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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.012666

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Paxillin-dependent stimulation of microtubule catastrophes at focal adhesion sites

¹ Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN, USA
² University of Innsbruck, Innsbruck, Austria
³ Erasmus University, Rotterdam, The Netherlands
⁴ SUNY Upstate Medical University, Syracuse, NY, USA
⁵ IMBA, Institute of Molecular Biotechnology, Vienna, Austria

View larger version (72K): [in this window] [in a new window]   Fig. 1. Microtubule catastrophes at focal adhesions (FA) are specific events. (A,B) Kymographs of microtubule dynamics at focal adhesions (A) and in adhesion-free cytoplasm (B). Upper panels show 3xGFP-EMTB (green), mCherry-paxillin (red) and microtubule life-history plot (white line); catastrophes at adhesions (arrows) and a catastrophe in an adhesion-free area (arrowhead) are indicated. Lower panels show microtubule images only. Microtubule shrinkage of 0.5 µm or more is considered to be a catastrophe. (C) Frame from a TIRF video sequence of fish fibroblasts co-transfected with 3xGFP-EMTB (green) to visualize microtubules and mCherry-paxillin (red) to mark focal adhesions. Bar, 10 µm. The boxed region is presented in the kymograph in A. (D) Microtubule catastrophe distribution in the ventral cell layer. A total of 292 catastrophes were quantified in five 3xGFP-EMTB- and mCherry-paxillin-co-transfected cells within 12 minutes. (E) The average microtubule elongation per catastrophe is reduced by a factor of seven at focal adhesions (blue, 0.71 µm/catastrophe) compared with elsewhere (red, 4.91 µm/catastrophe). A total of 24 microtubules were quantified in five 3xGFP-EMTB- and mCherry-paxillin-co-transfected cells within 12 minutes. Error bars are ± s.d.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Microtubule tip dynamics at focal adhesions. (A) Kymograph of a microtubule catastrophe event at a focal adhesion (FA). The microtubule is marked with mCherry-EB3 (red), and the focal adhesion is marked with GFP-paxillin (green). (B) Kymograph of a microtubule tip (mCherry-EB3, red) that does not undergo catastrophe at the adhesion (GFP-paxillin, green). The microtubule tip changes its growth dynamics at the focal adhesion. (C) An example of assigned area zones for microtubule growth paths near focal adhesions (GFP-paxillin, green). Cytoplasmic zones close to the cell center are encoded as zones `–2' and `–1'. The zone adjacent to a focal adhesion is encoded as zone `0'. A focal adhesion contains zones `1-3', according to its length. Cytoplasmic zones towards the cell periphery are encoded as zones `1out' and `2out'. Each zone is 1 µm in size. (D) The distribution between fast (growth rate >0.1 µm/second) and slow (growth rate <0.1 µm/second) microtubules for each zone. In the cytoplasm (yellow background), 70% of microtubules grow at a speed of 0.1 µm/second or faster. At focal adhesions (green background), the percentage of fast microtubules reduces down to 50%. (E) Dependence of the microtubule catastrophe ratio (percentage of approaching microtubules undergoing catastrophe) on the time a microtubule spends in the cytoplasm zone (zone –1, green), at the adhesion base (zone 0, blue) and in the adhesion (zone 1, red). (F) Total microtubule catastrophe ratio for each zone. The percentage of approaching microtubules undergoing catastrophe increases from 2% in the cytoplasm to 25% at focal adhesions. A total of 139 microtubules were quantified in three 3xGFP-EMTB- and mCherry-paxillin-co-transfected cells within 15 minutes. Bar, 1 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Microtubule catastrophes do not depend on the maturation stage of focal adhesions. (A,B) A frame from a video sequence of a cell co-transfected with mCherry-paxillin (A, red) and Cerulean-zyxin (B, cyan). Paxillin marks both early (hollow arrows) and late (filled arrows) focal adhesions. Zyxin is a marker for late focal adhesions only (filled arrows). Bar, 5 µm. (C) Microtubule catastrophe ratio at early versus late adhesions. Approximately 90% of approaching microtubules undergo catastrophe both at early and late adhesions. A total of 117 catastrophe events in four cells were quantified.

View larger version (82K): [in this window] [in a new window]   Fig. 4. Catastrophes can occur in the zyxin-free zone of focal adhesions. (A) A frame from a video sequence of a cell co-transfected with mCherry-paxillin (red) and GFP-zyxin (green). Bar 10 µm. (B,C) Enlargement of areas at the leading edge (B) and trailing edge (C) of the cell (paxillin, red; zyxin, green). The intensity profiles of paxillin (red lines) and zyxin (green lines) along a line (white) of width one pixel are shown. Paxillin extends more distantly towards the cell edge. The image is a representative example from 15 cells. (D) A frame from a video sequence of a cell co-transfected with GFP-EMTB (blue) to visualize microtubules, Cerulean-zyxin (green) to mark late adhesions and mCherry-paxillin (red) to mark both early and late adhesions. Bar, 10 µm. The boxed area is enlarged to the right (E,F). (E) Enlarged frame sequence of microtubules (green) and paxillin (pink). (F) Enlarged frame sequence of microtubules (green) and zyxin (red). A catastrophe occurred at the 20-second time-point at the distal zyxin-free end of a focal adhesion. Time, seconds. Arrows show the direction of microtubule movement. (G) Enlarged frame sequence of the focal adhesion from E and F, showing zyxin (green) and paxillin (red). The arrows point at the zyxin-free distal end of the focal adhesion.

View larger version (52K): [in this window] [in a new window]   Fig. 5. LIM2-LIM3 displaces full-length GFP-paxillin from focal adhesions. Cells microinjected with GST-LIM2 (A,B) and GST-LIM2-LIM3 (C,D) proteins were stained with antibodies against GST (A,C) and vinculin (B,D). Only GST-LIM2-LIM3 (C,D) localizes to focal adhesions. Bar 10 µm. Cells expressing GFP-paxillin (E,G) were microinjected with either GST-LIM2-LIM3 (F) or GST only (H). By 45 minutes after injection of GST-LIM2-LIM3, paxillin is displaced from existing focal adhesions (F, arrows) and does not appear in new adhesion sites. Microinjection of just GST changed neither the intensity level of paxillin in old adhesions (H, arrows) nor the ability of paxillin to incorporate into new adhesion sites (H, arrowheads). Bar, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. GST-LIM2-LIM3 inhibits microtubule catastrophe at focal adhesions. (A,B) Cell expressing GFP–β-tubulin (green) and mRFP-zyxin (red) before (A) and after (B) microinjection of GST-LIM2-LIM3 protein. After injection, the microtubule density increases as a result of catastrophe suppression at adhesion sites. Bar 10 µm. (C,D) Kymographs of microtubule dynamics at focal adhesions. A microtubule undergoes catastrophe at an adhesion before injection (C) but grows through adhesions after injection (D, arrows). (E,F) Examples of microtubule life-history plots in cells before (E) and 30 minutes after (F) injecting the GST-LIM2-LIM3 construct. The black line indicates the tracking position of individual microtubule tips in relation to focal adhesions (FA) marked with red, green and yellow lines. Note multiple catastrophes at adhesions in (E), whereas microtubules grow through several adhesions without catastrophe in (F). (G) Frequency of catastrophes per square micron before and after microinjection of GST-LIM2-LIM3 protein. Upon injection, the frequency of catastrophes reduces in focal adhesions (upper bracket) but stays the same in the cytoplasm (lower bracket). Data are shown for 271 catastrophes in five individual cells recorded both before injection and 30 minutes after injection. (H) The difference in microtubule catastrophe before (blue boxes) and after (red boxes) injection of GST-LIM2-LIM3 shown in a `box-and-whisker' plot. The P value was calculated by a t test (Microsoft Excel). The reduced frequency of catastrophes at focal adhesions after injection of GST-LIM2-LIM3 was statistically significant (P<0.001). At the same time, the frequency of catastrophes in the cytoplasm did not change (P=0.24).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Model of paxillin involvement in the induction of microtubule catastrophe at adhesion sites. In control cells: (1) Paxillin (light blue) binds to adhesion sites (blue) through LIM2-LIM3 domains. Catastrophe factor (`Cat F', red box) binds to paxillin through sites other than LIM2-LIM3 domains and in this way is enriched at adhesion sites. (2A) When a microtubule (green) approaches a focal adhesion, the catastrophe factor associated with paxillin induces microtubule catastrophe (red star) and depolymerization (green arrow). Alternatively (2B), when a microtubule (green) approaches a focal adhesion, the catastrophe factor associated with paxillin activates microtubule-associated catastrophe-inducing factor (`CIF', magenta box) to induce microtubule catastrophe (magenta star) and depolymerization (green arrow). In LIM2-LIM3-injected cells (3), exogenous LIM2-LIM3 (light blue) binds to the adhesion site and replaces full-length paxillin. The catastrophe factor cannot bind to the LIM2-LIM3 mutant protein and is excluded from adhesion sites. (4) When a microtubule approaches the focal adhesion, it does not undergo catastrophe and continues to polymerize (green arrow) because catastrophe factor is absent or not activated at the adhesion site.

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