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Fig. 5. GFP-Kif5c associates with a drug-stable subset of MTs. (A) Frames from a time-lapse sequence with addition of the MT depolymerising drug nocodazole at 2 minutes. Note the resistance of GFP-Kif5c-decorated MTs to the drug even 40 minutes after its addition. Scale bar, 10 µm. (B) Detail from a time-lapse sequence beginning 70 minutes after nocodazole addition. Arrows indicate GFP-Kif5c puncta translocating along nocodazole-resistant MTs (supplementary material Movie 8). Scale bar, 5 µm. (C) Frames from a time-lapse sequence (addition of nocodazole at 0 minutes) showing a cell coexpressing mCherry–
-tubulin (red) and GFP-Kif5c (green). Microtubules not labelled with GFP-Kif5c (arrows) disappear within 10 minutes, whereas a GFP-Kif5c-labelled MT (boxed region) is resistant to nocodazole and can be followed through the sequence. This MT gradually accumulates GFP-Kif5c with time, as the remaining MTs depolymerise (supplementary material Movie 9). (D) Nocodazole-resistant MTs are modified and preferentially decorated by Kif5c. Untransfected cells treated with nocodazole for 60 minutes, and then fixed and stained for tyrosinated, detyrosinated and acetylated MTs, show that the few remaining MTs are acetylated and detyrosinated, and stain only weakly for tyrosinated tubulin (boxed region). Cells transfected with GFP-Kif5c and stained for tyrosinated and either detyrosinated or acetylated tubulin show that GFP-Kif5c decorates these stable MTs (boxed regions).
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