Full-length human His₆-Kif15 (hKif15), His₆-Kif15-eGFP, and His₆-Tpx2 (hTpx2) were expressed in insect SF9-cells and the recombinant proteins purified to near homogeneity by sequential affinity chromatography using a cation-exchange and a Co-NTA matrix (Figure 1A). Analysis of His₆-hKif15 on native PAGE revealed that the protein migrates at ∼730 KDa (Figure 1B). Given the predicted molecular weight of His₆-hKif15 (164.8 kDa) our data suggest the presence of hKif15 tetramers, although hydrodynamic analysis of the frog (xKlp2) and Sea urchin (KRP₁₈₀) Kif15 orthologues indicated that the motor is dimeric with a sedimentation coefficient of 8.1S and 8.3S respectively (Wittmann et al., 1998; Rogers et al., 2000). To further characterise human Kif15, we subjected His₆-hKif15 to glycerol gradient ultracentrifugation on 5–40% glycerol gradients. At physiological ionic strengths (35 mM sodium phosphate buffer, 0-150 mM NaCl) both His₆-hKif15 and His₆-hKif15-eGFP appear to be monodispersed and have apparent sedimentation coefficients of ∼12S (Figure 1C). However, increasing the salt concentration to 300 mM NaCl converts hKif15 into a species that runs at ∼8S (Figure 1C). Taken together, our data show that hKif15 exists as a tetramer at physiological ionic strength that can be forced to dissociate into a dimer at high ionic strength. To further confirm that hKif15 can form tetramers we performed a size-exclusion chromatography combined with multi angle light scattering (SEC-MALS), which allows determination of the absolute molecular weight. This analysis confirmed the presence of tetrameric hKif15 motors (molecular weight of 744.4 ± 14.1 kDa) (Figure 1D, 1^st peak, blue line). We could also detect a dimeric population of motors (molecular weight of 360.1 ± 6.5 kDa) indicating that there is some degree of complex dissociation in solution under these conditions (Figure 1D, 2^nd peak, orange line).

Figure 1

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Next, we used total internal reflection fluorescence (TIRF)-microscopy, to observe the movement of single hKif15-eGFP motors on polarity-marked microtubules that were stabilised with the non-hydrolysable GTP-analogue GMP-CPP. These experiments were done in buffer conditions (BRB20, 50 mM KCl) that would be expected to preserve hKif15 in a tetrameric state (from here on hKif15-eGFP refers to single tetrameric motors). The majority hKif15 motors move either by directional movement towards the plus-end of individual microtubules (Figure 2A, ‘P’) or by bidirectional 1D-diffusion (Figure 2A, ‘D’, Figure 2B, right, Figure 2—figure supplement 1D, right) along the microtubule lattice. These types of movement are equally likely at all salt conditions tested (Figure 2—figure supplement 1A) and single motors can switch between both modes (Figure 2—figure supplement 1B). Processively moving, plus-end-directed motors move at a speed of 137.8 ± 4.1 nm•s⁻¹ median ± SEM, though we observed velocities up to 550 nm•s⁻¹ (Figure 2B, left). The run length of hKif15 motors is 1.9 ± 0.09 µm median ± SEM (max. 9.6 µm) (Figure 2B, middle) and their residency time 26.3 ± 2.8 s median ± SEM (max. >180 s) (Figure 2B, right). Processive runs can include pauses up to 20.5 s (median ± SEM: 5.0 ± 0.4 s) at a frequency of 0.30 ± 0.04 pauses•µm⁻¹ mean ± SD (n = 162, three different experiments) without motor-dissociation from the microtubule lattice (Figure 2—figure supplement 1B,D, middle). Additionally, when motors reach the microtubule end, more than 90% remain attached (Figure 2—figure supplement 1D, left note orange column). Motors dwell at the tip for 18.8 ± 3.5 s median ± SEM (max. >152 s) (Figure 2A, (asterix), Figure 2—figure supplement 1D, left) and may switch to diffusional movement on the end-proximal microtubule lattice (Figure 2—figure supplement 1C). While the majority of hKif15 motors either moves directionally to the plus-end or diffuses along the lattice, we also observed a small proportion of motors that move processively towards the minus-end of polarity-marked microtubules (87.5% plus-end-directed motors vs 12.5% minus-end-directed motors, n = 504) (Figure 2A, right kymograph, ‘MP’). Minus-end-directed motors move at a speed similar to plus-end-directed motors, but have a significantly reduced run length/residency time and do not undergo directional reversals (Figure 2C).

Figure 2 with 2 supplements see all

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To confirm that motile hKif15 motors are tetramers, we imaged hKif15-eGFP motors under conditions that caused photobleaching and measured the change in fluorescence intensity with time (‘Materials and methods’). This analysis showed that both processive and diffusive motors bleach in up to four steps, indicating the presence of four eGFP molecules per motor (Figure 2D). Furthermore, processive tetramers are evident throughout the velocity distribution (Figure 2—figure supplement 1E). These data are in line with our biochemical analysis (Figure 1) and confirm that the observed hKif15-eGFP motors are indeed tetramers.

Within the mitotic spindle, motors will experience varying loads and thus it is essential to understand how a motor responds to force. We therefore investigated the mechanical properties of hKif15 motors using our single-bead laser trap set-up (Carter and Cross, 2005). Single hKif15 motors were adsorbed onto 550-nm diameter polystyrene beads and steered to the microtubule surface with the laser trap at which point the motor can bind to the lattice (see ‘Materials and methods’ for details and Figure 2—figure supplement 2A). With the trap turned off (zero-load) the mean velocity of beads moving on the microtubule was consistent with our measurements of hKif15-eGFP by TIRF microscopy (Figure 2—figure supplement 2B). Next, we held the bead in the trap and allowed the motor to walk along the microtubule, away from the centre of the trap. The motion of the bead reports the stepping actions of the motor. Figure 2E shows two position traces in which hKif15 motors make a number of processive steps along the microtubule before detaching at relatively low loads between 1.5 and 3.5 pN. We also observed that beads move out the trap before moving back in the opposite direction without detaching (green arrowhead). These events may reflect the minus- and plus-end-directed diffusive motion observed in our TIRF experiments (Figure 2A). Together with our TIRF microscopy, we conclude that hKif15 is a homotetramer that undergoes either diffusive movement along microtubules, processive plus-end-directed stepping over long-distances or short duration movements to the minus-end.

Our single molecule TIRF experiments also revealed a remarkable property of hKif15 motors as they approach microtubule intersections. The well-studied kinesin-1 will normally pass intersections and only in rare cases the motor will pause or switch microtubule tracks (Ross et al., 2008). In contrast, hKif15 motors show significantly elevated pause and switch events at intersections. Every fifth motor that approaches an intersection switches its microtubule track (Figure 3A,B; Videos 1 and 2). In general all events—pass, switch, pause, and dissociation at intersections—are equally likely. In this way, hKif15 is able to roam microtubule networks and Figure 3C demonstrates that one tetramer (see bleaching steps, lower panel) can indeed navigate along several different microtubules: the motor starts processive movement on microtubule #1 (trace A), eventually dissociates and rebinds to microtubule #2. On this, the motor diffuses a short distance (trace B) and dissociates again, just to rebind to microtubule #1. It processively follows microtubule #1 (trace C) and sequentially switches to microtubules #3 (trace D) and #4 (trace E), ending at the plus-end of microtubule #4 without detaching from the microtubule lattice(s) once again (see also Video 3). Thus, Kif15 is a processive motor that is able to navigate microtubule networks by effectively switching microtubule tracks.

Figure 3

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Video 1

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Video 2

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The tetrameric organisation of hKif15 would support the hypothesis that the motor drives extensile sliding of anti-parallel microtubule overlaps like the tetrameric Kif11 motor (Tanenbaum et al., 2009). To establish, whether tetrameric hKif15 indeed is able to cross-link microtubules on its own, we carried out experiments, in which biotinylated HiLyte-647-labelled GMP-CPP-microtubules were attached to a cover slip followed by addition of short XRhodamin-labelled GMP-CPP-stabilized- ‘transport’ microtubules and 1.4 nM tetrameric hKif15-eGFP. By drawing kymographs, we found that hKif15 could mediate the cross-linking of microtubules (Figure 4) on its own, but that there was very little microtubule–microtubule sliding activity. Besides pivoting microtubules, we did observe episodes of bidirectional short duration sliding indicating a tug-of-war (Figure 4, right) and rarely the transport of short microtubules (Figure 4, left): behaviours that were recently reported for the yeast Kinesin-8 Kip3 (Su et al., 2013). However, we have no clear evidence to support the idea that hKif15 can drive continuous extensile sliding of anti-parallel microtubule bundles like hKif11. This is presumably because the motors frequently pause during processive excursions on the lattice and dwell at the ends for a long period (Figure 2—figure supplement 1B–D). Consistent with this, we only observed discontinuous short distance movement and pivoting of microtubules (around their ends) on surface-bound hKif15 motors, but not continuous microtubule sliding in microtubule gliding assays (Videos 4 and 5).

Figure 4

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So far our experiments show that hTpx2 is not required for hKif15 to bind microtubules or for the motors processivity and ability to crosslink microtubules (as a tetramer). However, cell-biological experiments showed that hTpx2 is essential for hKif15 to be recruited to the mitotic spindle (Tanenbaum et al., 2009; Vanneste et al., 2009). To resolve this contradiction, we first analysed possible complex formation of recombinant hKif15 and hTpx2 in solution by ultracentrifugation in glycerol gradients. With nanomolar concentrations of each protein, we could not observe any complex formation over a range of different salt conditions (50 mM HEPES pH7.5, 0/150/300/450 mM NaCl [data not shown] and 35 mM sodium phosphate buffer [Figure 5A]). However, a 30-fold excess of hTpx2 dimers (2.5 µM hTpx2 [dimer] + 80 nM hKif15 [tetramer]) allowed the formation of a hKif15-hTpx2 complex of ∼14.0S, which included approximately one third of the provided hKif15 (Figure 5A, lower gels). The dissociation constant of the hKif15–hTpx2 complex is likely to be in the µM range arguing that its formation in vivo is less likely. In contrast, using a microtubule sedimentation assay, we observed that at low nM concentrations, hTpx2 (30 nM, dimer) does enhance the affinity of hKif15 (15 nM, tetramer) binding to GMP-CPP stabilised microtubules when in the presence of the non-hydrolysable ATP analogue AMP-PNP (the fitted K_d increasing ∼twofold from 993 nM to 490 nM; Figure 5B). This observation could explain why cell-biological experiments show that association of hKif15 with the mitotic spindle depends on hTpx2 (Tanenbaum et al., 2009; Vanneste et al., 2009). It is, however, unclear why hTpx2 further increases the microtubule binding affinity of a motor that is already highly processive.

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To address the impact of hTpx2 on moving hKif15 tetramers, we added hTpx2 in equimolar (i.e., 5 nM) concentration to our hKif15-eGFP TIRF setup. Addition of hTpx2 decreased the proportion of motile motors on microtubules by 70%. The remaining motors still showed both directional and diffusive movement, indicating that inhibition by hTpx2 is not selective for either type of movement (Figure 5C, kymographs and left graph below). Further, the velocity distribution of remaining processive motors (compared to Figure 2A) now shows a bimodal distribution with a high proportion of slow motors (42.1 ± 1.7 nm•s⁻¹ median ± SEM) and fewer motors at the median speed of hKif15 motors in the absence of hTpx2 (114.9 ± 3.2 nm•s⁻¹ median ± SEM) (Figure 5C, right graph below). This may reflect the higher probability of a fast moving motor from engaging hTpx2 molecules that are bound on the microtubule lattice.

To further confirm the inhibition of hKif15 by hTpx2 and to estimate the functional impact of hTpx2 inhibition on hKif15 functions beside movement, we included hTpx2 in our optical trap experiment. To do this, we captured a hKif15-linked bead and confirmed that it underwent stepping on the microtubule (Figures 5D, 1. blue trace). We then exchanged the buffer to allow 18 nM dimeric hTpx2 access to the hKif15-microtubule complex and again recorded the bead position. The hKif15 motor could no-longer step processively, although it remained bound to the microtubule lattice (4/5 cases; Figures 5D, 2. blue trace). To rule out that the bead had not simply detached, we moved the microscope stage and observed an increase in force indicating an intact microtubule-motor connection (see silver asterisk in Figure 5D). During this period, the bead can maintain microtubule attachment at forces that—in the absence of hTpx2—would have forced dissociation of the motor (compare also with Figure 2E). Thus, hTpx2 increases the force-holding capability of microtubule-bound hKif15. We can rule out a generic ‘roadblock’ inhibition-mechanism: Firstly, kinesin-1 motility is unaffected by the presence of hTpx2 (n = 5; Figure 5D, green traces). Secondly, hTpx2 already inhibits hKif15 motility at low nanomolar concentrations when only single molecules are present on the lattice. Kinesin-1 motility in the presence of hTpx2 also shows that inhibition of bead motility in case of hKif15 is not simply mediated by hTpx2-bead-microtubule interactions. Thus, hTpx2 is a selective inhibitor of hKif15 motor stepping presumably by forming stable trimeric hKif15-hTpx2-microtubule complexes.

The hKIF15 ORF was amplified in five 800- to 1000-bp sized fragments from a cDNA library (derived from hTERT immortalised retinal pigment epithelial (hTERT-RPE1 cells)) and subsequently joined by overlap extension PCRs using Pfu Ultra AD polymerase (Agilent, Stockport, UK). The complete hKIF15 ORF was cloned via AseI/NotI into a modified (bases 2391–2426 of the pIEx/Bac1-vector coding the Strep-Tag II were replaced by the His₆/TEV-cleavage site element of p11 [DNASU Plasmid ID: EvNO00085126, DNASU Plasmid Repository at Arizona State University], flanked by a 5′ NcoI and a 3′ NdeI site. [gift form Miho Katsuki, Riken, Japan]) pIEx/Bac1-vector (MERCK, Darmstadt, Germany) opened with NdeI/NotI. For hKIF15-eGFP an AscI-site was introduced in front of the NotI-site and the eGFP inserted via AscI/NotI before the hKIF15 ORF was inserted as above. We observed that the hKIF15 ORF is toxic to Escherichia coli cells when antibiotics other than ampicillin (e.g., kanamycin, gentamycin) are used, so viral genome generation by the Invitrogen Bac-to-Bac system (Life Technologies, Paisley, UK) using DH10BAC cells is not possible. The hTPX2 ORF was amplified from I.M.A.G.E. 3509275 (ATCC clone MG1537) and cloned into the pFastBacM13 vector (MPI-CBG, Dresden, Germany) via SpeI/SalI.

Assembly of viral genomes was carried out according to manufacturer protocols and transfection competent baculovirus particles were generated and used for transfection of 500 ml–1 L SF9-cells expression cultures according to (Wasilko et al., 2009). Cells were harvested at 250-g in a SLA-3000 rotor (Thermo Scientific, Waltham, MA, USA) and resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 3 mM EGTA, 5% glycerol, 0.1% Tween-20, 0.1 mM ATP, complete protease inhibitor [Roche, Burgess Hill, UK]).

For hKif15 purification, lysates were cleared at 48k-g in a SS-34 rotor (Thermo Scientific), diluted 1:3 (to 50 mM NaCl final) with 50 mM sodium phosphate buffer and adjusted to pH 7.0. Protein was allowed to bind to SP-sepharose (GE Healthcare, Little Chalfont, UK) in batch for 2 hr at 4°C. hKif15 was directly eluted by applying a threefold bed volume of 50 mM sodium phosphate buffer pH 7.5, 50 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20, 0.1 mM ATP onto Talon-beads (Takara Biotech, Saint-Germain-en-Laye, France). Again, protein was allowed to bind 2 hr at 4°C in batch. Talon-beads where washed with 10-fold bed volume of 50 mM sodium phosphate buffer pH 7.5, 300 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20, 0.1 mM ATP and a 15-fold bed volume of 50 mM sodium phosphate buffer pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20, 10 mM imidazole, 0.1 mM ATP. Protein was eluted with 50 mM sodium phosphate buffer pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 10% glycerol, 50 mM imidazole, 0.1 mM ATP and purity as well as concentration determined by SDS-PAGE against a BSA standard using ImageJ. For each protein preparation, the oligomerisation state and its dispersity have been checked by glycerol gradients or native PAGE. Note that for the SEC-MALS experiment, protein was eluted with 35 mM sodium phosphate buffer pH 7.0, 1 mM MgCl₂, 50 mM imidazole, 0.05 mM ATP and snap frozen in the absence of glycerol in 500 µl aliquots and stored in liquid nitrogen.

For purification of hTpx2 lysates were cleared and diluted 1:3 (to 50 mM NaCl) with 50 mM sodium phosphate buffer pH 7.5 as done for hKif15. Protein was allowed to bind to SP-sepharose in batch for 2 hr at 4°C. Beads were washed with a fivefold bed volume of 50 mM sodium phosphate buffer pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20 and protein was eluted with a threefold bed volume of 50 mM sodium phosphate buffer pH 7.5, 300 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20, 10 mM imidazole onto Talon-beads. Protein was allowed to bind 2 hr at 4°C in batch and beads were washed with a 25-fold bed volume of 50 mM sodium phosphate buffer pH 7.5, 150 mM NaCl, 10 mM imidazole, 1 mM MgCl₂, 5% glycerol, 0.05% Tween-20. Protein was eluted with 50 mM sodium phosphate buffer pH 7.5, 75 mM NaCl, 150 mM imidazole, 10% glycerol. Purity and concentration was determined as above. All protein was snap frozen in 5–20 µl aliquots and stored in liquid nitrogen.

Glycerol gradients were essentially carried out as described in McClelland and McAinsh (2009) in the presence of 1 mM DTT and complete protease protein inhibitor at 4°C for 15 hr over night. We checked for hKif15/hTpx2 interactions in 50 mM HEPES pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM ATP, 0/150/300/450 mM NaCl (150 mM NaCl also ± free tubulin in ATP or AMP-PNP) and 35 mM sodium phosphate buffer pH7.0, 1 mM MgCl₂, 1 mM EGTA, 0/150/300/500 mM NaCl, 0.1 mM ATP or AMP-PNP. The 5-mlgradient was fractionated by hand in fractions of 200 µl each. The protein was TCA-precipitated and fractions 1–23, together with ¹/₂ input, analysed on the same SDS-PAGE gel stained with coomassie (SimplyBlue, Invitrogen). Protein bands were quantified with ImageJ and peak values were determined by fitting a Gaussian distribution to the data using Origin 8.5. Selected proteins from the High/low molecular weight gel filtration calibration kit (GE Healthcare) were used in standard calibration runs.

Protein samples were concentrated to ∼2 mg/ml in 35 mM sodium phosphate buffer pH 7.0, 1 mM MgCl₂ and loaded onto a size exclusion column (Wyatt, Santa Barbara, CA, USA) connected to a Dawn Heleos II MALS detector and Optilab T-rEX refractometer (Wyatt). A dn/dc value of 0.185 was used for all calculations. Please note that for the SEC-MALS experiment only, protein-purification was modified in that hKi15 was finally eluted in 35 mM sodium phosphate buffer pH 7.0, 1 mM MgCl₂, 50 mM imidazole, 0.05 mM ATP and snap frozen in the absence of glycerol, which is likely to be the reason for the appearance of dimeric hKif15 in the SEC-MALS analysis that otherwise are absent in the preparation used in all the other experiments (as shown by native PAGE and glycerol gradients, see Figure 1B,C).

Microtubules for the sedimentation assay were grown in presence of 1 mM GTP in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl₂, 1 mM EGTA) by stepwise increasing the taxol concentration in solution. Polymerised microtubules were washed once with BRB80+Taxol using a Beckman Airfuge and resuspended in 35 mM sodium phosphate buffer pH 7.0, 1 mM MgCl₂, 1 mM EGTA and 1 mM DTT. Samples were mixed in the same buffer at the indicated concentrations (16.5 nM tetrameric hKif15, 33 nM dimeric hTpx2) and incubated for 20 min at room temperature in the presence of 2 mM ATP or AMP-PNP. Samples were spun for 20 min at 45k rpm in a TLA100 rotor (Beckman Coulter, High Wycombe, UK) using thick-wall polyallomer tubes (Beckman Coulter). The supernatants were TCA precipitated and analysed together with the resuspended pellets by SDS-PAGE. Protein bands were quantified using ImageJ. Since taxol stabilised microtubules are less stable in 35 mM sodium phosphate over long time periods and that hTpx2 has a stabilising effect on microtubules, we also quantified the actual amount of precipitated tubulin in order to compare both setups with and without hTpx2. This gives rise to the horizontal error bars in Figure 5B.

Preparation of sialylated coverslips and flow chamber setup (with double sided tape) was essentially carried out as described in Bechstedt et al. (2011), except for the initial cleaning step with piranha solution, which had been substituted by incubation with 7.4% hydrochloric acid at 60°C over night. For single molecule assays, GMP-CPP (Jena Bioscience, Jena, Germany) stabilised, polarity-marked microtubules labelled with XRhodamin- and HiLyte-647-linked tubulin (Cytoskeleton, Denver, CO, USA) were adsorbed to the glass surface via anti beta tubulin antibodies (TUB 2.1, Sigma-Aldrich, St.Louis, MO, USA) after the flow chamber has been blocked with 1% Pluronic F-127 (Sigma-Aldrich) and 1 mg/ml kappa casein. The flow chamber was loaded with 1–10 nM His₆-hKIF15-GFP (tetrameric) in BRB20 (20 mM PIPES pH6.8, 1 mM MgCl₂, 1 mM EGTA), 50 mM KCl, 2 mM ATP, 0.1 mg/ml kappa casein, 80 µg/ml glucose, 40 µg/ml glucose-oxidase, 16 µg/ml catalase, 1 mM DTT, sealed with VALAP (vaseline, lanolin, paraffin mixed 1:1:1) and imaged at 25°C on a Olympus CELL^R/TIRF microscope (Olympus, Southend-on-Sea, UK) equipped with a ImagEM emCCD camera (Hamamatsu Photonics, Welwyn Garden City, UK), an environmental chamber and a stage-top-incubator (Okolab, Ottaviano, Italy) using a 100x NA 1.49 objective with 1.6x auxiliary magnification.

To visualise hKif15–eGFP movement, 3-min time-lapse videos were recorded at 2 frames per second (fps) with a 100 ms exposure using a 488 nm laser line. For the bleaching experiments, videos were recorded at 4 fps. The position of polarised microtubules before and after the time-lapse video was determined by capturing a single image with the 561 nm (100 ms exposure) and 640 nm (100 ms exposure) laser lines. In General, kymographs were generated and analysed with ImageJ and the MultipleKymograph plugin (http://www.embl.de/eamnet/html/body_kymograph.html).

For the hTpx2 inhibition experiments, equimolar dimeric hTpx2 was directly loaded together with hKif15 into the flow chamber. Imaging was started after a short incubation period of 5 min at room temperature. To prevent a strong dilution of both proteins on the microtubule lattice, a microtubule density of 150 µm tubulin per field of view (51.2 × 51.2 µm) was not exceeded.

For microtubule sliding experiments, biotinylated (Cytoskeleton) and HiLyte-647-labelled GMP-CPP-microtubules were adsorbed via streptavidin to a PEG-biotin-coated (Surface Solutions Switzerland, Dübendorf, Switzerland) cover slip that has been blocked with 1 mg/ml kappa casein. Short XRhodamin-labelled GMP-CPP-microtubules were added as cargo to the imaging-mix (as above). Again, 3 min time-lapse videos at 2 frames per second (fps) were recorded to track hKif15-GFP and cargo microtubule movement. Exposure times were 100 ms with the 488 nm laser line and 150/200 ms with the 561 nm laser. Positions of the HiLyte-647-labelled substrate microtubules were determined by capturing a single image (100 ms exposure) with a 640 nm laser line both before and after the time-lapse video.

For the most part, instrumentation and sample preparation was the same as described in Carter and Cross (2005) with the following changes: motors were non-specifically bound to polystyrene beads (550 nm, NIST calibration size standards, Polysciences, Warrington, PA, USA) by incubation in a solution of 80 mM Pipes, 2 mM MgSO₄, 1 mM EGTA, 1 mM DTT, 3 mg/ml D-Glucose, 0.2 mg/ml casein and 1 μM ATP at pH 7.0. Before addition to the flow cell, the incubated bead–motor solutions were diluted in an assay buffer: 20 mM Pipes, 2 mM MgSO₄, 1 mM ATP, 1 mM EGTA, 1 mM DTT, 3 mg/ml D-Glucose, 0.4 mg/ml casein, 4 μM taxol, and a glucose oxidase/catalase-based oxygen scavenging system. For improved stability, recordings were made in flow-cells sealed with Dow Corning high vacuum grease (Dow Corning, Barry, UK).

Motor concentration was reduced until no more than one third of beads showed any binding. A bead-binding event was defined as a bead that remained attached to the microtubule for at least 2 s after the laser trap has turned off. Each bead was tested for binding on two or more different microtubules. Bead-binding data was fitted with two poisson probability curves using a linear least-squares method (Svoboda and Block, 1994). This analysis showed that our experimental hKif15 and Kinesin-1 data fit a ≥1 poisson distribution (the probability that a bead binds by one or more motors), rather than the ≥2 distribution (the probability that a bead binds by two or more motors). These data confirm that we are observing single motor (i.e., tetramer) motility at the low concentrations we used in our trapping experiments (3.3 and 6.5 nM hKif15, see Figure 2—figure supplement 2A).

Some hKif15-linked beads bound in a diffusive non force-producing state to the microtubule, a behaviour that was rarely observed with kinesin-1-linked beads and fits to our observation of diffusive motion in our TIRF assays.

In the case of the hTpx2 flow-through recordings, the samples were not sealed at the sides to allow wash-through. A bead with active motor was recorded and a further 3–4 cell volumes of assay buffer with 18 nM dimeric hTpx2 was carefully passed through the cell whilst retaining the same bead in the trap. The trapped bead was focused close to the lower cover-glass surface during these solution changes, this offered some boundary layer protection during the solution flow and also greatly reduced the chances of a second bead entering the trap. Following recordings in the presence of hTpx2, a further 3–4 cell volumes of assay buffer were passed through the flow cell to make the final recordings following hTpx2 wash out. Bead position data were recorded at 88 kHz and averaged down to 22 kHz for analysis and storage.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We would like to thank Miho Katsuki for providing the modified pIEx/Bac1 expression plasmid and Rob Cross for critical reading of this manuscript. We thank Warwick Medical School and Marie Curie Cancer Care for financial support.

© 2014, Drechsler et al.

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