Tunnelling nanotube formation is driven by Eps8/IRSp53‐dependent linear actin polymerization

  1. J Michael Henderson
  2. Nina Ljubojevic
  3. Sevan Belian
  4. Thibault Chaze
  5. Daryl Castaneda
  6. Aude Battistella
  7. Quentin Giai Gianetto
  8. Mariette Matondo
  9. Stéphanie Descroix
  10. Patricia Bassereau
  11. Chiara Zurzolo

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Abstract

Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin‐mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer‐based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3‐dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I‐BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.

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  1. Version published to 10.15252/embj.2023113761
  3. Arcadia Science

    . (e)

    It would be helpful to include a heat map gradient/scale/legend under subpanels in C. It took me a while to realize that it was a heat map LUT and not a blue marker and a magenta marker.

  4. Arcadia Science

    Following CK-666 addition, we observed an increase of IRSp53 fluorescence at the plasma membrane

    In Fig 4 C,F) It would be helpful to have these fluorescent images merged with a brightfield image so we can see the outline of the TNTs and where these fluorescent proteins are localized in the cell.

  5. Arcadia Science

    this suggests an anti-correlation between regions with high Arp2/3 activity and a cell’s ability to form TNTs

    To really illustrate this it would be helpful to show a bivariate analysis between like number of lamellipodia per cell and number of TNTs per cell for 2B. Then you could actually graphically show this anti-correlation and provide a correlation coefficient to strengthen this conclusion. It would also be interesting to show this with CK-666 treatment as I wondered while reading this if the increase in TNTs/filopodia with CK-666 treatment was accompanied by a decrease in lamellipodia.

  6. Arcadia Science

    Our data suggest a shift in the equilibrium (and usage of common actin proteins players) between branched and linear actin polymerization to form different cell protrusions.

    What a cool paper! Beautiful images and I love papers that deal with this concept of a limited pool of actin in the cell that is directed to its different functions by its interactions with actin binding/interacting proteins.

  9. Arcadia Science

    (a)

    The TNTs in the WT here look drastically different from the WT/control TNTs in previous figures In both the GFP/mCherry and the Eps8-IRSp53, the TNT actin looks thinner when treated with CK-666 compared to Mock/DMSO. Have you done any diameter measurements of the TNTs?

  10. Arcadia Science

    background values adjacent to the measured bands.

    I noticed in figure 4A the tubulin loading control isn't super consistent. Did you normalize to your loading control? Might also be worth doing total protein as your loading control instead of tubulin, because it is possilbe that tubulin is being affected since you're probing other cytoskeleton related proteins.

  11. Arcadia Science

    we utilized an optical tweezer (OT) setup to pull nanotubes of comparable lengths to the TNTs observed on the micropatterns to monitor by confocal microscopy F-actin polymerization within the nanotube in control and CK-666-treated conditions

    This experiment is very cool, but I wonder if you could show a similar thing with naturally forming TNTs? Do you see more actin in natural TNTs when the Arp2/3 complex is inhibited? I worry that stretching the cell like this could be causing other things to happen in the cell and isn't fully representative of a TNT forming on its own.

  13. Arcadia Science

    Eps8 and IRSp53 are recruited to form longer protrusions upon Arp2/3 inhibition.

    If I understand correctly, the figure shows that IPSp53 and Eps8 interact and localize to these extension, but not that they are responsible for forming the longer protrustions, right? The figure text suggests they are responsible which isn't shown until Fig 5

  14. Arcadia Science

    Fig. 4:

    Fig 4 C & F) does the 00:00 time point indicate the time when the cells were plated? In the earlier experiments, cells were allowed to adhere for 4-6 hours before treatment for 16-18 hours. Do TNTs form within 30 min of plating?

    Also, minor point, but C is in hh:mm:ss time stamp, but F is hh:mm time stamp. Since all of the timepoints in C end it 00 sec it would be good to make C/F consistent as hh:mm (or hh:mm:ss)

    Also, after 30 min, CK-666 is added and the extended protrusions form shortly after. For the control quantified in d and g, was DMSO added at the 30 min mark? It would be nice too see the control images as well. The force from the flow of added liquid could cause morphological changes

  15. Arcadia Science

    Upon Arp2/3 inhibition with CK-666 (6, 48), we observed a significant increase in the percent of TNT-connected cells on D15, D20 and D30 micropatterns

    It would be really great to see representative images of the data quantified in Figure 2C.

  17. Arcadia Science

    In the case of overnight drug treatments for TNT counting and co-culture experiments, cells first adhered for 4–6 hr and were then treated with 50 μM CK-666 or 1 μM IMM-01 for 16–18 hr.

    Have you done any experiments where you pre-treat with CK-666 before adhering the cells? Its possible the branched actin networks are necessary for the initial membrane deformation, but after the TNT is formed, Arp2/3 complex inhibition frees up more G-actin which goes the the linear filaments in the TNT?

  19. Arcadia Science

    Competition between actin binding proteins for stabilizing different actin populations is a super interesting phenomenon. I always imagined this being due to some actin binding proteins acting as a sink for monomers dropping local actin concentration available for other ABPs or sequestering monomers to other spatial domains (something we’ve observed in other systems). However, I found it really interesting that an actin bundler forming presumably linear actin tunneling nanotubes that seems to be antagonistic to branched actin formation by ARP2/3 directly binds components of the ARP2/3 complex. Do you imagine that the higher branching ARP2/3 complex isoforms are also sequestered from the rest of the complex where lamellipodia would form? Are you able to localize those Eps8-interacting ARP2/3 components to nanotubes?

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

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    Referee #2

    Evidence, reproducibility and clarity

    This work is is distinguished by impressive technical feats and experimental breadth, and is another excellent contribution from the Zurzolo lab. My comments are advisory regarding more explicit descriptions and qualified conclusions.

    Introduction: Please define how filopodia are distinguished from TNTs - is it length only or are there other characteristics? Do filopodia and individual TNTs have the same diameter? There is presumably a functional difference as well?

    Please state the number of cells in each array spot?

    Paragraph 2 of Results would benefit from a general description of procedure and rationale for assessing protrusions in the artificial setups used in this study of isolated cells. It would help to explicitly state when protrusions were assessed after fixation and when the observations were made with unfixed cells. What are the issues of concern with these methods and what aspects are relevant to general cell behavior? Isn't it important to point out that the conclusions regarding Arp2/3 inhibition and TNT formation are operational for the conditions used?

    Ln 148: If filopodia are distinguished/defined by their shorter relative length, the observation that "filopodia lengths showed that a majority of filopodia were far shorter" is not informative. Do cells with TNTs also have filopodia?

    Does the negative effect of increasing array separation distance on frequency of TNTs suggest that the observations represent a steady state, and the possibility that the observed frequencies are measures of protrusion stability? If the experiments monitor the steady state, can the authors distinguish between stability and inherent ability to extend filopodia to longer distances? Is the conclusion (ln 151) "there seems to be an upper limit to F-actin-based elongation" justified if stability or relative rates of extension and retraction are factors? Another possibility is that the observations reflect protrusion:protrusion interactions that promote stable TNTs. There is the precedent of cytoneme:cytoneme interactions associated with stable signaling contacts (Gonzalez-Mendez et al PMID: 28825565) as well as previous work from the Zurzolo lab (Sartori-Rupp et al PMID: 30664666). A kinetic analysis in real time might be very informative.

    Ln 158: "cells displaying only lamellipodia accounted for 4.1% of [cells with?] TNTs examined"

    Significance

    This work offers new insights into the cytoskeletal processes that generate long cell protrusions. The implications for understanding cell:cell interactions and signaling are fundamental and important.

  22. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    I applaud the authors for the creative experimentation and intriguing findings presented in this work, but the narrative lacks a clear biological question and, as written, comes across more like a collection of thematically related results, rather than a logical point A > B > C progression of studies that significantly advances our understanding of TNT formation beyond the current published literature. In addition, there are a number of inconsistencies and ambiguities in the approach - at least as described - that make it hard to follow the logic flow of the authors. Below I list some of the major inconsistencies and questions I had after reading through the manuscript.

    1. From Figure 1b - the micro patterned substrates separated by different distances also appear to be printed as different diameters - is this true? I could find no mention of it in the text. My concern is that changing the contact area between the cell and substrate might impact the cell's ability to extend very long membrane protrusions towards its neighbors. Why not print patterns of exactly the same diameter separated by increasing distances?
    2. By using micropatterned substrates the Authors hope to force the extension of TNTs using actin driven mechanisms, while eliminating the possibility that cells are form these connections by "dislodging" from the substrate. The Authors also state that the cells do not wander into the region in between the circular patterns. However, in many images featured in multiple figures, I can find examples where cells clearly extend into the space between the circular patterns (see Fig. 2.a.i for an example). The primary concern here is that the authors did not fully eliminate the possibility that cells are making direct contact and then pulling away from each other/dislodging to form TNTs. In the supplemental material, there were movies showing TNTs that had already formed, but I was unable to find examples of TNTs in the act of forming. If the authors do have timelapse data that clearly shows this, it should be front and center in this data set.
    3. The authors make use of CK-666 to inhibit Arp2/3, which is thought to free up actin monomers that can be used to further elongate TNTs. Other orthogonal methods should be employed to corroborate the results from CK-666 treatment. There are other drugs that inhibit Arp2/3 (e.g. arpin, wiskostatin) and of course there is always genetic manipulation (shRNA KD).
    4. The authors also employ a formin agonist, IMM-01, and the results from those experiments (Fig. S4) suggest that activating formin mDia could facilitate TNT elongation, but the authors do not follow up with direct molecular manipulation to further test this idea. Are formins (specifically mDia, the target of IMM-01) needed to elongate TNTs?
    5. With the experiments shown in Figure 3, the authors apply optical tweezers to pull what they refer to as "nanotubes" from the cell surface under conditions where Arp2/3 is inhibited with CK-666. The panels appear to show that CK-666 allows actin to assemble out into a pulled nanotube more readily than control cells. However, the reporter for actin assembly in these experiments is F-Tractin, and it only partially fills the protrusion. I note here that F-Tractin appears more soluble under the CK-666 condition. Can the Authors rule out the possibility that there is just a larger soluble pool of F-Tractin probe under these conditions?
    6. Related to the previous point, it is not clear to me why the Authors need to invoke the application of external forces with an optical trap to study the enhanced elongation of TNTs under CK-666 conditions. Why don't the authors directly visualize actin accumulation and TNT elongation after treatment with this inhibitor?
    7. In Figure 4, the Authors move away from TNTs and instead focus on "longer protrusions" (filopodia?) to examine the effects of EPS8 and IRSp53 on growth of these structures. The general findings here are consistent with the role of these factors in protrusion growth from previous studies.
    8. The experiments in Figure 5 are performed with a truncated "bundling active" form of EPS8 allegedly lacking actin capping activity. The logic behind this choice is unclear. These experiments need to be repeated in parallel with full length WT EPS8 to allow for a full and clear interpretation of these results. Along these lines, could the Authors stain for endogenous Esp8/IRSp53 complex within TNT-connected cells?
    9. Data in Figure 5 show that the Eps8dCAP/IRSp53 complex increases the vesicle transfer within TNT-connected cells from Eps8dCAP/IRSp53 donors to EBFP-H2B acceptors indicating the functionality of TNT. Interestingly, cells with overexpression of Eps8dCAP/IRSp53 and CK-666 treatment do not increase TNT-connections. Could the authors examine the stability of TNTs under this condition? The lack of increased vesicle transfer may be due to the instability of the TNT structure rather the saturation of the system.
    10. Figure 5c. The data show that vesicle transfer from EpsdCAP/IRSp53 donors to EBFP-H2B acceptors increases under the overexpression of EpsdCAP/IRSp53. Are the EBFP-H2B acceptors able to form TNT? If so, could the authors show the vesicle transfer in those TNT-connected cells? The images indicate that there is no presence of TNT structures in these conditions.

    Significance

    This study from Henderson et al. seeks to understand the mechanisms that drive tunneling nanotube (TNT) formation. TNTs are essentially giant filopodia that extend many microns from the cell surface to contact protrusions extending from neighboring cells, to establish channels that allow for exchange of biological material. The authors use an approach that involves micro patterned substrates, various inhibitors/agonists, construct overexpression experiments, and biophysical measurements with optical tweezers. The major insights from these studies are that actin monomer availability is limiting for the formation of long TNTs, and that proteins that are well known to regulate the formation of filopodia and related linear actin structures (namely EPS8 and IRSp53) promote TNT formation. These are interesting findings that are certainly consistent with the previously published literature on actin-based protrusions, and as such they should be of interest to cell biologists.

  23. Version published to 10.1101/2022.08.24.504515v2 on bioRxiv
  26. Arcadia Science

    background values adjacent to the measured bands.

    I noticed in figure 4A the tubulin loading control isn't super consistent. Did you normalize to your loading control? Might also be worth doing total protein as your loading control instead of tubulin, because it is possilbe that tubulin is being affected since you're probing other cytoskeleton related proteins.

  27. Arcadia Science

    we utilized an optical tweezer (OT) setup to pull nanotubes of comparable lengths to the TNTs observed on the micropatterns to monitor by confocal microscopy F-actin polymerization within the nanotube in control and CK-666-treated conditions

    This experiment is very cool, but I wonder if you could show a similar thing with naturally forming TNTs? Do you see more actin in natural TNTs when the Arp2/3 complex is inhibited? I worry that stretching the cell like this could be causing other things to happen in the cell and isn't fully representative of a TNT forming on its own.

  29. Arcadia Science

    Upon Arp2/3 inhibition with CK-666 (6, 48), we observed a significant increase in the percent of TNT-connected cells on D15, D20 and D30 micropatterns

    It would be really great to see representative images of the data quantified in Figure 2C.

  30. Arcadia Science

    this suggests an anti-correlation between regions with high Arp2/3 activity and a cell’s ability to form TNTs

    To really illustrate this it would be helpful to show a bivariate analysis between like number of lamellipodia per cell and number of TNTs per cell for 2B. Then you could actually graphically show this anti-correlation and provide a correlation coefficient to strengthen this conclusion. It would also be interesting to show this with CK-666 treatment as I wondered while reading this if the increase in TNTs/filopodia with CK-666 treatment was accompanied by a decrease in lamellipodia.

  31. Arcadia Science

    Our data suggest a shift in the equilibrium (and usage of common actin proteins players) between branched and linear actin polymerization to form different cell protrusions.

    What a cool paper! Beautiful images and I love papers that deal with this concept of a limited pool of actin in the cell that is directed to its different functions by its interactions with actin binding/interacting proteins.

  33. Arcadia Science

    (a)

    The TNTs in the WT here look drastically different from the WT/control TNTs in previous figures In both the GFP/mCherry and the Eps8-IRSp53, the TNT actin looks thinner when treated with CK-666 compared to Mock/DMSO. Have you done any diameter measurements of the TNTs?

  34. Arcadia Science

    In the case of overnight drug treatments for TNT counting and co-culture experiments, cells first adhered for 4–6 hr and were then treated with 50 μM CK-666 or 1 μM IMM-01 for 16–18 hr.

    Have you done any experiments where you pre-treat with CK-666 before adhering the cells? Its possible the branched actin networks are necessary for the initial membrane deformation, but after the TNT is formed, Arp2/3 complex inhibition frees up more G-actin which goes the the linear filaments in the TNT?

  35. Arcadia Science

    Fig. 4:

    Fig 4 C & F) does the 00:00 time point indicate the time when the cells were plated? In the earlier experiments, cells were allowed to adhere for 4-6 hours before treatment for 16-18 hours. Do TNTs form within 30 min of plating?

    Also, minor point, but C is in hh:mm:ss time stamp, but F is hh:mm time stamp. Since all of the timepoints in C end it 00 sec it would be good to make C/F consistent as hh:mm (or hh:mm:ss)

    Also, after 30 min, CK-666 is added and the extended protrusions form shortly after. For the control quantified in d and g, was DMSO added at the 30 min mark? It would be nice too see the control images as well. The force from the flow of added liquid could cause morphological changes

  36. Arcadia Science

    Competition between actin binding proteins for stabilizing different actin populations is a super interesting phenomenon. I always imagined this being due to some actin binding proteins acting as a sink for monomers dropping local actin concentration available for other ABPs or sequestering monomers to other spatial domains (something we’ve observed in other systems). However, I found it really interesting that an actin bundler forming presumably linear actin tunneling nanotubes that seems to be antagonistic to branched actin formation by ARP2/3 directly binds components of the ARP2/3 complex. Do you imagine that the higher branching ARP2/3 complex isoforms are also sequestered from the rest of the complex where lamellipodia would form? Are you able to localize those Eps8-interacting ARP2/3 components to nanotubes?

  37. Arcadia Science

    Following CK-666 addition, we observed an increase of IRSp53 fluorescence at the plasma membrane

    In Fig 4 C,F) It would be helpful to have these fluorescent images merged with a brightfield image so we can see the outline of the TNTs and where these fluorescent proteins are localized in the cell.

  38. Arcadia Science

    . (e)

    It would be helpful to include a heat map gradient/scale/legend under subpanels in C. It took me a while to realize that it was a heat map LUT and not a blue marker and a magenta marker.

  39. Arcadia Science

    Eps8 and IRSp53 are recruited to form longer protrusions upon Arp2/3 inhibition.

    If I understand correctly, the figure shows that IPSp53 and Eps8 interact and localize to these extension, but not that they are responsible for forming the longer protrustions, right? The figure text suggests they are responsible which isn't shown until Fig 5

  42. Version published to 10.1101/2022.08.24.504515v1 on bioRxiv