Figure S3.

Paclitaxel alters the MT lattice preference of WDR62, and katanin disassembles WDR62-decorated curved MT lattices.(A) TIRF microscopy images of live MRC5 cells expressing WDR62 WD-GFP together with DCX-mCherry before (top panels) or after (bottom panels) addition of 1 µM paclitaxel. (B) Quantification of the fluorescence intensities of WDR62 WD-GFP on straight and curved segments of dynamic MTs without or with paclitaxel treatment shown in A. The values were normalized to the intensity of curved segments without paclitaxel treatment. n = 30 MTs from three experiments for all conditions. (C and D) Images and quantification of the binding of WDR62-GFP (15 nM) to the GMPCPP- or paclitaxel-stabilized MTs in vitro. The values were normalized to the intensity of GMPCPP-stabilized MTs. From left to right, n = 196 and 163 MTs from three experiments. (E) Model of the recognition of extended MT lattice by WDR62. In the absence of paclitaxel, the outside protofilaments of the curved segments are in a more extended state compared with the rest of the MT lattices, explaining the preferential binding of WDR62 (green cartoon) to curved MTs both in cells and in vitro. In the presence of paclitaxel, except the inside curvature, which was occupied by DCX (red cartoon), the remainder of the MT lattices are all in an extended state, explaining why paclitaxel could change the MT geometry preference of WDR62. (F and G) Time-lapse images of live MRC5 cells expressing WDR62 N2-GFP together with DCX-mCherry. F represents the more frequently observed phenomenon (∼90%) that no breakage occurred (indicated with arrows) in WDR62 N2-GFP–decorated curved MTs, while G represents the less frequently observed phenomenon (∼10%) in which breakage occurred (indicated with arrows). (H and I) Time-lapse images of live MRC5 cells expressing p60/TagBFP-p80 R615A mutant or p60 E309Q/TagBFP-p80 mutant together with WDR62 N2-GFP and DCX-mCherry. Arrows indicate that p60/TagBFP-p80 R615A (H), but not p60 E309Q/TagBFP-p80 (I), could disassemble curved MT lattices, although both katanin mutants were recruited there by WDR62 N2-GFP. DCX-mCherry was used to distinguish between straight and curved MT lattices. Scale bars, 2 µm. Data represent mean ± SD. **, P < 0.01; ***, P < 0.001; two-tailed t test.