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First published online 11 March 2008
doi: 10.1242/jcs.026492

Journal of Cell Science 121, 1085-1095 (2008)
Published by The Company of Biologists 2008
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Differential trafficking of Kif5c on tyrosinated and detyrosinated microtubules in live cells

Sarah Dunn1, Ewan E. Morrison2, Tanniemola B. Liverpool3,*, Carmen Molina-París3, Robert A. Cross4, Maria C. Alonso4 and Michelle Peckham1,{ddagger}

1 Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK
2 CRUK Clinical Centre at Leeds, Leeds Institute of Molecular Medicine, St James University Hospital, Leeds, LS9 7TF, UK
3 Applied Mathematics (School of Mathematics) University of Leeds, Leeds, LS2 9JT, UK
4 Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK


Figure 1
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  		Fig. 1. (A-D) GFP-Kif5c localisation in living cells. Inverted fluorescence images are shown for COS-7 cells (A,B,D) and a neuronal cell (C) expressing GFP-Kif5c (supplementary material Movies 1-4). GFP-Kif5c shows a diffuse localisation but is excluded from organelles that show up as white in these inverted fluorescent images (black arrowheads in A and B). Some GFP-Kif5c is seen as puncta (grey arrows in B and C) along faintly labelled MTs, and in peripheral accumulations (black arrows in A and D). Curved MTs are also faintly labelled by GFP-Kif5c (to the left of the asterisk in B).

 

Figure 2
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  		Fig. 2. (A-D) GFP-Kif5c behaviour in living cells. (A,C) Two different, cropped still images from a time-lapse recording of GFP-Kif5c puncta (green) moving along a MT labelled with mCherry–{alpha}-tubulin (red) in a live cell. The GFP-Kif5c puncta move from right to left. Scale bar, 2 µm (see also supplementary material Movies 5 and 6). (B,D) Graphs showing the respective velocity of the GFP-Kif5c puncta seen in A and C over time, demonstrates the rapid changes in velocity of those puncta.

 

Figure 3
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  		Fig. 3. Analysis of GFP-Kif5c dynamics in living cells. (A) Average velocity of GFP-Kif5c puncta, including stationary phases. (B) Run lengths of individual GFP-Kif5c puncta. Results for COS-7 and neuronal cells are similar.

 

Figure 4
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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.

 

Figure 5
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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–{alpha}-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).

 

Figure 6
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  		Fig. 6. GFP-Kif5c preferentially associates with the detyrosinated-MT subpopulation in COS-7 cells. (A) Triple immunostaining shows that COS-7 cells contain detyrosinated, acetylated and tyrosinated tubulin. Microtubules can contain all three types of tubulin (see the MT in the boxed region). Other MTs stain predominantly for either tyrosinated (blue), acetylated (green) or detyrosinated (red) tubulin (merged image). The double arrow is shown parallel to a MT that is both detyrosinated and acetylated, but has very little tyrosinated tubulin. Arrows show an MT that is predominantly detyrosinated. Scale bar, 5 µm. (B) COS-7 cells transfected with GFP-Kif5c, and fixed and co-stained with antibodies specific for Kif5c (green) and tyrosinated, detyrosinated or acetylated tubulin (red). Box 1: MT decorated with GFP-Kif5c, showing very little staining for tyrosinated tubulin or acetylated tubulin. Boxes 2 and 6: MTs decorated with GFP-Kif5c and tyrosinated or acetylated tubulin, respectively. Boxes 3 and 4: MTs decorated with GFP-Kif5c and are stained for detyrosinated tubulin. Box 5: acetylated MT showing low levels of decoration by GFP-Kif5c. Scale bar, 5 µm. (C) 30 cells were used for the analysis. Ten were co-stained for GFP-Kif5c and tyrosinated tubulin, ten for GFP-Kif5c and detyrosinated tubulin, and ten for GFP-Kif5c and acetylated tubulin. Ten MTs were then randomly selected from each cell and scored for the level of GFP-Kif5c decoration along the length of MT visible at <10% (light grey), 10-50% (mid grey) or >50% decoration (dark grey) and are shown in the graph. The data shows that detyrosinated MTs are most likely to be decorated by GFP-Kif5c at high densities (greater than 50%).

 

Figure 7
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  		Fig. 7. In vitro analysis of kinesin-1 interactions with post-translationally modified MTs. (A) Dot-blot assays for different post-translational modifications of tubulin in the three types of MT used in the in vitro MT-binding and -motility assays. A range of tubulin concentrations (500-10 ng) were probed with antibodies that specifically recognise tyrosinated, detyrosinated or acetylated tubulin. Microtubules purified from pig brain were extensively modified. Microtubules purified from untreated HeLa cells [HeLa (–CPA)] were predominantly unmodified (composed of at least 95% tyrosinated tubulin). Carboxypeptidase A (CPA) treatment of HeLa cells [HeLa (+CPA)] resulted in at least 95% of tubulin being detyrosinated. (B) Tubulin from pig brain, untreated HeLa cells (–CPA) and treated HeLa cells (+CPA) resolved on SDS gel and stained with Coomassie-Blue. The two bands corresponding to {alpha}- and β-tubulin can be seen in the modified and unmodified tubulin of HeLa cells. (C) Enhanced DIC images from in vitro motility assays for tubulin from pig brain, untreated HeLa cells (–CPA) and treated HeLa cells (+CPA). There is no discernable difference between the MTs. (D) Stills from an in vitro motility assay using unmodified tubulin from HeLa cell. Time (in seconds) is indicated in the top left hand corner. Black arrows indicate the front of the MT, asterisks show the starting position of the MT. Scale bar, 5 mm. (E) The velocity distribution of the Kif5c motor moving on the three types of MT in an in vitro motility assay. Bin sizes for the velocities are 0.02 µm second–1. Average velocities for the three types of MT were 0.55 µm second–1 (±0.05, mean ± s.d., n=207), 0.66 µm second–1 (±0.06, n=103) and 0.58 µm second–1 (±0.07, n=206) for MTs from pig brain, tyrosinated and detyrosinated MTs, respectively. Dashed curves show the calculated normal distribution for each dataset.

 

Figure 8
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  		Fig. 8. Model to investigate how detyrosination affects kinesin-1 behaviour on MTs. (A) Basis for the model. Circles represent binding sites along MTs, n=0 corresponds to the minus-end of the MT, and n=N is the plus-end. Kinesin-1 can bind to an MT with an on-rate of kon, detach with an off-rate of koff, and can either move towards the plus-end (taking unit steps) at a rate of k+ or towards the minus-end at a rate of k–. For details of the model see Materials and Methods. (B) Results obtained when using the model. Stationary number density distribution of kinesin-1 was obtained using equations 10, 11, 12; average number density, average flux and average velocity as a function of tyrosinated tubulin, were obtained using equations 13, 14, 15.

 




© The Company of Biologists Ltd 2008