CLASPs link focal-adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover

Abstract

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.

Figure 1. Mature FAs recruit CLASP2-decorated microtubules

(a) Spinning disk confocal microscopy of a migrating HaCaT epithelial cell expressing EGFP-CLASP2 (black) and paxillin-mCherry (magenta). In this and subsequent figures, images from live time-lapse sequences are contrast-inverted to better visualize intracellular protein dynamics. See methods for details. (b) EGFP-CLASP2 and paxillin-mCherry dynamics at the advancing cell edge in the region indicated in (a). Arrows indicate the birth of two FAs that are engulfed by CLASP2-decorated microtubules prior to disassembly. Elapsed time is in minutes. (c) Continuation of the same sequence showing only the EGFP-CLASP2 channel to highlight EGFP-CLASP2 particles that appear near the leading edge, increase in intensity while flowing retrograde, and often appear to capture growing microtubules (red arrowheads). (d) Turnover dynamics of paxillin-mCherry-labelled FAs and surrounding EGFP-CLASP2. Fluorescence intensity profiles measured as a function of time were normalized to the maximum paxillin-mCherry fluorescence intensity for each FA and aligned relative to each other (n = 20 FAs). The solid line is an exponentially-modified Gaussian curve fit, and dashed lines 95% confidence intervals. (e) Immunofluorescence of endogenous CLASP1 and CLASP2. Insets show only the CLASP channel. In this and subsequent figures, single channels are displayed with inverted contrast for improved display. (f) Immunofluorescence demonstrating isoform-specific shRNA-mediated CLASP depletion. (g) Scanning angle interference microscopy (SAIM) of microtubule height above the substrate in control and CLASP2-depleted cells. Bottom panels show areas from different cells at higher magnification and a shallower height map. Note the decrease of microtubule height specifically near FAs in control cells that is largely absent in CLASP2-depleted cells. (h) Quantification of microtubule height near FAs larger than 1 μm² in control and CLASP-depleted cells. n = 40 (control shRNA); 28 (CLASP1 shRNA); 28 (CLASP2 shRNA) cells from two experiments. Box-and-whisker plot shows median, 1^st and 3^rd quartile (box), and 95% confidence intervals (notches) with whiskers extending to the furthest observations within ±1.5 times the interquartile range. Dots are individual data points, and source data are included in Supplementary Table 3. p values were calculated by non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.

Figure 2. CLASPs facilitate FA disassembly in migrating epithelial cells

(a) Time-lapse sequences of paxillin-mCherry dynamics in control and CLASP-depleted migrating HaCaT cells. The regions indicated are shown at higher magnification. The maximum intensity projections (MIP) over the entire three hour time-lapse sequences on the right further illustrate FA turnover defects and sliding. Elapsed time is in minutes. (b) Examples of turnover dynamics of representative FAs in control and CLASP-depleted cells used for calculation of FA dynamics parameters in (c). Data points are 3-frame running averages of FA fluorescence intensity. The green solid line is a logistic fit of the FA assembly phase, the red line a single exponential decay fit of the disassembly phase. The dashed arrow indicates FA lifetime as defined by fluorescence intensity above the half-maximum of the fit. (c) Analysis of FA assembly rates, lifetime and disassembly rates in control and CLASP-depleted migrating HaCaT cells. n = 54 (control shRNA); 53 (CLASP1 shRNA); 55 (CLASP2 shRNA) FAs from three experiments. (d) Length of FA sliding measured from MIPs. n = 405 (control shRNA); 217 (CLASP1 shRNA); 404 (CLASP2 shRNA) FAs. Representative data set of three experiments. Only outliers are shown as individual data points. (e) Quantification of directed migration of control and CLASP-depleted HaCaT cells at the edge of a cell monolayer. n = 62 (control shRNA); 71 (CLASP1 shRNA); 71 (CLASP2 shRNA) cells. Representative data set of three experiments. Box-and-whisker plots show median, 1^st and 3^rd quartile (box), and 95% confidence intervals (notches) with whiskers extending to the furthest observations within ±1.5 times the interquartile range. Dots are individual data points, and source data for (c), (d), and (e) are included in Supplementary Table 3. p values were calculated by non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.

Figure 3. CLASP clusters around FAs do not depend on microtubules

(a) Time-lapse sequence of EGFP-CLASP2 (black) and paxillin-mCherry (magenta) dynamics in HaCaT epithelial cells treated with 3.3 μM nocodazole at t = 0 min. (b) Average dynamics of individual paxillin-mCherry-labeled FAs and surrounding EGFP-CLASP2 in nocodazole-treated cells (n = 16 FAs). The solid line is an exponentially-modified Gaussian curve fit, and dashed lines are 95% confidence intervals. (c) Time-lapse sequence of EGFP-CLASP2 (black) and paxillin-mCherry (magenta) dynamics after nocodazole washout, illustrating microtubule repolymerization and capture at FA-associated CLASP clusters, and subsequent FA disassembly. (d) Time-lapse sequence of EGFP-CLASP2 (black) and paxillin-mCherry (magenta) dynamics in HaCaT cells in which microtubules were depolymerized by 3.3 μM nocodazole for 90 minutes prior to the addition of 10 μM Rho-kinase inhibitor Y-27632, resulting in disassembly of both FAs and associated CLASP clusters. (e) Scatter plot of the correlation between FA size and microtubule-independent EGFP-CLASP2 cluster. (f) Structured illumination super-resolution microscopy (SIM) of EGFP-CLASP2 and paxillin-mCherry expressing nocodazole-treated HaCaT cells, illustrating close intercalation of CLASP clusters and FAs. (g) Localization of endogenous CLASP1 (left) and CLASP2 (right) around FAs in 3.3 μM nocodazole-treated HaCaT cells. (h) HaCaT cells expressing paxillin-mCherry and either phosphomimetic EGFP-CLASP2 8xS/D (left) or non-phosphorylatable EGFP-CLASP2 9xS/A (right). (i) Time-lapse sequence of EGFP-CLASP2 (black) and paxillin-mCherry (magenta) dynamics in HaCaT epithelial cells treated with 10 μM Y-27632 at t = 0 min. (k) Average dynamics of individual paxillin-mCherry-labeled FAs and surrounding EGFP-CLASP2 in 10 μM Y-27632-treated cells (n = 20 FAs). The solid line is an exponentially-modified Gaussian curve fit, and dashed lines are 95% confidence intervals. All elapsed time is in minutes.

Figure 4. LL5β is required for CLASP-mediated focal adhesion turnover

(a) Time-lapse sequence of EGFP-LL5β (black) and paxillin-mCherry (magenta) dynamics in a migrating HaCaT epithelial cell at the edge of a cell monolayer. The image on the right shows a maximum intensity projection (MIP) of only the LL5β channel over the entire 3 hour sequence, illustrating the complex dynamics of LL5β particles near FAs. Elapsed time is in minutes. (b) Total internal reflection (TIRF) microscopy of EGFP-LL5β and EGFP-CLASP2 near paxillin-mCherry labeled FAs, demonstrating similar punctate pattern at the ventral cell surface. (c) LL5β immunofluorescence in HaCaT cells expressing the indicated EGFP-CLASP2 constructs indicating increased overlap of LL5β and CLASPs in the absence of CLASP-microtubule binding. (d) Time-lapse sequences of paxillin-mCherry dynamics in control and LL5β-depleted migrating HaCaT cells. The regions indicated are shown at higher magnification. Elapsed time is in minutes. (e) Analysis of FA assembly rates, lifetime and disassembly rates in control and LL5β-depleted migrating HaCaT cells. n = 33 (control shRNA); 21 (LL5β shRNA #28); 32 (LL5β shRNA #39) FAs from three experiments. Box-and-whisker plots show median, 1^st and 3^rd quartile (box), and 95% confidence intervals (notches) with whiskers extending to the furthest observations within ±1.5 times the interquartile range. Dots are individual data points, and source data are included in Supplementary Table 3. p values were calculated by non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.

Figure 5. CLASPS are required for FA-associated ECM degradation

(a) Time-lapse sequence of Alexa488-gelatine degradation in a paxillin-mCherry-expressing cell illustrating spatiotemporal correlation with FA turnover. The region indicated is shown at higher magnification. Areas of matrix degradation appear as dark areas in the Alexa488-gelatine images. Elapsed time is indicated in minutes. (b) Average turnover dynamics of Alexa488-gelatine at individual paxillin-mCherry-labeled FAs. Fluorescence intensity profiles measured as a function of time were normalized to the maximum Alexa488-gelatine and paxillin-mCherry fluorescence intensities, and aligned relative to the half maximum of FA assembly (n = 20 FAs). Because FA lifetime is highly variable in spreading HaCaT cells, the FA disassembly phase was not included in this quantification. Solid lines are a logistic fit for FA assembly and a single exponential decay for ECM degradation. Dashed lines are 95% confidence intervals. (c) Matrix degradation by control and CLASP-depleted HaCaT cells after 24 hours of spreading on Alexa488-gelatine-coated coverslips (green). Cells were also immunostained for paxillin (magenta). (d) Quantification of the area of gelatin degradation in the periphery of control and CLASP-depleted cells. n = 64 (control shRNA); 50 (CLASP1 shRNA); 39 (CLASP2 shRNA) cells from three experiments. (e) Alexa488-gelatine degradation is inhibited in BB-94 treated cells. (f) Analysis of FA assembly rates, lifetime and disassembly rates in control and BB-94-treated migrating HaCaT cells. n = 48 (control); 53 (BB-94) FAs from three experiments. (g) Analysis of Y-27632-induced (10 μM) FA disassembly in control, BB-94-treated, and CLASP-depleted HaCaT cells. n = 43 (control); 43 (BB-94); 37 (CLASP1 shRNA); 22 (CLASP2 shRNA) FAs. Box-and-whisker plots show median, 1^st and 3^rd quartile (box), and 95% confidence intervals (notches) with whiskers extending to the furthest observations within ±1.5 times the interquartile range. Dots are individual data points, and source data for (d), (f), and (g) are included in Supplementary Table 3. p values were calculated by non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.

Figure 6. MT1-MMP dynamics in migrating epithelial cells

(a) Spinning disk confocal microscopy of a migrating HaCaT epithelial cell expressing MT1-MMP-EGFP and paxillin-mCherry. The regions indicated are shown at higher magnification. Elapsed time is in minutes. (b, c) High speed TIRF microscopy of MT1-MMP-EGFP dynamics. Coloured arrowheads indicate individual MT1-MMP-EGFP vesicles moving toward and rapidly disappearing near FAs. MT1-MMP-EGFP vesicle fusion events identified by coloured dots and arrowheads in (c) are shown magnified in the bottom panel. Elapsed time is in seconds.

Figure 7. Targeting of Rab6A-mediated exocytosis to FAs depends on CLASPs

(a) TIRF image of control HaCaT cell expressing EGFP-Rab6A (black) and paxillin-mCherry (magenta). (b) Kymograph and time-lapse sequence of the region in (a). Pausing vesicles generate vertical and moving vesicles diagonal tracks. Coloured arrowheads correlate specific vesicles in the kymograph and time-lapse sequence. Red arrow: Burst of EGFP-Rab6A diffusion characteristic for a plasma membrane fusion event. (c) Map of all observed (~200) EGFP-Rab6A fusion events in a 10 min time-lapse sequence overlaid onto the paxillinmCherry channel, illustrating clustering near FAs. (d) Histogram of EGFP-Rab6A fusion distance to the nearest FA compared with a uniform distribution (20x20 pixel grid) for the cell in (c). (e) Immunofluorescence showing endogenous Rab6 vesicles around FAs. (f) TIRF image of CLASP1-depleted cell expressing EGFP-Rab6A and paxillin-mCherry. (g) Kymographs and time-lapse sequences of the regions in (f), illustrating different types of aberrant EGFP-Rab6A vesicle dynamics. Black arrowhead: Paused FA-associated vesicle that fails to fuse and continues to move on. (h) Analysis of EGFP-Rab6A vesicle number in regions surrounding FAs (left; n = 40 (control shRNA); 48 (CLASP1 shRNA); 52 (CLASP2 shRNA)) or identically sized regions between FAs (right; n = 27 (control shRNA); 43 (CLASP1 shRNA); 38 (CLASP2 shRNA)) from three experiments. (i) Analysis of the distance of fusion events from the nearest FA. n = 399 (control shRNA); 347 (CLASP1 shRNA); 300 (CLASP2 shRNA) fusion events from three cells per condition. Six extreme outliers (> 5x the interquartile range from median) were removed from this dataset. (j) Quantification of vesicle dwell time, the duration between a EGFP-Rab6A vesicle stopping near a FA and vesicle disappearance, determined by measuring the length of vertical kymograph track segments. n = 254 (no shRNA); n = 298 (control shRNA); 234 (CLASP1 shRNA); 149 (CLASP2 shRNA) vesicles from two experiments. Box-and-whisker plots show median, 1^st and 3^rd quartile (box), and 95% confidence intervals (notches) with whiskers extending to the furthest observations within ±1.5 times the interquartile range. Dots are individual data points, outliers only in (i) and (j). Source data for (h), (i), and (j) are included in Supplementary Table 3. p values were calculated by non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.