Interplay between PLEKHG3-regulated actin dynamics and lysosomal trafficking in cell motility

  1. Rainer Ettelt
  2. Georg Vucak
  3. Sebastian Didusch
  4. Biljana Riemelmoser
  5. Karin Ehrenreiter
  6. Markus Hartl
  7. Lukas A. Huber
  8. Manuela Baccarini

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Abstract

Lysosomes are highly dynamic organelles that regulate metabolic signaling pathways by recruiting cytosolic molecules to protein platforms on the lysosomal membrane. We performed a proximity-dependent labeling screen to identify novel proteins recruited to the LAMTOR complex, which regulates lysosome positioning and key signaling pathways such as mTORC1, AMPK, and MEK/ERK. We identified a network of proteins involved in actin remodelling, including Pleckstrin homology domain-containing family G member 3 (PLEKHG3), an actin-binding Rho guanine nucleotide exchange factor enriched in protrusions. We show that GFP-PLEKHG3 accumulates in focal adhesion sites, where it colocalizes with peripheral lysosomes. Peripheral accumulation of lysosomes concentrates PLEKHG3 below the plasma membrane, inhibits protrusion formation and limits cell motility. Thus, subcellular positioning of lysosomes impacts PLEKHG3 subcellular localization and the cell’s protrusion activity, shape, and motility. The results shed new light on the interplay between lysosomes and actin dynamics and provides insights into the mechanisms controlling cellular processes such as shape regulation and motility of the plasma membrane.

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    Reply to the reviewers

    Manuscript number: RC-2023-01861

    Corresponding author(s): Manuela, Baccarini

    [The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

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    1. General Statements [optional]

    We were delighted to learn that all three reviewers found the paper novel and of interest for a cell biology audience. They especially highlighted the carefully conducted screen, whose results will be integrally published with this paper and will be of use for scientists interested in lysosome biology.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer 1

    The claim that "peripheral accumulation of lysosomes inhibits protrusion formation and limits cell motility" should be tested more rigorously using the RAMP method, preferably in living cells. Other approaches, such as overexpression/siRNA of Arl8b and other motor adaptors, such as SKIP/PLEKHM2, can be used to alter lysosome positioning and confirm this central finding of the manuscript. The authors could also consider including additional mechanistic data in order to comprehend how lysosome positioning controls cell motility. For instance, the RAMP approach could be employed to investigate cortical actin dynamics upon repositioning of lysosomes to the peripheral/perinuclear region.

    Answer: We have purchased the RAMP system from Addgene and are adapting it to our color setup to use it in HeLa cells expressing GFP-PLEKHG3 and (hopefully) in PLEKHG3 KO cells, adding LiveAct to investigate cortical actin dynamics upon lysosomal repositioning as well as PLEKHG3 KO.

    Reviewer 2

    5 - It is not clear if in cells KO for PLEKHG3, the overexpression of KIF1A leads to more lysosomes localizing close to the PM, as well as more protrusions and more cell motility, as the authors only compare cell overexpressing GFP or GFP-PLEKHGL3.

    Answer: Currently, we do not have a PLEKHG3 KO. We have, however, redoubled our efforts, so far unsuccessful, to generate a PLEKHG3 CRISPR-Cas KO in HeLa, going up to 10 sg-guides, and hope that we will be successful in the next future. In this case, we will be able to easily address this interesting question.

    Reviewer 3

    • Data presented in Figure 6 showing cell motility analysis is interesting and has potential to make the manuscript impactful. Similarly, data in Figure 4F (live cell imaging) looks attractive but is not informative in the absence of relevant genetic perturbations as comparisons. These types of experiments would benefit greatly from PLEKHG3 loss of function analysis, as well as mutational analysis in the over-expression setting.*

    Answer: We have redoubled our efforts to generate a PLEKHG3 CRISPR-Cas KO in HeLa, going up to 10 sg-guides, and hope that we will be successful in the next future. This cell line will be helpful in answering the Reviewer’s question.

    Mutational analysis cannot be performed, because of the lack of binding between LAMTOR3 and PLEKHG3, which leaves us without a read-out.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer 1:

    • The authors should also confirm the specificity of the PLEKHG3 antibody in immunofluorescence using control and PLEKHG3 siRNA in untransfected cells that have not been transfected with GFP-PLEKHG3 (as is shown in Fig. S2C). Numerous antibodies recognize the overexpressed protein but do not recognize the same protein at endogenous expression levels.

    Answer: To assess the specificity of the antibody for endogenous PLEKHG3 we have used HEK293T cells, which based on the fact that PLEKHG3 is most highly expressed in neuronal cells (https://www.proteinatlas.org/ENSG00000126822-PLEKHG3/tissue#expression_summary) should yield a clearer endogenous signal. The results of this experiment are shown in the revised Figure S2B-C. The pattern of PLEKHG3-positive bands is similar to that observed in HeLa cells, and only the band around 250 kD is clearly reduced by the siPLEKHG3. The IF images show a selective loss of the PLEKHG3 signal in correspondence of actin filaments close to the plasma membrane, whereas the nuclear signal is preserved, and therefore to be considered non-specific (Figure only shown in attached revision plan and revised Figure S2B-C). In addition, we have redoubled our efforts, so far unsuccessful, to generate a PLEKHG3 CRISPR-Cas KO in HeLa cells, going up to 10 sg-guides, and hope that we will be successful in the next future.

    Extract from revised Figure S2B-C: ____PLEKHG3 KD test in HEK293T cells: B) Western blot of HEK293T cells showing downregulation of PLEKHG3 expression upon siPLEKHG3 treatment compared to siScr. Bar plot shows quantification of PLEKHG3 bands from immunoblot above. Error bars = SEM, n=3. * = p values according to student's t-test. C) Immunofluorescence images of HEK293T cells. siPLEKHG3 shows drop in PLEKHG3 intensity in the periphery of the cell and less colocalization with Phalloidin. Scale bar = 50 µm. Line plots show intensity profiles of Phalloidin (green) and PLEKHG3 (red) along the white lines in the merged inset images. Scale bar = 10 µm.

    *The colocalization of endogenous PLEKHG3 and LAMP1 as depicted in figures 3B and 3C (data from fixed cells) is not convincing. PLEKHG3 appears to be present on cortical actin structures as opposed to being colocalized with LAMP1 on lysosomes. *

    And related to this point:

    There is no apparent colocalization of PLEKHG3 and lysotracker in the movie S5.

    Answer:

    We do not claim that the two structures always colocalize. The images in Figure 3C are a schematic representation of the colocalization analysis shown in the plot and were included to explain how we define PLEKHG3 high/low regions or LAMP1 high/low regions, respectively. We agree with the Reviewer and with the previous literature that PLEKHG3 main localization is to cortical actin structures, as shown in Figure 3F of the original version and in Figure S2C (HEK293T cells) and in Figure S3A in HeLa cells in the revised version. The claim is rather that PLEKHG3 has been identified as a vicinal protein of LAMTOR3, seen by a fraction of lysosomes when they traffic into protrusions. We have clarified the text referring to Figure 3F on page 13, line 7-10 as follows:

    "Immunofluorescence experiments showed the reported colocalization of endogenous PLEKHG3 (Figure S2C in HEK293T cells, Figure S3A in HeLa cells) and GFP-PLEKHG3 with cortical actin structures and the partial localization of LAMP1-positive vesicles to these structures in correspondence with vinculin-positive focal adhesions."

    This specific claim is also based on the observation made in GFP-PLEKHG3-expressing cells (including movie S5, and particularly the stills of the leading edge in Figure 4F). In the text describing Figure 4F, we now clearly state on page 14, line 15-17: “Following a single cell over time, we could observe that __a subset of __lysosomes appears to travel to PLEKHG3 accumulation sites and specifically move into developing protrusions.”

    It is not clear how the authors conclude that Figure 4E graph shows "the LAMP1 signal was stronger in paxillin-labeled FA compared to control regions". The 4E graph shows LAMP1 signal in GFP versus GFP-PLEKHG3 and shows a modest enrichment of LAMP1 in FAs in GFP-PLEKHG3 overexpression. LAMP1 enrichment in FAs is also not obvious in the image shown in Figure 4B.

    Answer: We stand corrected – the Figure we referred to was actually not in the manuscript. It has been inserted now, as a plot next to Figure 4B on page 16 Figure 4B (schematic representation of colocalization analysis) was designed to explain how we define focal adhesions (paxillin positive) and adjacent control regions (same size and shape, but paxillin-negative). The actual analysis was missing and has now been inserted. We apologize for this mistake.

    We do not claim that PLEKHG3 brings lysosomes to FAs. The enrichment of lysosomes in FA regions of cells expressing GFP-PLEKHG3 compared to GFP-expressing cells shown in 4E, as the Reviewer correctly notes, is marginal and is not highlighted anywhere in the text exactly for this reason.

    In Fig. 2B, there appears to be a labeling error. The lanes 2,4 and 7 appear to be transfected with L3-T-V5 but labeled as GFP-V5-cyto. Here the PLEKHG3 band should be indicated.

    AND -Fig. 2C is an IP experiment as per the manuscript text but it is labeled as pulldown.

    Answer: We stand corrected, and the necessary changes have been made in the revised version in Figure 2B on page 11.

    Reviewer 2

    *1 - Specificity of PLEKHG3 antibody: In Fig. S2, authors show that PLEKHG3 antibody recognizes 3 bands (above 100 kDa, above 130 kDa and 250 kDa) and all of them are reduced by the silencing of PLEKHG3. Then, in Fig. 2A and C, authors only show the band above 130 kDa, despite implying that the specific band should be "much higher than the 134 kDa calculated from the aminoacid sequence of the protein". *

    In Fig. 2 B, they show all the bands shown in Fig. S2 and presumably favor that the specific and is the 250 kDa one. Finally, in Fig. 2D, they show all bands and note that the band above 130 kDa is not specific. Therefore, authors need to conclude what is the specific band and always analyze the same one, and, possibly, use a different antibody or purify this one to remove non-specific binding. Without this, the main result of the paper, cannot be substantiated.

    Answer: We apologize for this misunderstanding. The antibody recognizes three bands, all reduced by siRNA treatment. These three bands are only resolved in the gels in Figure S2A and B, and in Figure 2B. The reason for this is the high molecular weight of the isoforms, that are resolved in these 8% gels, but collapse into one band in the 15% gels shown in Figure 2A and C. Therefore, the high molecular weight bands are not resolved under these conditions. 8% gels such as the ones in Figure 2B are needed to resolve the high molecular weight bands.

    Figure 2D shows an 8% gel, and therefore all bands are visible. The band marked by an arrow is only present in the streptavidin pulldowns but not in the input or in the supernatant and is therefore considered unspecific. This has been clarified in the revised figure legend on page 11. In addition, to assess the specificity of the antibody for endogenous PLEKHG3 we have used HEK293T cells, which based on the fact that PLEKHG3 is most highly expressed in neuronal cells (https://www.proteinatlas.org/ENSG00000126822-PLEKHG3/tissue#expression_summary) should yield a clearer endogenous signal. The results of this experiment are shown in the Figure S2B-C of the revised manuscript. The pattern of PLEKHG3-positive bands is similar to that observed in HeLa cells, and only the band around 250 kD is clearly reduced by the siPLEKHG3. The IF images show a selective loss of the PLEKHG3 signal in correspondence of actin filaments close to the plasma membrane, whereas the nuclear signal is preserved, and therefore to be considered non-specific.

    2 - In page 12, authors state that "These results indicated that PLEKHG3 is a transient interactor, or a proximal, not directly binding protein, of L3" and in page 14 that "... PLEKHG3 is a proximal L3 protein rather than a transient physical interactor". It is not clear at all how did the authors reach such conclusions, nor they have data to conclude this. Indeed, they would have to express the proteins in vitro and test their interaction to conclude about a direct binding. They also do not know what is the stability of the interaction.

    Answer: This is also a misunderstanding. Unfortunately, we mislabeled Figure 2C as “pulldown”, rather than “IP”, as it is characterized in the text. The fact that we cannot co-ip PLEKHG3 by immunoprecipitating L3 using a V5 antibody led us to conclude that the interaction between the proteins is not direct or stable enough to survive a co-ip. Therefore, the most likely conclusion is that PLEKHG3 is a vicinal protein rather than an interactor of L3 – we changed the labeling of Figure 2C to clarify the issue on page 11.

    Based on these negative data, we did not proceed to test the possibility of complex formation in vitro.

    3 - Still in page 12, authors state that "... two different membrane structures, protrusions and ruffles". What do the authors mean exactly by "protrusions", as there are several different ones (e.g., lamellipodia, filopodia, pseudopods)? And how can they distinguish between ruffles and, for example, lamellipodia? They need to use markers and more carefully analyze their morphology to be able to distinguish these. Like this, it is too preliminary.

    Answer: It was our intention to indicate with the arrows the trajectories in the figure along which we measured the MFI of LAMP1 and PLEKHG3. Although this is indicated in the figure legend, it had apparently given the impression that the arrows indicated specific membrane structures. Since we are focusing on different types of membrane protrusions rather than ruffles, we replaced the terms "ruffles" and "protrusions" with the terms "elongated protrusions" (Figure 3D upper panel) and then compared these with "non" elongated protrusions” (Figure 3D lower panel). Indeed, we note that PLEKHG3 accumulation is possible below and along the plasma membrane, but colocalization with lysosomes occurs preferentially in elongated protrusions. We therefore amended the text on page 12, line 24 – page 13, line 5 as follows:

    „More specifically, we found that PLEKHG3 colocalized more strongly with LAMP1-positive vesicles in elongated membrane structures (Figure 3D-E). Focal adhesion sites, which anchor the intracellular cortical actin network to the extracellular matrix and are remodeled with the help of late endosomes/lysosomes during protrusion formation and cell motility, can also be found in such elongated membrane protrusions (reviewed in Burridge and Burridge, 2017; Schiefermeier et al., 2014).”

    6 - Regarding the statistical analysis, authors assert that it was done using Student's t tests, unless otherwise stated. However, they never refer in figure legends other statistical analysis methods. If so, they cannot use such test, for example, in cases where more than two groups are compared.

    Answer: in all our experiments we performed two-group comparisons. We have now deleted “unless otherwise stated” from the Materials and Methods section on page 41, lines 1-2.

    *Minor comments: *

    *1 - In the abstract, authors refer that cytosolic proteins are recruited to platforms on the limiting membrane of lysosomes. What do they mean by "platforms"? Is it microdomains? *

    Answer: We apologize for this unclarity and have now changed the first sentence in the abstract on page 1 to “Lysosomes are key organelles involved in metabolic signaling pathways through their ability to recruit cytosolic molecules to protein platforms bound to the lysosomal membrane”. We refer to protein platforms as multifunctional protein complexes that can recruit and assemble signaling components (e.g., the recruitment of mTORC1 activating proteins by the LAMTOR complex).

    *2 - In the Introduction, there is a period before the reference at the end of the first paragraph. *

    Answer: We stand corrected. See changes on page 3, line 8.

    3 - In the results, Fig. 1E is mentioned before Fig. 1D and Figure S1F before Fig S1E, which can be confusing.

    Answer: Figure S1E on page 6 was mislabeled as Figure 1E and Figure S1K on page 9 was mislabeled as Figure 1K. We stand corrected. See changes on page 6, line 20 and page 9, line 4.

    4 - All the immunofluorescence images need to be bigger, in general, and have zoom-ins, except Fig. 3A, 4B, 4F, and S2C. Also, in Fig. S1F, the green channel has different intensities and the V5-lyso signal is clearly saturated. Finally, Fig. S1D, S1I and S3F must be enlarged, too.

    Answer: We appreciate the Reviewer's suggestion, but enlarging all the immunofluorescence images and including zoom-ins would make the manuscript overly crowded and could distract from the main findings. Regarding the expression levels of the baits, as mentioned in the manuscript, we aimed to express them at near-endogenous levels. However, TMEM192 is expressed at higher levels than LAMTOR3 in these cells, which may have resulted in the observed discrepancy. We hope the Reviewer will understand our decision and find the current presentation of the data clear and informative.

    5 - In page 9, where it reads "Figure 1K", should read "Figure S1K".

    Answer: See answer to minor point 3.

    6 - The observation that PLEKHG3 silencing leads to loss of the perinuclear clustering of LAMP1-positive vesicles, and increase in their accumulation at the cell tips, is not referred in the text.

    Answer: While this might seem the case in part of the cells shown in the representative image in Figure S2C, quantification of lysosome distribution did not show a significant difference throughout the population as displayed in the figure below (Figure only shown in attached revision plan).

    __Figure 1 for Reviewer 2: __Lysosomal distribution in HeLa cells transfected with either siScr or siPLEKHG3. X-axis is relative distance from the nucleus and Y-axis the normalized intensity of the LAMP1 channel. Results are averages of >30 cells from one experiment.

    7 - Fig. 2C is not referred in the legend.

    Answer: We stand corrected and have changed the legend of Figure 2 accordingly on page 11.

    8 - Figure S3A and B: authors should show the colocalization of endogenous PLEKHG3 with phalloidin and not only the GFP-tagged protein.

    Answer: We thank the Reviewer for this comment and have performed this experiment showing the colocalization of endogenous PLEKHG3 with F-actin structures stained by Phalloidin. Even though the endogenous PLEKHG3 staining in HeLa cells is rather weak, sites where membrane protrusions are formed are clearly marked with PLEKHG3 staining below the plasma membrane. These data confirm the specific colocalization of PLEKHG3 with Phalloidin shown in the revised Figure S3A. See also the extract from Figure S3A below (Figure only shown in attached revision plan and revised Figure S3A).

    Extract from revised Figure S3A: Immunofluorescence images of HeLa cells. A) HeLa cells stained with PLEKHG3 (red) and Phalloidin (green). The nucleus is indicated by DAPI staining (blue). Scale bar = 50 µm. Insets on the right as indicated by white box in image on the left. Scale bar = 10 µm. Line plot corresponds to white line in merged inset.

    *9 - In page 14, authors refer to Fig. 3G, which does not exist. *

    Answer: We stand corrected, the sentence on page 14, line 9 refers to Figure S3G.

    10 - In page 30 and page 32, different antibodies for LAMP1 and PLEKHG3 are mentioned, but in the figure legends authors do not refer which one they used.

    Answer: We tried different PLEKHG3 antibodies but ended up using only one. The other antibody has been excluded from the list on page 32, line 18. We have specified which LAMP1 antibodies were used in which Figure in the Material and Methods on page 30, lines 17.20-23.

    11 - In page 33, where it reads "300 µm protein", it should probably read "300 µg protein".

    Answer: We stand corrected and thank the Reviewer. See changes on page 33, line 17.

    Reviewer 3

    • A key issue … is that the authors focus solely on peripheral lysosomes as target compartments for PLEKHG3. This is not self-evident, particularly in light of images presented in Figures 2 and 3, where colocalization of PLEKHG3 with perinulcear lysosomes appears very likely. The authors should make differences/similarities they observe between effects on perinuclear versus peripheral lysosomes explicit both with data and in the text, if such differences exist.*

    Answer: The Reviewer is likely addressing the images in Figure 3, which have been obtained by staining endogenous PLEKHG3 and do show diffuse staining around the nucleus. This perinuclear staining is resistant to siPLEHG3 (revised Figure S2C) and is not observed with the GFP-PLEKHG3 fusion protein (revised Figure S2E-F; including PLEKHG3 knockdown), which gives a less diffuse signal. This is why we are confident about the colocalization of PLEKHG3 with peripheral lysosomes. This said, we have redoubled our efforts to generate a PLEKHG3 CRISPR-Cas KO in HeLa, going up to 10 sg-guides, and hope that we will be successful in the next future. This cell line will be helpful in answering the Reviewer’s question.

    Minor point: 1. Multicolor overlays with one of the channels in white is in my view not reader-friendly. Appreciating colocalization between endosomes/lysosomes, actin and G is very important for this study, and while is typically reserved to show overlay between green and magenta or green (standard for 2 channels), red and blue (standard for 3-channels). I therefore advise the authors to choose a different color combination throughout the figures when presenting microscopy images.

    Answer: White as a channel color has been substituted for with red (in the 2- and 3-color images) or with blue (in the 4-color images) throughout the images of the revised manuscript, except for the stills from the videos that have not been changed because no colocalization analysis has been performed in this case.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer 2

    4 - At least Fig. 2F and 3A need quantification. Regarding cell motility, there is no quantification and the authors must perform a quantitative assay (despite stating that "As another measure of cell motility, analysis of the number of forming protrusions and retracting membranes..."). Not only this is not a measure of cell motility, but there the issue of what are "protrusions" referred above. Therefore, authors need to quantify the distance that the cells move and/or perform quantitative motility/migration assays.

    Answer: We appreciate the Reviewer’s attention to detail and agree that the quantification of these figures is essential to understand the results. We believe that the Reviewer is referring to Figure 3F and Figure 4A, as there is no Figure 2F, and Figure 3A only confirms the localization of endogenous PLEKHG3, as previously reported in (Nguyen et al., PNAS 2016). If our assumption is correct, then the salient aspects of Figure 3F, which is a representative image, are quantified in Figure 3C-E (endogenous PLEKHG3 colocalization colocalization with LAMP1/lysosomes) and Figure 4E and 5F-G (FA with LAMP1/lysosomes). Figure 4A is quantified in Figure 4C-E (GFP-PLEKHG3 colocalization with FAs, this time labeled with paxillin, and LAMP1 colocalization with FAs).

    In response to the Reviewer's comment regarding the absence of quantification for cell movement/migration in our study, we apologize for any confusion that may have arisen from our use of the term "cell motility." We did not use this term to describe exclusively directed cell movement, but rather in a broader sense, to encompass the entirety of membrane remodeling processes involved in migration. In this context, our statement that lysosomal subcellular localization plays a role in cell motility remains valid. The relationship between membrane protrusive activity and motility is evident from our observations in cells overexpressing KIF1A-mCherry, where both membrane remodeling/protrusive activity and movement are significantly impaired compared to control cells (refer to Movie S7 vs. S6 and S10 vs. S9).

    To address the Reviewer's concern, we have clarified our definition of motility in the introduction by stating on page 5, lines 1-4: "We demonstrate that PLEKHG3 colocalizes with lysosomes at focal adhesion and protrusion sites, and that the localization and function of this protein, as well as cell motility – which we define as the sum of membrane remodeling processes involved in migration – depend on lysosomal dynamics." This revision ensures that our results are accurately described and minimizes any potential confusion. Additionally, we have removed the statement on page 23, line 1 of the original manuscript. We apologize for any confusion our original wording may have caused and appreciate the opportunity to clarify our intentions.

    Reviewer 3

    • The mechanism of PLEKHG3 action on lysosomes/late endosomes is underdeveloped in my view. In the absence of for instance mutational analyses to examine what drives the interaction of PLEKHG3 with LAMTOR3, as well as delineation of at least some molecular consequences of this binding, the study remains incomplete.*

    Answer: We are grateful for the Reviewer's feedback and concur that gaining insight into the mechanistic details of PLEKHG3's interaction with LAMTOR3 would be beneficial. However, our investigation revealed that PLEKHG3 is a transient interactor/proximal protein of LAMTOR3, and due to the absence of direct binding between LAMTOR3 and PLEKHG3 (Figure 2C on page 11), we are unable to perform a mutational analysis of this interaction, as it lacks a direct read-out.

    Furthermore, as demonstrated in Figure S3H-L, LAMTOR3 ablation does not affect PLEKHG3 subcellular localization, suggesting that delving deeper into the molecular consequences of their interaction may be highly complex and beyond the scope of this study. We kindly ask the Reviewer to bear with us on this point, considering the novelty of our findings, which illuminate the interplay between lysosomes and actin dynamics as well as the role of PLEKHG3 in regulating cell protrusions—findings not previously reported in the literature.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Ettelt et al describes the identification of PLEKHG3 as a collaborator of the LAMTOR complex on lysosomes using proximity-based biotinylation. The biotinylation screen is well executed and controlled. The authors choose to follow up on PLEKHG3, a protein involved in actin dynamics, which they refer to as understudied (I let the validity of the latter statement to be evaluated by the editor). Generally speaking, the data are of good quality, and the manuscript is clear and well written. However, much of the evidence on the role of PLEKHG3 on lysosomes is suggestive at best and further investigation into its mechanisms of action is warranted. Some important points to address prior to publication are detailed below.

    Major Points:

    1. The mechanism of PLEKHG3 action on lysosomes/late endosomes is underdeveloped in my view. In the absence of for instance mutational analyses to examine what drives the interaction of PLEKHG3 with LAMTOR3, as well as delineation of at least some molecular consequences of this binding, the study remains incomplete.
    2. A key issue possibly (but not necessarily) related to the point above is that the authors focus solely on peripheral lysosomes as target compartments for PLEKHG3. This is not self-evident, particularly in light of images presented in Figures 2 and 3, where colocalization of PLEKHG3 with perinulcear lysosomes appears very likely. The authors should make differences/similarities they observe between effects on perinuclear versus peripheral lysosomes explicit both with data and in the text, if such differences exist.
    3. Data presented in Figure 6 showing cell motility analysis is interesting and has potential to make the manuscript impactful. Similarly, data in Figure 4F (live cell imaging) looks attractive but is not informative in the absence of relevant genetic perturbations as comparisons. These types of experiments would benefit greatly from PLEKHG3 loss of function analysis, as well as mutational analysis in the over-expression setting.

    Minor point

    1. Multicolor overlays with one of the channels in white is in my view not reader-friendly. Appreciating colocalization between endosomes/lysosomes, actin and G is very important for this study, and while is typically reserved to show overlay between green and magenta or green (standard for 2 channels), red and blue (standard for 3-channels). I therefore advise the authors to choose a different color combination throughout the figures when presenting microscopy images.

    Significance

    In principle, I consider this study to be of interest to the community of cell biologists working on the endolysosomal system and/or the actin cytoskeleton and its relationship to intracellular membranes. However, the authors find themselves in a rather crowded field. I feel that developing the mechanism of action of PLEKHG3 on lysosomes beyond this first submission could help with boosting the impact of the study. There is clearly something interesting going on, but what that is exactly, remains unclear in my view.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: The authors use proximity-dependent labelling and mass spectrometry to identify cytoplasmic proteins that interact with lysosomes. They show that PLEKHG3 interacts with the LAMTOR complex; that PLEKH3 accumulates in focal adhesion sites, where it colocalizes with peripheral lysosomes; and that the increased translocation of lysosomes to the periphery leads to less "protrusions", as well as rounder cells and less motile cells.

    Major comments: While the study is generally carefully performed and thorough, there are major shortcomings that affect the conclusions taken, namely the specificity of the PLEKHG3 antibody, the identification of "protrusions" and ruffles, several quantifications missing, and the data used to conclude about cell motility. There are also conclusions for which there is no concrete or solid evidence.

    Specific issues:

    1. Specificity of PLEKHG3 antibody: In Fig. S2, authors show that PLEKHG3 antibody recognizes 3 bands (above 100 kDa, above 130 kDa and 250 kDa) and all of them are reduced by the silencing of PLEKH3. Then, in Fig. 2A and C, authors only show the band above 130 kDa, despite implying that the specific band should be "much higher than the 134 kDa calculated from the aminoacid sequence of the protein". In Fig. 2 B, they show all the bands shown in Fig. S2 and presumably favor that the specific and is the 250 kDa one. Finally, in Fig. 2D, they show all bands and note that the band above 130 kDa is not specific. Therefore, authors need to conclude what is the specific band and always analyze the same one, and, possibly, use a different antibody or purify this one to remove non-specific binding. Without this, the main result of the paper, cannot be substantiated.
    2. In page 12, authors state that "These results indicated that PLEKHG3 is a transient interactor, or a proximal, not directly binding protein, of L3" and in page 14 that "... PLEKHG3 is a proximal L3 protein rather than a transient physical interactor". It is not clear at all how did the authors reach such conclusions, nor they have data to conclude this. Indeed, they would have to express the proteins in vitro and test their interaction to conclude about a direct binding. They also do not know what is the stability of the interaction.
    3. Still in page 12, authors state that "... two different membrane structures, protrusions and ruffles". What do the authors mean exactly by "protrusions", as there are several different ones (e.g., lamellipodia, filopodia, pseudopods)? And how can they distinguish between ruffles and, for example, lamellipodia? They need to use markers and more carefully analyze their morphology to be able to distinguish these. Like this, it is too preliminary.
    4. At least Fig. 2F and 3A need quantification. Regarding cell motility, there is no quantification and the authors must perform a quantitative assay (despite stating that "As another measure of cell motility, analysis of the number of forming protrusions and retracting membranes..."). Not only this is not a measure of cell motility, but there the issue of what are "protrusions" referred above. Therefore, authors need to quantify the distance that the cells move and/or perform quantitative motility/migration assays.
    5. It is not clear if in cells KO for PLEKHG3, the overexpression of KIF1A leads to more lysosomes localizing close to the PM, as well as more protrusions and more cell motility, as the authors only compare cell overexpressing GFP or GFP-PLEKHGL3.
    6. Regarding the statistical analysis, authors assert that it was done using Student's t tests, unless otherwise stated. However, they never refer in figure legends other statistical analysis methods. If so, they cannot use such test, for example, in cases where more than two groups are compared.

    Minor comments:

    1. In the abstract, authors refer that cytosolic proteins are recruited to platforms on the limiting membrane of lysosomes. What do they mean by "platforms"? Is it microdomains?
    2. In the Introduction, there is a period before the reference at the end of the first paragraph.
    3. In the results, Fig. 1E is mentioned before Fig. 1D and Figure S1F before Fig S1E, which can be confusing.
    4. All the immunofluorescence images need to be bigger, in general, and have zoom-ins, except Fig. 3A, 4B, 4F, and S2C. Also, in Fig. S1F, the green channel has different intensities and the V5-lyso signal is clearly saturated. Finally, Fig. S1D, S1I and S3F must be enlarged, too.
    5. In page 9, where it reads "Figure 1K", should read "Figure S1K".
    6. The observation that PLEKHG3 silencing leads to loss of the perinuclear clustering of LAMP1-positive vesicles, and increase in their accumulation at the cell tips, is not referred in the text.
    7. Fig. 2C is not referred in the legend.
    8. Figure S3A and B: authors should show the colocalization of endogenous PLEKHG3 with phalloidin and not only the GFP-tagged protein.
    9. In page 14, authors refer to Fig. 3G, which does not exist.
    10. In page 30 and page 32, different antibodies for LAMP1 and PLEKHG3 are mentioned, but in the figure legends authors do not refer which one they used.
    11. In page 33, where it reads "300 µm protein", it should probably read "300 µg protein".

    Significance

    The study provides evidence that lysosome positioning can affect cortical actin cytoskeleton dynamics, as well as cell shape and motility. Experiments are in general thorough and data subjected to quantification. However, there are fundamental conclusions that are preliminary at this stage, as some of the data is not yet solid enough. Therefore, it needs to be further strengthened to be considered for publication. In general, it reads well but the amount of abbreviations (e.g. in the case of the constructs) makes it somehow difficult to follow. The study will be interesting for the cell biology, membrane trafficking and cytoskeleton dynamics communities.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Ettelt et al., describes identification of Rho guanine nucleotide exchange factor- PLEKHG3 as one of the positive hits from a TurboID proximity-dependent labeling screen using LAMTOR3 (one of the subunits of the pentameric LAMTOR complex/Ragulator) as a bait protein. The authors find that PLEKHG3 colocalizes with lysosomes at focal adhesions and that peripheral clustering of lysosomes promotes PLEKHG3 localization near the plasma membrane, and also inhibits protrusion formation and cell motility. The experiments, particularly the Turbo ID proximity-dependent labeling screen, are well-executed, and the imaging data is aptly quantified. The manuscript explores an exciting question of how lysosome positioning regulates cortical actin dynamics and thereby cell motility.

    Major comments:

    • The colocalization of endogenous PLEKHG3 and LAMP1 as depicted in figures 3B and 3C (data from fixed cells) is not convincing. PLEKHG3 appears to be present on cortical actin structures as opposed to being colocalized with LAMP1 on lysosomes. The authors should also confirm the specificity of the PLEKHG3 antibody in immunofluorescence using control and PLEKHG3 siRNA in untransfected cells that have not been transfected with GFP-PLEKHG3 (as is shown in Fig. S2C). Numerous antibodies recognize the overexpressed protein but do not recognize the same protein at endogenous expression levels.

    Moreover, do the authors observe colocalization between GFP-PLEKHG3 and lysotracker in living cells? There is no apparent colocalization of PLEKHG3 and lysotracker in the movie S5.

    • The authors observe that GFP-PLEKHG3 is concentrated at the cell's periphery when KIF1A is overexpressed, whereas RUFY3 overexpression results in more cytosolic staining. To bolster their conclusion that a change in lysosomal positioning alters the subcellular localization of PLEKHG3, it is preferable to employ inducible techniques, such as the recently described "reversible association with motor proteins" (RAMP) (PMID: 31100061). The method is a rapid and reversible method for altering organelle positioning. It is still unknown whether PLEKHG3 is associated with lysosomes and mechanism of how positioning of lysosomes affects PLEKHG3 localization.
    • Similarly to the preceding point, the claim that "peripheral accumulation of lysosomes inhibits protrusion formation and limits cell motility" should be tested more rigorously using the RAMP method, preferably in living cells. Other approaches, such as overexpression/siRNA of Arl8b and other motor adaptors, such as SKIP/PLEKHM2, can be used to alter lysosome positioning and confirm this central findings of the manuscript. The authors could also consider including additional mechanistic data in order to comprehend how lysosome positioning controls cell motility. For instance, the RAMP approach could be employed to investigate cortical actin dynamics upon repositioning of lysosomes to the peripheral/perinuclear region.
    • It is not clear how the authors conclude that Figure 4E graph shows "the LAMP1 signal was stronger in paxillin-labeled FA compared to control regions". The 4E graph shows LAMP1 signal in GFP versus GFP-PLEKHG3 and shows a modest enrichment of LAMP1 in FAs in GFP-PLEKHG3 overexpression. LAMP1 enrichment in FAs is also not obvious in the image shown in Figure 4B.
    • In Fig. 2B, there appears to be a labeling error. The lanes 2,4 and 7 appear to be transfected with L3-T-V5 but labeled as GFP-V5-cyto. Here the PLEKHG3 band should be indicated.
    • Fig. 2C is an IP experiment as per the manuscript text but it is labeled as pulldown.

    Significance

    Using a TurboID proximity-dependent labelling screen, the authors identified an interesting subset of actin-remodeling proteins that interact with the lysosomal protein LAMTOR3. The authors further characterized one of these proteins, PLEKGH3, and found that lysosome positioning regulates PLEKGH3 localization, as well as plasma membrane protrusion formation and cell motility. This study suggests that lysosome peripheral accumulation could regulate cortical actin remodelling and consequently cell migration by regulating PLEKGH3 localization (although this is not tested in the manuscript). This study adds to the previous findings that microtubule-based transport of late endosomes regulate delivery of late endosomal LAMTOR proteins to the vicinity of focal adhesions, which in turn, regulate focal adhesion dynamics. The mechanism of how lysosomes can influence actin remodeling will be important in the context of cancer cell migration. My area of expertise is lysosome fusion and motility and I have limited expertise in regulation of actin dynamics and how Rho family members regulate actin remodeling.

  5. Version published to 10.1101/2023.02.01.526449v3 on bioRxiv
  6. Version published to 10.1101/2023.02.01.526449v2 on bioRxiv
  7. Version published to 10.1101/2023.02.01.526449v1 on bioRxiv