SUPPLEMENTARY INFORMATION

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1 DOI: /NPLANTS The non-processive rice kinesin-14 OsKCH1 transports actin filaments along microtubules with two distinct velocities Figure S1 The OsKCH1 constructs used in this study Schematic representation of the domain organization in wt OsKCH1 and the recombinant constructs used in this study, OsKCH1(aa1-744), OsKCH1(aa4-744), and OsKCH1(aa ). The full-length protein OsKCH1 has a sequence of 954 amino acids with a central kinesin-14 motor domain flanked by putative coiled-coils and an N-terminal calponin-homology (CH) domain. The OsKCH1(aa1-744) and OsKCH1(aa4-744) constructs contain the kinesin motor, the dimerization, and the CH domains and are His-tagged at the C- or N-terminus, respectively. The OsKCH1(aa ) lacks the CH domain and has an N-terminal His-tag whereas the mfgfp-oskch1(aa ) has an N-terminal mfgfp 1. All constructs lack the C-terminal 210 amino acids that form a domain of unknown function. Figure S2 The transport velocity is independent of the actin filament length. The length of 201 actin filaments plotted against the respective transport velocity. The mean lengths of filaments transported with a velocity up to 50 nm s -1 (l 0-50 = 1.56 ± 1.31 µm, mean +/- SD, N = 97), with a velocity between 50 and 100 nm s -1 (l = 2.08 ± 1.53 µm, N = 62), and with a velocity between 100 and 150 nm s -1 (l = 1.92 ± 1.52 µm, N = 36) are indicated in red. NATURE PLANTS 1

2 DOI: /NPLANTS.2015.X Figure S3 Flipping of an actin filament at the microtubule end. Schematic representation of an actin filament reaching the microtubule end where it flips around by 180. Figure S4 OsKCH1 is a non-processive motor. (a) Schematic representation of the single -molecule stepping assay using mfgfp-oskch1(aa ) (see Methods for details). (b) Typical kymograph (derived from movie 4) showing the transient interactions of OsKCH1 with the microtubule indicative of the non-processivity of the motors. 2 NATURE PLANTS

3 DOI: /NPLANTS.2015.X SUPPLEMENTARY INFORMATION Figure S5 OsKCH1(aa4-744) transports actin filaments along microtubules with two distinct velocities. (a-b) Fluorescence micrographs of two representative actin filaments f1 and f2 (red) being transported by OsKCH1(aa4-744) along a microtubule (green) at different points in time. The starting positions of the transported actin filaments are marked by the white arrowheads. (c) Kymographs of the two actin filaments shown in (a-b). The slopes indicate two different transport velocities for the two actin filaments. (d) Histogram of the instantaneous frameto-frame transport velocities for 15 independent actin filaments. Two distinct velocity populations are visible (v 1 and v 2). A double Gaussian fit of the histogram delivers the velocities v 1 = 111 ± 13 nm s -1 and v 2 = 15 ± 23 nm s -1 (mean ± SD). SUPPLEMENTAL METHODS OsKCH1 expression and purification. The expression plasmid for OsKCH1(aa1-744) was obtained from Prof. Dr. Peter Nick (Karlsruher Institut für Technologie). The other constructs were derived from the latter using standard PCR and cloning techniques. C-terminally hexahistidine-tagged OsKCH1(1-744) and N-terminally octo-histidine-tagged OsKCH1(4-744) and OsKCH1( ) were expressed in E. coli BL21(DE3)-pRARE (Millipore) induced with 0.2 mm IPTG for 16 h at 16 C. Harvested cells were resuspended in buffer A (274 mm NaCl, 5.4 mm KCl, 16.2 mm Na 2HPO 4, 3.52 mm KH 2PO 4, 2 mm MgCl 2, 1 mm ATP, 1 mm dithiothreitol (DTT), and EDTA-free protease inhibitors (Roche), ph 7.4) and lysed using an EmulsiFlex high pressure homogenizer (Avestin) at 4 C. The crude lysate was centrifuged at 17,400 g at 4 C and loaded onto a HiTrap NiNTA column (GE Healthcare). The column was washed with 3 x buffer A containing 30 mm imidazole. Proteins were eluted in buffer A containing 500 mm imidazole, ph 8.0. Proteins were snap-frozen in liquid nitrogen and stored at -80 C. NATURE PLANTS 3

4 DOI: /NPLANTS.2015.X Microtubules and actin filaments. Microtubules were polymerized as described before 2 using a mixture of either digoxygenin-labeled, Cy5-labeled and unlabeled (6:1:5) tubulin for the transport motility assays, Alexa488-labeled tubulin for the rotation assays, or DyLight594-labeled tubulin for the gliding assays. Polarity-marked microtubules were grown as described previously 3 using rhodamine-labeled tubulin for the seeds and Alexa488-labeled tubulin for elongation. Actin filaments were polymerized from 3 µm rabbit skeletal muscle actin in 10 mm 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid/naoh ph7.0, 100 mm KCl, 2 mm ATP, and 5 µm rhodaminephalloidin (life technologies) for 60 min at 4 C. Gliding motility assay. Flow chambers with hydrophobic glass surfaces were prepared as described previously 2. To immobilize OsKCH1 to the glass surface, channels were washed with a series of buffers. First a penta-his antibody (Qiagen) at concentrations of 5, 10, or 20 µg ml 1 in BRB80 (80 mm piperazine-n,n'-bis(2-ethanesulfonic acid)/koh ph6.9, 1 mm MgCl 2, 1 mm ethylene glycol tetraacetic acid) was incubated for 1-2 min followed by 45 min incubation of 1% Pluronic F127 (Sigma) in BRB80. Then 1 µm OsKCH1(aa ) in BRB80, 10 mm DTT, 2 mm ATP were allowed to bind to the antibodies for 5 min. Microtubules diluted in BRB80, 10 µm taxol, 10 mm DTT, 2 mm ATP were allowed to bind to the motors before finally adding the imaging solution (BRB80, 10 µm taxol, 10 mm DTT, 2 mm ATP, 40 mm glucose, 40 µg ml 1 glucose oxidase, 16 µg ml 1 catalase). Because the applied motor concentration was in large excess of the applied antibody concentration, distinct surface densities of the OsKCH1 motors were obtained by varying the antibody concentration. Transport motility assay. Microtubules co-labeled with digoxygenin and Cy5 were immobilized to the glass surface via digoxygenin antibodies (Roche). After blocking with 1% Pluronic F127 in BRB80 for 45 min, 0.2 µm OsKCH1(aa1-744) in BRB80, 10 µm taxol, 10 mm DTT, 0.1 mg ml 1 casein, 0.1 % Tween20 were added to the microtubules in absence of ATP. After 5 min incubation actin filaments in imaging solution were allowed to bind to the motors. After 1 min unbound actin filaments were washed out with imaging solution containing 0.1 µm OsKCH1(aa1-744) 0.1 mg ml 1 casein, and 0.1 % Tween20. Single-molecule stepping assay. Microtubules labeled with DyLight594 were immobilized to glass surface blocked with Pluronic F-127 via tubulin antibodies. After 1 min unbound microtubules were washed out with imaging solution containing 10 nm mfgfp-oskch1(aa ). 4 NATURE PLANTS

5 DOI: /NPLANTS.2015.X SUPPLEMENTARY INFORMATION Single-molecule rotation assays. For the rotation assays OsKCH1(4-744) molecules were sparsely attached to the surface via penta-his antibodies (Qiagen) or actin filaments immobilized to the surface via rhodamine antibodies (abcam). The rotation angles θ (as defined in Fig. 3c) were measured using ImageJ (NIH). To avoid the inclusion of microtubules which were attached to more than one motor into the angle analysis the following criteria were applied: (i) microtubules needed to rotate around a single attachment point for the whole observation period and (ii) both microtubule ends needed to show rotational movement in a correlated manner. Imaging and data analysis. Cy5-, DyLight594, or Alexa488-labeled microtubules and rhodamine-labeled actin filaments were visualized using epi-illumination with the respective filter sets. Images were acquired by the MetaMorph (Universal Imaging) or NIS (Nikon) software packages using an EMCCD camera (Ixon DV 897, Andor) mounted on an inverted fluorescence microscope (Axiovert 200M, Zeiss or Ti-E, Nikon) equipped with an Alpha Plan-Apochromat 63xoil 1.46 NA DIC objective (Zeiss) or a CFI Apochromat 100xoil 1.49 NA objective (Nikon), and an autofocus system (Zeiss or Nikon respectively) at an acquisition rate of 1 frame per 2 or 3 seconds for gliding and transport assays. Single-molecule stepping assays were performed on the Nikon setup using a motorized TIRF slider (Nikon) and a 488 nm laser (Nikon) at 10 frames per second. Positions of microtubules and actin filaments were obtained using FIESTA tracking software as described before 4. Mean velocities were determined by fitting the velocity histograms to Gaussian functions using MatLab (Mathworks). SUPPLEMENTAL REFERENCES 1 Kobayashi, T. et al. Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PloS one 3, e3822, doi: /journal.pone (2008). 2 Fink, G. et al. The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding. Nature cell biology 11, , doi: /ncb1877 (2009). 3 Helenius, J., Brouhard, G., Kalaidzidis, Y., Diez, S. & Howard, J. The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441, , doi: /nature04736 (2006). 4 Ruhnow, F., Zwicker, D. & Diez, S. Tracking single particles and elongated filaments with nanometer precision. Biophysical journal 100, , doi: /j.bpj (2011). NATURE PLANTS 5

6 DOI: /NPLANTS.2015.X SUPPORTING MOVIES Movie 1: Movie 2: Movie 3: Movie 4: Movie 5: Movie 6: Movie 7: Representative example of two actin filaments (red) transported along a microtubule (green) by OsKCH1(aa1-744) with distinct velocities. Shown in the movie are two microtubules of presumably opposite polarity in close proximity. The described movement occurs on the lower one. The progressive lateral offset of the motile actin filaments from the microtubule(s) over time is due to spatial drift in conjunction with the microtubule image acquired only once at the beginning of the sequence. Representative example of an actin filament transported along a microtubule by OsKCH1(aa1-744) and flipping around at the end of a microtubule. Each flipping event coincides with a change in transport velocity (v 1 or v 2 ). OsKCH1(aa ) gliding motility assay with polarity-marked microtubules. mfgfp-oskch1(aa ) single-molecule assay showing non-processive interactions. Representative example of two actin filaments transported along microtubules by OsKCH1(aa4-744) with distinct velocities. Representative example of a microtubule rotating on a single OsKCH1(aa4-744) molecule attached to the surface via a penta-his antibody. Representative example of a microtubule rotating on a single OsKCH1(aa4-744) molecule attached to the surface via a surface-bound actin filament. 6 NATURE PLANTS