Research Article 1085 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,‡ 1Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK 2CRUK Clinical Centre at Leeds, Leeds Institute of Molecular Medicine, St James University Hospital, Leeds, LS9 7TF, UK 3Applied Mathematics (School of Mathematics) University of Leeds, Leeds, LS2 9JT, UK 4Molecular Motors Group, Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK *Present address: Department of Mathematics, University of Bristol, University Walk, Bristol, BS8 1TW, UK ‡Author for correspondence (e-mail: [email protected])

Accepted 11 December 2007 J. Cell Sci. 121, 1085-1095 Published by The Company of Biologists 2008 doi:10.1242/jcs.026492

Summary Kinesin-1 is a molecular transporter that trafficks along showed that the motor domain of Kif5c moved detyrosinated microtubules. There is some evidence that kinesin-1 targets microtubules at significantly lower velocities than tyrosinated specific cellular sites, but it is unclear how this spatial regulation (unmodified) microtubules. Mathematical modelling predicted is achieved. To investigate this process, we used a combination that a small increase in detyrosination would bias kinesin-1 of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of occupancy towards detyrosinated microtubules. These data kinesin-1) fused to GFP, in vitro analyses and mathematical suggest that kinesin-1 preferentially binds to and trafficks on modelling. GFP-Kif5c fluorescent puncta localised to a subset detyrosinated microtubules in vivo, providing a potential basis of microtubules in live cells. These puncta moved at speeds of for the spatial targeting of kinesin-1-based cargo transport. up to 1 m second–1 and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant Supplementary material available online at GFP-Kif5c bound to microtubules but did not move along them. http://jcs.biologists.org/cgi/content/full/121/7/1085/DC1 Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily Key words: Kinesin-1, Microtubules, Detyrosinated microtubules, composed of detyrosinated tubulin. In vitro motility assays Trafficking Journal of Cell Science Introduction Kinesin-1 is a processive motor. In vitro, it moves along MTs The kinesin superfamily is a structurally diverse group of ATP- and for several m, at a maximum velocity of ~0.8 m second–1 (Coy microtubule (MT)-dependent motors (Lawrence et al., 2004). et al., 1999b; Svoboda et al., 1993). Less is known about the motile Kinesin-1 (conventional kinesin) (Brady, 1985; Vale et al., 1985a) behaviour of kinesin-1 in vivo. Most of our knowledge has been moves towards the plus ends of MTs in vitro (Vale et al., 1985b). obtained indirectly, through the observation of putative cargoes. It is a tetramer consisting of two heavy chains and two light chains. Velocity measurements from those experiments ranged from 0.034 The globular N-terminal domain of the heavy chain contains the m second–1 for RNA transport granules (Kanai et al., 2004) to motor, which interacts with MTs in an ATP-dependent manner. In 0.33 m second–1 for dense-core vesicles in clonal -cells (Varadi the absence of MTs, ADP release is rate limiting (Hackney, 1988) et al., 2002). A recent study used a modified form of total internal and this release step is accelerated by the interaction of kinesin-1 reflection fluorescence microscopy to measure the dynamic with MTs. behaviour of a motor domain construct in live cells, and found that Folding of kinesin-1 regulates its activity (Hackney et al., 1992). average speeds were 0.78 m second–1 (Cai et al., 2007). The In the folded state, the tail of kinesin-1 binds to the head and ATPase processive nature of kinesin-1 enables it to function as a trafficker activity is low. Binding of kinesin-1 to cargo unfolds the molecule, in cells, transporting vesicles or other cargo over long distances. and allows it to bind to MTs and move along them in an ATP- It is still unclear how the cargo trafficking by kinesin-1 is spatially dependent manner (Coy et al., 1999a; Friedman and Vale, 1999). regulated in vivo. As it is a plus-end-directed MT motor, and the A 65 amino-acid-long region at the end of the C-terminal tail is plus ends of MTs are oriented towards the cell periphery, kinesin- responsible for this cargo binding (Coy et al., 1999a). Kinesin-1 1 will broadly direct trafficking towards the plasma membrane. heavy chain can bind to its cargo either directly through adaptor However, kinesin-1 can transport cargoes as diverse as membrane- , or indirectly through the light chains associated with this and signalling-molecules to specific intracellular regions, and this region of the (for reviews, see Hirokawa and Takemura, is likely to be crucial in processes such as cell migration and 2005; Schnapp, 2003; Vale, 2003). The amount of kinesin-1 neuronal differentiation. One possibility is that post-translational expressed in cells varies from 0.1-1 M in tissues (Hollenbeck, modifications of tubulin are important in directing trafficking to 1989), but it is mostly found in a cytoplasmic pool where it is likely specific regions at the cell periphery (reviewed in Lakamper and to be in a folded inactive form. A smaller amount of kinesin-1 is Meyhofer, 2006). Microtubules can be post-translationally modified bound to organelles. in a variety of ways, including through acetylation of -tubulin at 1086 Journal of Cell Science 121 (7)

lysine at position 40, and through detyrosination by removing the C-terminal tyrosine residue of -tubulin, which reveals a charged glutamate on the so-called ‘E-hook’ of -tubulin (reviewed in Luduena et al., 1992). A recent study has shown that the localisation of JNK-interacting protein 1 (JIP1), a protein trafficked by kinesin- 1, depends on MT acetylation (Reed et al., 2006). Trafficking of proteins by kinesin-1 has been shown to depend on detyrosination (Gyoeva and Gelfand, 1991; Gurland and Gundersen, 1995). Here, we have investigated the movement of kinesin-1 in live cells using a construct in which enhanced green fluorescent protein (eGFP) was fused to the N-terminal of the kinesin-1 isoform Kif5c (GFP-Kif5c). We then determined whether movement of GFP-Kif5c depends on post-translational modification of tubulin. Vertebrates contain three conventional kinesin-1 : Kif5a, Kif5b and Kif5c. Whereas Kif5b is ubiquitously expressed, Kif5a and Kif5c are enriched in neurons. The three proteins are highly homologous and functionally redundant (Kanai et al., 2000). We investigated the properties of GFP-Kif5c in neuronal and non-neuronal cells, and confirmed that it moves at a speed similar in vivo to that measured Fig. 1. (A-D) GFP-Kif5c localisation in living cells. Inverted fluorescence for the same motor in vitro. Moreover, we found that GFP-Kif5c 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 preferentially moves along a subset of MTs, these MTs are post- localisation but is excluded from organelles that show up as white in these translationally modified by detyrosination, and detyrosination inverted fluorescent images (black arrowheads in A and B). Some GFP-Kif5c affects velocity in vitro. Our results suggest that the spatial control is seen as puncta (grey arrows in B and C) along faintly labelled MTs, and in of kinesin-1-based trafficking can be effected by the tyrosination- peripheral accumulations (black arrows in A and D). Curved MTs are also detyrosination cycle of MTs. faintly labelled by GFP-Kif5c (to the left of the asterisk in B).

Results observe any movement of the puncta (data not shown). However, GFP-Kif5c distribution in living cells in cells expressing low levels of GFP-Kif5c, only a subset of MTs GFP-Kif5c was transfected into mammalian cells and its distribution was decorated with GFP-Kif5c and it was at the ends of these MTs examined using live cell imaging. The N-terminal GFP tag was not that clusters of GFP-Kif5c puncta were localised. It seems expected to interfere with the interaction of the motor with its normal reasonable to assume that these puncta represent GFP-Kif5c binding partners through the C-terminal tail (Coy et al., 1999a). associated with a variety of cargo within the transfected cells. The Two main cell types were used; COS-7 fibroblasts and, because size of these puncta varied. Judging from the size and the intensity

Journal of Cell Science Kif5c was originally identified as a neuronal motor, the neuronal of the puncta, the majority are likely to contain more than one cell line Hdh+/+. Only cells that expressed very low levels of GFP- molecule of GFP-Kif5c. Kif5c were used in this study to minimise any potential artifacts Co-imaging of GFP-Kif5c and fluorescently labelled MTs caused by expression of GFP-Kif5c at high levels. The morphology confirmed (by direct observation) that GFP-Kif5c moved along MTs of these cells, as well as the distribution of intracellular organelles, (Fig. 2; supplementary material Movies 5 and 6). Single fluorescent appeared normal when transfected cells were compared with puncta of GFP-Kif5c could be followed through sequential frames adjacent untransfected cells after fixation and immunostaining (data (Fig. 2A,C) as they moved along a single MT that had been not shown). Organelles, which appear white in an inverted fluorescently labelled using mCherry–-tubulin (Shaner et al., fluorescence image because they exclude GFP-Kif5c, are distributed 2004). The velocity of single puncta varied with time (Fig. 2B,D), throughout the cell. We confirmed that the majority of the larger suggesting that obstacles to movement exist in vivo that affect the organelles were mitochondria by using a mitochondrion-specific velocity of Kif5c as it moves along the MT. We also found that dye (mitotracker). We did not observe any differences in some puncta stop moving for a short period of time, but remained mitochondrial dynamics between transfected and untransfected cells attached to the MT, before resuming movement. (data not shown). We determined the velocity of GFP-Kif5c movement along MTs In cells whose expression levels of GFP-Kif5c were low, we by tracking GFP-Kif5c puncta in time-lapse images of COS-7 or observed several distinct distributions of GFP-Kif5c. GFP-Kif5c neuronal (Hdh+/+) cells. To ensure accurate tracking of fast-moving localised to fluorescent puncta along MTs and clustered at the ends puncta, we used a frame capture rate of two per second. We found of these MTs in specific cortical regions of the cell (Fig. 1). We that the average velocities for individual puncta, including any also found a diffuse cytoplasmic distribution of GFP-Kif5c, which stationary phases (Fig. 3A), were similar in Hdh+/+ cells (0.32±0.13 probably represents folded inactive GFP-Kif5c not bound to MTs. m second–1, mean ± s.d., n=58 puncta) and COS-7 cells (0.29±0.13 In addition, there was often a low level of diffuse labelling by GFP- m second–1, n=73 puncta). These average velocities are similar Kif5c along the MTs, which allowed us to see their outline. This to those obtained by Varadi et al., who had imaged the movement distribution was observed in both COS-7 (Fig. 1A,B,D, of dense core vesicles by Kif5b in -cells (Varadi et al., 2002). supplementary material Movies 1-3) and neuronal cells (Fig. 1C, Excluding any stationary phases in calculating the average velocity supplementary material Movie 4). In cells that expressed high levels (i.e. velocity=0 m second–1) slightly increased the average of GFP-Kif5c, this distribution along MTs was much brighter, puncta velocities to 0.39±0.14 m second–1 for Hdh+/+ cells and to 0.34± of GFP-Kif5c were able to crosslink MTs, and we were not able to 0.11 m second–1 for COS-7. The average maximum velocities Kif5c behaviour in live cells 1087

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–-tubulin (red) in a live cell. The GFP-Kif5c puncta move from right to left. Scale bar, 2 m (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.

Fig. 3. Analysis of GFP-Kif5c dynamics in living cells. (A) Average velocity measured for each of the puncta were higher in Hdh+/+ cells of GFP-Kif5c puncta, including stationary phases. (B) Run lengths of (0.78±0.31 m second–1) than in COS-7 cells (0.62±0.24 m individual GFP-Kif5c puncta. Results for COS-7 and neuronal cells are second–1). These maximum values are close to those reported for similar. in vitro motility studies of kinesin-1, and to the recently reported velocities for movement of a fluorescently tagged motor domain of the cell was bleached (Fig. 4C, supplementary material Movie

Journal of Cell Science in COS cells (Cai et al., 2007). We also measured the run lengths 7). After bleaching, the fluorescence intensity in peripheral (distance moved per individual GFP-Kif5c fluorescent puncta) (Fig. accumulations of GFP-Kif5c recovered (Fig. 4C) with a t1/2 of 16 3B). GFP-Kif5c puncta could be tracked for up to 8 m and we seconds (n=7). FRAP was slightly faster at the periphery than in did not find any difference in run length between COS-7 and the centre of the cell (Fig. 4D). By contrast, photobleaching neuronal cells. experiments with GFP-Kif5cmut showed very little recovery of To further test whether the observed distribution of GFP-Kif5c fluorescence after photobleaching (Fig. 4C,D). These data indicate was dependent on its motor function, we compared its distribution that the distribution of GFP-Kif5c in live cells is dependent on the with that of a non-motile mutant (GFP-Kif5cmut) in living cells. motor function of this protein. GFP-Kif5cmut contains a point mutation (T93N) in the motor domain that inhibits ATP hydrolysis (Nakata and Hirokawa, 1995). GFP-Kif5c colocalises with detyrosinated MTs This mutant can bind to MTs but cannot move along them. It has As we described above, the live-cell imaging experiments showed been used previously as a ‘roadblock’, and has been shown to that GFP-Kif5c preferentially associates with a subset of MTs in decorate the MT network in vivo but not to re-organize it cells (Fig. 1). We suspected that these were stable MTs and used (Krylyshkina et al., 2002; Crevel et al., 2004). In agreement with two approaches to investigate this further. First, stable MTs tend to this earlier work, we found that GFP-Kif5cmut heavily decorates be more resistant to the MT poison nocodazole (Kreis, 1987). MTs in the central region of the cell but does not appear as puncta Therefore, we treated cells expressing GFP-Kif5c with nocodazole or accumulate in cortical clusters or in the cytoplasm (Fig. 4A). (Fig. 5) and found that GFP-Kif5c-decorated MTs persist after this Neither wild-type nor mutant GFP-Kif5c affected MT organisation treatment (Fig. 5A). GFP-Kif5c was able to move along the few (Fig. 4B). Expression of GFP-Kif5cmut also inhibited movement remaining nocodazole-resistant microtubules (Fig. 5B, of vesicles, such as mitochondria in live cells (data not shown), as supplementary material Movie 8). To further investigate this, we previously shown for this mutant (Varadi et al., 2002). performed dual imaging of GFP-Kif5c and of microtubules labelled In addition, we found that accumulations of puncta at the cell with mCherry–-tubulin during and after the addition of nocodazole periphery that were identified by visualising GFP-Kif5c were not to the culture medium (Fig. 5C, and supplementary material Movie simply static aggregates of kinesin, but exhibited dynamic behaviour 9). We found that microtubules not decorated with GFP-Kif5c before by fluorescence recovery after photobleaching (FRAP). Cells were treatment with nocodazole disappeared rapidly, whereas MTs that imaged on a laser scanning (LSM) confocal microscope with a were decorated with GFP-Kif5c persisted for longer and, eventually, heated stage, and a small area either at the periphery or in the centre began to accumulate more GFP-Kif5c (Fig. 5C). 1088 Journal of Cell Science 121 (7)

Stable MTs accumulate a number of post- translational modifications that allow them to be identified by immunostaining. These include detyrosination, which creates MTs whose C-terminal tyrosine residue has been removed from the -tubulin subunit (also known as Glu-MTs) (Gundersen et al., 1984), and acetylation (Piperno et al., 1987), in which lysine residue at position 40 is acetylated. To determine whether the nocodazole-resistant MTs decorated by GFP-Kif5c are post-translationally modified, untransfected cells and cells expressing GFP-Kif5c were fixed and stained for acetylated, detyrosinated and tyrosinated tubulin after 60 minutes of nocodazole treatment (Fig. 5D). In untransfected cells, triple-stained for all three types of tubulin, nocodazole-resistant MTs stained for both acetylated and detyrosinated tubulin, and weakly for tyrosinated tubulin. GFP-Kif5c-expressing cells were additionally stained for tyrosinated and either detyrosinated or acetylated tubulin. Nocodazole-resistant MTs were decorated by GFP-Kif5c, and stained positively for both acetylated and detyrosinated tubulin but only weakly for tyrosinated tubulin. This confirms that GFP- Kif5c binds to stable MTs in nocodazole-treated cells. We next investigated the association of GFP-Kif5c with tyrosinated, detyrosinated or acetylated MTs in cells not treated with nocodazole to confirm that GFP- Kif5c-decorated MTs tended to be stable, post- translationally modified MTs. Untransfected cells were triple-stained for tyrosinated, detyrosinated and acetylated tubulin (Fig. 6A) to determine the relative amounts of modified and unmodified MTs. An analysis of 60 randomly selected MTs from ten cells showed that 95% of the MTs were predominantly tyrosinated

Journal of Cell Science (Fig. 6A), and the remaining 5% contained modified (acetylated and/or detyrosinated) tubulin. Of the modified MTs, 10% were predominantly detyrosinated, 27% were predominantly acetylated, and the remainder was both acetylated and detyrosinated. In GFP-Kif5c-expressing cells, GFP-Kif5c associated with all three types of MT examined (Fig. 6B). However, further analysis and quantification (Fig. 6C) showed that a MT in which 50% or more of its visible length was decorated with GFP-Kif5c was much more likely to be detyrosinated (70% of MTs) or acetylated (20%) than tyrosinated (<2%). Thus, GFP- Kif5c appears to associate predominantly with the 5% of modified MTs in the cell and to show a strong bias Fig. 4. Comparison of the behaviour of wild-type GFP-Kif5c and GFP-Kif5cmut in COS7 towards the MTs that are predominantly detyrosinated. 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 MT modifications affect kinesin-1 interactions in is localised to a subset of MTs, and MT organisation in the GFP-Kif5c-expressing cell (white vitro arrowhead) is similar to that of the adjacent untransfected cell. GFP-Kif5cmut localised to To further investigate the interaction of kinesin-1 MTs and has little effect on MT organisation (Scale bars, 10 m). (C) Series of time-lapse with modified MTs, we used in vitro motility assays 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 to determine whether kinesin-1 moved modified and image indicate bleached regions (see also supplementary material Movie 7). Arrows unmodified MTs differently. We used three different indicate the re-appearance of the GFP-Kif5c puncta after photobleaching. Scale bars, 2 m. sources of tubulin; predominantly tyrosinated tubulin (D) Plots of FRAP values for bleached regions towards the cell edge (11 regions from nine purified from HeLa cells (Cytoskeleton), tubulin 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 HeLa cells treated with carboxypeptidase A from five cells for GFP-Kif5cmut). These data confirm that recovery is much slower for the (CPA), which removes the C-terminal tyrosine and mutant. generates detyrosinated tubulin (Chapin and Kif5c behaviour in live cells 1089

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 m. (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 m. (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 Journal of Cell Science 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).

Bulinski, 1991), and highly modified tubulin isolated from pig significantly lower (0.58±0.07 m second–1, mean ± s.d., n=206; brain. The latter source of tubulin is most commonly used in in- P<0.05) than for unmodified (tyrosinated) MTs (0.66±0.06 m vitro motility assays. A dot-blot assay showed that tubulin from second–1, n=103). Interestingly, the velocity of highly modified untreated HeLa cells contained at least 95% tyrosinated tubulin MTs from pig brain was significantly slower than that on (Fig. 7A) as well as partially acetylated tubulin, whereas tubulin detyrosinated MTs (0.55±0.05 m second–1, n=207, P<0.5). It has from CPA-treated cells was at least 95% detyrosinated only (Fig. been reported previously (Liao and Gundersen, 1998) that purified 7A), and pig-brain tubulin was heavily modified containing high kinesin-1 from squid binds with a 2.8-fold greater affinity to levels of both detyrosinated and acetylated tubulin (Fig. 7A). tyrosinated tubulin from HeLa cells than to detyrosinated tubulin Tubulin from CPA-treated cells looked similar to that of untreated (Kd= 29 nM and Kd=81 nM, respectively). We performed a similar cells when analysed on SDS-PAGE (Fig. 7B) and, when binding assay to confirm that the binding of the Kif5c motor domain polymerised, all three types of tubulin formed MTs that were to tyrosinated and detyrosinated MTs from HeLa cells showed the indistinguishable using enhanced video differential interference same trend (data not shown). Without ATP, the Kif5c motor domain microscope (Fig. 7C). The MTs were used in in-vitro motility was incubated with taxol-stabilised tyrosinated and detyrosinated assays (Fig. 7D) together with a purified Kif5c-motor-domain MTs from HeLa cells or MTs from pig brain. Samples were then construct. Movement of MTs in these assays was tracked and centrifuged, and pellet and supernatant analysed by western their velocity calculated. blotting or SDS-PAGE. We found that ~1.5 times more Kif5c was The assays showed that the velocity of MTs depended on the bound to tubulin from pig brain and to detyrosinated tubulin than type of tubulin (Fig. 7E). The velocity for detyrosinated MTs was to tyrosinated tubulin, confirming that Kif5c has a higher steady- 1090 Journal of Cell Science 121 (7)

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 m. (B) COS-7 cells Journal of Cell Science 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 m. (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%).

state occupancy on detyrosinated than on tyrosinated MTs (data The model predicts that an increase from 0% detyrosination to not shown). 5% detyrosination, similar to the levels we observed in COS-7 cells, increased the levels of bound kinesin-1 (stationary number density Modelling Kif5c behaviour on tyrosinated and detyrosinated MT distribution of kinesin) by about 3% (Fig. 8B). The model also To determine whether a small difference in velocity and the predicts that the density of kinesin-1 on MTs, the average flux of reported 2.8-fold greater binding affinity of kinesin-1 to kinesin-1 along a MT and the average velocity depends on the degree detyrosinated MTs compared with tyrosinated MTs (Liao and of detyrosination (Fig. 8B). The change in stationary number density Gundersen, 1998) is sufficient to bias transport of Kif5c towards distribution of kinesin-1 as a result of detyrosination is small, and modified MTs, a simple mathematical model was used. The model one possible reason for this is that the kon value (or landing rate) is a ‘hopping’ model for the dynamics of kinesin-1 behaviour on a in the model is independent of detyrosination. If kon were to increase track of N+1 sites (Fig. 8A). It was used to predict the equilibrium when tubulin is detyrosinated, the difference between detyrosinated distribution (stationary number density distribution of kinesin-1) and tyrosinated MTs in our model would increase further. termed cn (for 0

GFP-Kif5c can bind to tyrosinated, detyrosinated and/or acetylated MTs in vivo, we found that it preferentially associated with detyrosinated MTs and, to a lesser extent, with acetylated MTs. Our in vitro work indicates that tubulin detyrosination directly influences the velocity of MTs moved by Kif5c. Why does GFP-Kif5c preferentially traffic along detyrosinated MTs in vivo? A clue might lie in the results of our in vitro assays. We observed slower sliding of detyrosinated MTs (0.58 m second–1 vs 0.66 m second–1), indicating that detyrosination causes a slower stepping/ATP-turnover rate (72.5 second–1 vs 82.5 second–1, assuming 8- nm steps). This observation is consistent with a model in which detyrosination of MTs shifts the steady-state binding equilibrium of kinesin-1 heads towards strong binding by reducing the detachment rate of the weak binding kinesin.ADP intermediate. This would reduce the stepping rate and increase the steady-state occupancy of MTs. In current models for the kinesin-1 stepping mechanism, reducing the detachment rate in the weak-binding kinesin.ADP state will decrease the probability of detachment following a step, giving rise, therefore, to longer processive runs. Detyrosination would then result in a slower motor that made longer processive runs, and increase the steady-state occupancy on MTs, as we observed. Our mathematical modelling

Journal of Cell Science confirms this relationship between the detachment rate, the run length and the steady-state occupancy. An analogy might be Fig. 7. In vitro analysis of kinesin-1 interactions with post-translationally modified MTs. (A) Dot-blot that detyrosination gives the motor ‘magnetic assays for different post-translational modifications of tubulin in the three types of MT used in the in vitro boots’, attaching it more reliably to the track, MT-binding and -motility assays. A range of tubulin concentrations (500-10 ng) were probed with but making stepping correspondingly more antibodies that specifically recognise tyrosinated, detyrosinated or acetylated tubulin. Microtubules difficult. 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). Detyrosination might also result in a Carboxypeptidase A (CPA) treatment of HeLa cells [HeLa (+CPA)] resulted in at least 95% of tubulin slower but more consistent and sustained being detyrosinated. (B) Tubulin from pig brain, untreated HeLa cells (–CPA) and treated HeLa cells delivery of kinesin-1 cargo to specific (+CPA) resolved on SDS gel and stained with Coomassie-Blue. The two bands corresponding to - and intracellular sites. That GFP-Kif5c is capable -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 of processive movement on heavily modified cells (+CPA). There is no discernable difference between the MTs. (D) Stills from an in vitro motility MTs in vivo was confirmed by our assay using unmodified tubulin from HeLa cell. Time (in seconds) is indicated in the top left hand corner. observations of motor movement along Black arrows indicate the front of the MT, asterisks show the starting position of the MT. Scale bar, 5 mm. nocodazole-resistant MTs in drug-treated (E) The velocity distribution of the Kif5c motor moving on the three types of MT in an in vitro motility cells (supplementary material Movie 8). assay. Bin sizes for the velocities are 0.02 m second–1. Average velocities for the three types of MT were 0.55 m second–1 (±0.05, mean ± s.d., n=207), 0.66 m second–1 (±0.06, n=103) and 0.58 m second–1 In support of this idea, it has been shown (±0.07, n=206) for MTs from pig brain, tyrosinated and detyrosinated MTs, respectively. Dashed curves previously that complete cleavage of the ‘E- show the calculated normal distribution for each dataset. hook’ reduced the velocity of a fungal kinesin-1 from 1.6 m second–1 to 1.0 m second–1 (Lakamper and Meyhofer, 2005; (Macioce et al., 2003), and also with the distribution of the kinesin- Skiniotis et al., 2004). Cleavage of the E-hook also shifted the 3 family member Kif1c expressed as a GFP fusion protein in normally weak binding kinesin.ADP state towards strong binding. macrophages (Kopp et al., 2006). The binding and movement of The E-hook is the glutamate-rich sequence at the C-terminus of GFP-Kif5c along MTs is dependent on the presence of an active both and tubulin that contributes to an electrostatic interaction motor, because a rigor mutant GFP-Kif5cmut (T93N) was able to between kinesin-1 and tubulin (Thorn et al., 2000). Both bind to, but not move along, MTs in the same system. Although detyrosination and E-hook cleavage could produce their effects 1092 Journal of Cell Science 121 (7)

by strengthening the binding of the kinesin-ADP intermediate. If correct, this idea would also account for our observation of higher steady-state occupancy of detyrosinated MTs compared with tyrosinated MTs. Kinesin-1 is known to bind with increased affinity to detyrosinated MTs (Liao and Gundersen, 1998), and this would be expected if detyrosination tightens the binding of the kinesin.ADP intermediate. Our work has focused on the importance of detyrosination, but other tubulin modifications probably also affect the interaction of kinesin-1 with MTs. Polyglutamination, another post-translational modification seen at the E-hook, influences the trafficking of Kif1a cargo in mouse brain (Ikegami et al., 2007). Acetylation also affects kinesin-1 trafficking. Drosophila kinesin-1 binds with a higher affinity and moves acetylated MTs from Tetrahymena faster (~20%) than MTs isolated from mutants in which acetylation was prevented (Reed et al., 2006). This latter finding is intriguing, because acetylation occurs on the lysine residue at position 40, which is positioned on the internal face of -tubulin and, therefore, within the lumen of the MT. This modification would not be expected to have a direct effect on binding affinity of kinesin-1, but could have an indirect effect by influencing the structure of the tubulin dimer. This might also mean that acetylation affects a different step in the kinesin ATPase cycle compared with detyrosination, and this might explain why detyrosination increases binding affinity and reduces Fig. 8. Model to investigate how detyrosination affects kinesin-1 behaviour on MTs. velocity, whereas acetylation increases both binding (A) Basis for the model. Circles represent binding sites along MTs, n=0 corresponds to the affinity and velocity. Further work is needed to minus-end of the MT, and n=N is the plus-end. Kinesin-1 can bind to an MT with an on-rate of k , detach with an off-rate of k , and can either move towards the plus-end (taking unit understand how these different tubulin modifications on off steps) at a rate of k+ or towards the minus-end at a rate of k–. For details of the model see

Journal of Cell Science affect kinesin-1 binding and movement. However, it Materials and Methods. (B) Results obtained when using the model. Stationary number is possible that both modifications are important in density distribution of kinesin-1 was obtained using equations 10-12; average number directing kinesin-1 trafficking, which may be related density, average flux and average velocity as a function of tyrosinated tubulin, were obtained to the large variety in MT acetylation and/or using equations 13-15. detyrosination between different cell types (Bulinski et al., 1988). The results described here, in combination with work by pathway – which terminates with APC-mediated MT stabilisation previous investigators (Kreitzer et al., 1999; Liao and Gundersen, – provides directional cues for kinesin-1-dependent cargo 1998), allow us to propose a biologically relevant explanation for transport. the phenomenon of tubulin detyrosination. We propose that the In conclusion, our observations confirm by direct in vivo targeting of MT-motor-dependent transport in vivo is the specific observation that the full-length heavy chain of kinesin-1 is associated reason for this tubulin modification. Preferential trafficking along with MTs where it forms part of a processive plus-end-directed detyrosinated MTs could provide targeting cues for intracellular motor complex that moves with a velocity comparable with that transport, such as those previously reported by others (Nakata and seen in vitro. Our results also define a mechanism that exploits post- Hirokawa, 2003). Detyrosinated MTs in migrating cells penetrate translational tubulin modifications associated with MT stability to into lamellipodia that extend in the direction of travel (Gundersen target kinesin-1 dependent transport to specific regions within cells. and Bulinski, 1988; Kupfer et al., 1982). Furthermore, signalling Finally, our data help to explain the 20-year old observation that events that leading to the generation of detyrosinated MTs are vesicles in lobster axons are found only on a small proportion of mediated by the activity of a variety of effectors downstream of the MTs, suggesting that distinct classes of MTs have distinct roles the small GTPase Rho (Cook et al., 1998; Palazzo et al., 2001). in either trafficking or maintaining structural integrity of the cell Recently, the final link in this pathway has been suggested to be (Miller et al., 1987). dependent upon the APC tumour suppressor protein (Bienz, 2001). Consistent with this, co-immunostaining of transfected cells indicated that APC and GFP-Kif5c distributions extensively Materials and Methods overlapped along MT distal segments as well as in clusters located Antibodies and reagents Rat anti--tubulin antibody (YL1/2) specific for the EEY epitope of tyrosinated MTs at the cell periphery (S.D., M.P. and E.E.M., unpublished data). was obtained from Serotec. Rabbit anti-detyrosinated-tubulin antibody (T12) specific This observation supports the hypothesis that this signalling for the xEE epitope of -tubulin was a kind gift from Holly Goodson, University of Kif5c behaviour in live cells 1093

Notre Dame, IN (Kreis, 1987). Mouse anti-acetylated-tubulin antibody was obtained BRB 80 was washed into the flow cell. The kinesin solution also contained 100 g from Sigma-Aldrich. To amplify the GFP-Kif5c signal in fixed cells, anti-Kif5c ml–1 casein as surface block. The motor was allowed to adsorb to the surface for 5 (Abcam, Ltd) or anti-KHC (Chemicon International) were used. Secondary antibodies minutes at room temperature. Non-adsorbed Kif5c-motor-domain was washed out used for immunofluorescence were Alexa-Fluor-488, -594 and -647 conjugates with 2-3 flow cell volumes of BRB 80 supplemented with 5 mM DTT and 20 M (Molecular Probes). Nocodazole was obtained from Sigma-Aldrich and used at a taxol. Two flow cell volumes of MTs (~2 M) in BRB 80 with 1 mM DTT, 20 M final concentration of 2.5 g ml–1. taxol and 1 mM ATP were then allowed to flow through. The slide was immediately transferred to the microscope and MT gliding was observed by video-enhanced Cloning differential interference microscopy (VEDIC). The measurement of MT velocities Two fluorescently tagged Kif5c fusion proteins were used in this study. GFP-Kif5c was performed using Nick Carter’s (MCRI, Oxted, Surry, UK) freeware RETRAC was constructed by cloning the full-length rat Kif5c cDNA (a kind gift from Scott program (http://mc11.mcri.ac.uk/retrac.html) for image capture and tracking. Brady, University of Illinois, IL) into pEGFP-C3 (BD Biosciences), creating an N- terminally tagged GFP-Kif5c fusion protein. The rigor kinesin-1 mutant, GFP- Mathematical model Kif5cmut was created using an overlap PCR approach to introduce the T93N mutation Details of the model and the equations used are given below. To solve the equations (Nakata and Hirokawa, 1995). The mCherry--tubulin expression vector was a kind detailed there, a C code was written that solves equations 1-3 by the Euler method. gift from Vic Small (Institute of Molecular Biotechnology, Vienna, Austria). Given the solution of the number distribution, the code then computes the flux and the velocity distributions, as well as the average values of the number density, the Cell culture and manipulation flux and the velocity at any given time (equations 7-9). The code has been run for COS-7 cells were cultured as described previously (Morrison et al., 1998). The long enough to obtain the steady-state solution (equations 10-12). Given the steady neuronal cell line Hdh+/+ was cultured as described previously (Zuccato et al., 2003) number density distribution, the code computes the steady-state average number- Cells were transiently transfected with pGFP-Kif5c using GeneJuice (Novagen) density, average flux and average velocity as a function of the tyrosination f (equations according to the manufacturer’s protocol. 13-15). Kinesin ’walks’ along the MT – taking unit steps – in a stochastic manner starting Immunocytochemistry from the minus end of the filament (site n=0; Fig. 8A) towards the plus-end of the MT (site n=N; Fig. 8A). The starting point of our model is that detyrosination of the For immunofluorescence studies cells were cultured on acid-washed glass coverslips tubulin dimers results in a stronger binding of kinesin as the reported K decreases (VWR International) coated with 0.02% gelatine (Sigma). Cells were either fixed in d from 81 nM to 29 nM (Liao and Gundersen, 1998), and in a slower rate of motion cold methanol (Morrison et al., 1998; Musa et al., 2003) or paraformaldehyde (Swailes along the MT by kinesin molecules (as described here). et al., 2006), immunostaining was performed as described previously. Fluorescence In this section, we develop a hopping model for the dynamics of kinesin on a track imaging was performed using a deconvolution microscope (Deltavision) or confocal of N+1 sites. The density of kinesin (number of molecules per unit volume) at the laser scanning microscope (LSM) 510 Meta (Zeiss). binding site n and at time t is given by cn(t), with n ʦ {0,1,2…., N–1,N}. We can write a dynamical equation for the number density of the nth site cn(t), (1nN–1), Live cell imaging which is given by: Cells were grown in 35-mm glass-bottomed culture dishes (Iwaki brand; Asahi Techno Glass Corporation, Japan; supplied by Bibby Sterilin). 18-48 hours post-transfection dcn(t)/dt = k+cn–1 (t) + k–cn+1(t) – (k++k–+koff)cn(t) + kon ,(1) the growth medium was exchanged for CO2-independent medium (Invitrogen) –1 supplemented with 10% foetal calf serum (FCS), 100 g ml penicillin-streptomycin where =V0, and the bulk concentration of dissociated kinesin molecules in a and 4 mM glutamine for imaging. volume V0 that is assumed to be constant. The density of kinesin motors is low enough Imaging at the slower frame rate of one frame every 5 seconds was performed so that we can ignore the interactions between motors in this mean field model. At essentially as described previously (Langford et al., 2006). Cells were transferred to the ends of the MT, the equation becomes: a Zeiss Axiovert 200 inverted microscope with the stage enclosed by a heated chamber (Solent Scientific, UK), and the temperature maintained at 37°C. Cells were examined dc0(t)/dt = k–c1(t) – (k++koff)c0(t) + kon (2) by fluorescence microscopy using a Zeiss Plan Apochromat 63/1.4NA oil- and

immersion lens. An excitation-emission filterset optimised for eGFP imaging was dcN(t)/dt = k+cN–1(t) – (k–+koff)cN(t) + kon .(3) used (Chroma Technology Corp., Brattleboro, VT; filterset ID 86007). Time-lapse images were obtained using Ludl shutters and a Hamamatsu Orca II ER camera. Equations 1-3 can be obtained from the following continuity argument (Edelstein- Journal of Cell Science Microscope, camera, filter wheels and shutters were controlled by Kinetic Imaging Keshet, 2005): the rate of change in the number of kinesin molecules at the binding AQM 6 software. site n per unit time dcn(t)/dt is equal to the rate of entry of kinesin molecules into To perform imaging at higher frame rates (two frames per second) or to perform site n per unit time k+cn–1(t)+k+cn+1(t) minus the rate of departure of kinesin molecules multicolour time-lapse live cell imaging, cells were transferred to an Olympus from site n per unit time –(k++k–cn(t) plus the rate of binding of kinesin molecules microscope enclosed in a heated chamber (Solent Scientific, as above). Cells were at site n per unit time kon minus the rate of detachment of kinesin molecule at site imaged using an Olympus 60 /1.4NA oil-immersion lens, and camera, shutters and n per unit time koffcn(t). software were controlled using Softworx software package (Deltavision, USA). The Our experimental data show that, the fraction of MT-binding sites that are image size was set to 256256 pixels, using 11 binning to obtain the highest tyrosinated is 95%, kinesin moves along MTs with a velocity of 0.66 m second–1 resolution of puncta. Only sequences captured at this faster frame rate and at this on tyrosinated MTs and at 0.58 m second–1 on detyrosinated MTs. Furthermore it higher spatial resolution were used in the tracking analysis to calculate GFP-Kif5c has been shown previously that the Kd for detyrosinated MTs (29 nM) is about 3 velocities. Particle tracking analyses were conducted on original datasets using Kinetic times lower than that for tyrosinated MTs (81 nM) (Liao and Gundersen 1998). Finally, Imaging Motion Analysis software, or using GMview (kindly provided by Gregory the on-rate is diffusion limited and was described as 2107 M–1 second–1, the Mashanov, NIMR, London, UK; (Mashanov et al., 2004). GFP-Kif5c puncta were detachment rate as 20 second–1 (Johnson and Gilbert, 1995, and Table 1 within), and tracked from frame to frame. When puncta temporarily stopped moving, velocity the forward rate of motion for a kinesin molecule on tyrosinated MTs is ~82.5 second–1, dropped to zero; these phases were counted as ‘stationary’. The average velocity for given the size of the steps (8 nm) (Svoboda et al., 1993) and its velocity (0.66 m each punctum was calculated either by including these stationary phases or by second–1). excluding them. As expected, excluding them increased velocities slightly. Time- Given these experimental data, a mean field model (Edelstein-Keshet, 2005) is an lapse image series were cropped and converted into Quicktime or AVI movies using appropriate description of the kinetics of kinesin walking on a MT, where a fraction Image J, Adobe ImageReady 7, Photoshop CS3 or Fast Movie Processor. Some movies f of the sites are tyrosinated and (1–f) are detyrosinated. According to our experiments t g are presented using an inverted greyscale colour look-up table to increase perceived f is 95%. We introduce k + and k +, the forward rates for fully tyrosinated and fully g t contrast. detyrosinated MTs, respectively. We have a=k +/k +=0.58/0.66=1/1.379 (from the two measured velocities of kinesin movement on tyrosinated and detyrosinated MTs). We In vitro motility assays have introduced the parameter a=1/1.379 that implies slower forward walking along Tubulin purified from HeLa cells (Cytoskeleton) was modified to produce pure detyrosinated MTs. For a partially detyrosinated track, the forward rate is given by t g t t –1 populations of detyrosinated and tyrosinated subunits as described (Chapin and k+=f k ++(1–f)k +=k +[f+a(1–f)], with k +=82.5 second . The on-rate is given by kon, 7 –1 –1 –7 Bulinski, 1991). Dot-blot assays were used to assess the purity of the tyrosinated with kon=210 M second and =10 M. The detachment rate, koff depends on and detyrosinated MTs, and to determine the extent of post-translational modifications f as follows. koff is approximately three times lower for fully detyrosinated than for of tubulin purified from pig brain. All tubulin was polymerised according to the fully tyrosinated MTs (Liao and Gundersen, 1998). We can therefore write this in t t methods of Lockhart and Cross (Lockhart and Cross, 1996) and MTs were the mean field description koff=fk off+(1–f), where k off is the detachment rate for fully t –1 resuspended in BRB 80 (80 mM PIPES-KOH pH 6.9, 1 mM MgCl2, 1 mM EGTA) tyrosinated MTs and is equal to k off =20 second . Finally, the backward rate of motion –1 containing 20 M taxol. The kinesin-1-motor-domain construct used was K430 can be parametrised in terms of the bias b=k+/k– so that k–=b k+, which we have [430aa; as described in Crevel et al. (Crevel et al., 1997)]. Flow cells were constructed set to 100. The probability that kinesin motors take a backwards step is very low, by sealing together two ethanol-washed glass coverslips using two parallel strips of but increasing the bias to 1000 or even larger only has very small effects on the silicone grease (Dow Corning). An aliquot of 5 M Kif5c-motor-domain diluted in results presented here. 1094 Journal of Cell Science 121 (7)

The previous equations for cn(t) allow us to define a ‘current’ or flux of kinesin Bulinski, J. C., Richards, J. E. and Piperno, G. (1988). Posttranslational modifications at the binding site n and at a time t, given by: of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in clultured cells. J. Cell Biol. 106, 1213-1220. jo(t) = 0 (4) Cai, D., Verhey, K. J. and Meyhofer, E. (2007). Tracking single Kinesin molecules in the cytoplasm of mammalian cells. Biophys. J. 92, 4137-4144. Chapin, S. J. and Bulinski, J. C. (1991). Preparation and functional assay of pure jn(t) = x[k+cn–1(t) – k–cn(t)] for all 1 n N , (5) populations of tyrosinated and detyrosinated tubulin. Meth. Enzymol. 196, 254-264. and a velocity at site n and time t defined by: Cook, T. A., Nagasaki, T. and Gundersen, G. G. (1998). Rho guanosine triphosphatase mediates the selective stabilization of microtubules induced by lysophosphatidic acid. v (t) = j (t)/c (t) . (6) J. Cell Biol. 141, 175-185. n n n Coy, D. L., Hancock, W. O., Wagenbach, M. and Howard, J. (1999a). Kinesin’s tail We can compute the average number density of kinesin molecules on the MT, the domain is an inhibitory regulator of the motor domain. Nat. Cell Biol. 1, 288-292. average flux of kinesin and the average velocity of kinesin as follows: Coy, D. L., Wagenbach, M. and Howard, J. (1999b). Kinesin takes one 8-nm step for each ATP that it hydrolyzes. J. Biol. Chem. 274, 3667-3671. N Crevel, I. M., Lockhart, A. and Cross, R. A. (1997). Kinetic evidence for low chemical Ό ΍ 1 c(t) = cn(t) (7) processivity in ncd and Eg5. J. Mol. Biol. 273, 160-170. N+1 n=0 Crevel, I. M., Nyitrai, M., Alonso, M. C., Weiss, S., Geeves, M. A. and Cross, R. A. (2004). What kinesin does at roadblocks: the coordination mechanism for molecular walking. EMBO J. 23, 23-32. Edelstein-Keshet, L. (2005). Mathematical Models in Biology. Philadelphia, USA: SIAM. N Friedman, D. S. and Vale, R. D. (1999). Single-molecule analysis of kinesin motility 1 reveals regulation by the cargo-binding tail domain. Nat. Cell Biol. 1, 293-297. Όj(t)΍ = jn(t) (8) Gundersen, G. G. and Bulinski, J. C. (1988). Selective stabilization of microtubules N+1 n=0 oriented toward the direction of cell migration. Proc. Natl. Acad. Sci. USA 85, 5946- 5950. N Gundersen, G. G., Kalnoski, M. H. and Bulinski, J. C. (1984). Distinct populations of 1 Ό ΍ microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in v(t) = vn(t) . (9) N+1 n=0 vivo. Cell 38, 779-789. Gurland, G. and Gundersen, G. G. (1995). Stable, detyrosinated microtubules function to localize intermediate filaments in fibroblasts. J. Cell Biol. 131, 1275-1290. We are interested in the stationary solution of our system of equations 1-3 that Gyoeva, F. K. and Gelfand, K. I. (1991). Coalignment of vimentin intermediate filaments corresponds to the equilibrium (time independent) distribution of kinesin molecules with microtubules depends on kinesin. Nature 353, 445-448. on the MT. In order to do so, we have solved the dynamical equations numerically Hackney, D. D. (1988). Kinesin ATPase: rate-limiting ADP release. Proc. Natl. Acad. Sci. and have taken N=100. Note that the stationary solutions correspond to solving the USA 85, 6314-6318. following system of equations: Hackney, D. D., Levitt, J. D. and Suhan, J. (1992). Kinesin undergoes a 9 S to 6 S conformational transition. J. Biol. Chem. 267, 8696-8701. 0 = k–c1 – (k++koff)c0 + kon , (10) Hirokawa, N. and Takemura, R. (2005). Molecular motors and mechanisms of directional transport in neurons. Nat. Rev. Neurosci. 6, 201-214. Hollenbeck, P. J. (1989). The distribution, abundance and subcellular localization of kinesin. 0 = k+cn–1 + k–cn+1 – (k++k–+koff) cn +kon for all 1 n N(11) J. Cell Biol. 108, 2335-2342. Ikegami, K., Heier, R. L., Taruishi, M., Takagi, H., Mukai, M., Shimma, S., Hatanaka, K., Morone, N., Yao, I., Campbell, P. K. et al. (2007). Loss of alpha-tubulin 0 = k+cN–1 –(k–+koff) cN +kon . (12) polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A We have solved the stationary number density distribution of kinesin cn (for and modulated synaptic function. Proc. Natl. Acad. Sci. USA 104, 3213-3218. n={0,1,2,N–1, N}), the equilibrium average number density c, the equilibrium average Johnson, K. A. and Gilbert, S. P. (1995). Pathway of the microtubule-kinesin ATPase. velocity v and the equilibrium average flux j. Bars over given quantities indicate the Biophys. J. 68, 173S-179S. quantity evaluated in the steady-state (equilibrium, time independent) solution. Note Kanai, Y., Okada, Y., Tanaka, Y., Harada, A., Terada, S. and Hirokawa, N. (2000). Journal of Cell Science that the end points are included. These three quantities are computed as follows: KIF5C, a novel neuronal kinesin enriched in motor neurons. J. Neurosci. 20, 6374-6384. Kanai, Y., Dohmae, N. and Hirokawa, N. (2004). Kinesin transports RNA: isolation and N characterization of an RNA-transporting granule. Neuron 43, 513-525. c 1 c = n (13) Kopp, P., Lammers, R., Aepfelbacher, M., Woehlke, G., Rudel, T., Machuy, N., Steffern, N+1 n=0 W. and Linder, S. (2006). The kinesin KIF1C and microtubule plus ends regulated podosome dynamics in macrophages. Mol. Biol. Cell 17, 2811-2823. N Kreis, T. E. (1987). Microtubules containing detyrosinated tubulin are less dynamic. EMBO j 1 j J. 6, 2597-2606. = n (14) N+1 Kreitzer, G., Liao, G. and Gundersen, G. G. (1999). Detyrosination of tubulin regulates n=0 the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism. Mol. Biol. Cell 10, 1105-1118. N Krylyshkina, O., Kaverina, I., Kranewitter, W., Steffen, W., Alonso, M. C., Cross, R. v 1 v A. and Small, J. V. (2002). Modulation of substrate adhesion dynamics via microtubule = n (15) N+1 n=0 targeting requires kinesin-1. J. Cell Biol. 156, 349-359. Kupfer, A., Louvard, D. and Singer, S. J. (1982). Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental and the solutions are plotted in Fig. 8B. wound. Proc. Natl. Acad. Sci. USA 79, 2603-2607. We also considered whether we can model the effects of acetylation. It has been Lakamper, S. and Meyhofer, E. (2005). The E-hook of tubulin interacts with kinesin’s head to increase processivity and speed. Biophys. J. 89, 3223-3234. recently reported (Reed et al., 2006) that acetylation affects the binding and velocity Lakamper, S. and Meyhofer, E. (2006). Back on track – on the role of the microtubule of conventional kinesin. Lack of acetylation reduces binding substantially, to ~10% for kinesin motility and cellular function. J. Muscle Res. Cell Motil. 27, 161-171. of wild-type levels, and velocity to ~20% of wild-type levels. However, because the Langford, K. J., Askham, J. M., Lee, T., Adams, M. and Morrison, E. E. (2006). extent of detyrosination of the MTs used in those experiments was not measured, it Examination of and microtubule dependent APC localisations in living mammalian is unclear how this may have affected the results. cells. BMC Cell Biol. 7, 3. Lawrence, C. J., Dawe, R. K., Christie, K. R., Cleveland, D. W., Dawson, S. C., Endow, This work was funded by the Wellcome Trust, Cancer Research UK S. A., Goldstein, L. S., Goodson, H. V., Hirokawa, N., Howard, J. et al. (2004). A standardized kinesin nomenclature. J. Cell Biol. 167, 19-22. and the Marie Curie Foundation. S.D. was an MRC-funded PhD student. Liao, G. and Gundersen, G. G. (1998). Kinesin is a candidate for cross-bridging We thank Holly Goodson for antibodies used in this work, and Nikolai microtubules and intermediate filaments. Selective binding of kinesin to detyrosinated Belyaev for providing us with the neuronal cell line. tubulin and vimentin. J. Biol. Chem. 273, 9797-9803. Lockhart, A. and Cross, R. A. (1996). Kinetics and motility of the Eg5 microtubule motor. Biochemistry 35, 2365-2373. References Luduena, R. F., Banerjee, A. and Kham, I. A. (1992). Tubulin structure and biochemistry. Bienz, M. (2001). Spindles cotton on to junctions, APC and EB1. Nat. Cell Biol. 3, E67- Curr. Opin. Cell Biol. 4. 53-57. E68. Macioce, P., Gambara, G., Bernassola, M., Gaddini, L., Torreri, P., Macchia, G., Brady, S. T. (1985). A novel brain ATPase with properties expected for the fast axonal Ramoni, C., Ceccarini, M. and Petrucci, T. C. (2003). Beta-dystrobrevin interacts transport motor. Nature 317, 73-75. directly with kinesin heavy chain in brain. J. Cell Sci. 116, 4847-4856. Kif5c behaviour in live cells 1095

Mashanov, G. I., Tacon, D., Peckham, M. and Molloy, J. E. (2004). The spatial and Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E. and temporal dynamics of pleckstrin homology domain binding at the plasma membrane Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins measured by imaging single molecules in live mouse myoblasts. J. Biol. Chem. 279, derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572. 15274-15280. Skiniotis, G., Cochran, J. C., Muller, J., Mandelkow, E., Gilbert, S. P. and Hoenger, A. Miller, R. H., Lasek, R. J. and Katz, M. J. (1987). Preferred microtubules for vesicle (2004). Modulation of kinesin binding by the C-termini of tubulin. EMBO J. 23, 989-999. transport in lobster axons. Science 235, 220-222. Svoboda, K., Schmidt, C. F., Schnapp, B. J. and Block, S. M. (1993). Direct observation Morrison, E. E., Wardleworth, B. N., Askham, J. M., Markham, A. F. and Meredith, of kinesin stepping by optical trapping interferometry. Nature 365, 721-727. D. M. (1998). EB1, a protein which interacts with the APC tumour suppressor, is Swailes, N. T., Colegrave, M., Knight, P. J. and Peckham, M. (2006). Non-muscle associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17, 2A and 2B drive changes in cell morphology that occur as myoblasts align and 3471-3477. fuse. J. Cell Sci. 119, 3561-3570. Musa, H., Orton, C., Morrison, E. E. and Peckham, M. (2003). Microtubule assembly Thorn, K. S., Ubersax, J. A. and Vale, R. D. (2000). Engineering the processive run in cultured myoblasts and myotubes following nocodazole induced microtubule length of the kinesin motor. J. Cell Biol. 151, 1093-1100. depolymerisation. J. Muscle Res. Cell Motil. 24, 301-308. Vale, R. D. (2003). The molecular motor toolbox for intracellular transport. Cell 112, 467- Nakata, T. and Hirokawa, N. (1995). Point mutation of adenosine triphosphate-binding 480. motif generated rigor kinesin that selectively blocks anterograde lysosome membrane Vale, R. D., Reese, T. S. and Sheetz, M. P. (1985a). Identification of a novel force- transport. J. Cell Biol. 131, 1039-1053. generating protein, kinesin, involved in microtubule-based motility. Cell 42, 39-50. Palazzo, A. F., Cook, T. A., Alberts, A. S. and Gundersen, G. G. (2001). mDia mediates Vale, R. D., Schnapp, B. J., Mitchison, T., Steuer, E., Reese, T. S. and Sheetz, M. P. Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 3, 723- (1985b). Different axoplasmic proteins generate movement in opposite directions along 729. microtubules in vitro. Cell 43, 623-632. Piperno, G., LeDizet, M. and Chang, X. J. (1987). Microtubules containing acetylated Varadi, A., Ainscow, E. K., Allan, V. J. and Rutter, G. A. (2002). Molecular mechanisms alpha-tubulin in mammalian cells in culture. J. Cell Biol. 104, 289-302. involved in secretory vesicle recruitment to the plasma membrane in beta-cells. Biochem. Reed, N. A., Cai, D., Blasius, T. L., Jih, G. T., Meyhofer, E., Gaertig, J. and Verhey, Soc. Trans. 30, 328-332. K. J. (2006). Microtubule acetylation promotes kinesin-1 binding and transport. Curr. Zuccato, C., Tartari, M., Crotti, A., Goffredo, D., Valenza, M., Conti, L., Cataudella, Biol. 16, 2166-2172. T., Leavitt, B. R., Hayden, M. R., Timmusk, T. et al. (2003). Huntingtin interacts Schnapp, B. J. (2003). Trafficking of signaling modules by kinesin motors. J. Cell Sci. with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. 116, 2125-2135. Nat. Genet. 35, 76-83. Journal of Cell Science