Localized Mechanical Stress Promotes Microtubule Rescue Hélène de Forges, Antoine Pilon, Isabelle Cantaloube, Antoine Pallandre, Anne-Marie Haghiri-Gosnet, Franck Perez, Christian Poüs  Current Biology  Volume 26, Issue 24, Pages 3399-3406 (December 2016) DOI: 10.1016/j.cub.2016.10.048 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 3399-3406DOI: (10.1016/j.cub.2016.10.048) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 GTP Islands Occur at Microtubule Crossings upon Mechanical Constraints (A) GTP island staining after cytosol removal in PtK2 cells stably expressing GFP-tubulin. Cells were extracted in a MT-stabilizing buffer containing glycerol and Taxol and then processed without fixation for labeling with hMB11 antibody. The arrows indicate hMB11-positive stretches at MT bundles. (B) GTP islands and MT crossings of the MT outlined in (A). (C) Mean percentages (±SD) of MT crossings that displayed GTP islands compared to that estimated for a stochastic distribution as determined by Monte Carlo simulation. The simulation parameters were the spatial frequencies of apparent crossings and GTP island distribution along MTs in PtK2 cells. (D) 2D-constrained MT growth assay. Taxol-stabilized MT seeds (bright red) were stuck on coverslips that were coated with recombinant KHC in the presence of AMPPNP. The removal of Taxol and addition of green fluorescent tubulin and GTP allowed for MT elongation and binding to the motors. After removal of the residual tubulin and GTP, MTs were processed for the immunolabeling of GTP islands. Circles indicate the MT crossings that displayed GTP islands. Arrowheads indicate the GTP islands that are juxtaposed to MT crossings. (E) Comparison between the mean percentage (±SD) of GTP island-positive MT crossings measured by 2D-constrained growth assays and estimated by Monte Carlo simulation for a stochastic distribution. (F) MT collision and MT sedimentation assays on microfabricated pillars. The protocol used for the collision assays (upper panels) was the same as that described for (D), except the experiments were performed on landscapes of pillars of controlled height (nm). The contours of the pillars are outlined in gray. Note that the top of the pillars (High) often appears to have a reddish background, whereas the valley background was green (Low). Sample MT crossings over the pillar edges are shown in the insets. The white arrows indicate hMB11-positive MT-MT crossings. The protocol used for the sedimentation assays (lower panels) was the same as that used for collisions, except the MT seeds were elongated in suspension prior to spontaneous sedimentation over the KHC-coated landscapes. (G) Quantification of the proportion of MT crossings over the pillar edges that displayed GTP islands as a function of the thickness of the pillar (circles). The adjacent numbers indicate the total number of crossings that were measured at each pillar height. The fraction of MT-MT crossings that also displayed GTP islands (crosses) was used as a control. Scale bars, 10 μm. See also Figure S1 for the fabrication of the pillar landscapes. Current Biology 2016 26, 3399-3406DOI: (10.1016/j.cub.2016.10.048) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Repaired Damage at Microtubule Crossings and Microtubule Lattice Defects Are Efficiently Detected as GTP Islands (A) MT elongation and repair assay. MTs that had been polymerized in suspension with unlabeled tubulin and stabilized with Taxol were sedimented onto KHC-coated coverslips by centrifugation, through a Taxol-containing sucrose cushion used to remove any residual tubulin and GTP. MT elongation and repair were induced by the addition of green fluorescent tubulin and GTP. Samples were processed directly for the labeling of GTP islands (red), and MTs were then fixed with ice-cold methanol and subjected to total tubulin immunofluorescence labeling (blue). (B) GTP islands and MT repair islands were counted at the location of MT crossings and away from the crossings in the repair experiments. The histograms show the percentage of total islands found at each location. (C) Annealing protocol of MTs assembled with GMPCPP and those assembled with GDP and Taxol or GDP and Taxotere. MTs were assembled prior to the assay using GDP-bound tubulin with different fluorescent labels. GMPCPP MTs were mixed with GDP/taxane MTs for 2–4 hr at room temperature to allow spontaneous annealing of MTs. After sedimentation on the KHC-coated coverslips, MTs were processed for the labeling of GTP islands. The images show four sample fields containing chimeric MTs for each condition. The white arrows indicate the GMPCPP/taxane interfaces that present GTP islands. (D) Negative control formed by annealing two populations of GMPCPP MTs labeled with different colors. The yellow arrows indicate the location of GMPCPP/GMPCPP annealing interfaces. (E) Histogram showing the proportion of annealed MT interfaces that displayed GTP islands, as well as the percentage of expected positive interfaces if the distribution of GTP islands were stochastic (as determined by Monte Carlo simulation). Scale bars, 5 μm. Current Biology 2016 26, 3399-3406DOI: (10.1016/j.cub.2016.10.048) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Rescues and GTP-like Islands Occur at Microtubule Crossings in Living Cells (A and D–H) The MT dynamics in PtK2 cells that stably express GFP-tubulin were recorded. Where appropriate, cells were permeabilized with Triton X-100 in MT-stabilizing buffer containing glycerol and Taxol and then processed for the labeling of GTP-like islands without fixation (right panels in D, E, and G). MTs that crossed other MTs are highlighted in blue, and MTs that were crossed are highlighted in yellow. The crossings of interest are indicated by yellow arrowheads, and the rescues are indicated by blue arrowheads. (B) Kymograph composed from a series of images. For each image in the series, the fluorescence intensity of the crossing MT (blue) along its growth trajectory (red dashed line) was adjusted to form consecutive lines on the kymograph image. The blue trace represents the life history of the crosser MT length (horizontal) versus time (vertical). The crossed MT (yellow) appears as a vertical trace. The kymograph displayed in (H) was built along the crossed MT (yellow) to show its rescue. Scale bars, 5 μm. (C) Histogram showing the rescue percentage (mean ± SD) at MT crossings compared to that estimated by Monte Carlo simulations. See Movie S1 for the occurrence of rescues at crossings and for the presence of GTP-like islands at a crossing where a rescue took place, Movie S2 for the occurrence of rescues and GTP-like islands in crosser MTs, and Movie S3 for similar occurrences in crossed MTs. See also Figure S2 for the promotion of rescues at MT crossings in vitro and Figure S3 for the impact of MT crossings and network organization on MT dynamics. Current Biology 2016 26, 3399-3406DOI: (10.1016/j.cub.2016.10.048) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 CLIP-170 Is Retained at GTP-like Islands and at Microtubule Crossings after Comet Passage (A) PtK2 cells stably expressing GFP-tubulin and transiently expressing the dominant-negative CLIP-DH were extracted and processed for the labeling of GTP-like islands without fixation. (B) Top: histograms showing the extent (mean ± SD) of MT depolymerization following catastrophes in PtK2 cells. Bottom: frequency of distribution of GTP-like islands. (C) Time-lapse images of RPE-1 cells transiently expressing GFP-tubulin and mCherry-CLIP-170 were recorded, and cells were then extracted in MT-stabilizing buffer containing glycerol and Taxol and processed for the labeling of GTP islands without fixation. Colored arrowheads indicate the locations of CLIP-170 retention after passage of the comets, which are reported for the retrospective labeling of tubulin and GTP-like islands (right). (D) Percentage (mean ± SD) of the CLIP-170 comet-trailing dots that colocalized with MT crossings. (E) Sample RPE-1 cells were fixed and processed for immunolabeling of CLIP-170 and tubulin. The magnified insert shows CLIP-170 dots that colocalize with MT crossings (circles). (F) Quantification of the percentage (mean ± SD) of CLIP-170-positive intersections in three cell types. Scale bars, 5 μm. See also Movie S4 for the registration of CLIP-170 comet-trailing dots with GTP-like islands and MT crossings. Current Biology 2016 26, 3399-3406DOI: (10.1016/j.cub.2016.10.048) Copyright © 2016 Elsevier Ltd Terms and Conditions