Optogenetic and chemical genetic tools for rapid repositioning of vimentin intermediate filaments

  1. Milena Pasolli
  2. Joyce C.M. Meiring
  3. James P. Conboy
  4. Gijsje Koenderink
  5. Anna Akhmanova

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Abstract

Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, organelle positioning, and modulating cell signaling. Current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Rapid perinuclear clustering of vimentin had no major immediate effects on the actin or microtubule organization, cell spreading, or focal adhesion number, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were reorganized due to vimentin clustering, whereas lysosomes were only briefly displaced and rapidly regained their normal distribution. Keratin moved along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate and local effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

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  1. Version published to 10.1101/2024.11.21.624725v2 on bioRxiv
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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments

    Reviewer #1 (Evidence, reproducibility and clarity):

    SUMMARY: The manuscript is well written, with excellent explanation and documentation of experimental approaches. All conclusions are well supported by the data. The discussion is balanced and appropriate. The data, including images and movies, are of high quality and beautifully presented. The experimental design and analysis, including quantification of parameters in the images, is rigorous. Additional rigor is provided by comparing different cell types. The rapalog and iLID dimerization strategies have been described previously, as has their use to recruit kinesin motors to membranous organelles. However, this is the first application of these strategies to recruit motors to intermediate filaments. The evidence that vimentin filaments can be redistributed locally is clear and convincing and offers appealing potential for future experimentation. The redistribution was not fully reversible in all cells, but this is not surprising given the entanglement that must result from the action of motors along the length of these long flexible polymers.

    In terms of the biology of intermediate filaments, the authors show that vimentin redistribution had negligible effect on microtubule or F-actin organization, cell area, or the number of focal adhesions. Depletion of vimentin filaments locally reduced cell stiffness. Both ER and mitochondria segregated with vimentin filaments, but not lysosomes. These findings are consistent with published reports (e.g. comparing vimentin null and wildtype cell lines), but the acute and reversible nature of the motor recruitment strategy is a more elegant experimental approach, and the selectivity of the observed effects is evidence of its specificity. It is interesting that the ER network segregated with vimentin even in the absence of RNF26. While this is not explored further, it points to the potential power of this motor recruitment strategy for future studies on intermediate filament interactions.

    The following are some major and minor issues, which should all be easy for the authors to address.

    MAJOR COMMENTS:

      • Fig. S1 shows that the Vim-mCherry-FKBP construct coassembles with endogenous vimentin, but similar data for the iLID constructs appears to be lacking. I would like to see data demonstrating the incorporation of the Vim-mCherry-SspB constructs into the vimentin filaments. This should include high magnification images of single filaments in the cytoplasm of the cells.*

    Response:

    We have included a new Figure 2D, which illustrates the incorporation of the vimentin-mCherry-SspB construct into the vimentin network stained for endogenous vimentin.

      • The authors do not discuss the density of motor recruitment along the filaments. To address this, I'd like to see images showing the extent of recruitment of motors to the filaments using the rapalog and LID strategies. This should include high magnification images of single filaments in the cytoplasm of the cells.*

    Response:

    We have included new Figure S1B,C and Figure S2A, which illustrate the recruitment of kinesin motors to vimentin filaments upon induction with rapalog or light, respectively, by using super-resolution imaging with an Airyscan microscope. The motors were stained with antibodies against GFP. These data are discussed in the text, lines 126-132 and 165-168.

      • For the experiments on vimentin and keratin organization, the authors do not explain that these proteins form distinct networks and do not coassemble. The authors should show this in the cell types examined. This should also be explained explicitly in the body of the manuscript, though the data could be placed in the supplementary data. This is important because many intermediate filaments can coassemble freely, and coassembled proteins would be expected to segregate together.*

    Response:

    To address this important comment, we have now included images of vimentin and keratin in the three studied cell types using super-resolution imaging, both for cells expressing vimentin constructs (updated Figure 5) and endogenous filament staining in untransfected cells (updated Figure S4). These images illustrate that vimentin and keratin mostly form distinct filaments in HeLa cells. However, we do observe some degree of co-assembly of vimentin and keratin in COS-7 and U2OS cells. We were really surprised by this observation as, to our knowledge, it has not been clearly documented in the literature. These data help to explain why vimentin pulling causes keratin co-clustering in COS-7 and U2OS cells. We note that in a study where kinesin-1 mediated transport of vimentin and keratin has been previously investigated by the Gelfand lab in RPE1 cells, the two networks also appear to overlap quite strongly (Robert et al, 2019, FASEB J). Since no super-resolution microscopy was performed in that study, potential co-assembly of keratin and vimentin filaments was not discussed. Colocalization and coprecipitation of vimentin and keratin have been also described by Velez-delValle et al. in epithelial cells (Sci Rep 2016). Cell type-specific co-assembly of keratin and vimentin would require more investigation, and we make no strong conclusions about it, but we think that our data illustrate the usefulness of our methodology to address the co-dependence of different types of intermediate filaments.

    MINOR COMMENTS:

      • The authors refer to selecting cells within an "optimized expression range" for their transiently expressed recombinant proteins. They should state the proportion of the cells that met this criterion in their transient transfection experiments as this is important information for other researchers that might wish to use this approach in their own studies*. Response:

    These numbers are now included in lines 137 -142 and 173-176 of the revised paper. For the FRB-FKP system, ~50% of transfected cells could be used for analysis, for the light-induced system, ~40% were in the optimal range.

      • In Fig. 1F there should be a statistical comparison between cells transfected with the Kin14 construct and control (untransfected) cells in the absence of rapalog*

    Response:

    This comparison has been added.

      • In Fig. 1G there should be a statistical comparison between cells expressing Kin14 and KIF5A in the absence of rapalog.*

    Response:

    This comparison has been added.

      • The depletion of the ER network in the cell periphery is not evident in Fig. 7B, though the perinuclear accumulation is evident. Perhaps the authors could select another example or explain to the reader what exactly to look for in these images.*

    Response:

    We note that Figure 7B is a line scan of the image shown in Figure 7A. We assume that the reviewer meant Figure 7C, which is discussed in detail below.

      • In Fig. 7C, the intensity of the mCherry declines markedly over time. This is presumably due to photobleaching but should be explained in the legend.*

    Response:

    We have now improved Figure 7 by adding additional quantifications of ER and vimentin intensity and distribution in Figures 7D and E. We also extended the corresponding text (lines 288-297), which now reads; “Using the optogenetic tool, we observed that ER sheets and matrices, but not tubules, were pulled along with vimentin, confirming their previously described direct connections (Cremer et al., 2023) (black arrows, Figure 7C; Video S5). Most of the vimentin and ER repositioning occurred within approximately 10 minutes (Figure 7C, D, Video S5). While initially this resulted in a sparser tubular ER network at the cell periphery, over time, the network became denser, with smaller polygonal structures. This effect could also be observed in the ratio of perinuclear to peripheral intensity, where a subset of ER initially follows vimentin to the perinuclear region but then redistributes again towards the cell periphery (Figure 7D). It should be noted that while photobleaching of the ER channel was negligible, there was a 40% reduction in total Vim-mCh-SspB intensity over the course of the experiment due to photobleaching (Figure 7E).”

    Reviewer #1 (Significance):

    SUMMARY: The authors show that chemical-induced and light-induced dimerization strategies can be used to recruit microtubule motors to vimentin filaments, allowing rapid and reversible experimental manipulation of vimentin filament organization either locally or globally in cells. These strategies provide an experimental approach for investigating the physical interaction of intermediate filaments with organelles and other cytoskeletal component, as well as a method for probing the role of intermediate filaments in cell mechanics, cytoskeletal dynamics, etc. This is a technical improvement over previous experimental strategies, which have relied largely on chronic manipulation such as global disassembly or genetic deletion of intermediate filaments, e.g. comparison of vimentin null and wild type cells.

    The principal weakness of this study is that it offers limited insight into intermediate filament biology. As such, it might be most appropriate for a tools or techniques section of a journal. The dimerization strategies have been reported previously, so that is not new, but the application to intermediate filaments is novel.

    Response:

    We agree that our paper is primarily of technical nature and thus would be most appropriate for the tools and techniques section of a journal. We also agree that we used motor recruitment strategies that we and others have employed previously. However, we would like to emphasize that the demonstration that the tools work very well for intermediate filaments is entirely novel, as are the observations that these tools can be used to very rapidly alter cell stiffness or probe the links between intermediate filaments and organelles. Most importantly, the intermediate filament field currently lacks rapid specific manipulation strategies, and our tools will allow revisiting many important pending questions in the field. For example, they will allow to distinguish short-term and direct effects of intermediate filaments on cell polarity, adhesion and migration from their function in signaling and gene expression. We also report some new biology, such as evidence of some degree of co-assembly of vimentin and keratin.

    AUDIENCE: This paper will be of interest to cell biologists who study cytoskeletal interactions, particularly the interaction of intermediate filaments with other cellular organelles or cytoskeletal polymers, or the role of intermediate filaments in cellular mechanics.

    REVIEWER EXPERTISE: This reviewer has expertise on the cytoskeleton, cytoskeletal dynamics, and intracellular transport including intermediate filament biology.

    __ __

    Reviewer #2 (Evidence, reproducibility and clarity):

    Summary: The manuscript presents a novel methodology for acute manipulation of vimentin intermediate filaments (IFs) using chemical genetic and optogenetic tools. By recruiting microtubule-based motors to vimentin via inducible dimerization systems, the authors achieve precise temporal and spatial control over vimentin distribution. Apart from the significant advancement in terms of methods development, key findings include:

    * Vimentin's role in organelle positioning: Mitochondria and ER are repositioned with vimentin, while lysosomes are less dependent on its organization.

    * Cytoskeletal interactions: Vimentin clustering minimally impacts actin and microtubule networks in the short term.

    * Cell stiffness: Vimentin repositioning reduces cell stiffness, indicating its significant role in cellular mechanics.

    * Cell-type-specific keratin interactions: The study highlights diverse interactions between vimentin and keratin-8 across cell lines.

    The study demonstrates methodological advancements enabling rapid vimentin manipulation and provides insights into vimentin's interactions with cellular structures.

    A major shortcoming is the unclear narrative, what do the authors want to present? This aspect requires significant attention.

    Response:

    By “unclear narrative” the reviewer meant that we should have provided a more balanced discussion of the insights that could be obtained using our new method compared to previously published literature, and we have modified our narrative accordingly.

    * *

    General Comments and Overall Assessment

    The manuscript represents an interesting contribution to the cytoskeletal field, addressing limitations of long-term perturbation methods. The tools developed are innovative, allowing controlled and reversible vimentin reorganization with minimal off-target effects. The findings are robust and provide important insights into the role of vimentin in cellular mechanics and organelle positioning.

    * *

    Strengths:

    Methodological novelty with broad applicability - this is the most exciting aspect.

    Comprehensive validation of the tools in multiple cell lines.

    Clear differentiation between vimentin's short- and long-term roles.

    Addressing gaps in understanding vimentin-organelle interactions.

    * *

    Limitations:

    * The manuscript is a little bit all over the place. While the method development is clear, the manuscript makes claims way beyond the method development. The message and narrative needs to be improved, and in the respect the whole structure needs an overhaul.

    Response:

    We have carefully modified the manuscript to avoid the impression that we make any claims that go beyond the immediate and quantifiable effects of vimentin repositioning on different cellular structures.

    * Unclear how much the differences in expression levels impact results and reproducibility.

    Response:

    Quantifications of expression levels and their discussion are included in Figures 1G-I, 2G-H, S2B and lines 137-142 and 173-176.

    * Would be good to discuss some findings that are specific to a given experimental cell line. How generalizable are these results?

    Response:

    Cell line-specific findings concerned mostly the co-displacement of keratin together with vimentin, which occurred in COS-7 and U2OS cells but in in HeLa cells. This interesting finding is discussed in the text, lines 246-269 and 375-383 (see also our answers on page 3 above and page 7 below).

    * *

    Major Comments

    Evidence and Claims:

    * While the methodological aspect is very strong the balance between presenting a novel method and presenting specific cell biological findings needs to be improved. Now it is quite unclear what the manuscript wants to present.

    * The abstract needs a complete overhaul. From reading the abstract, it is not clear what the manuscript wants to present.

    Response:

    We have modified the abstract to make it more clear that we do not make any general claims on the impact of vimentin on the interactions and functions of different organelles, but rather describe what can be directly observed after the acute displacement of vimentin and which conclusions can be made from these observations.

    Regarding the research findings there are a number of things for the authors to consider. Since the methods aspect is, in the eyes of this reviewer, in focus, I have not stringently assessed the experimental findings. Hence, the comments below are things to be considered in order to make the findings related to IF research stronger:

    * Cell-specific keratin interactions: The manuscript could benefit from some further validation of the physical interactions between vimentin and keratin-8 across different cell types.

    Response:

    We have improved the images of keratin and vimentin by using super-resolution (Airyscan) microscopy to show that they indeed form distinct filaments in HeLa cells, whereas in COS-7 and U2OS cells, where their co-displacement occurs, they can also incorporate into the same filaments. This observation was very surprising but agrees with the data published by the Gelfand lab on similarity in the distribution pattern and co-transport of vimentin and keratin in RPE1 cells (Robert et al, 2019, FASEB J). Colocalization and coprecipitation of vimentin and keratin has been also described by Velez-delValle at al. in epithelial cells (Sci Rep 2016).

    * Impact on microtubules: The disorganization of stable microtubules in cells expressing KIF5A was attributed to overexpression effects. It would be helpful to include additional controls, such as expressing KIF5A without vimentin constructs, to confirm this claim.

    Response:

    This control has been included in the new Figure S3. We note that this observation fully aligns with data published by another lab (Andreu-Carbó et al, 2024, Nat Comm).

    * ER-vimentin linkages: The observation that ER-vimentin interactions persist in RNF26 knockout cells is intriguing. The manuscript would benefit from a discussion on possible candidates for alternative linkers.

    Response:

    We have added a short discussion (lines 394-398) about the potential involvement of nesprins, such as nesprin-3, because they can connect the nuclear envelope to intermediate filaments, and might also partly participate in ER sheet-IF connections because ER and nuclear membranes are continuous and show some overlap in proteome.

    * Construct variability: Do the authors have some data on how much Expression level differences significantly affect the outcomes (e.g., incomplete recovery)?

    Response:

    We have added a figure (Figure S2B), which shows that incomplete recovery of vimentin clustering does not correlate with protein expression levels and likely depends on other factors, which could possibly be the cell cycle phase or degree of vimentin entanglement after repositioning. This point is discussed in revised text, lines 194-197.

    Reviewer #2 (Significance):

    Significance

    General Assessment: The study represents a significant technical advance in the study of cytoskeletal dynamics. The tools developed address critical limitations of traditional vimentin perturbation methods, allowing for spatiotemporally precise manipulation without long-term effects on gene expression or signaling pathways.

    Novelty:

    This is, to my knowledge, the first demonstration of reversible and acute vimentin repositioning using optogenetics. The study extends understanding of vimentin's short-term mechanical and organizational roles, distinguishing them from compensatory effects observed in knockdown models.

    Audience and Impact:* The manuscript will appeal to researchers in cytoskeletal dynamics, cell mechanics, and organelle biology. The tools have broader applicability in studying other cytoskeletal systems and could inspire translational applications, such as investigating the role of vimentin in cancer or fibrosis.*

    *The reference list provide a relatively representative selection of articles relevant for the article. However, the authors may consider whether there could be relevant information in the relatively recent special edition of Current Opinion in Cell Biology, which focused on IFs, specially featuring vimentin *https://www.sciencedirect.com/special-issue/10TFHK2QCKW

    Response:

    We thank the reviewer for this excellent suggestion, and we have included some additional references from this issue.

    Field of Expertise

    I specialize in cell biology, intermediate filaments, post-translational modifications, cytoskeletal dynamics, and advanced microscopy techniques.

    * *

    Reviewer #3 (Evidence, reproducibility and clarity):

    Summary:

    This is an excellent paper describing the use of chemical and light-induced heterodimerization of microtubule-based motors to rapidly disrupt the distribution of the vimentin cytoskeletal network. Rapid clustering of vimentin did not significantly affect the microtubule or actin networks, cell spreading or focal adhesions. Other organelles were repositioned together with vimentin. Interestingly, in some cell lines, keratin networks were displaced along with vimentin while in other cells they were not.

    Major comments:

    The conclusions are well supported by the data presented and appropriate controls are included.

    Optional comments:

      • The authors should expand on why they think the plus end directed KIF5A gives such a strong localization of vimentin to the perinuclear area.* Response:

    We think that two factors can contribute to this counterintuitive effect. First, vimentin is strongly concentrated and entangled in the perinuclear region, and displacement of some vimentin filaments to the cell periphery can cause the collapse of the rest to the cell center, with kinesins being unable to pull the perinuclear network apart. Second, kinesin-1 KIF5A is a motor that strongly prefers stable, post-translationally modified microtubules, and our previous study has shown that a significant proportion of such microtubules are located with their minus ends facing towards the cell periphery (Chen et al., Elife 2016). This could contribute to the accumulation of vimentin in the cell center upon KIF5A recruitment. These considerations were added to the revised text, lines 344-347.

    • Consideration should be given to the idea that the pulling of ER and mitochondria along with the vimentin could be due to trapping of these organelles within the vimentin matrix and not necessarily due to direct interactions. Such reasoning could explain the transient localization of lysosomes with the center aggregate since lysosomes are generally not thought to significantly bind to vimentin networks.*

    Response:

    This is an excellent point, and we have included it in the revised article, lines 333-335 and 405.

    Reviewer #3 (Significance):

    This study describes some valuable tools that should be useful to cell biologists interested in determining the role of the cytoskeleton and possibly other organelles in a variety of cellular contexts. It overcomes some of the existing shortcomings of the pharmacological reagents currently available for studying intermediate filament biology and will provide a useful adjunct to other more long-term manipulations of the cytoskeleton. While much of the data presented confirm results obtained by other methods, this is a significant technical advance as it provides a short time scale, and in one instance, reversible manipulation of the cytoskeleton.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This is an excellent paper describing the use of chemical and light-induced heterodimerization of microtubule-based motors to rapidly disrupt the distribution of the vimentin cytoskeletal network. Rapid clustering of vimentin did not significantly affect the microtubule or actin networks, cell spreading or focal adhesions. Other organelles were repositioned together with vimentin. Interestingly, in some cell lines, keratin networks were displaced along with vimentin while in other cells they were not.

    Major comments:

    The conclusions are well supported by the data presented and appropriate controls are included.

    Optional comments:

    1. The authors should expand on why they think the plus end directed KIF5A gives such a strong localization of vimentin to the perinuclear area.
    2. Consideration should be given to the idea that the pulling of ER and mitochondria along with the vimentin could be due to trapping of these organelles within the vimentin matrix and not necessarily due to direct interactions. Such reasoning could explain the transient localization of lysosomes with the center aggregate since lysosomes are generally not thought to significantly bind to vimentin networks.

    Significance

    This study describes some valuable tools that should be useful to cell biologists interested in determining the role of the cytoskeleton and possibly other organelles in a variety of cellular contexts. It overcomes some of the existing shortcomings of the pharmacological reagents currently available for studying intermediate filament biology and will provide a useful adjunct to other more long-term manipulations of the cytoskeleton. While much of the data presented confirm results obtained by other methods, this is a significant technical advance as it provides a short time scale, and in one instance, reversible manipulation of the cytoskeleton.

  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 #2

    Evidence, reproducibility and clarity

    Summary: The manuscript presents a novel methodology for acute manipulation of vimentin intermediate filaments (IFs) using chemical genetic and optogenetic tools. By recruiting microtubule-based motors to vimentin via inducible dimerization systems, the authors achieve precise temporal and spatial control over vimentin distribution. Apart from the significant advancement in terms of methods development, key findings include:

    • Vimentin's role in organelle positioning: Mitochondria and ER are repositioned with vimentin, while lysosomes are less dependent on its organization.
    • Cytoskeletal interactions: Vimentin clustering minimally impacts actin and microtubule networks in the short term.
    • Cell stiffness: Vimentin repositioning reduces cell stiffness, indicating its significant role in cellular mechanics.
    • Cell-type-specific keratin interactions: The study highlights diverse interactions between vimentin and keratin-8 across cell lines.

    The study demonstrates methodological advancements enabling rapid vimentin manipulation and provides insights into vimentin's interactions with cellular structures. A major shortcoming is the unclear narrative, what do the authors want to present? This aspect requires significant attention.

    General Comments and Overall Assessment

    The manuscript represents an interesting contribution to the cytoskeletal field, addressing limitations of long-term perturbation methods. The tools developed are innovative, allowing controlled and reversible vimentin reorganization with minimal off-target effects. The findings are robust and provide important insights into the role of vimentin in cellular mechanics and organelle positioning.

    Strengths:

    Methodological novelty with broad applicability - this is the most exciting aspect. Comprehensive validation of the tools in multiple cell lines. Clear differentiation between vimentin's short- and long-term roles. Addressing gaps in understanding vimentin-organelle interactions.

    Limitations:

    • The manuscript is a little bit all over the place. While the method development is clear, the manuscript makes claims way beyond the method development. The message and narrative needs to be improved, and in the respect the whole structure needs an overhaul.
    • Unclear how much the differences in expression levels impact results and reproducibility.
    • Would be good to discuss some findings that are specific to a given experimental cell line. How generalizable are these results?

    Major Comments

    Evidence and Claims:

    • While the methodological aspect is very strong the balance between presenting a novel method and presenting specific cell biological findings needs to be improved. Now it is quite unclear what the manuscript wants to present.
    • The abstract needs a complete overhaul. From reading the abstract, it is not clear what the manuscript wants to present.

    Regarding the research findings there are a number of things for the authors to consider. Since the methods aspect is, in the eyes of this reviewer, in focus, I have not stringently assessed the experimental findings. Hence, the comments below are things to be considered in order to make the findings related to IF research stronger:

    • Cell-specific keratin interactions: The manuscript could benefit from some further validation of the physical interactions between vimentin and keratin-8 across different cell types.
    • Impact on microtubules: The disorganization of stable microtubules in cells expressing KIF5A was attributed to overexpression effects. It would be helpful to include additional controls, such as expressing KIF5A without vimentin constructs, to confirm this claim.
    • ER-vimentin linkages: The observation that ER-vimentin interactions persist in RNF26 knockout cells is intriguing. The manuscript would benefit from a discussion on possible candidates for alternative linkers.
    • Construct variability: Do the authors have some data on how much Expression level differences significantly affect the outcomes (e.g., incomplete recovery)?

    Significance

    General Assessment: The study represents a significant technical advance in the study of cytoskeletal dynamics. The tools developed address critical limitations of traditional vimentin perturbation methods, allowing for spatiotemporally precise manipulation without long-term effects on gene expression or signaling pathways.

    Novelty:

    This is, to my knowledge, the first demonstration of reversible and acute vimentin repositioning using optogenetics. The study extends understanding of vimentin's short-term mechanical and organizational roles, distinguishing them from compensatory effects observed in knockdown models.

    Audience and Impact: The manuscript will appeal to researchers in cytoskeletal dynamics, cell mechanics, and organelle biology. The tools have broader applicability in studying other cytoskeletal systems and could inspire translational applications, such as investigating the role of vimentin in cancer or fibrosis.

    The reference list provide a relatively representative selection of articles relevant for the article. However, the authors may consider whether there could be relevant information in the relatively recent special edition of Current Opinion in Cell Biology, which focused on IFs, specially featuring vimentin https://www.sciencedirect.com/special-issue/10TFHK2QCKW

    Field of Expertise

    I specialize in cell biology, intermediate filaments, post-translational modifications, cytoskeletal dynamics, and advanced microscopy techniques.

  5. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary The manuscript is well written, with excellent explanation and documentation of experimental approaches. All conclusions are well supported by the data. The discussion is balanced and appropriate. The data, including images and movies, are of high quality and beautifully presented. The experimental design and analysis, including quantification of parameters in the images, is rigorous. Additional rigor is provided by comparing different cell types. The rapalog and iLID dimerization strategies have been described previously, as has their use to recruit kinesin motors to membranous organelles. However, this is the first application of these strategies to recruit motors to intermediate filaments. The evidence that vimentin filaments can be redistributed locally is clear and convincing and offers appealing potential for future experimentation. The redistribution was not fully reversible in all cells, but this is not surprising given the entanglement that must result from the action of motors along the length of these long flexible polymers.

    In terms of the biology of intermediate filaments, the authors show that vimentin redistribution had negligible effect on microtubule or F-actin organization, cell area, or the number of focal adhesions. Depletion of vimentin filaments locally reduced cell stiffness. Both ER and mitochondria segregated with vimentin filaments, but not lysosomes. These findings are consistent with published reports (e.g. comparing vimentin null and wildtype cell lines), but the acute and reversible nature of the motor recruitment strategy is a more elegant experimental approach, and the selectivity of the observed effects is evidence of its specificity. It is interesting that the ER network segregated with vimentin even in the absence of RNF26. While this is not explored further, it points to the potential power of this motor recruitment strategy for future studies on intermediate filament interactions.

    The following are some major and minor issues, which should all be easy for the authors to address.

    Major Comments:

    • Fig. S1 shows that the Vim-mCherry-FKBP construct coassembles with endogenous vimentin, but similar data for the iLID constructs appears to be lacking. I would like to see data demonstrating the incorporation of the Vim-mCherry-SspB constructs into the vimentin filaments. This should include high magnification images of single filaments in the cytoplasm of the cells.
    • The authors do not discuss the density of motor recruitment along the filaments. To address this, I'd like to see images showing the extent of recruitment of motors to the filaments using the rapalog and LID strategies. This should include high magnification images of single filaments in the cytoplasm of the cells.
    • For the experiments on vimentin and keratin organization, the authors do not explain that these proteins form distinct networks and do not coassemble. The authors should show this in the cell types examined. This should also be explained explicitly in the body of the manuscript, though the data could be placed in the supplementary data. This is important because many intermediate filaments can coassemble freely, and coassembled proteins would be expected to segregate together.

    Minor Comments:

    • The authors refer to selecting cells within an "optimized expression range" for their transiently expressed recombinant proteins. They should state the proportion of the cells that met this criterion in their transient transfection experiments as this is important information for other researchers that might wish to use this approach in their own studies.
    • In Fig. 1F there should be a statistical comparison between cells transfected with the Kin14 construct and control (untransfected) cells in the absence of rapalog
    • In Fig. 1G there should be a statistical comparison between cells expressing Kin14 and KIF5A in the absence of rapalog
    • The depletion of the ER network in the cell periphery is not evident in Fig. 7B, though the perinuclear accumulation is evident. Perhaps the authors could select another example or explain to the reader what exactly to look for in these images.
    • In Fig. 7C, the intensity of the mCherry declines markedly over time. This is presumably due to photobleaching but should be explained in the legend.

    Referees cross-commenting

    This session contains comments of Reviewer 1 and Reviewer 2

    Reviewer 1:

    I don't understand what Reviewer 2 means by "A major shortcoming is the unclear narrative, what do the authors want to present? This aspect requires significant attention." I found the narrative, purpose and conclusions of this study very clear to me. I also do not understand Reviewer 2's concern with the abstract. I re-read it and it still seems very clear and appropriate to me. For example, the authors state "Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light". This seems clear and correct to me. It would be helpful if Reviewer 2 could point to specific language and explain why it is problematic.

    Reviewer 2:

    The strength of this paper is clearly the strong methods development and I find this aspect very intriguing and attractive. There is an imbalance in the narrative presenting on one hand the method and on the other hand presenting concrete research results. In my view, although interesting, the different experimental results serve more as proof-of-concept and they should not be presented as bona fide evidence of an existing or lacking bilateral interrealtionship.

    Indeed, the cited sentence makes sense: "Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light." as it features the methods aspect of the paper. However, the following sentences: "Perinuclear clustering of vimentin had no strong effect on the actin or microtubule organization, cell spreading, and focal adhesions, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were repositioned together with vimentin, whereas lysosomes were only briefly repositioned and rapidly regained their normal distribution. Keratin was displaced along with vimentin in some cell lines but remained intact in others. " embraces everything from actin to microtubules to cell spreading to focal ahdesions to cell stiffness to mitochondrial function to lysosomes to interactions with other IF family members etc. This gives the impression that the authors want to make claims on how vimentin affects or does not affect these cellular functions and structures and once just cannot make such sweeping claims with so little evidence. With the experimental setting included, non of these claims can be really made without rigidly examining each and every interaction (which has been done separately for many of these bilateral interactions during the past 20 years or so).

    Hence, it should be made clear that these observations are used and mentioned as proof of concept that the tool is working, not as evidence that this or that interaction takes place or does not take place. As I indicated in my review, such claims on any of these bilateral interactions would require a lot more evidence to be properly substantiated.

    My comment is to be regarded as a positive one. If I would judge the paper based on how one could interpret the abstract and the text regarding, for example, that vimentin does not affect focal adhesions but changes cellular stiffness, my review would be significantly more stringent. However, I would really like to see this paper being published, but the claims on revealing new vimentin functions or disproving earlier observations based on these very limited data are just not sufficiently substantiated to be acceptable. Hence, I urge the authors to adjust the narrative to be clear on the methods development, which is also the focus of the title. I believe this is a justified recommendation and also, overall, a fair shake of the study and a constructive approach on how to publish this manuscript without extensive experiments.

    Reviewer 1:

    I thank Reviewer 2 for this explanation. I do understand their point. However, while not the end of the story, I do feel the authors' data are a bit more than just a proof of principle and do offer important insights into the biology which the field will need to grapple with. Each graph includes measurements on dozens of cells from multiple experiments and there is clearly selectivity to what segregates with the vimentin filaments and what does not. I would just ask the authors to be a bit more nuanced in their interpretation and conclusions about the biology to address Reviewer 2's concerns. Reviewer 2:

    That sounds like a fair assessment. Main thing is that this data is presented in a balanced way, with emphasis on the model development. Some of the presented data are in contradiction with quite established concepts by several researchers and the data presented here does not substantiate a paradigm shift. Regardless of this, some pieces of the data are intriguing, for example, the live cell imaging.

    Significance

    Summary: The authors show that chemical-induced and light-induced dimerization strategies can be used to recruit microtubule motors to vimentin filaments, allowing rapid and reversible experimental manipulation of vimentin filament organization either locally or globally in cells. These strategies provide an experimental approach for investigating the physical interaction of intermediate filaments with organelles and other cytoskeletal component, as well as a method for probing the role of intermediate filaments in cell mechanics, cytoskeletal dynamics, etc. This is a technical improvement over previous experimental strategies, which have relied largely on chronic manipulation such as global disassembly or genetic deletion of intermediate filaments, e.g. comparison of vimentin null and wild type cells.

    The principal weakness of this study is that it offers limited insight into intermediate filament biology. As such, it might be most appropriate for a tools or techniques section of a journal. The dimerization strategies have been reported previously, so that is not new, but the application to intermediate filaments is novel.

    Audience: This paper will be of interest to cell biologists who study cytoskeletal interactions, particularly the interaction of intermediate filaments with other cellular organelles or cytoskeletal polymers, or the role of intermediate filaments in cellular mechanics.

    Reviewer Expertise This reviewer has expertise on the cytoskeleton, cytoskeletal dynamics, and intracellular transport including intermediate filament biology.

  6. Version published to 10.1101/2024.11.21.624725v1 on bioRxiv