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Armadillo repeat-containing kinesin represents the versatile plus-end-directed transporter in Physcomitrella

Nature Plants volume 9, pages 733–748 (2023)Cite this article

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Abstract

Kinesin-1, also known as conventional kinesin, is widely used for microtubule plus-end-directed (anterograde) transport of various cargos in animal cells. However, a motor functionally equivalent to the conventional kinesin has not been identified in plants, which lack the kinesin-1 genes. Here we show that plant-specific armadillo repeat-containing kinesin (ARK) is the long sought-after versatile anterograde transporter in plants. In ARK mutants of the moss Physcomitrium patens, the anterograde motility of nuclei, chloroplasts, mitochondria and secretory vesicles was suppressed. Ectopic expression of non-motile or tail-deleted ARK did not restore organelle distribution. Another prominent macroscopic phenotype of ARK mutants was the suppression of cell tip growth. We showed that this defect was attributed to the mislocalization of actin regulators, including RopGEFs; expression and forced apical localization of RopGEF3 partially rescued the growth phenotype of the ARK mutant. The mutant phenotypes were partially rescued by ARK homologues in Arabidopsis thaliana, suggesting the conservation of ARK functions in plants.

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Fig. 1: ARK disruption causes severe growth defects.
Fig. 2: ARK transports multiple organelles in protonemal cells.
Fig. 3: ARK promotes the movement of RabA2b-marked secretory vesicles.
Fig. 4: ARKb shows processive motility and is enriched around the nucleus in a tail-dependent manner.
Fig. 5: Cell tip growth was severely suppressed in the absence of ARK.
Fig. 6: ARK is necessary for the tip localization of RopGEFs.
Fig. 7: AtARK2 and AtARK3 expression partially rescues moss ARKabc-1.

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All materials and data are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We are grateful to M. Bezanilla, P. Yi and M. Yamada for providing the moss lines and plasmids; N. Oguri, C. Koketsu and R. Inaba for media preparation; and H. Motose for communicating unpublished data. This work was funded by the Japan Society for the Promotion of Science KAKENHI (17H06471, 18KK0202, 22H04717 and 22H02644 to G.G.). M.W.Y. is a recipient of the Japan Society for the Promotion of Science predoctoral fellowship.

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  1. Department of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan

    Mari W. Yoshida, Maya Hakozaki & Gohta Goshima

  2. Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Toba, Japan

    Gohta Goshima

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  1. Mari W. Yoshida
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M.W.Y. and G.G. designed the research. M.W.Y. performed the experiments and analysed the data. M.W.Y. and M.H. selected the transgenic lines. M.W.Y. and G.G. wrote the paper.

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Correspondence to Gohta Goshima.

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Extended data

Extended Data Fig. 1 Establishment of Physcomitrium patens ARK mutants.

(a)Three-week-old ARK mutant moss. mCherry-α-tubulin line was used as control. Scale bar: 5 mm. (b) Sequencing revealed frameshift mutations in the ARKabc-1 and ARKabcd-1 sequences used in this study (displayed as SnapGene sequence files). The altered amino acid sequences are shown in Supplementary Table 1. (c) Rhizoid images. The images presented in Fig. 1a are cropped and magnified. Scale bar: 1 mm. (d) Rhizoid length comparison. The mean length (mm) was 2.53 ± 0.132 (control, ±SEM, n = 40), 0.510 ± 0.0406 (ARKabc-1, ±SEM, n = 34), 2.21 ± 0.138 (ARKabc-1/ARKb-mNG full-length OX, ± SEM, n = 37), 0.683 ± 0.0427 (ARKabc-1/ARKb (T169N) -mNG OX, ± SEM, n = 38), 0.387 ± 0.0275 (ARKabcd-1, ±SEM, n = 30). P values were calculated using two-sided Steel–Dwass test; P < 0.0000001 (control—ARKabc-1), P < 0.0000001 (ARKabc-1 - ARKabc-1/ARKb-mNG OX), P = 0.0295 (ARKabc-1 - ARKabc-1/ARKb(T169N)-mNG OX). P = 0.3144 (ARKabc-1 - ARKabcd-1).

Extended Data Fig. 2 ARK deletion does not affect microtubule polymerization dynamics.

(a) Comparison of microtubule polymerization dynamics. Upper row, control (mCherry-α-tubulin) and ARKabc-1; lower row, control (GFP-α-tubulin/Histone-mCherry), and ARKabcd-1. The mean ± SEM, number of samples, and P values are shown in Supplementary Table 2. (b) Comparison of microtubule orientation in apical cells based on EB1-Citrine tracking. The quantification method is described in the Methods section. The frequency of tip-directed movement (%) was 88.5 ± 2.61 (mCherry-α-Tubulin, ±SEM, n = 16) and 97.0 ± 1.16 (ARKabc-1, ±SEM, n = 18). The P value was calculated using two-sided Mann–Whitney U-test; P = 0.0366.

Extended Data Fig. 3 Organelle motility rate.

(a) Rate of nuclear motility in apical cells of the control and ΔKCHabcd lines. Rate of nuclear anterograde motility in apical cells of the ΔKCHabcd line was measured to exclude retrograde motility. Mean rate: 11.6 ± 3.44 nm/s (control, ±SD, n = 23), 11.1 ± 6.49 nm/s (ΔKCHabcd, ±SD, n = 24). (b) Rate of chloroplast motility in apical cells of the control and ΔKCBPabcd lines. Rate of chloroplast anterograde motility in apical cells of the ΔKCBPabcd line was measured to exclude retrograde motility. The mean rate was 35.7 ± 1.91 nm/s (control, apical direction, ±SD, n = 101) or 42.9 ± 24.2 nm/s (ΔKCBPabcd, apical direction, ±SD, n = 160). (c) Rate of mitochondrial motility in apical cells of the control line (mCherry-α-tubulin). The mean rate was 219 ± 110 nm/s (apical direction, ±SD, n = 15). (d) Rate of motility of RabA2b-positive vesicle in apical cells of the control line (mCherry-α-tubulin). The mean rate was 922 ± 674 nm/s (apical direction, ±SD, n = 85).

Extended Data Fig. 4 The truncation-rescue assay reveals the essentiality of ARM repeats.

(a) Truncated ARKb-mNG constructs used in this experiment. The protein domains were predicted using NCBI’s conserved domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart.embl.de). (b) Area comparison of 3-week-old moss. A single protoplast was cultured for three weeks on BCDAT medium. The datasets of ARKabc-1/FL (full-length) are identical to those shown in Fig. 1b. The mean area (mm2) was 4.88 ± 0.651 (ARKabc-1/ΔARM, ± SEM, n = 18), 7.04 ± 1.07 (ARKabc-1/ΔTail, ±SEM, n = 18). (c) Relative intensity of chloroplasts along the apical cell at 150 min after anaphase onset (mean ± SEM). The chloroplasts remained accumulated near the basal cell wall after the expression of the ΔARM (n = 7) or ΔTail (n = 12) construct.

Extended Data Fig. 5 ARK deletion affects cell length and Arp3a localization, but not the cell cycle duration or Rop4 localization.

(a) Comparison of cell cycle durations. The mean duration (h) was 7.43 ± 0.307 (control, ±SEM, n = 24), 7.58 ± 0.232 (ARKabc-1, ±SEM, n = 13), 6.93 ± 0.220 (ARKabc-1/ARKb-mNG OX, ± SEM, n = 36). (b) Comparison of tip growth rates in the presence of 500 pM or 50 nM latrunculin A (LatA). The mCherry-α-tubulin line was used for drug treatment. The mean rate (µm/h) was 9.69 ± 1.20 (ARKabc-1, ±SEM, n = 5), 23.0 ± 1.00 (control [+0.5% DMSO], ± SEM, n = 5), 16.7 ± 1.20 (+500 pM LatA, ±SEM, n = 6), 9.64 ± 0.702 (+50 nM LatA, ± SEM, n = 17). (c) Representative images and comparison of intensities of functional Rop4-mNG at the cell tip. Images were acquired using a spinning-disc confocal microscope with a z-series taken every 0.5 µm for a range of 15 µm. The best focal plane is presented. Scale bar: 5 µm. The mean intensity was 1.29 ± 0.0724 (control, ±SEM, n = 6), 1.54 ± 0.140 (ARKabc-1, ±SEM, n = 6). (d) Representative images and comparison of intensities of Arp3a-mNG at the cell tip. Images were acquired using a spinning-disc confocal microscope with a z-series taken every 0.5 µm for a range of 15 µm. The best focal plane is presented. Scale bar: 5 µm. The mean intensity was 1.75 ± 0.0814 (control, ±SEM, n = 29), 0.726 ± 0.107 (ARKabc-1, ±SEM, n = 20). P value was calculated using Student’s two-sample t-test: P < 0.0000001 (Control—ARKabc-1). (e) The apical accumulation of RopGEF3 and RopGEF6 depends on the microtubules. Snapshots of the same cell before and 50-min after oryzalin addition are shown. DMSO was used as a control. mCherry-α-tubulin-expressing RopGEF3-mNG or RopGEF6-mNG was used in this experiment. Images were acquired with a spinning-disc confocal microscope using a z-series taken every 1.5 µm for a range of 6 µm. The best focal plane is presented. Scale bar: 5 µm. (f) Temporal changes in RopGEF3-mNG or RopGEF6-mNG intensity at the cell tip after drug addition. A decrease in localization was observed after microtubule disruption with oryzalin. Intensities relative to those before drug treatment are plotted (±SEM). DMSO control (RopGEF3-mNG), n = 12. +Oryzalin (RopGEF3-mNG), n = 6. DMSO control (RopGEF6-mNG), n = 8. +Oryzalin (RopGEF6-mNG), n = 13. (g)- (I) Relative intensity between tip and cytoplasm. Mean ±SEM. The identical dataset to Fig. 6d–f was used. (G) For2A-mNG; 4.28 ± 0.222 (n = 36), 2.98 ± 0.222 (n = 36), 4.71 ± 0.184 (n = 36). P values were calculated using two-sided Games–Howell test; P = 0.0003 (control—ARKabc-1), P < 0.0000001 (ARKabc-1 - ARKabc-1/ARKb OX). (H) RopGEF3-mNG; 1.30 ± 0.0274 (n = 48), 1.11 ± 0.0247 (n = 85), 1.43 ± 0.0231 (n = 74). P values were calculated using two-sided Steel–Dwass test; P < 0.0000001 (control—ARKabc-1), P < 0.0000001 (ARKabc-1 - ARKabc-1/ARKb OX). (I) RopGEF6-mNG; 3.43 ± 0.236 (n = 43), 2.03 ± 0.159 (n = 29), 3.26 ± 0.295 (n = 30). P values were calculated using two-sided Steel–Dwass test; P < 0.0000001 (control—ARKabc-1), P < 0.0000001 (ARKabc-1 - ARKabc-1/ARKb OX).

Extended Data Fig. 6 RNAi of ARKd in ARKabc-1 causes abnormal outgrowth.

(a) Representative image sequences of abnormal cell growth after ARKd RNAi induction by β-estradiol. Left, outgrowth in the apical cell; right, outgrowth in the subapical cell. Scale bars, 50 µm. Images were acquired using a wide-field microscope with transmission light. (b) Comparison of apical cell shape after 1 µM β-estradiol or control 0.1% DMSO treatment. The numbers shown in the bar graphs indicate the actual number of counted cells.

Extended Data Fig. 7 ARK deletion does not affect RopGEF or For2A expression level.

(a) (left) A test result of Western blot following immunoprecipitation (IP). Moss lines overexpressing MO25A2-mNG-FLAG100 and the RopGEF3-mNG-FLAG (endogenously-tagged) lines were used. Anti-FLAG-conjugated magnetic agarose beads were used for IP, whereas anti-mNeonGreen antibody was used for western blotting. The negative control represents a moss line without mNG expression. Red arrowheads indicate the RopGEF3-mNG-FLAG protein size. Detection of MO25A2 with much brighter signals than RopGEF3 indicated that the antibody attached to the beads was not saturated during IP of RopGEF3-mNG-FLAG. (right) Loading control. The moss extracts used for IP were subjected to SDS–PAGE, followed by Coomassie Brilliant Blue staining. (b) - (d) (top) Western blot following IP (mNG-FLAG-tagged For2A, RopGEF3, and RopGEF6). The identical method to (A) was applied. The negative control represents a moss line without mNG expression. Red arrowheads indicate the target protein size; smaller bands indicate degradation products or cross-reactions. The results of band intensity quantification are shown in Supplementary Table 4. (bottom) Coomassie staining of the extracts.

Source data

Extended Data Fig. 8 AtARK2 and AtARK3 expression rescued rhizoid development, but did not alter microtubule polymerization dynamics.

(a) Representative images of gametophores in the indicated lines. The image of the mutant line is identical to that presented in Fig. 1a. Scale bar: 1 mm. (b) Rhizoid length comparison. The mean length (mm) was 0.510 ± 0.0406 (ARKabc-1, ±SEM, n = 34), 1.14 ± 0.0793 (ARKabc-1/ARK2-mNG OX, ± SEM, n = 41), 1.37 ± 0.0992 (ARKabc-1/ARK3-mNG OX, ± SEM, n = 43). The dataset of ARKabc-1 is identical to that shown in Extended Data Fig. 1d. P values were calculated using the two-sided Steel–Dwass test: P = 0.00000003 (ARKabc-1 - ARKabc-1/ARK2-mNG OX), P < 0.0000001 (ARKabc-1 - ARKabc-1/ARK3-mNG OX). (c) Comparison of microtubule polymerization dynamics. The mean ± SEM, number of samples, and P values are shown in Supplementary Table 3; none of the P values were <0.05.

Extended Data Fig. 9 Confirmation of the moss lines established in this study.

Genotyping PCR strategy (left) and PCR results (right) for the moss lines established in this study. (a) Exogenous integration, (b) C-terminal tagging, (c) N-terminal tagging, and (d) knockout. Band-size markers are shown at the leftmost lane of each panel, and the actual band sizes are described in (e). Asterisks indicate non-specific bands. The number in each lane indicates the line ID, and the genotype of each ID is shown in Supplementary Table 5. ‘C’ indicates the control (parental line). Primers used are listed in Supplementary Table 8.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: Mutant alleles used in this study. Supplementary Table 2: Statistical analysis of microtubule dynamics in ARK mutants. Supplementary Table 3: Statistical analysis of microtubule dynamics in moss lines expressing AtARK2 or AtARK3. Supplementary Table 4: Band intensity after immunoprecipitation. Supplementary Table 5: Moss lines used in this study. Supplementary Table 6: Plasmids used in this study. Supplementary Table 7: Primers used for plasmid construction and sequencing. Supplementary Table 8: Primers used for genotyping PCR.

Supplementary Video 1

Overall basal motility of chloroplasts after cell division in ARKabc-1. Time-lapse video of microtubules (mCherry-α-tubulin, magenta) and chloroplasts (autofluorescence, cyan) in apical cells after anaphase onset (0.0 min). The bright magenta signals on the left represent the anaphase spindles and phragmoplasts. White arrowheads indicate the positions of the nuclei. Control (expressing mCherry-α-tubulin) and ARKabc-1 lines are also shown. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (2.5 µm ×3 sections). Scale bar, 20 µm.

Supplementary Video 2

Motility of mitochondria along cytoplasmic microtubules. Bidirectional movement of a mitochondrion (γF1ATPase-mNG, green) along the microtubule (mCherry-α-tubulin, magenta). Images were acquired using oblique illumination fluorescence microscopy. The faint green signals on the left represent chloroplast autofluorescence. Scale bar, 1 µm.

Supplementary Video 3

Cytoplasmic movement of mNG-RabA2b-marked vesicles. Time-lapse video of cytoplasmic movement of RabA2b-marked vesicles (mNG-RabA2b) in the control, ARKabc-1 mutant, and rescue lines.Videos were acquired using spinning-disc confocal microscopy. Autofluorescent chloroplasts are also faintly visualized. Scale bar, 20 µm.

Supplementary Video 4

mNG-RabA2b-marked vesicles at the protonemal cell tip. Time-lapse video of the cluster of mNG-RabA2b-positive vesicles (green) at the cell tip. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (0.5 µm ×35 sections). Scale bar, 10 µm.

Supplementary Video 5

Dynamics of cytoplasmic microtubules. Time-lapse video of cytoplasmic microtubules (GFP-α-tubulin) in interphase protonemal cells of control and ARKabdc-1 lines. Microtubule dynamics were comparable between these lines (Supplementary Table 2). Videos were acquired using oblique illumination fluorescence microscopy. Scale bar, 2 µm.

Supplementary Video 6

Motility of ARKb-mNG on cytoplasmic microtubules. Time-lapse video of ARKb-mNG obtained using oblique illumination fluorescence microscopy. White arrowheads indicate the puncta of ARKb-mNG that move processively and unidirectionally on the microtubules. Magenta, tubulin; green, ARKb-mNG. Scale bar, 1 µm.

Supplementary Video 7

In vitro motility of ARKb motor on microtubules. Time-lapse video of recombinant ARKb (1–613 aa)-GFP on microtubules obtained using TIRF microscopy (three microtubules are shown). Magenta, tubulin; green, ARKb-GFP. Scale bar, 5 µm.

Supplementary Video 8

Motor- or tail-deficient ARKb does not suppress chloroplast mispositioning or reduced tip growth in ARKabc-1 mutant. Time-lapse video of protonemal cells expressing truncated or mutated ARKb constructs. Magenta, mCherry-α-tubulin; cyan, chloroplast. Videos were acquired using an epifluorescence (wide-field) microscope at a single focal plane. Scale bar, 100 µm.

Supplementary Video 9

RNAi of ARKd in ARKabc-1 causes abnormal outgrowth. Time-lapse video of protonemal cells after ARKd RNAi induction by β-estradiol treatment. Left: outgrowth in the apical cell. Middle: outgrowth in the subapical cell. Right: no RNAi induction (control). Videos were acquired using a wide-field microscope with transmission light (×20 0.75 lens). Scale bar, 50 µm.

Supplementary Video 10

Instability of actin foci at the cell tip in ARKabc-1 mutant. Time-lapse video of actin near the cell tip (lifeact-mNG). Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (0.5 µm ×7 sections). Scale bar, 5 µm.

Supplementary Video 11

Actin foci disappear with cells expansion. Time-lapse video of lifeact-mNG (green) at the cell tip. Four cells are shown. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (1 µm ×23 sections). Scale bar, 10 µm.

Supplementary Video 12

RopGEF3 at the protonemal cell tip. Time-lapse video of RopGEF3-3×mNG (green) at the cell tip. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using a spinning-disc confocal microscope with a z-series taken every 0.5 µm for a range of 17 µm. The best focal plane is presented. Scale bar, 10 µm.

Supplementary Video 13

RopGEF6 at the protonemal cell tip. Time-lapse video of RopGEF6-3×mNG (green) at the cell tip. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using a spinning-disc confocal microscope with a z-series taken every 0.5 µm for a range of 17 µm. The best focal plane is presented. Scale bar, 10 µm.

Supplementary Video 14

For2A at the protonemal cell tip. Time-lapse video of For2A-mNG (green) at the cell tip. Magenta, mCherry-α-tubulin. Note that autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using a spinning-disc confocal microscope with a z-series taken every 0.5 µm for a range of 17 µm. The best focal plane is presented. Scale bar, 10 µm.

Supplementary Video 15

Dynamics of filamentous actin in the endoplasm. Time-lapse video of filamentous actin (lifeact-mNG) in interphase protonemal cells obtained using oblique illumination fluorescence microscopy. Tracking of individual F-actin ends revealed a slightly reduced growth rate in the mutant line (Fig. 5f). Scale bar, 5 µm.

Supplementary Video 16

Apical RopGEF3 signals diminish with cell expansion. Time-lapse video of RopGEF3-3×mNG (green) at the cell tip. Three cells are shown. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (1 µm ×23 sections). Scale bar, 10 µm.

Supplementary Video 17

Apical RopGEF6 signals diminish with cell expansion. Time-lapse video of RopGEF6-3×mNG (green) at the cell tip. Three cells are shown. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (1 µm ×23 sections). Scale bar, 10 µm.

Supplementary Video 18

Apical For2A signals diminish with cell expansion. Time-lapse video of For2A-mNG (green) at the cell tip. Three cells are shown. Magenta, mCherry-α-tubulin. Autofluorescent chloroplasts in the cytoplasm are also visible in the green channel. Videos were acquired using spinning-disc confocal microscopy and processed using maximum z-projection (1 µm ×23 sections). Scale bar, 10 µm.

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Source Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed gels.

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Yoshida, M.W., Hakozaki, M. & Goshima, G. Armadillo repeat-containing kinesin represents the versatile plus-end-directed transporter in Physcomitrella. Nat. Plants 9, 733–748 (2023). https://doi.org/10.1038/s41477-023-01397-x

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