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Self-repair promotes microtubule rescue

Nature Cell Biology volume 18, pages 1054–1064 (2016)Cite this article

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Abstract

The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events can arrest the depolymerization and restore microtubule elongation. However, the origin of these rescue events remains unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints.

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Figure 1: Microtubule self-repair in living cells.
Figure 2: Self-repair sites are rescue sites in living cells.
Figure 3: Photodamaged sites are repair and rescue sites in living cells.
Figure 4: Microtubule self-repair induces rescue events in vitro.
Figure 5: Incorporation and hydrolysis of free tubulin.
Figure 6: Self-repair biases microtubule dynamic instability in vitro.
Figure 7: Self-repair biases microtubule dynamic instability in vivo.
Figure 8: Microtubule self-repair and rescue.

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Acknowledgements

We thank G. Montagnac for sharing his unpublished data on tubulin photo-activation in cells; and F. Perez and C. Poüs for sharing their unpublished data on the microtubule rescue by CLIP170. This work has been supported by HFSP (RGY0088/2012), ANR funding (ANR-12-BSV5-0014) and ERC (Starting Grant 310472).

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Author notes

  1. Charlotte Aumeier and Laura Schaedel: These authors contributed equally to this work.

Authors and Affiliations

  1. CytoMorpho Lab, Biosciences & Biotechnology Institute of Grenoble, UMR5168, CEA/INRA/CNRS/Université Grenoble-Alpes, 38054 Grenoble, France

    Charlotte Aumeier, Laura Schaedel, Jérémie Gaillard, Laurent Blanchoin & Manuel Théry

  2. Laboratoire Interdisciplinaire de Physique, CNRS/Université Grenoble-Alpes, 38402 Saint-Martin-d’Hères, France

    Karin John

  3. CytoMorpho Lab, Hopital Saint Louis, Institut Universitaire d’Hematologie, UMRS1160, INSERM/Université Paris Diderot, 75010 Paris, France

    Laurent Blanchoin & Manuel Théry

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Contributions

C.A. performed experiments in cells. L.S. and J.G. performed the experiments in vitro. L.B. and M.T. directed the work. C.A., L.S., K.J., L.B. and M.T. analysed the data. M.T. wrote the paper.

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Correspondence to Laurent Blanchoin or Manuel Théry.

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Integrated supplementary information

Supplementary Figure 1 Effect of laser intensity on photo damage induced on microtubules.

(a) Examples of microtubules damaged with different laser intensities. Left panel: Microtubule damaged at intermediate laser intensity. The microtubule was structurally damaged, as shown by the kinked shape it displayed when submitted to flow. No severing occurred. Right panel: At high laser intensity, microtubules were cut. (b) Histogram of the distance of the photo-damage site with respect to the tip (set to zero).

Supplementary Figure 2 Microtubules self-repair at photo damage sites.

Microtubules were grown in the device shown in Fig. 4a. Green microtubule seeds were elongated with red free tubulin (step I). Photo damage (yellow star) was induced in the presence of green tubulin (step II). After a two-minute delay, the medium was changed again for red tubulin (step IV). Free fluorescent tubulin dimers were removed and replaced with non-fluorescent dimers to visualize microtubules. The white arrow point at the incorporation of green tubulin along the pre-existing red microtubule following laser-induced damage.

Supplementary Figure 3 Microtubules rescue at photo damage sites.

Kymographs of microtubules showing rescue or depolymerization at photo damage sites (yellow star), analogous to Fig. 4e.

Supplementary Figure 4 Effect of laser power and frequency on microtubule photo-damaging.

(a) Red microtubule seeds were elongated with green free tubulin (step I). A GMPCPP cap was grown at the microtubule tip to avoid spontaneous depolymerization (step II). Photo damage was induced in the absence of free tubulin to avoid microtubule self-repair (step III). Depolymerization was initiated by removing the GMPCPP cap with a laser pulse at high intensity (step IV). In the absence of free tubulin, microtubule could not grow but only depolymerize. The laser-induced damage could only temporally interrupt microtubule depolymerisation (pause). High laser power could break the microtubule. (b) At low laser power and frequency (I), such as the one used throughout the study, photo damaged microtubules depolymerized without pausing. At high laser power and low frequency (II), microtubules broke at the laser damage site and immediately started to depolymerize after fracture. At low laser power and high frequency (III), most microtubules either broke, pausing for a short time before depolymerization (left panel), or paused at the laser damage site (right panel). At high laser power and frequency (IV), microtubules usually paused at the damage site before resuming depolymerization, suggesting that the laser power had a cauterizing effect on the microtubule lattice. (c) Quantification of the events shown in b. Between 17 and 24 events were monitored per condition in 4 independent experiments.

Supplementary Figure 5 EB1 recruitment at microtubule repair sites.

(a) EB3 recruitment at repair sites in the presence of free tubulin. Red microtubules were photodamaged in the presence of red-fluorescent free tubulin dimers and EB3-GFP. Images show microtubule fluorescence in the red (left) and green (right) channels, before (top) and after (bottom) the laser-induced damage. The graph shows fluorescent linescans along this microtubule before and after the damage. The yellow star represents the damaged site. (b) Absence of EB3 recruitment at repair sites in the absence of free tubulin. Same experiment in the absence of free tubulin. (c) Quantification of the effect of laser damage on the ratio of EB3 intensity after/before.

Supplementary Figure 6 Laser photo damage promotes microtubule growth.

(a) Time lapse and kymograph of a microtubule in vivo with multiple photo-damage sites. Microtubule was photo-damaged (yellow stars) close to the tip, promoting multiple rescue events (red arrowhead). Though catastrophe events were frequent, this microtubule was protected from complete depolymerization by the photo damage. Scale bar is 5 μm. (b) Time lapse of a growing microtubule in vitro without or with laser damage. Left, a growing microtubules in absence of laser damage. Right, a growing microtubule in presence of repeated laser damages. Image sequences corresponding to the kymographs shown in Fig. 6a.

Supplementary Figure 7 Self-repair biases microtubule dynamic instability in vivo.

The sheme illustrates laser induced photo-damages either on the microtubules, in the red region, or next to the microtubules, in the green region. The images show the cell before (top) and after (bottom) the shooting. Comparison of the left and right cell margin pre- and post-photo-damage. Scale bar is 5 μm.

Supplementary Figure 8 Laser-induced photo damage on microtubules influences cell migration.

Time series montage of a migrating PtK2 GFP-tubulin cell. Cell migrated 33 min prior photo-damage to the left upper corner of the field of view. Photo-damage at the retracting cell edge (yellow stars) from t = 33 min to t = 75 min changed migration direction towards the photo-damage site. Even after the laser-induced photo-damage was stopped the cell continued the motion towards the bottom right corner of the field of view.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1495 kb)

PtK2 cell expressing mEos2 post photo-conversion.

The microtubule tips grow by incorporating converted tubulin dimers (magenta). (MOV 1498 kb)

PtK2 cell expressing mEos2 post photo-conversion.

Converted free tubulin dimers (magenta) diffused through the cytoplasm. Signal of converted dimers (magenta) was observed at the growing tips as well as in spot-like structures along the length of pre-existing microtubules (green). Video related to Fig. 1a. (MOV 2902 kb)

Self-repair sites are rescue sites. Overview and detail of PtK2 cell expressing mEos2 post photo-conversion.

Incorporation (arrow) of converted tubulin dimers (magenta) in pre-existing microtubule (green). Rescue event occurs exactly at the site of incorporation (arrow). Video related to Fig. 2a. (MOV 1248 kb)

The localization of rescue events in cells are frequently at microtubule crossing site.

White arrow indicates the microtubule tip. Red circle highlights the rescue at microtubule crossing site. PtK2 cells stable expressing GFP-tubulin. (MOV 661 kb)

Representative sites of laser induced photo-damage (red circle) within the cell margin and near the nucleus of PtK2 GFP-tubulin cells.

Microtubules undergo rescue at photo-damage sites. Arrow highlights the dynamic microtubule tip pre- and post-rescue. Video related to Fig. 3c. (MOV 14283 kb)

Detail of microtubule rescue at photo-damage site in vivo.

Multiple rescues at photo-damage site are occurring occasionally, bottom video. Red circle highlights the rescue at photo-damage site. Arrow highlights the dynamic microtubule tip pre- and post-rescue. Video related to Fig. 3d, e. (MOV 1495 kb)

Microtubule rescuing at a photo damage site in vitro.

The upper panel shows a microtubule without laser damage; in these conditions, microtubules rarely undergo spontaneous rescue. The lower panel shows a microtubule rescuing several times at a laser damage site (red circle) before complete depolymerization. Video related to Fig. 4e. (MOV 2148 kb)

Microtubule rescue at cross-over.

The video shows the growth of non-micropatterned and non-photo-damaged microtubules. Microtubule depolymerisation was stopped at the site where microtubules cross each other. (MOV 794 kb)

Laser damage increases microtubule length and life-time.

The upper panel shows a microtubule without laser damage and infrequent rescue events. In the lower panel, the microtubule was damaged repeatedly close to the tip (red circles), inducing rescue and making the microtubule grow longer and longer. Video related to Fig. 6a. (MOV 6823 kb)

Microtubules rescue and over growth upon repeated photo-damages.

PtK2 GFP-tubulin cell. Photo-damage on single microtubule (red dots) or next to microtubules (green dots). Microtubule number and length increased at the site of microtubule photo-damage (red dots) and decreased on the opposite site (green dots). The cell spread toward the site where microtubules were photo-damaged. Video related to Fig. 7. (MOV 3555 kb)

Reoriented migration by photo-damaged microtubules.

PtK2 GFP-tubulin cell migrated 34 min prior photo-damage to the left upper corner of the field of view. Photo-damage of single microtubule (red dots) at the retracting cell edge from t = 33 min to t = 75 min changed migration direction towards the photo-damage site. Even after the offset of photo-damage cell continued the motion towards the right. Video related to Supplementary Fig. 8. (MOV 26223 kb)

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Aumeier, C., Schaedel, L., Gaillard, J. et al. Self-repair promotes microtubule rescue. Nat Cell Biol 18, 1054–1064 (2016). https://doi.org/10.1038/ncb3406

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