 10 μg/ml α-factor. For nocodazole arrest in SC-complete medium, cells were incubated with 1% peptone and 15 µg/ml nocodazole. For auxin-depletion experiments, proteins were tagged with 3HA-IAA and depleted with an IAA  concentration of 1 mM at 30°C. For drop tests, cells derived from S288C , rabbit anti-GFP , goat anti-GST , mouse anti-HA , rabbit anti-Spc97 , rabbit anti-Stu2 , goat anti-Tub4 , rabbit anti-Spc72  and mouse anti-tubulin . Secondary antibodies were donkey anti-rabbit IRDye 800CW , donkey anti-goat A680 , donkey anti-mouse A680 , goat anti-mouse HRP , rabbit anti-goat HRP , donkey anti-rabbit HRP  and goat anti-rabbit HRP .2) for 2 min and then washed twice with water. Cells were allowed to attach for 5 to 15 min, subsequently washed with water, and finally SC medium was added. For imaging, a DeltaVision RT system  equipped with a Photometrics CoolSnap HQ camera (Roper Scientific), a 100 × 1.4 NA Super–Plan Apochromat oil objective (Olympus), a four-color Standard InsightSSI module light source including a laser-based hardware autofocus, and a Workstation with a CentOS operating system and SoftWoRx software (Applied Precision Ltd.) was used. For photobleaching experiments, a 50 mW 488 nm laser system was used on the same microscope. For deconvolution of images, the softWoRx software package of DeltaVision was used. For live-cell imaging, the system was prewarmed to 30°C and imaging was done with binning 2 × 2. Image analysis was performed with ImageJ 1.46 r  and GraphPad Prism (GraphPad Software).Microscopy was performed in sterile filtered synthetic complete (SC) medium at 30°C. Log-phase cells were treated as annotated and immobilized on a glass bottom dish . In advance, the dish was coated with 100 μl of ConA .For line scans of detached cMTs of kar1Δ15 cells and CSE4-yeGFP Δste2 cells were α-factor treated and mixed. First, all 21 stacks of the yeGFP wavelength were acquired followed by all stacks of the second fluorescence signal in order to localize the yeGFP signal on the detached cMT. A reference image of an empty Matek dish was acquired with the imaging buffer and the same acquisition settings to correct for variations of the field illumination in the GFP-channel. After subtracting the reference image from all the GFP-stacks, the brightest signal intensity from single z-planes of the 21 stacks was analysed in ImageJ and corrected for the background close to the measured signal as published *e(–Kd*x)+Plateau (One-phase decay in GraphPad Prism). The constant Kd was extracted from the control measurement and the recovery curve was calculated with y=y0+(Plateau–y0)*(1-e–(K+Kd)x) (as in One-phase association in GraphPad Prism). The half-time was extracted from that curve with ln(2)/K.For FRAP experiments examining The γ-TuSC was purified as described by 2, 5% glycerol[(w/v]) together with one tablet of Protease Inhibitor Cocktail  and 1 mM DTT. Cells were lysed on ice with a Polytron  for 5 min at 4000 rpm. The lysate was incubated with 0.25% Brij35 for 10 min at 4°C and centrifuged at 4°C for 30 min at 15,000 rpm in a SS-34 rotor. The supernatant was bound to 500 mg equilibrated Ni-beads  for 2 hr at 4°C. Beads were washed four times with HB200, and proteins were eluted in 500 mM imidazole. The eluate was injected into an ÄKTA system , which was equilibrated with HB200 and purified through size exclusion chromatography  at 4°C. The fractions were analysed by SDS-PAGE and those containing Stu2 were pooled, concentrated and stored at −80°C. The bar graphs were designed with DOG1.0  overnight, diluted to OD600 0.2 in LB-Amp-Cam and incubated at 22°C for 1 hr at 100 rpm. Induction was carried with 200 µM IPTG for 20 hr at 22°C. Cell pellets of 1 l culture were harvested by centrifugation at 3000 g for 15 min and stored at −80°C. The pellet was resuspended in lysis buffer  together with one tablet of Protease Inhibitor Cocktail and 1 mM PMSF. Cells were lysed on ice by sonication (Bandelin Sonopuls GM 200 equipped with a Bandelin electronic UW200) with a KE76 tip five times for 30 s with 50% power. The lysate was centrifuged for 30 min at 4°C at 26,890 g in a SS-34 rotor. The supernatant was incubated with 750 μl equilibrated GST-beads  for 2 hr at 4°C. The beads were washed three times with lysis buffer and the protein was eluted with 30 mM L-glutathione in lysis buffer. The eluate was injected into an ÄKTA system  and purified through size-exclusion chromatography  at 4°C. The chromatography was done with TB150 . The fractions were analysed by SDS-PAGE and those containing Spc72 were pooled, concentrated and stored with 10% glycerol at −80°C.The purification of GST–Spc72N followed a variation of the protocol published by E. coli pellet of 1 l from GST–Spc72N-expressing cells was resuspended in HB200 together with one tablet of Protease Inhibitor Cocktail and 1 mM DTT. Lysis and centrifugation were carried out as for Spc72 purification. The supernatant containing GST–Spc72N was bound to equilibrated GST-beads for 2.5 hr at 4°C. Meanwhile, a pellet of 800 ml SF21 insect cells expressing 6His–Stu2 was resuspended in HB200 together with one tablet of Protease Inhibitor Cocktail and 1 mM DTT. Cells were lysed with the Polytron, 0.25% Brij35 was added for 10 min and the lysate was centrifuged as mentioned in Stu2 purification. The GST–Spc72N beads were washed five times with HB200 and the 6His–Stu2 lysate was added after dilution with HB200 containing 0.125% Brij35. After incubation overnight at 4°C, the beads were washed five times with HB200 containing 0.125% Brij35 and proteins were eluted with 50 mM L-glutathione in HB200 with 0.125% Brij35. The elutions were loaded on an ÄKTA system  with an HiLoad 16/60 Superdex 200 column , which was equilibrated with HB200 at 4°C. Gel filtration fractions were analysed by SDS-PAGE and those containing GST–Spc72N and 6His–Stu2 were pooled. The purified complexes were stored at −80°C with 0.125% Brij and 5% glycerol.An 1-220) and its mutant version Spc110NCM1 (GST–Spc1101-220-QA) were performed as described by Purification of the N-terminal version of Spc110N (GST–Spc110N2) and incubated with GST-beads for 30 min on ice. The supernatant was mixed with GST or GST–Spc72N to a final concentration of 2 μM for Stu2, 150 nM for γ-TuSC, 1 μM for GST and 1 μM for GST–Spc72N. The GST–Stu2TOG (aa 1–590 of Stu2) and GST–Stu2MT (aa 451–684 of Stu2) were purified as described previously  and transferred to a new tube. Beads were washed six times and the remaining buffer was removed with a squeezed pipette tip. Samples were incubated with sample buffer at 65°C for 5 min and analysed by immunoblotting.The pulldown protocol is a variation of a protocol published by eviously . Pig bra2, 1 mM EGTA), precleared by centrifugation in a S100AT3 rotor at 4°C for 5 min at 353,000 g, and half of the supernatant was incubated with Ni-beads (Qiagen) equilibrated with HB300  for 30 min at 4°C. The tubulin supernatant was taken without transferring any beads. 2 μM of Stu2 proteins  were incubated together with 2 μM tubulin from the supernatant on equilibrated Ni-beads in HB300 for 1 hr at 4°C. Beads were washed once with HB300 supplemented with 0.5% Nonidet P40 (Biochemica) and 20 mM imidazole, and transferred to a new tube. Then beads were washed two more times and the remaining buffer was removed with a squeezed pipette tip. Beads were treated with sample buffer at 65°C for 5 min and the supernatant was analysed by immunoblotting.Pig brain tubulin was diluted to 30 μM together with 30 μM nocodazole in BRB80 , centrifuged at 4°C for 5 min at 353,000 g in a S100AT3 rotor, and half of the supernatant was used for the experiments. Stu2  and tubulin were diluted together to 5 μM in HB100 supplemented with 10% glycerol and incubated for 40 min on ice. Size-exclusion chromatography was performed with a Superose six column  on an ÄKTA system . Fractions were treated with sample buffer and analysed by immunoblotting.For in vitro nucleation of MTs, unlabelled and Cy3-labelled (following protocol by CM1 or GST–Spc110N complex in 1:4 molar ratio (4.6 µM:18.6 µM) in HB100-50 buffer  with salt as indicated in the figure legends. After incubating for 15 min at 4°C, the protein mixture was applied to a gel filtration column . Elution profiles were recorded at absorbance at 280 nm. Proteins were detected by Coomassie Blue or immunoblotting. For WT or GST–Spc72N–6His–Stu2TOGAA to a final concentration of 4.6 µM for γ-TuSC and 2.3 µM for the other proteins in HB100 buffer with 10% glycerol. Proteins were incubated on ice for 40 min, centrifuged at 20,817 g for 10 min at 4°C and 50 µl were loaded on a gel filtration column (Superose 6 PC 3.2/30) equilibrated with HB100. Proteins were detected by immunoblotting. Electron microscopy analysis was performed as described by Purified γ-TuSC was mixed with GST–Spc72Nd calculation were performed with Nanotemper software. In all cases, MST data were fitted on the based of a single-site model.MST assays were carried out with a Monolith NT.115 instrument . Each titration curve consisted of 16 points prepared from a serial dilution of analytes and a constant concentration of the fluorescein-labelled ligand. Purified γ-TuSC and Stu2 were fluorescently labelled with GREEN-NHS . To measure the affinity between γ-TuSC and its binding partners, 2X serial diluted recombinant Spc110N, Spc72N, Stu2 or Spc72N-Stu2 complexes were titrated against 50 nM of labelled γ-TuSC. To measure the binding affinity between Stu2 and Spc72N, increasing concentrations of recombinant Spc72N were titrated against 150 nM of labelled Stu2. Experiments were carried out in HB100-50 buffer supplemented with 0.05% (w/v) Tween-20. The samples were loaded into standard glass capillaries . MST assays were performed with 60% and 80% LED power using a green filter and 40%, 60% and 80% MST power. The normalized fluorescence readings (Thermophoresis + T jump or T-jump only) were plotted as a function of analyte concentration and the curve fitting and dissociation constant KStatistical analysis of the data was carried out with GraphPad Prism and performed as two-tailed unpaired t-tests with a confidence interval of 95%. For MST measurements, the statistical analysis was done in Excel. The number of repeated experiments is indicated in the figure legend together with the sample size and the P-values. In the MST measurements, when the data points of a single concentration value were deviated from normal intensity range, they were considered as outliers and excluded from the analysis. Otherwise no data points were excluded. Experiments are technical replicates if not stated differently. The experiments were not randomized and the investigators were not blinded to allocation during the experiments and outcome assessment. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "TOG domain independent microtubule nucleation activity of Stu2 protein at centrosomes" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in As you will see from the comments below all three reviewers had significant concerns about controls, methods, and interpretations for both the biochemical and in vivo experiments. Together, this led them to a decision that this paper should be rejected.Reviewer #1:Gunzelmann et al., present a study on Stu2, a member of the TOG domain-containing microtubule (MT) polymerase family. Stu2 is well characterized as acting at MT plus ends. In this work the authors explore the role of Stu2 at the centrosome/ yeast spindle pole body (SPB), where Stu2 localizes in yeast cells. On the nuclear side of the nuclear membrane Spc110 serves as the Ɣ-TuSC receptor, while on the cytoplasmic face Spc72 is the Ɣ-TuSC receptor. This work presents the novel finding that Stu2 facilitates Spc72 binding to Ɣ-TuSC and possibly promotes Ɣ-TuSC oligomerization in vitro. The authors then look at Stu2 localization in vivo using mutants with detached MT minus ends. They find that Stu2 localizes to MT minus ends in the absence of the Stu2 binding site in Spc72 and that Kar9 plays a role in this recruitment. The authors then present additional in vitro experiments to look at microtubule aster formation. They suggest that Stu2 can promote MT aster formation in the absence of functional TOG domains in the presence of Spc72.eLife. I recommend that the authors revise their work based on the following comments to submit elsewhere.While this work contains several interesting observations, many experiments are lacking important controls to support the conclusions made. In addition, in its current form I do not see that it dramatically advances our understanding of TOG domain-containing proteins nor is it of sufficient quality for Essential revisions:1) Does Stu2 directly interact with Ɣ-TuSC? This should be addressed.2) The gel filtration experiment in Figure 2D is missing standards and the void volume is not labeled. How many times was this experiment repeated? This should be listed in the legend. What concentrations of proteins were used?3) Ɣ-TuSC oligomerization appears to be enhanced in the presence of Spc72N-Stu2TOGAA (defective in tubulin binding). This experiment should also be done with Spc72 and wildtype Stu2.3) The authors should quantify Stu2 fluorescence at the cMT minus end in the spc72Δstu2/Kar9-depletion conditions. Is Stu2 completely absent or are factors beyond Spc72/Kar9 also recruiting Stu2 to the minus end?4) As the authors currently present it, the mechanism of Stu2-dependent MT assembly is unclear. The experiments in Figures 6 and 7 are difficult to interpret because controls are lacking. For example, the Stu2 mutants alone were not analyzed in Figure 6. Is there any affect of Stu2 or the Stu2 mutants in the presence of Ɣ-TuSC, but in the absence of Spc72 ?5) The concentrations of tubulin, and all other proteins used in Figures 6 and 7 are not listed in the methods or legends. This is critical to include. Also, were different concentrations tested? How were the concentrations that were used chosen?6) It is unclear what distinct roles the authors propose for the MT-binding and TOG-binding domains of Stu2 to have in enhanced MT nucleation at the centrosome/ SPB. Are cells with Stu2 MT-binding and TOGAA mutations viable?7) The authors reference two "nucleation activities" of Stu2, but then posit that the TOG domains may be required for extending Ɣ-TuSC-nucleated seeds. This seems contradictory.8) The manuscript could benefit greatly from additional editing for clarity and conciseness. There are many grammatical errors as well. The Figure Legends need to be improved for clarity and completeness.Reviewer #2:eLife. The Dis1/Stu2/XMAP215 family of microtubule polymerases are complex, multi-domain proteins that play roles at microtubule plus ends as well as at microtubule organizing centers (MTOCs). While the family's conserved N-terminal tubulin-binding TOG domains play critical roles in microtubule polymerization, less is known about the C-terminal regions that engage MTOC components. Yeast members have significantly diverged from non-yeast members across their C-terminal region. In this manuscript, the authors focus on elucidating the role of Stu2 (the S. cerevisiae XMAP215 member) on microtubule nucleation and stability at the spindle pole body (SPB), and how it uses specific determinants to engage other factors and drive microtubule formation/stabilization. The authors show that Stu2 potentiates the interaction between Spc72 and γ-TuSC and in turn promotes γ-TuSC oligomerization which serves to template and nucleate microtubules. While Spc72 recruits Stu2 to the microtubule minus end, the authors identify Kar9 as another factor capable of recruiting Stu2 to the microtubule minus end. The authors probe the role of the Spc72-Stu2 and Kar9-Stu2 interactions on spindle morphology and microtubule stability and length in vivo and find that both sets of interactions play key roles. The authors then analyze the in vitro effects of Spc72 and Stu2 on microtubule nucleation in vitro with and without addition of the γ-TuSC complex and map key regions within Stu2 that are required for microtubule nucleation. The manuscript adds to our understanding of Stu2 and its role in yeast mitosis using a nice blend of in vitro and in vivo analyses. In its current state it lacks some critical experiments that, if present, would best unite disparate pieces in the paper, and help the authors arrive at some of their conclusions.This is a review of the article titled "TOG domain independent microtubule nucleation activity of Stu2 protein at centrosomes" by Gunzelmann et al., submitted to 1) Gel filtration is used to determine whether Spc72 (plus or minus Stu2) causes oligomerization of γ-TuSC, as does Spc110. The results show that Spc72 does shift γ-TuSC to the void volume, and that Stu2 enhances this shift. However, Stu2 also causes a new peak to arise at fraction A11 that could be accounted for by Stu2-mediated dimerization of a γ-TuSC-Spc72 complex. This then calls into question the increase of the γ-TuSC fraction in the void volume: is this really oligomerization as seen with Spc110 which generated γ-TuSC filaments, or is it due to cross-linking mediated by dimers of Spc72 and dimers of Stu2? To resolve this, the authors should determine if the Spc72-Stu2 complex does drive γ-TuSC oligomerization – this could be achieved using negative stain EM analysis, similar to work done by Kollman et al., 2010, DIC microscopy, or fluorescence microscopy.2) Figure 7, the authors should conduct a Stu2 WT +/- γ-TuSC control to best determine how much Spc72-Stu2 augments the MT nucleation activity in vitro.3) The authors delineated independent roles for Spc72 and Kar9 on Stu2 recruitment to the MT minus end, analyzed the effects of these respective complexes on cMT nucleation, number, and length in cells. However, when the authors then move to in vitro analyses, they concentrated exclusively on the Spc72-Stu2 interaction (plus and minus γ-TuSC) and its effects on microtubule nucleation in vitro. To be comprehensive, it would greatly strengthen the paper to conduct a comparative analysis of the effects of Kar9 and Kar9-Stu2 (plus and minus γ-TuSC) on MT nucleation in vitro with that of Spc72-Stu2.Reviewer #3:Review of Gunzelmann et al., TOG domain independent microtubule nucleation activity….This paper describes a novel nucleation activity within the spindle pole component, Spc72 that recruits Stu2 (TOG domain protein) to the cytoplasmic face of the spindle pole body, where it increases MT density and plays a role in nucleating MT asters.The binding data in Figure 1 and Figure 2 was very convincing. The data in Figure 3 were problematic. Firstly, they state that incubation in a kar1 mutant results in detached cMTS . I looked at both of these papers and was unable to find the percent of detached vs attached cytoplasmic microtubules in the mutant. It is important to quantitate what fraction of the microtubules are detached to differentiate pole binding from minus-end binding. An alternative interpretation for Figure 3c is that observation of Stu2 in the absence of the stu2 binding site in Spc72 is that these microtubules are not detached from the pole.The FRAP recovery are nicely done and convincing that there are multiple binding sites. It is interesting that in this and panel b the mt minus ends seem to be in the shmoo tip. Since there are known plus-end binding complexes in the shmoo tip, is there concern that the co-localization reflects the juxtaposition of the minus-end to the shmoo tip. This is easily addressed by quantitating the fraction of spc72 or stu2 spots in the tip vs. the presence or absence of mt minus ends.Figure 4D is a critical panel for interpretation of aforementioned experiments. It is stated that a-g are kar1Δ15. Approximately 50% of the cMT are attached in these mutants. Thus, the reviewer needs to redraw Figure 3a to reflect the fact that 50% are detached and 50% are released. In addition, the reviewer needs to include the attachment/detachment state of the microtubules for the co-localization. Are they only examining detached microtubules, if so, what is the metric for the data in Figure 3? The metric in Figure 4 is clear, mCherry-Tub1, but if 50% are attached, the data without Tub1 become significantly ambiguous.In summary, this paper documents an important second TOG-independent mechanism in Stu2 for MT formation. Stu2 has critical roles in spindle orientation and kinetochore function, and thus this second site is important to understand. The authors should ensure that for all reports of minus-end binding, that these are indeed detached cytoplasmic microtubules.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Anna Akhmanova (Senior Editor), a Reviewing Editor, and three reviewers. The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.Thank you for resubmitting your work entitled "The TOG domain protein Stu2 promotes γ-TuSC oligomerization in cytoplasmic microtubule nucleation" for further consideration at Gunzelmann et al., present a study on Stu2, a member of the TOG domain-containing microtubule (MT) polymerase family. Stu2 is well characterized as acting at MT plus ends. Here the authors explore the role of Stu2 at MT minus ends and its interactions with Ɣ-TuSC. On the nuclear side of the nuclear membrane Spc110 serves as the Ɣ-TuSC receptor, while on the cytoplasmic face Spc72 is the Ɣ-TuSC receptor. Using a combination of in vitro characterization and in vivo work the experiments presented here strongly suggests a role for Stu2 in both the stabilization of the Ɣ-TuSC-Spc72 interaction as well as in assembling detached MTs in the cytoplasm. The study expands our understanding of MT nucleation in yeast. It adds to the field in a unique way and complements the recently published XMAP215/γ-tubulin work from the Petry lab.However, before the paper can be published, we would like to ask you to address some remaining issues which concern data analysis, paper organization and writing.Data analysis and interpretation:1) The authors state that the aster density is lower in Spc72N-Stu2TOGAAA asters than in Spc72N-Stu2 asters. However, the quantification used to determine aster density is 'MT formation activity', or the mean intensity fluorescence of the entire field. That type of measurement encompasses the entirety of MT assembly. Therefore, a better quantification of aster density is required.Also, it appears that the example images shown in Figure 3E are saturated, making aster density difficult to assess.2) Is there a suspected reason for the increase in Stu2 fluorescence in spc72Stu2Δ versus Spc72 WT cells ? If you remove the CM1 region of Spc72 in yeast, is there a decrease in Stu2 fluorescence at the cMT minus end?3) The authors state that the Stu2 signal in spc72Stu2Δ KAR9-AID +IAA cells is similar to spc72Stu2Δ. The value stated (~0.9) is actually even more similar to the Stu2 fluorescence in Spc72 WT cells . This quantification, even if it is for a small number of cMTs, should be shown in the figure.Additionally, it is suggested that the KAR9-AID was not fully degraded under these conditions, and that is why Stu2 is still recruited to the cMT minus end. However, a complete knockdown of KAR9-AID in another yeast strain  is shown. The authors should demonstrate the extent of KAR9-AID degradation in spc72Stu2Δ KAR9-AID +IAA cells. Alternatively, the authors should fluorescently tag the KAR9-AID protein to determine if the few numbers of cMTs that still maintain Stu2 also have Kar9 at the cMT minus ends. A possible interpretation is that there may be an additional factor recruiting Stu2 to cMT minus ends.4) Comments on the FRAP analysis: The authors claim that there are two pools, a stable and unstable fraction of Stu2 at the minus-ends of microtubules. There is concern that there is a limited pool of soluble Stu2, and the recovery curves  potentially misleading. The lack of recovery in one sample could be due to limited protein pool (so there is not enough unlabeled protein around to populate the FRAP zone. The authors could report the average fluorescence intensity of the spots (in arbitrary units) to examine whether spots of the same fluorescence are being targeted in each sample.Improving the organization and writing of the paper:1) The authors have a lot of interesting data, but because of the complex nature of Stu2's multiple roles as well as the complexity of some of the experiments, a lot of the intricacies are lost upon first reading. It would be specifically helpful to have a section in the introduction that clearly outlines each putative role and interaction of Stu2 that will be tested, and the general strategy used to test each of these roles/interactions.2) Before each set of experiments, the authors should clearly explain why they are performing the experiment and what they expect to occur (a hypothesis/model). In many areas this information is lacking or vague. For example, subsection “Stu2 stabilizes γ-tubulin at cMT ends” "To further elevate the role of Stu2 at the cMT minus end…". It's unclear what the authors mean by that and it is difficult to follow their line of logic. As much as possible, the authors should reference back to their original models so that the reader can more easily follow.3) It appears that the authors fit their MST data to a single-site model, but this should be clearly stated in the Materials and methods section.4) The grammar, primarily in the Introduction, needs some additional work.eLife, we suggest that this phrase be rewritten in a more active form so that people, who do not speak English as a first language, do not confuse the statement with "independent of.."5) The authors frequently use the uncommon term: "in dependence of" referring to an effect that is "dependent". Given the wide international readership of 6) Subsection “Purified Stu2 in complex with Spc72 assembles MT aster” fifth paragraph is confusing and should be rewritten.7) Discussion section, ninth paragraph, the authors probably mean "ratios" rather than "rations".8) Figure 7—figure supplement 1D and E are important and should probably be included in a main figure.9) Figure 8 needs a figure legend and the objects within the figure need to be clearly labeled.10) Figure 7A, B need labels indicating what is fluorescently tagged.11) Several times protein constructs or yeast strain acronyms aren't described upon first mention. Upon the first introduction of a new protein construct that is given an abbreviation, a description and the amino acid modifications (if applicable) should be stated. [Editors’ note: the author responses to the first round of peer review follow.]We would like to thank the three reviewers for their helpful and constructive comments. As outlined below, we have addressed all the points that were raised by the reviewers. The manuscript was heavily modified and new data have been added. The title, conclusions and discussion were changed accordingly.Reviewer #1:[…]While this work contains several interesting observations, many experiments are lacking important controls to support the conclusions made. In addition, in its current form I do not see that it dramatically advances our understanding of TOG domain-containing proteins nor is it of sufficient quality for eLife. I recommend that the authors revise their work based on the following comments to submit elsewhere.The reviewer acknowledges that the work contains interesting observations. However, he/she also points out that controls are missing. We followed the suggestion of reviewer 1 and have revised our manuscript addressing the points raised by this reviewer. We also included new data  to increase the impact of our manuscript.This manuscript for the first time compares in vitro and in vivo activities of TOG domain proteins with and without Ɣ-tubulin complexes. Our manuscript shows that Stu2 has a structural role in the oligomerization of Ɣ-TuSC and the attachment of Ɣ-TuSC to the MT minus end. It explains why proteins such as Spc72 with an imperfect CM1 can function in the oligomerization of Ɣ-TuSC and how Stu2 bound to Spc72 facilitates MT nucleation. In addition, we provide evidences that the MT binding site in Stu2 interacts with the Ɣ-TuSC. This probably promotes the assembly of the Spc72-Ɣ-TuSC-Stu2 complex on the cytoplasmic side of the SPB. We further show that the Ɣ-tubulin Tub4 is mainly important to restrict cMT nucleation to the SPB but not for cMT nucleation per se. In contrast, Stu2 is essential for Tub4-dependent and independent cMT nucleation. Taken this together, our manuscript will have a significant impact on the MT nucleation field in general.Essential revisions:1) Does Stu2 directly interact with Ɣ-TuSC? This should be addressed.TOG and Stu2MT . Indeed, Ɣ-TuSC binds to Stu2 via Stu2MT. These data are consistent with the recent publication by Petry and co-workers in Nat Cell Biol indicating that human Ɣ-tubulin interacts with the MT binding site of XMAP215 in gel filtration experiments.We have addresses this point by MST measurements  and in vitro binding experiments with Stu22) The gel filtration experiment in Figure 2D is missing standards and the void volume is not labeled. How many times was this experiment repeated? This should be listed in the legend. What concentrations of proteins were used?WT or GST-Spc72N-6HisStu2TOGAA to a final concentration of 4.6 µM for γ-TuSC and 2.3 µM for the other proteins in HB100 buffer with 10% glycerol.”We have added the size markers to Figure 2D  (and Dextan blue to other runs to indicate the void). Dextran blue marks the void volume. As outlined in the figure legend to Figure 2D, we show one representative experiment of three independent experiments. All of them showed a much higher oligomerization activity for Ɣ-TuSC + Spc72N-Stu2 comapred to Ɣ-TuSC + Spc72N or only Ɣ-TuSC. The concentration of proteins is described in Materials and methods section: “For Figure 2E and Figure 2—figure supplement 2D, purified γ-TuSC was mixed with GST-Spc72N, GST-Spc72N-6His-Stu23) Ɣ-TuSC oligomerization appears to be enhanced in the presence of Spc72N-Stu2TOGAA (defective in tubulin binding). This experiment should also be done with Spc72 and wildtype Stu2.TOGAA experiment is shown in Figure 2—figure supplement 2D. In all three experiments Stu2TOGAA behaved as Stu2.We now have performed this experiment with WT Stu2  as requested by reviewer 1. The Stu23) The authors should quantify Stu2 fluorescence at the cMT minus end in the spc72Δstu2/Kar9-depletion conditions. Is Stu2 completely absent or are factors beyond Spc72/Kar9 also recruiting Stu2 to the minus end?We have addressed this point in subsection “Stu2 stabilizes γ-tubulin at cMT ends”:∆Stu2spc72cells should further decrease Stu2-yeGFP intensity at the minus end of detached cMTs. However, as outlined below, these measurements were complicated by the instability of cMTs in ∆Stu2 KAR9-AID kar1∆15spc72 cells and the observation that of the surviving detached cMTs only half carried Tub4 . The normalized Stu2-yeGFP signal intensity at these rare Tub4-positive cMT minus ends was measured with ~0.9, which is similar to what we determined in ∆Stu2spc72cells but clearly higher than the signal in KAR9-AID cells . This suggests that Kar9-AID was not fully degraded in these ∆Stu2 KAR9-AIDspc72 cells with detached cMTs. Indeed, variations in the efficiency of the auxin-induced degradation of proteins between yeast cells have been reported . ““Depletion of Kar9 in 4) As the authors currently present it, the mechanism of Stu2-dependent MT assembly is unclear. The experiments in Figures 6 and 7 are difficult to interpret because controls are lacking. For example, the Stu2 mutants alone were not analyzed in Figure 6. Is there any affect of Stu2 or the Stu2 mutants in the presence of Ɣ-TuSC, but in the absence of Spc72 ?We have now analysed the Stu2 mutants alone . In addition, besides aster formation ability, we have quantified MT formation ability as was done by Roostalu and Surrey in Nat Cell Biol. In response to reviewer 1’s suggestion, we also combined Ɣ-TuSC with Stu2 . In this combination, we see the activity of Stu2. Moreover, we show that Stu2 has to be in the preformed Spc72N-Stu2 complex since addition of individual Spc72N and Stu2 into the reaction mix (which dilutes the proteins and disfavours complex formation) did not show the activity of the Spc72N-Stu2 complex.Thus, we hope that the data clarify the mechanisms of Stu2 in cMT nucleation. We intensively discuss the function of Stu2 in cMT in the Discussion section.5) The concentrations of tubulin, and all other proteins used in Figures 6 and 7 are not listed in the methods or legends. This is critical to include. Also, were different concentrations tested? How were the concentrations that were used chosen?The concentration of tubulin was described in the Materials and methods section (not the legends – I am sorry for this). The final concentration was 12 µM. This concentration was chosen because the Spc110-Ɣ-TuSC rings are able to nucleate MTs at this tubulin concentration. Our tubulin concentration is similar to what Roostalu and Surrey have used in NatCell Biol.To clarify this point, we write in subsection “Purified Stu2 in complex with Spc72 assembles MT asters”:“The α/β-tubulin concentration of 12 µM was chosen because it enables the oligomerized Spc110N-γ-TuSC to nucleate microtubules . A similar α/β-tubulin concentration has been used in MT nucleation assays with purified TPX2 and chTOG . Assembled MTs were spun onto cover slips and analysed by fluorescence microscopy. “6) It is unclear what distinct roles the authors propose for the MT-binding and TOG-binding domains of Stu2 to have in enhanced MT nucleation at the centrosome/ SPB. Are cells with Stu2 MT-binding and TOGAA mutations viable?STU2 mutants. Moreover, we have heavily modified the Discussion section addressing the role of the TOG domains in Stu2. I agree that the title of the first manuscript version was misleading. TOG domains of Stu2 have an important role in cMT nucleation as indicated by our experiments and outlined in the text.This is a good point and we should have added this already in the first submission. Now we show in Figure 3—figure supplement 3F the growth test of the different For example, in the Discussion section we write:“The TOG domains in the Kar9-Stu2 layer are most likely positioned more distantly away from the cMT minus end than those within Spc72-Stu2 . The two Stu2 layers probably function in a sequential order: Spc72-Stu2 for the addition of the first α/β-tubulin subunits followed by Kar9-Stu2 that adds the next α/β-tubulin molecules. A single XMAP215 molecule bound via its MT binding site to γ-tubulin has with five TOG domains a greater reach to add α/β-tubulin subunits to the growing MT than a single Stu2 layer with only two TOG domains . Binding of Stu2 to Spc72 and Kar91 probably compensates for the smaller number of TOG domains in Stu2.”7) The authors reference two "nucleation activities" of Stu2, but then posit that the TOG domains may be required for extending Ɣ-TuSC-nucleated seeds. This seems contradictory.As outlined above, we have modified the Discussion section and now better explain the role of Stu2 in MT nucleation.8) The manuscript could benefit greatly from additional editing for clarity and conciseness. There are many grammatical errors as well. The Figure Legends need to be improved for clarity and completeness.Thank you very much for this suggestion. I hope that we have done a better job this time.Reviewer #2:[…] The manuscript adds to our understanding of Stu2 and its role in yeast mitosis using a nice blend of in vitro and in vivo analyses. In its current state it lacks some critical experiments that, if present, would best unite disparate pieces in the paper, and help the authors arrive at some of their conclusions.The reviewer states: “The manuscript adds to our understanding of Stu2 and its role in yeast mitosis using a nice blend of in vitro and in vivo analyses.” He/she also points out that the paper lacks critical experiments that should unite different experimental parts.We have addressed this point of reviewer 2 by re-arrange the manuscript and adding critical experiments for example the new Figure 7.1) Gel filtration is used to determine whether Spc72 (plus or minus Stu2) causes oligomerization of γ-TuSC, as does Spc110. The results show that Spc72 does shift γ-TuSC to the void volume, and that Stu2 enhances this shift. However, Stu2 also causes a new peak to arise at fraction A11 that could be accounted for by Stu2-mediated dimerization of a γ-TuSC-Spc72 complex. This then calls into question the increase of the γ-TuSC fraction in the void volume: is this really oligomerization as seen with Spc110 which generated γ-TuSC filaments, or is it due to cross-linking mediated by dimers of Spc72 and dimers of Stu2? To resolve this, the authors should determine if the Spc72-Stu2 complex does drive γ-TuSC oligomerization – this could be achieved using negative stain EM analysis, similar to work done by Kollman et al., 2010, DIC microscopy, or fluorescence microscopy.We have performed the negative staining EM as suggest by reviewer 2. These data are in Figure 2—figure supplement 2E. They clearly show that Spc72-Stu2 oligomerizes Spc72Stu2 into rings that are different in structure compared to the Spc72-Ɣ-TuSC rings. Additional high-resolution EM analysis is needed to understand the structural differences between the Spc72-Stu2-Ɣ-TuSC and Spc72-Ɣ-TuSC rings. This will be done in a future study using cryo-EM.2) Figure 7, the authors should conduct a Stu2 WT +/- γ-TuSC control to best determine how much Spc72-Stu2 augments the MT nucleation activity in vitro.We have extended the analysis that was shown in Figure 7  by adding the controls that have been asked for by reviewers 1 and 2. The Stu2 WT +/- γ-TuSC control is now shown in Figure 3—figure supplement 3A/B.3) The authors delineated independent roles for Spc72 and Kar9 on Stu2 recruitment to the MT minus end, analyzed the effects of these respective complexes on cMT nucleation, number, and length in cells. However, when the authors then move to in vitro analyses, they concentrated exclusively on the Spc72-Stu2 interaction (plus and minus γ-TuSC) and its effects on microtubule nucleation in vitro. To be comprehensive, it would greatly strengthen the paper to conduct a comparative analysis of the effects of Kar9 and Kar9-Stu2 (plus and minus γ-TuSC) on MT nucleation in vitro with that of Spc72-Stu2.We appreciate this suggestion. Adding Kar9 in addition to Stu2, Spc72 and γ-TuSC bears the challenge of purifying Kar9 (as Kar9 or Kar9-Bim1 complex – ?) in a native functional state and find buffer conditions that are compatible with all components. This was already challenging enough for the three-component system. I guess, reviewer 2 is suggesting this because we did not explain too well the function of the second Kar9-Stu2 layer in comparison to Spc72-Stu2. We have done this for the new submission in the Discussion section:“The situation in cells is even more complex since two proteins, Spc72 and Kar9, recruit Stu2 to the minus end of cMTs. The recruitment role of Kar9 is consistent with the reported interactions of Kar9 with Stu2 and γ-tubulin Tub4  .”“The TOG domains in the Kar9-Stu2 layer are most likely positioned more distantly away from the cMT minus end than those within Spc72-Stu2 . The two Stu2 layers probably function in a sequential order: Spc72-Stu2 for the addition of the first α/β-tubulin subunits followed by Kar9-Stu2 that adds the next α/β-tubulin molecules. A single XMAP215 molecule bound via its MT binding site to γ-tubulin has with five TOG domains a greater reach to add α/β-tubulin subunits to the growing MT than a single Stu2 layer with only two TOG domains . Binding of Stu2 to Spc72 and Kar91 probably compensates for the smaller number of TOG domains in Stu2.”Reviewer #3:Review of Gunzelmann et al., TOG domain independent microtubule nucleation activity.This paper describes a novel nucleation activity within the spindle pole component, Spc72 that recruits Stu2 (TOG domain protein) to the cytoplasmic face of the spindle pole body, where it increases MT density and plays a role in nucleating MT asters.The binding data in Figure 1 and Figure 2 was very convincing. The data in Figure 3 were problematic. Firstly, they state that incubation in a kar1 mutant results in detached cMTS . I looked at both of these papers and was unable to find the percent of detached vs attached cytoplasmic microtubules in the mutant. It is important to quantitate what fraction of the microtubules are detached to differentiate pole binding from minus-end binding. An alternative interpretation for Figure 3c is that observation of Stu2 in the absence of the stu2 binding site in Spc72 is that these microtubules are not detached from the pole.kar1∆15 cells as outlined in Figure 5A. We were quite careful in identifying detached cMTs. We followed the entire cMT to ensure that the cMT was not attached to the SPB.40% of cMTs detach from the SPB in In cases where we used Tub4-mCherry as marker, the Tub4 signal at the SPB was strong; Tub4 at the cMT minus end was weaker . Stu2-yeGFP decorated the wall of the cMT. Thus, we are quite confident that we have only analysed minus ends at detached cMTs.To make this point clearer, we have now added cartoons to the images  illustrating for each case the position of the detached cMT.The FRAP recovery are nicely done and convincing that there are multiple binding sites.Thank you.It is interesting that in this and panel b the mt minus ends seem to be in the shmoo tip. Since there are known plus-end binding complexes in the shmoo tip, is there concern that the co-localization reflects the juxtaposition of the minus-end to the shmoo tip. This is easily addressed by quantitating the fraction of spc72 or stu2 spots in the tip vs. the presence or absence of mt minus ends.SPC72 and ∆Stu2spc72 cells (n=57) frequently localized (~70%) within the schmoo tip periphery (2 µm radius). Detached cMTs of SPC72 and ∆Stu2spc72 cells that lacked Stu2 at the minus end showed a similar schmoo tip preference (11 out of 16 cMT). This orientation is probably a reflection of the association of the minus end motor Kar3 with the schmoo tip {Maddox, 2003}. Kar3 may pull detached cMT minus ends to this localization. In response to this comment of reviewer 3, we now point out in subsection “Spc72 and Kar9 target Stu2 to the cMT minus end” that the cMT minus end frequently localizes within the schmoo tip periphery.We have done this analysis. The minus end of detached cMTs of “This cap is the Spc72-γ-TuSC complex  that in ~70% of cells localized within the schmoo tip region (2 µm radius). This orientation is probably a reflection of the association of the minus end motor Kar3 with the schmoo tip  pulling the detached cMT minus ends into the schmoo.”Figure 4D is a critical panel for interpretation of aforementioned experiments. It is stated that a-g are kar1Δ15. Approximately 50% of the cMT are attached in these mutants. Thus, the reviewer needs to redraw Figure 3a to reflect the fact that 50% are detached and 50% are released.kar1∆15 cells have detached cMTs; 60% do not have detached cMTs. This is now indicated in the Figure 5A.We have modified Figure 5A  as suggested by reviewer 3. 40% of In addition, the reviewer needs to include the attachment/detachment state of the microtubules for the co-localization. Are they only examining detached microtubules, if so, what is the metric for the data in Figure 3?kar1Δ15 cells treated with α-factor and analysis was done on single, detached cMTs.” Similarly, we state in the legend to Figure 6A-C and g that detached cMTs were measured.In Figure 5 and Figure 6A-C and G we only have analysed detached cMTs because we specifically were interested in the minus end of these cMTs. This analysis is not possible with cMTs that are attached to the SPB. A SPB signal may come from e.g. the binding of Stu2 to a SPB component or its association with the cMT end. In the legend to Figure 5 we state: “(b-f) Experiments were performed with In Figure 6D and E we have included all cMT categories: (i) SPB attached cMTs, (ii) detached cMTs, and (iii) no cMTs. This analysis gives us an overview how the different populations behave in mutant cells and over time.The metric in Figure 4 is clear, mCherry-Tub1, but if 50% are attached, the data without Tub1 become significantly ambiguous.TUB4-mCherry STU2-GFP cells. The Tub4-mCherry signal gives the position of Stu2 at the cMT minus end. Stu2 also decorates the detached cMT . We only analysed cells where we unambiguously could identify the nuclear MT bundle and the single detached cMT to be sure that only the minus end of detached cMTs was analysed.I do not fully understand the concern of reviewer 3. Figure 4  shows mostly cells with mCherry-Tub1 signal . Figure 6A shows co-localization of Kar9 and Spc72 (minus end marker) at the detached cMT minus end. Kar9 decorates in part the detached cMT. Figure 6—figure supplement 4B confirms Kar9-GFP at a detached cMT labelled with mCherry-Tub1. The quantification in Figure 6B was done in In summary, this paper documents an important second TOG-independent mechanism in Stu2 for MT formation. Stu2 has critical roles in spindle orientation and kinetochore function, and thus this second site is important to understand. The authors should ensure that for all reports of minus-end binding, that these are indeed detached cytoplasmic microtubules.Thank you.[Editors’ note: the author responses to the re-review follow.][…] The study expands our understanding of MT nucleation in yeast. It adds to the field in a unique way and complements the recently published XMAP215/γ-tubulin work from the Petry lab.However, before the paper can be published, we would like to ask you to address some remaining issues which concern data analysis, paper organization and writing.Data analysis and interpretation:1) The authors state that the aster density is lower in Spc72N-Stu2TOGAAA asters than in Spc72N-Stu2 asters. However, the quantification used to determine aster density is 'MT formation activity', or the mean intensity fluorescence of the entire field. That type of measurement encompasses the entirety of MT assembly. Therefore, a better quantification of aster density is required.We now included an additional quantification of aster fluorescence intensity, which reflects the aster density .Also, it appears that the example images shown in Figure 3E are saturated, making aster density difficult to assess.All image settings were chosen to be exactly the same and they are optimised to visualise single MTs. This is why some images in the centre are saturated. However, for quantification  we only analysed images in the linear range without saturation.2) Is there a suspected reason for the increase in Stu2 fluorescence in spc72Stu2Δ versus Spc72 WT cells ? If you remove the CM1 region of Spc72 in yeast, is there a decrease in Stu2 fluorescence at the cMT minus end?ΔCM1Spc72 is complicated to realise. As we have shown in Figure 1B this mutation is lethal. Instead, we tested the possibility that enhanced recruitment of Kar9 could be responsible for the increased recruitment of Stu2 in Δstu2spc72 mutant cells. Indeed, it turned out that there is a higher availability of Kar9 at the minus end of detached cMTs in Δstu2Spc72 cells compared to SPC72 cells as it is now shown in the new Figure 6—figure supplement 1C.The suggested experiment using the 3) The authors state that the Stu2 signal in spc72Stu2Δ KAR9-AID +IAA cells is similar to spc72Stu2Δ. The value stated (~0.9) is actually even more similar to the Stu2 fluorescence in Spc72 WT cells . This quantification, even if it is for a small number of cMTs, should be shown in the figure.We now included the quantification as Figure 5E. However, as the number of quantified cells is so low we did not performed a statistic analysis.Additionally, it is suggested that the KAR9-AID was not fully degraded under these conditions, and that is why Stu2 is still recruited to the cMT minus end. However, a complete knockdown of KAR9-AID in another yeast strain  is shown. The authors should demonstrate the extent of KAR9-AID degradation in spc72Stu2Δ KAR9-AID +IAA cells. Alternatively, the authors should fluorescently tag the KAR9-AID protein to determine if the few numbers of cMTs that still maintain Stu2 also have Kar9 at the cMT minus ends. A possible interpretation is that there may be an additional factor recruiting Stu2 to cMT minus ends.KAR9 cells, probably even more. This reduction will be below the detection limit of the immunoblot. To address this point of the reviewers, we added in subsection “Stu2 stabilizes γ-tubulin at cMT ends” following sentence.Depletion of a human centrosomal protein by siRNA appears very efficient based on an immunoblot analysis of cell extracts. However, in IF quite often a residual signal is observed at the centrosome. Γ-Tubulin siRNA is a good example for such behaviour. According to our analysis about 5% of cells carry some residual amount of Kar9-AID at the minus end of cMTs. We do not know how much Kar9 is needed for the recruitment of functional levels of Stu2. In any case, Kar9-AID levels are reduced by at least 95% compared to ∆Stu2 KAR9-AIDspc72 cells with cMTs was, however, below the detection limit of the immunoblot in the new Figure 6—figure supplement 1D.”“This residual amount of Kar9-AID in the small number of 4) Comments on the FRAP analysis: The authors claim that there are two pools, a stable and unstable fraction of Stu2 at the minus-ends of microtubules. There is concern that there is a limited pool of soluble Stu2, and the recovery curves  potentially misleading. The lack of recovery in one sample could be due to limited protein pool (so there is not enough unlabeled protein around to populate the FRAP zone. The authors could report the average fluorescence intensity of the spots (in arbritrary units) to examine whether spots of the same fluorescence are being targeted in each sample.Δstu2Spc72 compared to SPC72 cells in the beginning of the experiment.To rule out the possibility that a limitation of freely available cytoplasmic Stu2-yeGFP is the reason for incomplete recovery, we quantified the fluorescence signal intensity of the examples shown in Figure 5F before bleaching. It should be noted, that the examples shown and that have been used for the analysis have a higher intensity in Stu2-yeGFP for Improving the organization and writing of the paper:1) The authors have a lot of interesting data, but because of the complex nature of Stu2's multiple roles as well as the complexity of some of the experiments, a lot of the intricacies are lost upon first reading. It would be specifically helpful to have a section in the introduction that clearly outlines each putative role and interaction of Stu2 that will be tested, and the general strategy used to test each of these roles/interactions.We are grateful for the suggestion of the reviewer to improve the overall comprehensibility of the paper. We now included a section at the beginning of each result paragraph that refines the question that we wanted to address and the strategy how to test for possible roles/interactions.In the Introduction we write: “However, there are still the following open questions: (1) Do TOG domain proteins have the ability to nucleate MTs in vivo independently of γ-tubulin complexes? (2) Do TOG domain proteins have structural functions at the MT minus end? (3) Is the role of TOG domain proteins in MT nucleation context specific ?“2) Before each set of experiments, the authors should clearly explain why they are performing the experiment and what they expect to occur (a hypothesis/model). In many areas this information is lacking or vague. For example, subsection “Stu2 stabilizes γ-tubulin at cMT ends” "To further elevate the role of Stu2 at the cMT minus end…". It's unclear what the authors mean by that and it is difficult to follow their line of logic. As much as possible, the authors should reference back to their original models so that the reader can more easily follow.This goes in line with the first point and as suggested we tried to improve the understandability by rephrasing/rewriting the mentioned paragraph and the results section in general.3) It appears that the authors fit their MST data to a single-site model, but this should be clearly stated in the Materials and methods section.Indeed, we used a single-site model for fitting our MST data and we now sate this in the Materials and methods section.4) The grammar, primarily in the Introduction, needs some additional work.We tried to improve this point as suggested and modified the Introduction.5) The authors frequently use the uncommon term: "in dependence of" referring to an effect that is "dependent". Given the wide international readership of eLife, we suggest that this phrase be rewritten in a more active form so that people, who do not speak English as a first language, do not confuse the statement with "independent of.."We changed this term to avoid confusion, as implied.6) Subsection “Purified Stu2 in complex with Spc72 assembles MT aster” fifth paragraph is confusing and should be rewritten.We rephrased this paragraph in order to make it clearer.7) Discussion section, ninth paragraph, the authors probably mean "ratios" rather than "rations".We appreciate the notion of this typo and changed it accordingly.8) Figure 7—figure supplement 1D and E are important and should probably be included in a main figure.We changed the appearance of the Figure as suggested by the reviewer and moved the Figure 7—figure supplement 1D and E to Figure 7E and F.9) Figure 8 needs a figure legend and the objects within the figure need to be clearly labeled.We added a figure legend and all components shown in this Figure are now labelled.10) Figure 7A, B need labels indicating what is fluorescently tagged.We have added the missing label.11) Several times protein constructs or yeast strain acronyms aren't described upon first mention. Upon the first introduction of a new protein construct that is given an abbreviation, a description and the amino acid modifications (if applicable) should be stated.We carefully checked the entire manuscript and introduced missing descriptions or abbreviation at the first time of appearance.


Global Engagement works with health partnerships to establish workforce and educational translation on a global scale to support the National Health Service (NHS). There is growing evidence on how international experiences  benefit the NHS through an innovative workforce that develops international best practice and promotes lifelong learning. Most of this evidence has been captured though surveys to returned international volunteers. However, there is limited evidence about how to quantify the value that returned international healthcare volunteers bring back to their country of residence.This paper identifies the various benefits to the NHS from returned international healthcare volunteers. The outcomes from returned international volunteers, which have been identified as relevant form a NHS perspective, are linked to three key areas in a multisector analytical framework used by the World Bank to evaluate labour market programmes: (1) Investment climate and Infrastructure, (2) Labor market regulations and institutions, and (3) Education and skills development. The monetary value of these outcomes is quantified through productivity indices which capture the economic value that the achievement of these outcomes have on the quality of the NHS labor force. This model is applied to a dataset of international volunteers provided by the Global Engagement health partnerships.The results suggest that international volunteering generates average productivity gains of up to 37% for doctors and up to 62% for nurses. Average productivity gains estimated from health partnerships data vary depending on duration of volunteering periods and occupational category mix.Our analysis offers a value for money rationale for international volunteering programmes purely from a domestic and NHS perspective. The valuation method considers only one of the aims of international volunteering programmes: the development of the existing and future NHS workforce. Broader benefits for health system strengthening at a global level are acknowledged but not accounted for. Overall, we conclude that if the acquisition of volunteering outcomes is realised, the NHS can accrue a productivity increase of between 24 and 41% per volunteer, with a value ranging from £13,215 to £25,934 per volunteer.The online version of this article (10.1186/s12992-019-0473-y) contains supplementary material, which is available to authorized users. The Global Engagement (GE) is a Health Education England (HEE) programme working to establish workforce and educational translation on a global scale to support the National Health Service (NHS). This is being done through programmes focused on international recruitment, global placements, evaluation and research, international volunteering and health partnerships.GE is committed to evaluating the benefits of these programmes as a part of the solution for today’s global health challenges. These benefits extend beyond health delivery in low- and middle-income countries (LMICs). International volunteering, exchanges and structural placements benefit the NHS through an innovative workforce that develops international best practice and promotes lifelong learning . The proThis paper discusses several models in the existing literature that could be used to measure the monetary value of the benefits from healthcare volunteering. There is an extensive literature that recognises a broad range of outcomes, mostly beneficial, which returned international volunteers bring to high income countries, including three systematic reviews –5. MoreoBenefits to the NHS from returned international healthcare volunteers are analysed within a multisector framework that encompasses all the key aspects of the economic, political, and institutional context for job creation. This analytical framework, designed by the World Bank, is known as MILES  . Only thThe quantitative model of productivity indices are applied using data from health partnerships organised by King’s College London (KCL) and the Royal College of Paediatrics and Child Health (RCPCH) which placed 279 volunteers mainly in sub-Saharan Africa, including during the Ebola outbreak in 2013–2016. The model relies on the assumption that international volunteers from these partnerships are comparable with NHS staff in terms of professional occupation, and some of the volunteers develop their skills during the placement period. And additional assumption is that these volunteers return to their previous health professional category after the placement so that their learning outcomes are captured by the NHS instead of resulting in job promotion. The limitation of these assumptions is discussed.The rest of the paper is organised as follows. Section 2 describes and maps the outcomes from international volunteers according to the MILES framework and presents the productivity valuation method used to monetise the outcomes. Section 3 explains the data and how they are applied in the quantitative model. Section 4 presents the results. The limitations of the scope of the study and from the underlying assumptions are discussed in Section 5. Finally, a discussion and concluding remarks are presented in Sections 6 and 7, respectively.The first step towards defining and monetising productivity gains rendered to the NHS by returned international volunteers is to understand the competencies that affect their productivity. The concept of productivity is broad but limited to the NHS perspective. This underlying productivity captures how the NHS marshals their available inputs to improve patients’ health. The main input is labor, but as well as considering the economic value of working hours, we consider outcomes that affect the organizational context and the skills of NHS staff. We discuss these outcomes within the MILES analytical framework.The World Bank uses the MILES framework in designing labor market strategies by identifying key constraints for job creation within five components : (M) MacIn particular, MILES was used to evaluate World Bank and International Finance Corporation (IFC) programmes on youth employment . We consWe have searched literature focusing on outcomes of international volunteering in the health sector. Two recent peer-reviewed systematic reviews analyse the outcomes reported by international volunteers and how these outcomes render benefits in the UK. The study by Jones et al.  reviews Figure Given its link with professional competencies, all outcomes from international volunteering could be transmitted to benefits for health professionals, organisations, and ultimately to overall NHS service delivery and health outcomes for patients. This transmission of outcomes has been mapped for different domains except the domain of “personal satisfaction and interest” . NonetheOn the I domain, outcomes are captured by healthcare organisations and help to shape relationships with donors and define the organisational role in the demand for volunteers. Recognised outcomes include improving health care service delivery in rural or difficult to reach populations, bringing healthcare management closer to the needs of local populations through decentralised management, and improving patient-provider relationships, in particular by helping to bridge the communication gap between professionals and patients. Analysed the role of international volunteering sending agencies on volunteering outcomes depending on how these agencies negotiate local dynamics and transnational governance. According to the Measuring the Outcomes for Volunteering for Education (MOVE) study, these outcomes correspond to the organisational and contextual variables facilitating or hindering the optimal acquisition and mobilisation of knowledge and core outcomes such as “appreciation of clinical governance within NHS” .On the L domain, returned volunteers help improve outcomes related to career progression and attitudes of employers to apply the learning from the volunteering experience . For exaOn the E domain, the specifics of the acquired knowledge and attitudes reported by returned volunteers broadly map onto the strategic objectives of the NHS with emphasis on resourcefulness, cost efficiency, flexibility and inter-professional working , 11–13. The outcomes explained above and included in all studies analysed in systematic reviews , 5 are pThe development and acquisition of competencies from the volunteering experience is complex and may take longer than 1 year to develop, varying by amount of individual experience and prior experience , 16. TheFigure On the negative side, international volunteers have reported some risks and negative outcomes associated with the experience. Some volunteers have reported that they have “lost ground” and find their skills out-of-date, and especially the loss of networks, with some UK employers concerned about this knowledge loss or not valuing the international experience. Tyler et al.  find thaDifferent theories have been put forward to measure the value of volunteering, both community and international, although these theories seek to measure value captured by the country where the volunteering activities are delivered. In particular, the International Labour Organisation’s Manual on the measurement of volunteer work recommends capturing the “economic value” of volunteering, which derives from the domestic product accounting concept applied to the “replacement theory”; basically, volunteers are considered as employment substitutes. This concept of economic value is very narrow and would only capture a part of the value of good and services supplied over the amount of volunteering time. The replacement theory overlooks two other main components of economic value: organisational benefits beyond staff wages, and quality improvements of goods and services. These components also pertain to economic value from an added value or GDP perspective and should be accounted for.Beyond the economic value, the well-being theory and social capital theories capture wider societal benefits for the volunteers and the community. Nonetheless, none of these theories can be applied to specifically measure the value from returned volunteers from an NHS perspective. As pointed before, the outcome category under which these benefits are considered is “personal satisfaction and interest”  which isThe “volunteering onion” as named by Haldane  describeMeasuring productivity within the health sector is complex, including difficulties in measuring variation in skills  and the We propose a direct measurement of NHS labor growth which has been used to quantify the impact on NHS employment in a given year. The key assumption is that the international volunteering experience causes changes in the staffing mix towards higher levels of productivity which can be captured by the earnings differentials between adjacent staff groups. Even though we have reported above how international volunteering benefits career progression and employability, we assume the productivity gain is retained by the NHS as additional economic value instead of being fully captured by returned volunteers through job promotion. Table To measure labor growth attributed to international volunteers we use the proposed method of an index of input growth which weights the number of staff of each type by their respective wages. We use baseline constant wages in 2017 reported in the NHS Staff Earnings statistics as weights for both the pre- and post-volunteering periods. By holding wages constant, our index of input growth is a Laspeyres index which measures “real” changes in the weighted volume of NHS staff caused by the international volunteering experience; we do not measure how much these volunteers are paid. Nonetheless, this real or quality-adjusted growth in volume of NHS staff can be monetised according to average wages to provide a monetary value of the benefits for the NHS from returned international volunteers.The Laspeyres volume index, in which inputs are measured directly, takes the following form:znv denotes the number of international volunteers whose NHS staff group is n, and znr denotes the number of returned international volunteers as ascribed to a productivity level of staff group n. Wages for each staff group n, wnb, are measured for the annual period b . The “real” or “quality adjusted” changes applied to NHS staff from their international volunteering experience is (∆Z − 1)% in terms of increase in quality-adjusted staff volume. This percentage increase can be measured separately for different staff groups and a monetary value can be calculated in proportion to the average earnings of the staff group. The monetary benefit could be measured as the product of the baseline value by the productivity growth index: Where znv), so some sensitivity analysis is carried out from alternative definitions, in particular to the adjustment of FTE from observed volunteer days so that the productivity gain of a given volunteer is increasing with the volunteering time, which corresponds to findings of increased achievement outcomes as predicted by competencies acquired during the volunteering experience [We do not have an exact definition of FTE volume of volunteers of type n (perience .The productivity gain corresponding to each of the three scenarios when converting volunteering days (D) to annual FTE time, are calculated as follows:This scenario assumes that the gains accrued from international volunteering are proportional to the volunteering time and no larger than annual productivity premium in cases of volunteering periods longer than 1 year. Therefore, the index of annual productivity growth (1) is calculated as:This scenario becomes a pessimistic or conservative assumption when valuing productivity gains for short-term volunteering experiences.This scenario assumes that all volunteers achieve full productivity gains independently of their volunteering time. In this sense it is an optimistic scenario for short-term volunteering experiences.In this case, the index is applied in its simple expression  considerThere are arguments on how the length of volunteering time affects the returns for returned volunteers both positively and negatively . Given oznv to znr. Volunteers who stay less than 22 days are inputted as All productivity gains are attributed to volunteers in the input This scenario functions as an intermediate assumption, even though it is more conservative than the first scenario for very short stays which are more common among senior staff.Three health partnerships from King’s College London (KCL) and one from the Royal College of Paediatrics and Child Health (RCPCH) have provided data of volunteers. The KCL partnerships placed volunteers in Sierra Leone (King’s Sierra Leone Partnership - KSLP), Somalia , and Democratic Republic of Congo . RCPCH operates in a wide range of LMICs; the sample of volunteers were placed in six countries . Details of the activities of these partnerships are provided in Additional file Table Our dataset of international volunteers from KCP and RCPCH includes information on the number of volunteers, volunteering function, and volunteering time in days, which are the variables needed to compute the productivity indexes. The allocation of a NHS staff group, occupation and care setting requires some ad-hoc assumptions to match the information in the dataset with the definitions of staff group, occupation code, level and care setting available from NHS Staff Earnings statistics. For example, in the KCSLP the volunteer function describes the volunteer role in the host country  and we make several assumptions to match these roles with the NHS staff groups and occupations. Therefore, although the matching of these levels has considered the role definitions and HEE core skills and competencies, some margin of arbitrariness remains. On the contrary, the KKCP database exactly specifies the volunteering role as a defined NHS staff group , which we adopt as the pre-volunteering category.On the volunteering time, some missing information in the KCSLP programme, has been replaced with average days in the corresponding category from non-missing data, with three considered categories: Hospital and Community Health Services (HCHS) doctors, Nurses, and Others. In total, volunteering time has been imputed for 30 volunteers with missing information from a total of 128 volunteers in this programme. We tested the sensitivity of our results to this imputation by undertaking the calculations without including these 30 missing observations.A top-level comparison of our sample of volunteers to that from the MOVE study  in termsRegarding volunteering time, just a few of our sample volunteers (from KSP) can be considered as short term (under 1week) volunteers according to MOVE definition. The average days of volunteering in our sample is 248.7 days, and this average is above 3 months in our three staff categories, corresponding to a larger representation of long-term volunteering than in MOVE. This is due to long-term placements by KCSLP and RCPCH volunteers.Annual wages data for different staff groups, occupations, levels, and care settings have been collected from NHS Digital, Provisional NHS Staff Earnings Estimates. Wages presented for the annual period October 2016 to September 2017 , 23.The estimations of productivity indexes (1), (2), and (3) are presented separately for the four group of volunteers and for three broad staff groups: HCHS doctors, Nurses, and Other. Comparisons across groups are presented in terms of the percentage point increase in “real” volume of NHS staff and of average monetary value of productivity gain per volunteer per year. These comparisons will shed light on the effect of different scenarios depending on the observed volunteering time and staff mix allowing an assessment of which is the most plausible scenario and results.Table With regard to the monetisation of volunteering benefits, Tables The RCPCH programme is the most beneficial for HCHS doctors whose increase in productivity is 41%, valued at £26,099. The explanation of this advantage with respect to other programmes is due to the case-mix of HCHS doctors which in the RCPCH programme is assumed to be homogeneous for Specialty Doctors  whose productivity gains are imputed by new skills equivalent to the upper Associate Specialist productivity level . For other programmes, the average productivity gain of HCHS doctors in the optimistic scenario 2 ranges between 19 and 37%, with average benefits between £11,326 and £21,780, according to a staff mix which includes doctors in levels below those of Specialty doctors.Volunteer nurses have larger benefits than doctors except in the KSP programme. This benefit implies an annual productivity increase of up to around 60%, valued at £19,034, for the KKCP partnership in scenario 2. The differences in productivity gain are due to the nurse occupational category and levels. At the lower level, the KSP programme includes several nurses in the educational care setting, and some 2nd level nurses for which adjacent levels to 1st level nurses or even to nurse consultant implies annual salary increases below £3000. At the upper level, all nurses participating in the KKCP program are ascribed to the Acute, Elderly & General care setting and their volunteering experience is assumed to benefit initial 1st level nurses with the skills of nurse managers. The development of these skills is valued at £19,034 per nurse according to the difference in annual earnings between nurse managers and 1st level nurses.The only programme which represents volunteers in clinical staff and NHS infrastructure support roles is the KCSLP programme which includes 37 volunteers in this group, mainly in scientific and technical positions. These volunteers accrue around 30% in productivity gains to the NHS, which represents £10,245 per year.These results are very slightly affected by the imputation of volunteering time for missing data in the KCSLP. The maximum absolute effect of this imputation of time is an overestimation of 5% in the value per volunteer in scenario 1, such that is missing data are not replaced with average time per category, the overall value at £10,399 per volunteer would decrease to £9862.Overall, we could conclude that if the acquisition of volunteering outcomes is realised, the NHS can accrue between 24 and 41% of productivity increase per volunteer with a value between £13,215 and £25,934 per volunteer.Limitations of this study refer to the identification of achievement outcomes and consequent monetary measure as particular from international volunteering programmes. That is, to what extent is the achievement of these outcomes, and the associated monetary value of the productivity improvement different from other training programmes, e.g. domestic programmes in the UK?Regarding the achievement, we have cited some longitudinal and panel data studies from the U.S.  and JapaThe monetary value of the productivity improvement we have presented does not aim to be especially designed for international volunteers. Yet, it follows the logic of measuring quality improvement for labor applied to a minimum skill improvement as defined by promotion to adjacent categories. These productivity gains are captured by the NHS through improved services at no extra cost in terms of salaries. We do not rule out a positive effect of international volunteering on career progression, but this is not a frequently reported outcome. Any ad hoc assumption on career progression would not change the quantitative results. Yet, the interpretation of productivity gain should be monetised as a private return for international volunteers instead of a benefit for the NHS.An additional limitation is the blurred boundary between the societal and the NHS perspectives. We have argued on a ceiling on accountability so that the NHS is not accountable for competencies related to personal satisfaction and interest. Nonetheless, we have also mentioned that these personal traits are also related to desirable competencies (the 6Cs). Moreover, each of the outcomes can have societal spillovers over and above economic value and for non-health sectors, as it happens with any outcome contributing to health gains.The study results suggest that international volunteering generates average productivity gains of up to 37% for doctors and un to 62% for nurses. Average productivity gains estimated from health partnerships data vary depending on the duration of the volunteering period as well as in the occupational category mix. These gains are monetised based on 2017 NHS staff earnings data.Overall, we conclude that if the acquisition of volunteering outcomes is realised, the NHS can accrue a productivity increase of between 24 and 41% per volunteer, with a monetary value ranging from £13,215 to £25,934 per volunteer.To understand the role of health partnerships and its benefits, it is important to understand the Global Engagement strategy of HEE (HEE: forthcoming) within the global health strategy and the role of official development assistance (ODA) and into the UK Aid strategy. This global health strategy is also linked to the current trends in NHS competencies and the importance of international volunteering outcomes within them.Lancet Commission on Investing in Health [The n Health , 25 demon Health .Health is only entirely targeted in one (third SDG) out of 17 SDGs. However, this does not mean that health has been deprioritised in the development agenda considering that three of the MDGs were dedicated to health. All SDGs are connected, and most can be linked back to health . In partThe implementation of Universal Health Coverage (UCH) as a target of the SDG 3 has been organised around international health partnerships. The fundaments of the partnership between institutions of LMICs are defined by the health strategy put forward by LMICs, and donors remain accountable on its achievements. Therefore, LMICs are empowered in defining their health strategies and accountability by health partnerships removes the element of discretion which can result from earmarking donor funds to specific programmes. International Health Partnerships for UHC 2030 has evaluated performance of partnerships in this sense . One of By 2013, Jones et al. .We have discussed some studies which report outcomes perceived by international volunteers as these benefit the sender developed country patients and organisations. All these studies capture different qualitative dimensions for outcomes reported by returned international volunteers. These dimensions match learning outcomes pursued in the capacity building programmes of HEE, whose overarching aim is the development of a workforce with the skills, values and behaviors to respond to future needs and drive innovation and improvement. Evidence on the development of these skills will be collected and monitored through a psychometric tool developed by HEE and this evidence can be used to measure quality improvements in staff and organisations.In this paper we have presented a rationale to place the global learning outcomes from international volunteering into a framework which emphasises changes in labor market outcomes for both the individual (e.g. skills and attributes) and the organisations . We present a method to monetise these outcomes based on measures of quality-adjusted changes in NHS staff. In contrast to the method applied to real changes in staff configuration, we apply a hypothetical scenario analysis which assumes the same staff configuration as in the pre-volunteering period but a quality improvement for returned international volunteers whose monetary value is the productivity gain implicit in a superior salary level within each staff category .In summary, the analysis presented here offers a valuation method only for one of the aims of international volunteering programmes: the development of the existing and future NHS workforce. Broader benefits for health system strengthening at global level are recognised but not accounted for such that our analysis offers a value for money rationale for international volunteering programmes purely from a domestic and NHS perspective.Under the current financial pressures and shortages of healthcare staff, it is important to assess the opportunity costs of international volunteering and placements realized by NHS staff and by the potential healthcare labor force. Our analysis offers this assessment providing a value for money rationale for international volunteering programmes from a domestic and NHS perspective. The valuation method considers only one of the aims of international volunteering programmes: the development of the existing and future NHS workforce. Broader benefits for health system strengthening at a global level are acknowledged but not accounted for.Overall, we conclude that if the acquisition of volunteering outcomes is realised, the NHS can accrue a productivity increase of between 24 and 41% per volunteer, with a value ranging from £13,215 to £25,934 per volunteer.Additional file 1:Background on health partnerships –34. (DOC


Acinetobacter nosocomialis is an opportunistic human pathogen that is part of the Acinetobacter calcoaceticus/Acinetobacter baumannii (ACB) complex. Here, we report the complete genome sequence of Acinetobacter nosocomialis strain M2. Acinetobacter nosocomialis is an opportunistic human pathogen that is part of the Acinetobacter calcoaceticus/Acinetobacter baumannii (ACB) complex. Here, we report the complete genome sequence of Acinetobacter nosocomialis strain M2. Acinetobacter nosocomialis is a Gram-negative coccobacillus that is a member of the Acinetobacter calcoaceticus/Acinetobacter baumannii (ACB) complex. While A. baumannii predominates over all other members of the ACB complex in terms of incidence, poorer clinical outcomes, and antibiotic resistance rates, A. nosocomialis remains a clinically relevant human pathogen.A. nosocomialis strain M2  of 0.05, as measured by a BioMate 3 spectrophotometer (Thermo Fisher Scientific). This was incubated at 37°C and 180 rpm until an OD600 of 0.7 was reached. DNA extraction was performed using 5 ml of this culture and the Genomic-tip 500 kit (Qiagen) per the manufacturer’s instructions. Purified genomic DNA (gDNA) was analyzed using a NanoDrop spectrophotometer and Qubit fluorometer (Thermo Scientific), the latter using a double-stranded DNA (dsDNA) high-sensitivity (HS) kit per the manufacturer’s instructions. A 1D native gDNA barcoded library was generated using an Oxford Nanopore Technologies (ONT) native barcoding expansion kit  and ligation sequencing kit  per the manufacturer’s instructions. The DNA library containing the NB01 barcode was sequenced on a FLO-MIN106 flow cell using the MinION device (ONT). This sequencing run was multiplexed with 1 other bacterial gDNA library tagged with a different barcode. All computation, other than data collection, was performed using Amazon’s Elastic Compute Cloud (EC2) service. Base calling was performed using Guppy v3.0.3 (ONT) with default settings and graphics processing unit (GPU) acceleration (EC2 p3.2xlarge instance). Demultiplexing was performed first using Guppy, which yielded 176,357 reads totaling ∼2.9 Gbp. An additional round of demultiplexing, with adapter and barcode trimming, was performed with Porechop v0.2.4 (github.com/rrwick/Porechop) with the discard_middle option enabled. Reads were filtered using Filtlong v0.2.0 (github.com/rrwick/Filtlong) with standard settings, except for a minimum length of 20,000 bp, to keep only the top 90% of reads, and with a target output of 500 Mbp. Canu v1.8  plate and incubated at 37°C for ∼16 h. A 14-ml round-bottom tube containing 2 ml of Luria broth was inoculated with a single colony picked from this plate and incubated at 37°C and 180 rpm for ∼16 h. This overnight culture was used to inoculate a 250-ml Erlenmeyer flask containing 25 ml of Luria broth prewarmed to 37°C to a starting optical density at 600 nm (ODanu v1.8 , with stanu v1.8  with staanu v1.8  and Bowtols v1.9 . The comA. nosocomialis strain M2 reported here was deposited in GenBank under accession number CP040105. Base-called reads can be found in the Sequence Read Archive under accession number SRR9018422.The complete genome sequence of