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First published online 23 August 2005
doi: 10.1242/jcs.02531

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Quantification of microtubule nucleation, growth and dynamics in wound-edge cells

Department of Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003, USA

View larger version (140K): [in a new window]   Fig. 1. Microtubule organization is asymmetric in wound-edge cells. (A,B) Maximum intensity projections showing the distribution of microtubules in wound-edge LLCPK1 (A) and CHO (B) cells fixed and stained with antibodies to tubulin. (C) CHO and (D) LLCPK1 cells expressing GFP-tubulin shows the asymmetric distribution of microtubules. (E,F) Microtubule distribution in wound-edge CHO cells expressing a constitutively active form of Cdc42 (L61Cdc42) (E, right cell, marked with asterisk) and uninjected control cells (E, left cell) and expressing dominant negative N19 RhoA (F, injected cell marked with asterisk). Microtubule distribution is asymmetric in control and N19 RhoA-expressing cells but not in L61 Cdc42-expressing cells. Scale bars: 10 µm.

View larger version (136K): [in a new window]   Fig. 2. Microtubule nucleation at the centrosome is not biased towards the wound edge. Confocal images show non-wound-edge controls (A,E) and wound-edge cells (B,F). (A-D) CHO fibroblasts and (E-H) LLCPK1 epithelial cells expressing EB1-GFP to mark the plus ends of growing microtubules; not all cells in the culture express the construct, and these cells cannot be detected in the fluorescence micrographs. The asterisk (*) marks the position from which the growing plus ends originate. Arrows indicate direction of migration into the wound. The boxed region from individual frames of the corresponding movie sequences are shown in C, D, G and H. Movies of the cells shown are in supplementary material Movies 1 and 2. Time is indicated in minutes and seconds in the upper right corner. Scale bars: 20 µm (A,B), 10 µm (E F) and 5 µm (C,D,G,H).

View larger version (78K): [in a new window]   Fig. 3. Microtubule growth events are uniform at the cell periphery. Confocal micrographs of a CHO fibroblast (A) and an LLCPK1 epithelial cell (B) expressing EB1-GFP. The average number of growth events per unit area is not statistically different at the leading and trailing edges of either CHO or LLCPK1 cells. Scale bars: 5 µm.

View larger version (42K): [in a new window]   Fig. 4. Microtubule dynamic instability differs in leading and trailing edges of CHO and LLCPK1 cells. Solid bars, leading edges; hatched bars, trailing edges. (A) Dynamicity, (B), catastrophe frequency, (C) pause duration, (D) Percentage time in pause. Microtubules in trailing edges have more catastrophes and greater overall dynamicity (A,B); microtubules in leading edges spend more time in pause and have longer pause durations (C,D). Error bars show standard error of the mean.

View larger version (29K): [in a new window]   Fig. 5. RhoA regulates microtubule dynamics in trailing edges. Solid bars, control cells, hatched bars, C3-injected cells; Front, leading edges; Back, trailing edges. (A) growth rate, (B) shortening rate, (C) growth distance, (D) shortening distance, (E) dynamicity and (F) pause duration.

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