Figure 5.
CSLD6 moves in linear trajectories on the plasma membrane, which are specifically inhibited by DCB. (A) Moss protonemata expressing mEGFP-CESA10, mEGFP-CSLD6, or mEGFP-CSLD6-TEN imaged with VAEM. mEGFP-CSLD6-TEN did not move in the membrane. 20 µM isoxaben did not affect CSLD6 particle movement, but treatment with 10 µM DCB treatment inhibited CSLD6 particle motility. Scale bar for all images in A, 2 µm (horizontal) and 1 min (vertical). Kymographs were generated along a trajectory in the time projection. Also see Video 5. (B) Histogram of particle speed as determined by particle tracking with the Fiji plugin TrackMate. On average CSLD6 particles moved faster than CESA10. (C) Speed measurements from kymographs (CESA10, n = 44 trajectories from two cells; CSLD6, n = 119 trajectories from two cells) or from particle tracking (CESA10, n = 72 trajectories from two cells; CSLD6, n = 198 trajectories from two cells) were the same. n.s. denotes no significant difference as determined by a Kruskal–Wallis statistical test. (D) Images of phyllids from gametophores of the indicated genotype show that the CSLD6-TEN allele (containing the D867N mutation) exhibits abnormally shaped cells similar to phyllids from csld2/6KO plants (see Fig. 2). Scale bar for all images in D, 100 µm. (E) Confinement ratio shows that CESA10 trajectories from ten cells were straighter than CSLD6 trajectories (CESA10, n = 271 from ten cells; CSLD6, n = 682 trajectories from five cells, CSLD6+isoxaben, n = 486 trajectories from six cells). Asterisks denote P < 0.001 and n.s. denotes no significant difference as determined by a Kruskal–Wallis statistical test. Scale bar for all images in E, 2 µm. (F) Histogram of CSLD6 particle speed with or without isoxaben treatment as determined by particle tracking with the Fiji plugin TrackMate. (G and H) Single focal plane confocal time-lapse image of protonemata expressing mScarlet-CSLD6 with no drug (G), 20 µM DCB, or 20 µM isoxaben (H). Scale bar for all images in G and H, 5 µm. Also see Video 6. (G) CSLD6 accumulated at the cell apex and appeared as punctae (yellow arrowheads). (H) With DCB treatment, CSLD6 still accumulated at the cell apex but the tip cell ruptures (blue arrowhead). Isoxaben treatment did not cause cell rupture and did not affect CSLD6 localization to the cell tip.

CSLD6 moves in linear trajectories on the plasma membrane, which are specifically inhibited by DCB. (A) Moss protonemata expressing mEGFP-CESA10, mEGFP-CSLD6, or mEGFP-CSLD6-TEN imaged with VAEM. mEGFP-CSLD6-TEN did not move in the membrane. 20 µM isoxaben did not affect CSLD6 particle movement, but treatment with 10 µM DCB treatment inhibited CSLD6 particle motility. Scale bar for all images in A, 2 µm (horizontal) and 1 min (vertical). Kymographs were generated along a trajectory in the time projection. Also see Video 5. (B) Histogram of particle speed as determined by particle tracking with the Fiji plugin TrackMate. On average CSLD6 particles moved faster than CESA10. (C) Speed measurements from kymographs (CESA10, n = 44 trajectories from two cells; CSLD6, n = 119 trajectories from two cells) or from particle tracking (CESA10, n = 72 trajectories from two cells; CSLD6, n = 198 trajectories from two cells) were the same. n.s. denotes no significant difference as determined by a Kruskal–Wallis statistical test. (D) Images of phyllids from gametophores of the indicated genotype show that the CSLD6-TEN allele (containing the D867N mutation) exhibits abnormally shaped cells similar to phyllids from csld2/6KO plants (see Fig. 2). Scale bar for all images in D, 100 µm. (E) Confinement ratio shows that CESA10 trajectories from ten cells were straighter than CSLD6 trajectories (CESA10, n = 271 from ten cells; CSLD6, n = 682 trajectories from five cells, CSLD6+isoxaben, n = 486 trajectories from six cells). Asterisks denote P < 0.001 and n.s. denotes no significant difference as determined by a Kruskal–Wallis statistical test. Scale bar for all images in E, 2 µm. (F) Histogram of CSLD6 particle speed with or without isoxaben treatment as determined by particle tracking with the Fiji plugin TrackMate. (G and H) Single focal plane confocal time-lapse image of protonemata expressing mScarlet-CSLD6 with no drug (G), 20 µM DCB, or 20 µM isoxaben (H). Scale bar for all images in G and H, 5 µm. Also see Video 6. (G) CSLD6 accumulated at the cell apex and appeared as punctae (yellow arrowheads). (H) With DCB treatment, CSLD6 still accumulated at the cell apex but the tip cell ruptures (blue arrowhead). Isoxaben treatment did not cause cell rupture and did not affect CSLD6 localization to the cell tip.

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