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Fig. 4. Comparison of the behaviour of wild-type GFP-Kif5c and GFP-Kif5cmut in COS7 cells. (A) GFP-Kif5cmut in a live cell is bound to MTs, shows very little cytosolic localisation and is not visible as puncta at the periphery. This is in contrast to wild-type GFP-Kif5c (see Fig. 1). (B) Fixing and staining transfected cells for tyrosinated tubulin shows that GFP-Kif5c is localised to a subset of MTs, and MT organisation in the GFP-Kif5c-expressing cell (white arrowhead) is similar to that of the adjacent untransfected cell. GFP-Kif5cmut localised to MTs and has little effect on MT organisation (Scale bars, 10 µm). (C) Series of time-lapse images before and after photobleaching, showing significant FRAP in cells expressing GFP-Kif5c but very little recovery in cells expressing GFP-Kif5cmut. Circles in the pre-bleach image indicate bleached regions (see also supplementary material Movie 7). Arrows indicate the re-appearance of the GFP-Kif5c puncta after photobleaching. Scale bars, 2 µm. (D) Plots of FRAP values for bleached regions towards the cell edge (11 regions from nine cells for GFP-Kif5c, and seven regions from six cells for GFP-Kif5cmut) and for bleached regions at the cell centre (seven regions from seven cells for GFP-Kif5c, and nine regions from five cells for GFP-Kif5cmut). These data confirm that recovery is much slower for the mutant.

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