The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics

Abstract The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine‐tune motor activity in time and space. These motor–adaptor–cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling‐based proteomics strategy to map the interactome of the unique minus end‐directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6‐adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG‐Induced F‐actin for Tethering) complex that controls endosome positioning and motility through RHO‐driven actin polymerisation; and the DISP (DOCK7‐Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes.

Thank you for the submission of your research manuscript to our journal. We have now received the full set of referee reports that is copied below.
As you will see, the referees acknowledge the potential interest of the findings and consider the BioID data for MYO6 largely convincing. However, all referees also suggest several experiments to strengthen the data and conclusions on the further characterization of the novel protein complexes CART and DISP or suggest to tone down the conclusions accordingly. Moreover, all control experiments need to be provided and the experimental details for Fig4 and EV3 added.
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As part of the EMBO publication's Transparent Editorial Process, EMBO reports publishes online a Review Process File to accompany accepted manuscripts. This File will be published in conjunction with your paper and will include the referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.
You are able to opt out of this by letting the editorial office know (emboreports@embo.org). If you do opt out, the Review Process File link will point to the following statement: "No Review Process File is available with this article, as the authors have chosen not to make the review process public in this case." I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. ***************************** REFEREE REPORTS Referee #1: This manuscript describes the use of a proximity-labelling, mass spectrometry approach, BioID, to identify near-neighbours of myosin 6. The authors identify a series of new neighbours/interactors, and perform further BioID with some of these components. In doing so, they identify and partially characterise several new myosin 6-associated complexes. Importantly, two of the complexes contain RHO GEFs that regulate the actin or septin cytoskeleton, meaning that MYO6-associated components can potentially regulate cytoskeletal and endosome dynamics in a co-ordinated fashion. Overall, this is a convincing study that is very well presented and executed and adds considerably to our understanding of MYO6 and its interactors. I have only minor criticisms. 1. A BioID study of dynein has been published (Redwine et al. (2017). The human cytoplasmic dynein interactome reveals novel activators of motility. eLife, 6. http://doi.org/10.7554/eLife.28257), so this is the first BioID investigation of a myosin, not of a motor. The text needs to be changed in several places to take account of that. 2. It would be helpful to provide a brief description of the BioID approach in the introduction or results for readers who don't already know it, and references should be provided. 3. The data presented show that the BirA*-MYO6 localises as expected in the RPE cell line, but it would be useful to know if it is fully functional. Does it rescue a knockdown, for example? Also, what is the relative level of expression of the tagged version in comparison to the endogenous protein? 4. On page 7, the authors state that CADR10 is a high-confidence MYO6 interactor that was present in the GIPC1 data set, however it does not appear in the list in Fig. 3A. 5. The authors use an antibody to SH3BP4 for IF and immunoblotting, but only show a knockdown control for blotting. Since the IF labelling is fairly non-descript, it would be good to show that knockdown also removes that staining. 6. Do the authors have a positive control for the septin IP data in Fig. EV5A? 7. On p11 the authors state that over-expression of LRCH3 led to the displacement of septins from the actin cytoskeleton, as they clearly take up unusual ring shaped structures. However, actin labelling is not shown, so it is formally possible that actin is rearranged too. It would be useful to show actin labelling in parallel as a control. 8. The enhancement of septin rings when LRCH3 and the DOCK7 DHR2 domain are co-expressed is dramatic. Is this MYO6 dependent? 9. The authors coin a new name for one of the complexes they identify: CART. One concern with this is that it is very similar to CARTS, which is also a trafficking-related name (Wakana et al. (2012). A new class of carriers that transport selective cargo from the trans Golgi network to the cell surface. EMBO Journal, 31, 3976-3990). 10. The methods are clearly defined, except that there is no description of the HA-surface labelling protocol used for the LPA stimulation experiments. This means it is hard to understand exactly what has been done in Fig. 4 and EV3. 11. It is hard to see the difference between the fine and thicker grey lines in Fig. 2. What is a forcedirectly layout? 12. How many cells were analysed in Fig. 4D?
This is an ambitious effort using BioID to identify proteins that interact directly or indirectly with myosin-6 in human cells. After a first round of BioID, the authors confirmed the interactions with BioID from the initial partners and IP's to expand the network and verify the partners. The text and figures are clear and easy to follow. This is a valuable addition to the literature, but can be improved by attention to the following points.
Page 3: It would be helpful to spell out or define in a short phrase the meaning of "DAB2, GIPC1, TOM1, LMTK2, OPTN, TAX1BP1 and NDP52". What are the meanings of "RRL and WWY?" Page 8: Reword "Together these data show".... The micrographs are so small that it is difficult to appreciate that "actin structures" are filopodia at the cell surface (Fig. 3D)." Consider showing higher magnification details. Also the concept of "filopodia protruding from the endosome surface" is difficult to understand. Is filopodium an appropriate word to describe these structures? Page 9: It is essentially impossible to confirm the "colocalisation of APPL1 with actin upon LPA stimulation ( Fig. 4C and Fig EV3B)" because the image is so small (even when enlarged 4 times). The used of dark blue for actin does not help. The text might explain how is Myo6 related to the conclusion "CART complex is an actin regulatory module, which functions downstream of GPCRs such as LPAR1 to drive RHO-mediated actin reorganisation at the early endosome surface, regulating organelle positioning and motility." Page 12: Overall the data support the authors' conclusions, but they may go a bit too far with "We show that the GEF activity of LARG or LPAR1-LARG-RHO signalling have profound effects on the positioning and motility of MYO6-GIPC1-positive endosomes." True interesting things happen with the reverse mutant myo6+, but that may not be enough to establish "profound effects". "Actin remodeling" as the output of this pathway in Fig. 4A is appropriately vague, since the mechanisms are not established.
Page 13: "These (septin) filaments are intimately linked to the actin cytoskeleton" may also be an overstatement. The experiment showing overexpression of the DISP complex results in septin rings is only indirect (and possibly misleading) evidence for the DISP complex regulating septins under normal conditions, given that "the precise mechanistic details" are missing.
Page 18: You did not fix the cells with paraformaldehyde. It is a solid, which you converted to formaldehyde to fix the cells.
Referee #3: The authors perform a proteomic study of the unconventional minus-end directed actin motor Myo6. They identify over 50 new potential interactions using the biotin ligase BirA fused to two different Myo6 constructs. They do further pulldowns and proteomic analysis on some of the prominent hits to confirm and identify networks. The proteomic data are nicely presented and appear to be carefully done. New binding partners for Myo6 give insight into the function of this motor and will open up new areas of study. Unfortunately, the specific studies of the two complexes are less convincing and the data surrounding these studies seems to be somewhat over-interpreted. Specific comments follow:

Major Points
What is the significance of the two different Myo6 constructs CBD-LI and CBD NI used for the pulldowns? They were described as interacting with different populations of vesicles but then no data are shown to address whether they have different binding partners or not? Pulldowns (Fig 3) do not really provide sufficient evidence to claim that a complex of MYO6, GIPC1, LARG and SH3BP4 exists -just that these proteins cross-interact. Can the authors use a method such as blue native PAGE or gel filtration to show that an actual complex exists? If not, then this needs to be toned down as the interactions may be transient and this complex may not actually be present in cells. Figure 3E-Are these filopodia? Maybe actin clusters is a better term? How was an actin cluster or filopodium defined? This seems unclear and the data presented are not very convincing. Figure 4 is not convincing and the importance of the colocalization with actin is not clear. When the cells are stimulated by LPA or LARG, endosomes align along actin fibers and are less mobile in the cytoplasm. But is this just because the cytoplasm is more crowded and so the endosomes are trapped in the stress fibers? Are endosomes normally associated with actin stress fibers? What is the function or reason for this? To me it seems like these data are over-interpreted.
It is pretty unclear from Figure 6 how directly Myo6 is involved with Sept7, as these are all pretty indirect experiments showing that disruption of the actin network that normally holds the Sept7 on stress fibers causes them to instead assemble rings. Maybe again the interpretation just needs qualification and toning down a bit. We have amended the text in the abstract, the end of the introduction and at the beginning of the discussion, which now states correctly that our study provides the first interactome for a myosin motor protein.

It would be helpful to provide a brief description of the BioID approach in the introduction or results for readers who don't already know it, and references should be provided.
A short description of BioID is now included on page 4 of the introduction.

The data presented show that the BirA*-MYO6 localises as expected in the RPE cell line, but it would be useful to know if it is fully functional. Does it rescue a knockdown, for example? Also, what is the relative level of expression of the tagged version in comparison to the endogenous protein?
In our experiments we only used the C-terminal cargo-binding domain (CBD) of MYO6, which is sufficient for cellular targeting and contains the major protein and lipid binding motifs. As BioID naturally has a limited range of approximately 10 nm (Kim et al., PNAS, 2014), and the full-length MYO6 is between 15-20 nm (Lister et al., EMBO J, 2004), we obtained the highest quality data using only the CBD. We now included a sentence explaining our rationale (the limited reach of BioID) in the results on page 6.
Therefore, beyond targeting and binding to known adaptor proteins, we cannot test whether the CBD is fully functional, as this domain alone will not be able to rescue MYO6-depleted cells.
In addition, determining the relative expression of our BirA* construct versus the endogenous is difficult to assess as we don't have antibodies (despite trying to raise one...) which react with both the full-length endogenous protein as well as the CBD used in our study. Furthermore, due to the differences in size or other properties between endogenous MYO6 (~150 kDa) and BirA*-MYO6 CBD (~62 kDa) we would argue any such comparison would be difficult to interpret. For example, differences in membrane transfer due to differing size/charge or possible auto-inhibitory back folding of the endogenous MYO6 altering antigen availability. We are satisfied that the BirA* constructs localised clearly to their relevant compartments which appeared morphologically normal. Fig. 3A.

On page 7, the authors state that CADR10 is a high-confidence MYO6 interactor that was present in the GIPC1 data set, however it does not appear in the list in
This oversight has been amended and CARD10 is now present in the list in Fig. 3A.
5. The authors use an antibody to SH3BP4 for IF and immunoblotting, but only show a knockdown control for blotting. Since the IF labelling is fairly non-descript, it would be good to show that knockdown also removes that staining.
A figure showing immunofluorescence staining of mock and SH3BP4 siRNA treated cells with the SH3BP4 antibody is now included in figure 3D, replacing the previous image. We have also included images through the Z-stack to highlight the specificity of the actin labelling. Fig. EV5A?

Do the authors have a positive control for the septin IP data in
GFP-LRCH3 and several truncation mutants were immunoprecipitated with GFP-nanobodies and the same eluants were blotted for DOCK7, MYO6 and also SEPT7. Our results show that DOCK7 and MYO6 successfully co-immunoprecipitate with LRCH3 (shown in Fig. 5C), however, we were not able to pull down a complex of GFP-LRCH3 and SEPT7 (shown in Fig. EV5B). The positive control for the septin IP from figure EV5A is therefore shown in figure 5C. We have highlighted the fact that the same IP was blotted for DOCK7, MYO6 as well as SEPT7 in the results on page 12.  Fig. 4 and EV3.
The missing method has now been included on page 20 in the Materials and Methods. Fig. 2. What is a forcedirectly layout?

It is hard to see the difference between the fine and thicker grey lines in
The fine lines have now been changed to red.
A force-directed layout is a type of graph drawing algorithm, which assigns forces (which can be attractive or repulsive) to the nodes and edges in the plot and then simulates their movement to create the lowest energy state. This leads to, for example, minimisation of overlapping edges or the clustering of nodes in regions of highly interconnected data. Details of the algorithms can be found within the Cytoscape software manual or, alternatively, an overview of force-directed layout algorithms generally can be viewed here: https://en.wikipedia.org/wiki/Force-directed_graph_drawing. Fig. 4D? Figure 4D depicts the mean Pearson's correlation coefficient calculated from n=4 independent experiments. In each experiment 1-7 cells per field were quantified from ≥7 randomly selected fields giving a total of 106 mock cells & 137 GFP-GEF expressing cells. We have also added clarifying comments to the legends of all figures containing quantification.

Reviewer #2
Page 3: It would be helpful to spell out or define in a short phrase the meaning of "DAB2, GIPC1, TOM1, LMTK2, OPTN, TAX1BP1 and NDP52". What are the meanings of "RRL and WWY?" These protein names are now spelled out on page 3 of the introduction. The RRL and WWY motif is a short amino acid sequence, named after their amino acid composition. Again, this has been highlighted in the text on page 3. amines in proximal proteins which can then be isolated using the high affinity interaction between the newly generated biotin tag and streptavidin. As the biotin is covalently attached to its target this permits lysis and purification under harsh, denaturing conditions while still preserving weak or transient interactions." In addition, we have highlighted the limitations in reach of the enzyme and therefore the need to fuse the BirA* enzyme to defined protein domains in our result section on page 6: "As BioID has a limited labelling radius, we used the truncated CBD in our experiments which is sufficient for adaptor and lipid binding and therefore subcellular targeting. Attaching BirA* at the N-terminus of the full-length protein (before the motor domain) largely failed to identify cargo interactions at the C-terminus, presumably due to the limited range of the biotinylation reaction (data not shown)." The figure reference in the text has now been corrected.

Page 8:
Reword "Together these data show".... The micrographs are so small that it is difficult to appreciate that "actin structures" are filopodia at the cell surface (Fig. 3D)." Consider showing higher magnification details. Is filopodium an appropriate word to describe these structures?
The text has been corrected.
We appreciate that it is very difficult to determine whether the SH3PB4-positive actin structures are filopodia protruding from the cell surface and have now included images through the Z-stack to clarify the colocalisation in this dimension. In addition, we have included immunofluorescence images from SH3BP4-depleted cells to further highlight the specificity of this staining.
Also the concept of "filopodia protruding from the endosome surface" is difficult to understand.
The term "filopodia protruding from the endosome surface" is not very precise and now has been change to "filopodia protruding from the cell surface above a cortical cluster of endosomes" on page 9 of our results section.

Is filopodium an appropriate word to describe these structures?
We have characterised these spike-like actin protrusions induced by MYO6+ in great detail in our recent PNAS paper (Masters et al., 2017) which is referenced in the manuscript. In that study we show these structures contain both myosin X and fascin, classic marker proteins for filopodia.

Page 9:
It is essentially impossible to confirm the "colocalisation of APPL1 with actin upon LPA stimulation ( Fig. 4C and Fig EV3B)" because the image is so small (even when enlarged 4 times). The used of dark blue for actin does not help.
We hope the submission of high quality eps/tiff files over the low quality jpegs of the initial submission will help to clarify this point. To make the colocalisation of APPL1 and actin more obvious, we have changed the colour of the actin channel to green as suggested and now show several images of enlarged areas. However, these are only select examples of the results quantified in three independent experiments (n=3) on more than 80 different cells.

The text might explain how is Myo6 related to the conclusion "CART complex is an actin regulatory module, which functions downstream of GPCRs such as LPAR1 to drive RHO-mediated actin reorganisation at the early endosome surface, regulating organelle positioning and motility."
We  Our results are based on overexpression of the DOCK7 GEF, which in the presence of LRCH3 induces a dramatic reorganisation of the septin cytoskeleton without inducing a visible change in actin organisation. So far very few proteins have been identified that induce such an obvious change in septin localisation and which can be assessed and quantified. We agree, at present we cannot present any insight into "precise mechanistic details", however, the regulation of septin function is likely to involve a number of different mechanisms and regulators dependent of the different cellular processes that require septin activity. We therefore feel that it is beyond the scope of this project to determine the details of septin cytoskeleton regulation, however, our discovery of a RhoGEF involved in this process is important.

Page 18: You did not fix the cells with paraformaldehyde. It is a solid, which you converted to formaldehyde to fix the cells.
Sorry, this has been changed now.

Reviewer #3
What is the significance of the two different Myo6 constructs CBD-LI and CBD NI used for the pulldowns? They were described as interacting with different populations of vesicles but then no data are shown to address whether they have different binding partners or not?
A detailed comparative analysis is now included in figure 1 and we have amended the manuscript to discuss these results as follows: "Comparison of the NI and LI showed 39 shared interactions and 16 or 47 specific interactions for the LI and NI isoforms respectively. Many of the known direct binding partners of MYO6 appear in the shared pool of interactions for the two isoforms (Fig. 1E). This confirms our previous observations that binding of DAB2 and other adaptors is not isoform specific [8], [10], [14], but targeting of the LI isoform to clathrin-coated structures is directed by the large insert [27]. As a result, the LI still appears to show enrichment for CCS proteins such as AP2 subunits, SYNJ1 and PICALM, whereas the NI specific interactions are less well annotated but are likely to link it to diverse cellular localisations and functions". (Fig 3) do not really provide sufficient evidence to claim that a complex of MYO6, GIPC1, LARG and SH3BP4 exists -just that these proteins cross-interact. Can the authors use a method such as blue native PAGE or gel filtration to show that an actual complex exists? If not, then this needs to be toned down as the interactions may be transient and this complex may not actually be present in cells.

Pulldowns
We have shown the reciprocity of the interactions between MYO6, GIPC1, LARG and SH3BP4 by multiple methods including BioID (with MYO6, GIPC1 and LARG), immunoprecipitations and mammalian two-hybrid assays. Together, we feel our data provides strong evidence for the presence of this complex. Of course, the referee is correct to point out that the components may not form a stable complex and we agree with the possibility that the complex only forms transiently in living cells. We do not feel we have overstated the stability/transience of this complex in the manuscript but we are happy to change the text and emphasise this point if required.
3. Figure 3E-Are these filopodia? Maybe actin clusters is a better term? How was an actin cluster or filopodium defined? This seems unclear and the data presented are not very convincing.
We have described these actin structures, which are induced by the plus-end directed MYO6 protruding from the cell surface, in great detail in our recent PNAS paper (Masters et al., 2017). We used a range of different markers and show that they are indeed filopodia-like actin protrusions, which contain myosin X and also fascin, proteins associated with filopodia. We inserted the reference (Masters et al. PNAS 2017) in the result section on page 9 to refer to our published characterisation of these MYO6+-induced filopodia. Figure 4 is not convincing and the importance of the colocalization with actin is not clear. When the cells are stimulated by LPA or LARG, endosomes align along actin fibers and are less mobile in the cytoplasm. But is this just because the cytoplasm is more crowded and so the endosomes are trapped in the stress fibers? Are endosomes normally associated with actin stress fibers? What is the function or reason for this? To me it seems like these data are over-interpreted.

4.
We have previously performed a very careful characterisation of the association of different types of endosomes with actin filaments using high-resolution structured illumination microscopy. Our results demonstrate that the APPL1 endosomes that are positive for MYO6 align along actin filament bundles whereas EEA1-positive endosomes, which do not colocalize with MYO6, are surrounded by actin filament patches (Masters et al., Cell Reports 2017). These results indicate that while both APPL1-and EEA1-positive endosomes are associated with actin, the architecture and geometry of these interactions are highly divergent and therefore we feel that it is highly unlikely that "the endosomes are trapped in the stress fibers". We observe the APPL1 endosomes associate with actin filament bundles of different sizes concentrating in the cortical actin network, however, sometimes these endosomes also align along larger actin bundles, which could be termed stress fibers.
In light of our previous published findings that MYO6 has an important role in tethering APPL1 endosomes to cortical actin filaments, we feel that our data is not over-interpreted. Depletion of MYO6 affects endosome localisation and leads to displacement of these endosomes from the cell cortex into the perinuclear space. Furthermore, we recently demonstrated (Masters et al. PNAS 2017) that expression of the plus end directed MYO6+, thus reversing the direction of MYO6, leads to accumulation and clustering of APPL1 endosomes at the base of filopodia. Figure 6  We now have amended figure 6 and have included actin staining, which clearly shows that the overexpression of the DOCK7 GEF, in the presence of LRCH3, induces a dramatic reorganisation of the septin cytoskeleton, without inducing a visible change in actin organisation. So DOCK7 and LRCH3 cause a highly specific reorganisation of the septin cytoskeleton, which is not caused by simply depolymerisation of actin filaments.

It is pretty unclear from
Unfortunately, so far we are not able to show a direct requirement for MYO6 in septin cytoskeleton regulation, however, as shown in figure 6C, MYO6 is recruited to septin ring structures in the cytoplasm along with LRCH3 and DOCK7.
2nd Editorial Decision 14 December 2017 Thank you for the submission of your revised manuscript to our editorial offices. We have now received the reports from the referees that were asked to re-evaluate your study (you will find enclosed below). As you will see, all three referees now support the publication of your manuscript in EMBO reports.
Before we can proceed with formal acceptance, I have the following final editorial requests: The manuscript is currently rather long (more than 75000 characters including spaces), even for an article. Thus, I would ask you to shorten the manuscript to around 65000. Especially the figure legends are rather wordy and contain methods information and detailed descriptions of the results, which could be removed if redundant.
For the references, please use 'et al' for those references with ten or more authors. Please also list the references with simple numbering (without the square brackets) in the reference section. Square brackets should only be used for the call outs. See also: http://embor.embopress.org/authorguide#referencesformat Please add a reference or an accession number for the data deposited at the PRIDE proteomics data repository.
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Please rename the movie file as "Movie EV1" and then combine the movie file with a simple text file of the legend in a ZIP archive file, and upload the ZIPped file. Then please update the callout for the movie in the text and remove the legend from the main manuscript text.
Finally, could statistics be provided for the bar diagrams in Figs. 4C and EV3B?
We now strongly encourage the publication of original source data, in particular of Western blots, with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure.
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