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

Nature Cell Biology volume 16, pages 558–570 (2014)Cite this article

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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.

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Figure 1: Mature FAs recruit CLASP2-decorated microtubules.
Figure 2: CLASPs facilitate FA disassembly in migrating epithelial cells.
Figure 3: CLASP clusters around FAs do not depend on microtubules.
Figure 4: LL5β is required for CLASP-mediated FA turnover.
Figure 5: CLASPS are required for FA-associated ECM degradation.
Figure 6: MT1–MMP dynamics in migrating epithelial cells.
Figure 7: Targeting of RAB6A-mediated exocytosis to FAs depends on CLASPs.

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Acknowledgements

This work was supported by National Institutes of Health grant R01 GM079139 to T.W., and American Heart Association postdoctoral fellowship 10POST3870021 to S.J.S. Research was conducted in a facility constructed with support from the Research Facilities Improvement Program grant C06 RR16490, and on a microscope system funded by shared equipment grant S10 RR26758 from the National Center for Research Resources of the National Institutes of Health. We also thank C. O’Connell at Nikon for imaging on the Nikon structured illumination super-resolution microscopy system, B. Webb for help with the gelatin degradation assay, A. Akhmanova, D. Bryant, K. Hu, M. McNiven and J. Norman for reagents, and D. Barber, D. Bryant, A. Yap and members of the Wittmann and Barber laboratories for discussions and comments on the manuscript.

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  1. Samantha J. Stehbens & Matthew Paszek

    Present address: Present addresses: Institute of Health Biomedical Innovation (IHBI), Queensland University of Technology Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia (S.J.S.); School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York (M.P.).,

Authors and Affiliations

  1. Department of Cell and Tissue Biology, University of California San Francisco, 513 Parnassus Avenue San Francisco, California 94143, USA,

    Samantha J. Stehbens, Hayley Pemble, Andreas Ettinger, Sarah Gierke & Torsten Wittmann

  2. Department of Surgery and Center for Bioengineering and Tissue Regeneration, University of California San Francisco, 513 Parnassus Avenue San Francisco, California 94143, USA,

    Matthew Paszek

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Contributions

S.J.S. and T.W. conceived the project. S.J.S., M.P., H.P., A.E., S.G. and T.W. generated reagents, conducted experiments and analysed data. M.P. contributed SAIM data and analysis. S.J.S. and T.W. wrote the manuscript and assembled figures.

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Correspondence to Torsten Wittmann.

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Integrated supplementary information

Supplementary Figure 1 Validation and phenotypes of CLASP depletion.

(a) Representative images of Golgi fragmentation in CLASP-depleted cells stained for GM130 to identify the Golgi apparatus, and Sytox orange as a nuclear stain. (b) Cytoskeleton phenotypes of CLASP depletion. Peripheral microtubules are sparser and disorganized. F-actin stress fibres anchoring into FAs are more predominant in CLASP-depleted cells. (c) Immunofluorescence of phospho-myosin (ppMLC) and E-cadherin indicate that CLASP depletion increases cell contractility. (d) Representative images of migrating HaCaT cells at the edge of a cell monolayer stained for paxillin (magenta) and F-actin (green), expressing control (non-targeting), CLASP1 or CLASP2 shRNA. Only one of the two shRNA sequences used for each CLASP isoform is shown. Single channels of the indicated regions are displayed with inverted contrast. (e) Immunoblot of lysates from cells expressing different shRNA constructs after 7 days of puromycin selection. Tubulin was used as loading control. Blots were probed with isoform-specific antibodies to either CLASP1 or CLASP2. Uncropped blots are shown in Supplementary Fig. 8. (f) Quantification of the degree of Golgi fragmentation around the nucleus in HaCaT cells migrating at the edge of a cell monolayer. The Golgi fragmentation angle α was defined as the angle through the center of the nucleus that encompasses all Golgi structures. n = 73 (control shRNA); 35 (CLASP1 shRNA 32); 47 (CLASP1 shRNA 33); 65 (CLASP2 shRNA 55); 43 (CLASP2 shRNA 58) cells. Representative data set of three experiments. (g) Quantification of FA size in CLASP-depleted cells. Each data point is the average FA size from one image which contained 3–5 cells. n = 28 (control shRNA); 32 (CLASP1 shRNA 32); 35 (CLASP1 shRNA 33); 27 (CLASP2 shRNA 55); 34 (CLASP2 shRNA 58) images. Representative data set of three experiments. Box-and-whisker plots show median, first and third 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.

Supplementary Figure 2 CLASPs are required for directional migration.

(a) Example phase images of HaCaT cells migrating at the edge of a cell monolayer expressing control, CLASP1 or CLASP2 shRNAs. (b) Plots of representative cell migration paths in control and CLASP-depleted cells from 20 cells per condition. Data is representative of 3 independent experiments. Phase contrast time lapse sequences were rotated such that the wound edge was aligned with the vertical image axis, and cells are migrating to the right. The positions of dark features in the nucleus (nucleoli) were tracked over time with the ‘Time Measurements’ function in NIS Elements using ‘Auto Detect ROI’. Migration paths were normalized to the starting position.

Supplementary Figure 3 Validation and phenotypes of LL5β depletion.

(a) Localization of endogenous LL5β (green) around paxillin-labeled FAs (magenta) in control HaCaT cells, and cells treated with the indicated drugs. Insets show only the LL5β channel of the indicated regions with inverted contrast. (b) Immunoblot of lysates from cells expressing different shRNA constructs after 7 days of puromycin selection. Tubulin and GAPDH were used as loading controls. Uncropped blots are shown in Supplementary Fig. 8. (c) Immunofluorescence of LL5β and paxillin, in wound-edge HaCaT cells expressing control (non-targeting), or the indicated LL5β shRNAs. (d) Immunofluorescence of CLASP2, in HaCaT cells expressing control or the indicated LL5β shRNAs. LL5β-depletion abolishes peripheral CLASP accumulations. (e) Immunofluorecence of paxillin and LL5β in HaCaT cells expressing control or CLASP2 shRNA. CLASP-depletion in HaCaT cells does not affect LL5β localization to adhesion sites. (f) Immunofluorescence of tubulin or (g) F-actin (phalloidin) in HaCaT cells expressing control or LL5β shRNA. Cytoskeleton phenotypes of LL5β depletion are qualitatively similar to CLASP-depletion. Peripheral microtubules are sparser and disorganized. F-actin stress-fibers anchoring into FAs are more predominant in LL5β-depleted cells.

Supplementary Figure 4 CLASPs do not co-localize with endocytic vesicles.

(a) Nocodazole washout experiment in cells expressing EGFP–CLASP2. Cells were pre-treated for 90 min with 3.3 μM nocodazole prior to washout, and immunostained for endocytic markers clathrin heavy chain (HC) or dynamin II. Images were acquired by spinning disc confocal microscopy. No obvious colocalization is observed between CLASP2 and either clathrin HC or dynamin II. (b) Nocodazole washout experiment in cells immunostained for clathrin HC and paxillin. Cells were pre-treated for 90 min with 3.3 μM nocodazole prior to washout. Samples were imaged using total internal reflection microscopy (TIRF).

Supplementary Figure 5 MT1–MMP–mCherry–TM and ER dynamics in migrating HaCaT cells.

(a) Diagram of fluorescently tagged MT1–MMP constructs used. SP, signal peptide; HPX, hemopexin domain (b) Spinning disk confocal microscopy of a migrating HaCaT epithelial cell at the edge of a cell monolayer expressing MT1–MMP–mCherry–TM. Red arrowhead indicates the evolution of a bright, luminal labelled vesicle through macropinocytosis indicative of cleavage of the mCherry tag from the transmembrane domain. (c) Dynamics of AcGFP-tagged Sec61 as an endoplasmic reticulum marker1 in a migrating HaCaT cell expressing paxillin–mCherry. Although ER tubules are dynamically pulled forward into the leading lamella, there was no obvious correlation between ER dynamics and FA turnover. Elapsed time is in minutes.

Supplementary Figure 6 EGFP–Rab6A vesicle fusion with the plasma membrane.

(a) High speed TIRF imaging of EGFP–Rab6A vesicles. Red arrowheads indicate two independent fusion events associated with lateral spreading of EGFP–Rab6A signal in the plasma membrane. Elapsed time is in seconds. (b) Analysis of fluorescence intensity across EGFP–Rab6A vesicles at different time points during fusion normalized to vesicle intensity before fusion (n = 11 vesicles). Solid lines are Gaussian fits. The integrated intensity remains high while the signal spreads in the TIRF plane demonstrating lateral diffusion of EGFP–Rab6A indicative of fusion with the plasma membrane and thus exocytosis. Error bars are 95% confidence intervals.

Supplementary Figure 7 Workflow of generating kymographs to quantify EGFP–Rab6A vesicle transport.

(a) Rotated raw and filtered data, cropped region around a FA (dashed box), and single x-t kymograph at the orange line. (b) Set of all twenty x-t kymographs for each horizontal row of pixels in the cropped image. (c) Maximum intensity projection of all twenty x-t kymographs. See methods for additional details.

Supplementary Figure 8 Uncropped immunoblots shown in supplementary Figures 1 and 3.

Membranes were cut along the dotted lines before incubation with primary antibodies in order to probe shRNA targets and loading controls (GAPDH and tubulin) on the same blots.

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EGFP–CLASP2 and paxillin–mCherry dynamics in a migrating HaCaT cell.

Spinning disk confocal microscopy time lapse sequence of a migrating HaCaT epithelial cell at the edge of a cell monolayer expressing EGFP–CLASP2 (black) and paxillin–mCherry (magenta). EGFP–CLASP2-decorated microtubules engulf FAs before FA disassembly underneath the advancing cell body. Images were acquired every 2 min. The video plays at 15 frames s−1 and is thus accelerated 1800 times. (MOV 8090 kb)

Focal adhesion turnover dynamics in control and CLASP-depleted cells.

Spinning disk confocal microscopy time lapse sequences of migrating HaCaT epithelial cells at the edge of a cell monolayer expressing paxillin–mCherry. Left panel: Control shRNA; middle panel: CLASP1 shRNA; right panel: CLASP2 shRNA. FA disassembly underneath the advancing cell body is disrupted in CLASP-depleted cells. Images were acquired every 3 min. The video plays at 15 frames s−1 and is thus accelerated 2700 times. (MOV 8312 kb)

Focal adhesion-associated CLASP clusters are microtubule independent.

Spinning disk confocal microscopy time lapse sequence of a HaCaT epithelial cell expressing EGFP–CLASP2 (black) and paxillin–mCherry (magenta). 3.3 μM nocodazole was added after 20 min, when EGFP–CLASP2-labeled growing microtubule plus ends abruptly disappear. Images were acquired every 2 min. The video plays at 15 frames s−1 and is thus accelerated 1800 times. (MOV 12138 kb)

Focal adhesion-associated EGFP–CLASP2 dynamics after nocodazole washout.

Spinning disk confocal microscopy time lapse sequence of a HaCaT epithelial cell expressing EGFP–CLASP2 (black) and paxillin–mCherry (magenta). 3.3 μM nocodazole was washed out at the beginning of the time lapse sequence. Re-growing microtubules associate with CLASP clusters prior to FA disassembly. Note that other FAs that did not have associated CLASP clusters do not disassemble. Images were acquired every 30 s. The video plays at 15 frames s−1 and is thus accelerated 450 times. (MOV 1742 kb)

CLASP clusters depend on focal adhesions.

Spinning disk confocal microscopy time lapse sequence of a HaCaT epithelial cell expressing EGFP–CLASP2 (black) and paxillin–mCherry (magenta). 10 M Y-27632 was added after 20 min, which induces FA and subsequent EGFP–CLASP2 cluster disassembly. Images were acquired every 2 min. The video plays at 15 frames s−1 and is thus accelerated 1800 times. (MOV 9486 kb)

EGFP–LL5β and paxillin–mCherry dynamics in a migrating HaCaT cell.

Spinning disk confocal microscopy time lapse sequence of a migrating HaCaT epithelial cell at the edge of a cell monolayer expressing EGFP–LL5β (black) and paxillin–mCherry (magenta). Similar to CLASPs, EGFP–LL5β punctae surround FAs before FA disassembly. Images were acquired every 3 min. The video plays at 15 frames s−1 and is thus accelerated 2700 times. (MOV 4223 kb)

Focal adhesion-associated matrix degradation in a spreading HaCaT cell.

Spinning disk confocal microscopy time lapse sequence of a paxillin–mCherry (magenta) expressing HaCaT epithelial cell spreading on Alexa 488-labeled gelatin. Images were acquired every 5 min. The video plays at 10 frames s−1 and is thus accelerated 3000 times. (MOV 4920 kb)

MT1–MMP–EGFP vesicle dynamics near focal adhesions.

Total internal reflection microscopy time lapse sequence of HaCaT epithelial cells expressing MT1–MMP–EGFP (black) and paxillin–mCherry (magenta) after initial photobleaching of membrane-bound signal. The same three fusion events as shown in Fig. 6c are highlighted by coloured squares. Images were acquired every 0.3 s. The video plays at 24 frames s−1 and is thus accelerated 7 times. (MOV 6307 kb)

EGFP–Rab6A vesicle dynamics in a migrating HaCaT cell.

Total internal reflection microscopy time lapse sequence of HaCaT epithelial cells expressing EGFP–Rab6A (black) and paxillin–mCherry (magenta). A number of closely FA-associated membrane fusion events are highlighted in the magnified region on the right. Images were acquired every second. The video plays at 30 frames s−1 and is thus accelerated 30 times. (MOV 20372 kb)

EGFP–Rab6A vesicle dynamics in CLASP-depleted cells.

Total internal reflection microscopy time lapse sequence of HaCaT epithelial cells expressing EGFP–Rab6A (black) and paxillin–mCherry (magenta). Left panel: CLASP1 shRNA; right panel: CLASP2 shRNA. Images were acquired every second. The video plays at 30 frames s−1 and is thus accelerated 30 times. (MOV 18926 kb)

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Stehbens, S., Paszek, M., Pemble, H. et al. CLASPs link focal-adhesion-associated microtubule capture to localized exocytosis and adhesion site turnover. Nat Cell Biol 16, 558–570 (2014). https://doi.org/10.1038/ncb2975

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