HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes
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Abstract
The dynein motor complex mediates polarised trafficking of a wide variety of organelles, intracellular vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dynein’s tail region and activates motor movement. How dynein and dynactin orchestrate trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the AP1 clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B is one of several proteins recovered in a biochemical screen for proteins whose association with the human dynein tail complex is augmented by dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the HEATR5B homologue in Drosophila is an essential gene that promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, our data point to a non-canonical process orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes in a dynein-independent manner. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.
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Reply to the reviewers
We thank the three reviewers for their thorough evaluation of our work and for their positive and constructive feedback. Below we provide an initial response to these comments, including descriptions of several changes incorporated in our preliminary revision. Please note that introductory and significance comments are not repeated here:
Reviewer 1
In my opinion, the main strength of this work is in the development and use of the original assay for adapter identification. As I already indicated, this is a biologically very important problem for cytoplasmic dynein. Another important strength of the paper is the extension of the work to Drosophila. …
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Reply to the reviewers
We thank the three reviewers for their thorough evaluation of our work and for their positive and constructive feedback. Below we provide an initial response to these comments, including descriptions of several changes incorporated in our preliminary revision. Please note that introductory and significance comments are not repeated here:
Reviewer 1
In my opinion, the main strength of this work is in the development and use of the original assay for adapter identification. As I already indicated, this is a biologically very important problem for cytoplasmic dynein. Another important strength of the paper is the extension of the work to Drosophila. Demonstration of the fact that Heatr5B is an essential gene, and that the product of this gene is involved in dynein-dependent trafficking in fly embryos makes the results significantly more important.I do not think there are many problems with the results in this manuscript. Generally speaking, the data on biochemical interactions are not as strong as I would like them to be. This is explained mainly by the fact that the authors do not have an expressed recombinant Heatr5B that they can use in biochemical experiments, and they limit their biochemistry by pulling down the protein from cell extracts.
Whilst we are very grateful to the reviewer for their thorough evaluation of our work, we do not understand this particular comment. We did include data with recombinant HEATR5B showing binding in vitro to purified dynein and dynactin complexes. The results are shown in Fig. 2B. We have now made it clearer (from line 153 of the preliminarily revised manuscript) that these experiments used recombinant HEATR5B. We hope in the future to determine the biochemical and structural basis of HEATR5B’s interaction with dynein and dynactin but feel that this goes well beyond the scope of this initial study (which already covers a lot of ground), especially as we have not yet found a way to express HEATR5B fragments (line 151).
This creates one of the few experimental problems with the paper. The authors claim that dynein and dynactin do not compete for Heatr5B binding, and therefore they can bind to both components of the complex at the same time. Unfortunately, I do not think that this claim is justified because concentrations of dynein and dynactin in their pull-down assay are much higher than the concentration of GFP-HEATR5B, and likely that HEATR5B does not saturate the binding sites on the motor complex. Therefore, it is unclear whether dynein and dynactin compete for Heatr5B binding. In any case, the conclusion about the competition cannot be seriously made without analysis of saturation curves.
The purified dynein and dynactin were not in excess to recombinant HEATR5B in this assay (80 pmol HEATR5B, 20 pmol dynein tail and 10 pmol dynactin). Nonetheless, the reviewer makes a very good point that we cannot draw strong conclusions about competition unless we generate saturation curves. We have therefore toned down the interpretation of this experiment and included the caveat raised by the reviewer (from line 157):
‘Compatible with this notion, we did not observe competition between the purified dynein tail and dynactin for HEATR5B binding in our in vitro binding assay when both complexes were added simultaneously to the beads (Figure 2B). However, we cannot rule out the possibility that a competitive interaction was masked by binding sites on one of the components not being saturated. Nonetheless, we can conclude from this set of experiments that HEATR5B complexes with endogenous dynactin and dynein in cell extracts and can interact with both complexes directly.’
Please note that this was only a minor point in our manuscript.
My second concern with this paper is the quality of imaging in mammalian cells. Unfortunately, not much can be done with live cell imaging because GFP-HEATR5B is expressed in cells at a low level (see, for example, Fig. 3A). However, in fixed cells GFP-HEATR5B signal could be easily amplified using anti-GFP antibodies.
The fixed cell images of GFP-HEATR5B cells are stained with anti-GFP antibodies and are the result of extensive optimisation of staining and imaging conditions. Due to its low expression and presence in both cytoplasm and membrane-bound pools, the signal for GFP-HEATR5B is not as striking as, for example, RAB11A and AP1γ. Nonetheless, the punctate signals are sufficiently strong to confidently evaluate co-localisation with membrane markers. We have now added to the relevant legends that the GFP signal is obtained via GFP antibody staining. Please note that the association of GFP-HEATR5B with AP1γ (Fig. 3A, B) was also confirmed by immunoprecipitation (Fig. 2A).
A minor problem with movie presentations is that the authors should include both a timer and a scale bar for all their live cell sequences, especially because the movies are looped. The authors did it for Movie 5, and they should do it for the rest of their live cell sequences.
Although information on the duration of movies (including loops) was included in the legends, we agree that it would be helpful to incorporate timers and scale bars in the movies. We have not been able to include this change in the preliminary revision as we have to co-ordinate with our visual aids team to reapply labels and arrows to the edited movies. This change will be made to the full revision.
In my opinion, the main novelty of this paper is in its pull-down assay, I would like to have it discussed more extensively. The authors state that they "were particularly drawn to Heatr5B". Is there an objective reason for this choice? If so, it should be specified.
We included our two reasons for focusing on HEATR5B in the previous submission, namely that it was the only protein to be enriched on the tail by dynactin in both the N- and C-terminal tethering configuration and that a previous study found it was one of a number of proteins present on dynactin-associated vesicles. We have modified the language in this section (which starts on line 120 of the preliminary revision) by using the connective ‘because’. This change makes it clearer that there were objective reasons for focusing on HEATR5B in the first instance.
Furthermore, I would like to see the authors discuss the other hits. Their list of hits includes a large number of ribosomal proteins. Do ribosomes really interact with dynein? Can the authors speculate on the number of true hits? Finally, it is likely that dynein interacts with some of the cargoes only transiently. How can the assay be modified to capture these transient interactions?
This is another very good suggestion. As requested, we have added a comment to the Discussion (from line 441) about how transient interactions might be captured using a variation of our strategy. We have now added a comment in the Discussion (from line 435) about the capture of ribosomes and other RNA-associated proteins in our screen, as well as the potential significance of this observation. We have also highlighted in this section another dynactin-stimulated hit, Wdr91, which we are following up. We also discussed the STRIPAK complex, which warrants further study, in the Results (line 106). We do not have space to discuss other hits but their identities are listed in Tables S1 and S2 together with a summary of their known functions for easy reference.
Reviewer 2:
Major points:
As a view of non-expert of light microscopy cellular imaging, some confocal images are difficult to accept as proofs of their conclusion that mutation to decrease HEAT5B/AP1 interaction results in diffusion from perinucleolar surface. For example, fluorescent signals in Control of Fig.4A seem more diffused than HR5B KO, which have fluorescence clearly localized on the surfaces of nuclei. Can they have explanation how it ends up with their statistical analysis in Fig.4E?
Fig. 4A is representative of what we typically see in mutant cells, with dispersion of the dimmer AP1γ signal in the cytoplasm and less disturbed localisation of the brighter AP1γ signal at the TGN (see Fig. S6B for quantification of AP1γ signal at the TGN in control and mutant cells). We should have made it clear in the Results that the unbiased image analysis pipeline used to produce Fig. 4E detects the total AP1γ signal not just bright signals (this feature of the pipeline is important given the differences in fluorescent intensity of puncta in the two genotypes). We have now clarified this issue in the Results (line 257) and the Fig. 4E legend. We have also added arrowheads to Fig. 4A to highlight the dispersed dim signal in the mutant cells. We thank the reviewer for leading us to improve the description of this experiment__.__
When they mention statistically more distance between target molecules and the perinuleolar surface, are dynein/dynactin connected to AP1 via HEAT5B stalled on the microtubule before reaching the minus end, or dissociate from the microtubule? Clarifying this will improve impact of this work. If the current data is not enough to answer, this reviewer will propose another confocal microscopy with also tubulin labeled. With this, the location of HEAT5B, AP1 etc. with respect to both nuclei and microtubule cytoskeleton will be clarified.
We would love to know the answer to the question of whether HEATR5B disruption reduces the association of AP1γ with microtubules. We have looked into co-localisation of microtubules and dynein’s cargoes previously using advanced light microscopy and found that it is not possible to draw conclusions about meaningful versus coincidental associations because of the density of the microtubule network. In the case of our current study, this approach would be further confounded by the difference in size in fluorescent AP1 puncta in control and HEATR5B mutant cells. We have also in the past attempted to purify recycling endosomal membranes from cells to determine how loss of HEATR5B influences dynein-dynactin association. However, even after extensive efforts we could not reproduce selective purification of recycling endosomes using the published protocol, or indeed variants of it. What is more, we find in general that there is rapid dissociation of motors during purification of membranes from cells, which would confound our results even if we could purify the recycling compartment. We therefore feel that the only way to address the question of how HEATR5B modulates dynein function at the molecular level is to reconstitute the transport machinery with pure proteins (including the as-of-yet unidentified activating adaptor) and microtubules in vitro, which is beyond the scope of this study. We have discussed the future aim of in vitro reconstitution to dissect mechanism in the Discussion (from line 494).
In Line168-169, they concluded AP1gamma associated with TGN rarely overlapped with HEATR5B, based on Fig.3A (where HR5B and AP1 seem overlapped in HeLa cells), Fig.S2A (where AP1gamma and TGN46 seem overlapped in U2OS cells) and Fig.S2B (where HR5B and TGN46 are not overlapped in HeLa cells). Is Fig.3A not contradictory to their conclusion (AP1gamma and HEATR5B not overlapped)? Why did they not directly check the overlap between AP1gamma and HR5B in the same cell in U2OS cells?
We don’t understand why our co-localisation data might be contradictory to our conclusions. Fig. 3A, together with the associated insets and quantification in Fig. 3B, show overlap of HEATR5B with AP1γ puncta in the cytoplasm of HeLa cells but not the AP1γ that is strong enriched in the perinuclear region, as we stated in the results. Absence of enrichment of HEATR5B with the TGN is additionally shown in Fig. S2B. These observations are commented on further in the Discussion (from line 517). We do, however, agree with the reviewer that is was not ideal that we did not show AP1 and TGN association in a HeLa cell (even though it has been documented in the literature). We have now corrected this oversight by showing HeLa cell data in Fig. S2A. We could not check the overlap of AP1 and HEATR5B in U2OS cells as we do not have a GFP-HEATR5B stable U2OS cell line.
Minor points:
Line100-105 and Fig.1EF are not clear. Is it correct that proteins in red bold letters and in blue letters in Fig.1EF are 28 proteins enriched on the dynactin tail?
We should have been clearer here and thank the reviewer for spotting this. We have modified the figure call outs in the text to include the labelling scheme, which we think helps significantly. We have also clarified the labelling system in the legend. To summarise, bold labelling indicates interactors of the dynein tail that are not core components of the dynein-dynactin machinery (such proteins are labelled in non-bold and italics); the blue bold text shows those ‘none core’ interactors that were only enriched on the dynein tail when exogenous dynactin was spiked into the lysates.
Do authors have any idea why the "dynactin-stimulated" ones (in blue) are localized at left end of this group (relatively less significance of dynactin tail binding, if this reviewer understands correctly)?
This question appears to indicate some confusion about whether we are capturing the dynein tail or dynactin. We believe the changes made in response to the previous comment about the labelling scheme should help clear this up. Being positioned to the left of this grouping shows a lower degree on enrichment vs the control (although still greater than 10 fold), rather than a difference in statistical significance. The observation that core dynein-dynactin subunits are more enriched on the dynein tail indicates that these interactions are the most stable or the most frequent.
Fig.S7: More explanation how to conclude that HR5B KO is dimmer than Ctrl based on this plot would be helpful.
We have added a line to the legend to Figure S7C to clarify this matter.
Reviewer 3:
Minor comments
HEATR5B overexpression in U2OS cells increased perinuclear clustering of Rab11A/AP1/dynactin-associated membrane. To which compartment are these vesicles directed and associated, the Golgi apparatus? Could the authors show which compartment it is?
We plan to perform new experiments to address this minor comment in the full revision. However, given their location near the microtubule organising centre, it is likely that the relocalised membranes will be in the vicinity of the Golgi apparatus.
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Referee #3
Evidence, reproducibility and clarity
Summary:
The goal of the authors is to identify dynein regulators which control how dynein and dynactin complexes orchestrate trafficking of diverse cargoes. To do so, the authors have performed a well thought proteomic screen for novel interacting proteins of the dynein tail potentially enhanced by dynactin. These pull-down experiments identified about 50 new dynein tail-interacting proteins, many of which were enhanced by dynactin.
The authors focused on one candidate, HEATR5B, because it was robustly isolated from the screens and its association with the dynein tail was stimulated by exogenous dynactin. HEATR5B is known to …
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Referee #3
Evidence, reproducibility and clarity
Summary:
The goal of the authors is to identify dynein regulators which control how dynein and dynactin complexes orchestrate trafficking of diverse cargoes. To do so, the authors have performed a well thought proteomic screen for novel interacting proteins of the dynein tail potentially enhanced by dynactin. These pull-down experiments identified about 50 new dynein tail-interacting proteins, many of which were enhanced by dynactin.
The authors focused on one candidate, HEATR5B, because it was robustly isolated from the screens and its association with the dynein tail was stimulated by exogenous dynactin. HEATR5B is known to interact with AP1 complex, as adaptors that orchestrate cargo loading of clathrin-coated vesicles from intracellular membranes.
The authors further show that HEATR5B complexes with endogenous dynactin and dynein as reveal by immuno precipitation from human cells extracts and can interact with both complexes directly. Then by using Hela cell line stably expressing GFP-HEATR5B, they show that HEATR5B is selectively enriched on the AP1 structure, some of which can be subjected to long-distance transport. They provide evidences that a large proportion of the HEATR5B-positive structures are associated with endosomal recycling membranes, as revealed by colocalization with RAB11A. They further show that the HEATR5B/ AP1 and HEATR5B/ RAB11 membrane structures show similar dynamics, indicating that HEATR5B associate with endosomal membranes that are capable of directed movement. SiRNA depletion of DYNC1H1 reveals that dynein promotes retrograde trafficking of AP-1 associated endosomal membranes.
The authors then investigate the contribution of HEATR5B to AP1-associated membrane trafficking by CRIPR/cas9-mediated mutagenesis in human U2OS cells that disrupt HEATR5B protein expression. They provide evidences that in HEATR5B mutant cells, there is a reduction in the amount of AP1 signal associated with RAB11A-positive structures indicating that disrupting HEATR5B reduces the association of AP1with endosomal membranes. This indicates that HEATR5B promotes AP1 recruitment to endosomal membranes. HEATR5B overexpression in U2OS cells increased perinuclear clustering of Rab11A/AP1/dynactin-associated membrane, suggesting that HEATR5B can stimulate retrograde trafficking of AP1-associated endosomal membranes by dynein- dynactin.
To assess the importance of HEATR5B function at the organismal level, as well as in polarized cell type the authors investigate its function in Drosophila in which there is a single HEATR5B homologue (Heatr5). They generated via crisper an Heatr5 mutant strain. Heatr5 homozygous mutants are zygotic lethal that died in second larval instar stage. They further provide evidence by investigating nos-cas9 gRNA-Hr51+2 mothers, that Heatr5 plays maternal function essential for embryogenesis. They further show that in early embryos from nos-cas9 gRNA-Hr5 females AP1 puncta are strongly reduced and dimmer.
Next, to understand the effect of Heatr5 disruption on AP1-based trafficking in Drosophila, they used the syncytial blastoderm embryo in which the microtubule cytoskeleton is highly polarized with less apically nucleated ends above the nuclei and more basally extended ends. In this system, the activity of minus end-directed motor, such as dynein, and minus end-directed motor, such as kinesin, can be distinguished by the direction of cargo movement.
By injecting AP1 antibodies into wild-type and Heatr5 mutant embryos, they provide evidence that AP1 undergoes net apical transport in the Drosophila embryo and that this process is strongly promoted by Heatr5. They further show that this process is microtubule and dynein dependent and that Heatr5 selectively promotes dynein-mediated transport of AP1 structures in the embryo. They then show that Heatr5-dependent AP1 trafficking pathways in the embryo involves the endosomal and Golgi membranes and that Heatr5 is also required for Golgi organization.
Major Comments
This study is very comprehensive and multi-scale. It ranges from the identification of a dynein motor adaptor for membrane trafficking by a proteomic screen, to its functional characterization in human cells and then during development with Drosophila embryo as model organism. The data are of high quality and are supported by very convincing quantitative analyses. The results are conclusive and the experiments have been carried out and presented in a very constructive way. This combination makes the manuscript very interesting.
Minor comments
HEATR5B overexpression in U2OS cells increased perinuclear clustering of Rab11A/AP1/dynactin-associated membrane. To which compartment are these vesicles directed and associated, the Golgi apparatus? Could the authors show which compartment it is?
Significance
This study is important in two aspects. Firstly, it has identified HEATR5B as a new adaptor of the dynein motor for intracellular membrane trafficking. It is important to mention that this motor is involved in many transport processes and it is still unclear how a single motor orchestrates the traffic of so many cargoes. Second, this work shed new light on the retrograde trafficking from endosomal material to the Golgi apparatus, in particular with HEATR5B, a known interactor of the AP1 clathrin adapter complex. This study highlights a role of HEATR5B in a novel dynein-based process for retrograde trafficking of AP1-associated endosomal vesicle to the Golgi apparatus. It also indicates that HEATR5B promotes association of AP1 with endosomal membrane in a dynein-independent manner.
This work is particularly important for the cell biology field.
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Referee #2
Evidence, reproducibility and clarity
Major points:
As a view of non-expert of light microscopy cellular imaging, some confocal images are difficult to accept as proofs of their conclusion that mutation to decrease HEAT5B/AP1 interaction results in diffusion from perinucleolar surface. For example, fluorescent signals in Control of Fig.4A seem more diffused than HR5B KO, which have fluorescence clearly localized on the surfaces of nuclei. Can they have explanation how it ends up with their statistical analysis in Fig.4E?
When they mention statistically more distance between target molecules and the perinuleolar surface, are dynein/dynactin connected to AP1 via HEAT5B …
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Referee #2
Evidence, reproducibility and clarity
Major points:
As a view of non-expert of light microscopy cellular imaging, some confocal images are difficult to accept as proofs of their conclusion that mutation to decrease HEAT5B/AP1 interaction results in diffusion from perinucleolar surface. For example, fluorescent signals in Control of Fig.4A seem more diffused than HR5B KO, which have fluorescence clearly localized on the surfaces of nuclei. Can they have explanation how it ends up with their statistical analysis in Fig.4E?
When they mention statistically more distance between target molecules and the perinuleolar surface, are dynein/dynactin connected to AP1 via HEAT5B stalled on the microtubule before reaching the minus end, or dissociate from the microtubule? Clarifying this will improve impact of this work. If the current data is not enough to answer, this reviewer will propose another confocal microscopy with also tubulin labeled. With this, the location of HEAT5B, AP1 etc. with respect to both nuclei and microtubule cytoskeleton will be clarified.
In Line168-169, they concluded AP1gamma associated with TGN rarely overlapped with HEATR5B, based on Fig.3A (where HR5B and AP1 seem overlapped in HeLa cells), Fig.S2A (where AP1gamma and TGN46 seem overlapped in U2OS cells) and Fig.S2B (where HR5B and TGN46 are not overlapped in HeLa cells). Is Fig.3A not contradictory to their conclusion (AP1gamma and HEATR5B not overlapped)? Why did they not directly check the overlap between AP1gamma and HR5B in the same cell in U2OS cells?Minor points:
Line100-105 and Fig.1EF are not clear. Is it correct that proteins in red bold letters and in blue letters in Fig.1EF are 28 proteins enriched on the dynactin tail? Do authors have any idea why the "dynactin-stimulated" ones (in blue) are localized at left end of this group (relatively less significance of dynactin tail binding, if this reviewer understands correctly)?
Fig.S7: More explanation how to conclude that HR5B KO is dimmer than Ctrl based on this plot would be helpful.
Significance
In this work, Madan and colleagues studied dynein adaptor proteins, which are stimulated by dynactin, using proteomics, fluorescent microscopy, live cell imaging techniques for U2OS and fly embryo cells. They especially focused on HEATR5B and proved its role to bind AP1 membrane associate protein for intracellular transport. They first conducted proteomic studies and presented novel lists of dynein-associated proteins and proteins stimulated by dynactin. Among them they decided to prioritize HEATR5B protein (it would be interesting to know their motivation to choose this protein) and carried on fluorescent microscopy studies to characterize roles of HEATR5B in microtubule-based motility. Their approach using U2OS cells is to correlate distribution of HEATR5B and such proteins as AP1gamma, TGN46, RAB11A, which they expect interaction with HEATR5B, between WT and mutants. They remarkably demonstrated distance from perinucleolar membrane is heavily influenced by defect of adaptor function of HEATR5B, by fluorescent microscopy and statistical analysis. Next they made HEATR5B depleted Drosophila embryo by CRSPR-CAS9. They proved its influence on AP1 trafficking to Golgi, which is another novel finding of this study, consistent with the case of U2OS cells.
In general the whole study proved importance of HEATR5 proteins on AP1 trafficking. Many data are presented in convincing way and carefully statistically analyzed. This work will attract attention of wide audience from the field of cytoskeleton, motor proteins and membrane trafficking. After addressing a few points, the manuscript will be ready for publication.
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Referee #1
Evidence, reproducibility and clarity
This work is the first systematic attempt to identify and characterize a diverse set of adapters that attach cytoplasmic dynein to its different cargoes and thus activate the motor. It is an important work because in animal cells dynein is the only efficient motor that can perform processive transport toward the minus ends of microtubules, and therefore the specificity of transport for multiple cargoes along microtubules is determined by these adapters.
The authors use the recombinant tail of dynein for pulling down interacting proteins from the cell extract. This is a straightforward approach, but its main problem is the large …
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Referee #1
Evidence, reproducibility and clarity
This work is the first systematic attempt to identify and characterize a diverse set of adapters that attach cytoplasmic dynein to its different cargoes and thus activate the motor. It is an important work because in animal cells dynein is the only efficient motor that can perform processive transport toward the minus ends of microtubules, and therefore the specificity of transport for multiple cargoes along microtubules is determined by these adapters.
The authors use the recombinant tail of dynein for pulling down interacting proteins from the cell extract. This is a straightforward approach, but its main problem is the large number of non-specific proteins that bind to the column. To solve the problem, the authors use a very smart approach. It is based on the fact that in all known cases so far dynein does not transport cargoes without dynactin, and, therefore, potential adaptors are unlikely to bind to the affinity column very efficiently. They compare pull-downs in the presence and absence of dynactin paying specific attention to proteins that bind in the presence of both dynein and dynactin but not dynein alone.
Among the proteins that have been identified by this assay is Heatr5B, the protein known to associate with AP1 clathrin adaptor. Functional characterization of the protein can be divided into two parts, work with mammalian Heatr5B in tissue culture cells and analysis of its function in Drosophila.
In my opinion, the main strength of this work is in the development and use of the original assay for adapter identification. As I already indicated, this is a biologically very important problem for cytoplasmic dynein. Another important strength of the paper is the extension of the work to Drosophila. Demonstration of the fact that Heatr5B is an essential gene, and that the product of this gene is involved in dynein-dependent trafficking in fly embryos makes the results significantly more important.
I do not think there are many problems with the results in this manuscript. Generally speaking, the data on biochemical interactions are not as strong as I would like them to be. This is explained mainly by the fact that the authors do not have an expressed recombinant Heatr5B that they can use in biochemical experiments, and they limit their biochemistry by pulling down the protein from cell extracts. This creates one of the few experimental problems with the paper. The authors claim that dynein and dynactin do not compete for Heatr5B binding, and therefore they can bind to both components of the complex at the same time. Unfortunately, I do not think that this claim is justified because concentrations of dynein and dynactin in their pull-down assay are much higher than the concentration of GFP-HEATR5B, and likely that HEATR5B does not saturate the binding sites on the motor complex. Therefore, it is unclear whether dynein and dynactin compete for Heatr5B binding. In any case, the conclusion about the competition cannot be seriously made without analysis of saturation curves.
My second concern with this paper is the quality of imaging in mammalian cells. Unfortunately, not much can be done with live cell imaging because GFP-HEATR5B is expressed in cells at a low level (see, for example, Fig. 3A). However, in fixed cells GFP-HEATR5B signal could be easily amplified using anti-GFP antibodies.
A minor problem with movie presentations is that the authors should include both a timer and a scale bar for all their live cell sequences, especially because the movies are looped. The authors did it for Movie 5, and they should do it for the rest of their live cell sequences.
In my opinion, the main novelty of this paper is in its pull-down assay, I would like to have it discussed more extensively. The authors state that they "were particularly drawn to Heatr5B". Is there an objective reason for this choice? If so, it should be specified. Furthermore, I would like to see the authors discuss the other hits. Their list of hits includes a large number of ribosomal proteins. Do ribosomes really interact with dynein? Can the authors speculate on the number of true hits? Finally, it is likely that dynein interacts with some of the cargoes only transiently. How can the assay be modified to capture these transient interactions?
Significance
The bottom line is very clear. For me, it is an excellent technical paper with biological results that clearly demonstrate the validity of the technique. As such, it can and should be published.
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