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Files in this Data Supplement:
Fig. S1. Mechanical obstacles do not necessarily cause microtubule catastrophe. (A) Successive video frames of a cell transfected with GFP−β-tubulin (green) and Cherry-EB3 (red). Microtubule tip movement is blocked by an obstacle in the cell; however, the microtubule continues to polymerize, as indicated by the presence of EB3 (arrowhead) at the tip. This process causes microtubule bending (arrow) but not catastrophe. Bar 1 µm. (B,C) Successive video frames of a cell transfected with GFP-EMTB to mark microtubules (cyan), Cerulean-EB3 (red) and Cherry-paxillin (green). Examples of when a focal adhesion (B) or another microtubule (C) serves as a mechanical obstacle preventing the movement of polymerizing microtubule tips (red, arrowheads). Bar 5µm. Arrows show microtubule bending. Time, seconds. (D) Kymograph illustrating microtubule bending at a focal adhesion. The microtubule was photobleached behind the polymerizing plus-end to create a reference point (hollow arrowheads) for measurements of microtubule length. When the plus-end reached a focal adhesion (time points 20, 25, 30 seconds), it stopped proceeding forwards (arrowheads) but continued to polymerize, as indicated by the increased length (indicated in micrometres, shown above) and microtubule bending (arrows). Later, the microtubule proceeds forwards without catastrophe (time-points 35 seconds and beyond). Left panel, microtubule (green) and focal adhesion (red) at the 15-second time-point.
Fig. S2. Injection of LIM2-LIM3 paxillin domains results in an increase in the number of growing microtubules. (A,B) Selected video-frames of a cell expressing GFP-EB1 before (A) and after (B) the injection. This is an inverted image. Time, minutes, bar, 5 µm. (C) Velocity of GFP-EB1-marked microtubule tips before injection of LIM2-LIM3 and 8 minutes after the injection. Ten microtubules per cell from eight cells were analyzed. (D) The number of GFP-EB1-marked microtubule tips per cell before injection of LIM2-LIM3 and 8 minutes after the injection. Note the increase in number of growing microtubule tips. 25 cells were analyzed.
Movie 1. A microtubule labelled with 3xGFP-EMTB undergoes multiple catastrophes at a focal adhesion marked with mCherry-paxillin (chevron). Time-lapse movie of duration 4 minutes 30 seconds.
Movie 2. Peripheral area of a fish fibroblast coexpressing GFP−β-tubulin and GFP-zyxin (inverted image) before (left) and after (right) microinjection of paxillin LIM2-LIM3 domains. Microtubules undergo repetitive catastrophes at an adhesion in controls but grow through adhesions in the presence of LIM2-LIM3 (arrows). Time-lapse movie of duration 8 minutes 20 seconds.
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