Clustering of a kinesin-14 motor enables processive retrograde microtubule-based transport in plants

Abstract

The molecular motors kinesin and dynein drive bidirectional motility along microtubules (MTs) in most eukaryotic cells. Land plants, however, are a notable exception, because they contain a large number of kinesins but lack cytoplasmic dynein, the foremost processive retrograde transporter. It remains unclear how plants achieve retrograde cargo transport without dynein. Here, we have analysed the motility of the six members of minus-end-directed kinesin-14 motors in the moss Physcomitrella patens and found that none are processive as native dimers. However, when artificially clustered into as little as dimer of dimers, the type-VI kinesin-14 (a homologue of Arabidopsis KCBP (kinesin-like calmodulin binding protein)) exhibited highly processive and fast motility (up to 0.6 μm s^-1). Multiple kin14-VI dimers attached to liposomes also induced transport of this membrane cargo over several microns. Consistent with these results, in vivo observations of green fluorescent protein-tagged kin14-VI in moss cells revealed fluorescent punctae that moved processively towards the minus-ends of the cytoplasmic MTs. These data suggest that clustering of a kinesin-14 motor serves as a dynein-independent mechanism for retrograde transport in plants.

Figure 1. Four kinesin-14 subgroup members exhibit minus-end-directed motor activity

a, Gene maps of the six subgroups of kinesin-14s in Physcomitrella patens. b, Coomassie blue staining after SDS-PAGE of the purified proteins used in the motility assays (FL stands for full length). c, An example of a gliding assay used to determine motor velocity and directionality (in this case kin14-Ia). MT minus-ends are labelled in red. Scale bar, 5 μm (left) or 2 μm (right). d, Gliding velocities. Bars represent the mean velocity of two independent experiments utilizing different protein purifications; solid and filled circles show the results from each preparation. Each circle represents the mean velocity of at least 100 motile MTs.

Figure 2. Artificially tetramerized kin14-VIb showed processive motility

a, A kymograph for dimeric kin14-VIb motors reveals no processive movement. b, A kymograph for the GFP-kin14-VIb GCN4 tetramer construct exhibits clear processive movement (diagonal lines) and long run lengths. Movie frames show two separate GFP spots (arrows), moving along a MT (blue). c, Representative traces of the photobleaching of kin14-VIb FL and the kin14-VIb GCN4 tetramer. d, Quantitation of the number of photobleaching steps. e, Velocity histogram of the kin14-VIb GCN4 tetramer with mean of 336 ± 97 nm s^{−1 (}mean ± s.d., n=267). f, 1 – cumulative frequency for run lengths of the GCN4 tetramer construct, which were fitted to an exponential yielding a fit parameter of λ = 1.27±0.03 μm (error was determined from goodness of fit parameters; R² = 0.995, n=267 particles).

Figure 3. Kin14-VIb transports liposomes along MTs

a, The attachment of histidine-tagged motor to Ni-NTA lipids incorporated into liposomes. b, Transport of liposomes. Two separate motor-coated fluorescently-labelled liposomes (red, indicated by arrows) can be seen moving along a Cy5-labelled MT (blue). Bar, 2 μm. c, A kymograph shows long run lengths of kin14-VIb-coated liposomes. d, Velocity histograms for liposomes coated with kin14-VIb (266 ± 69 nm s⁻¹; mean±s.d., n = 100 particles) and kin14-VIb FL (597 ± 134 nm s⁻¹; mean±s.d., n = 116 particles).

Figure 4. Minus-end-directed motility of kin14-VIb clusters in vivo

a, Protonemal cells were imaged in this study. Scale bars, 5 mm (left) and 100 μm (right). b, The endoplasm close to the cell cortex was observed with oblique illumination fluorescence microscopy (green; Citrine-kin14-VIb, magenta; mCherry-tubulin). Note that most of the MTs visualized in this area are single MTs, not bundles. Scale bar, 5 μm. c, Citrine-kin14-VIb signals moved away from the growing plus-end (white arrow in the kymograph). (Right) An example of Citrine movement (yellow arrows). Scale bar, 2 μm. d, The velocity of Citrine-kin14-VIb motility (n = 29). Static signals were not counted. e, 1 – cumulative frequency for run lengths of Citrine-kin14-VIb which were fit to an exponential yielding a fit parameter of λ = 1.01 ± 0.31 μm (error was determined from goodness-of-fit parameters; R²=0.942, n = 26 particles).