Figure 2.

Myosin inhibitor treatments reduce motility in hypocotyl epidermal cells. A, Representative time projections show the trajectories of Golgi motility from apical epidermal cells of 3-d-old etiolated hypocotyls expressing YFP-Mannosidase I. Seedlings were treated for 15 min with mock (0.2% DMSO), 30 mm 2,3-butanedione monoxime (BDM), 10 µm pentabromopseudilin (PBP), or 20 µm MyoVin-1 followed by imaging with variable-angle epifluorescence microscopy. Time projections were generated with maximum intensity of 61 frames collected at 0.5-s intervals. Bar, 10 µm. B, Trajectories of Golgi motility detected with the ImageJ plug-in “TrackMate” for images shown in A. Heat map of trajectories indicate the average speed from 0.1 to 2 µm s⁻¹. C, Box-whisker plots show average velocity of Golgi in inhibitor-treated epidermal cells. Red circles show the mean velocity. The rate of average Golgi velocity was significantly decreased after BDM and PBP treatments, whereas MyoVin-1 had little or no effect. Values given are means ± se (n > 3,000 trajectories from 10 hypocotyls per treatment; one-way ANOVA with Tukey’s post-hoc test, P < 0.001). D, Representative single-frame images of the cortical cytoplasm in hypocotyl epidermal cells expressing MYOSIN XIK-YFP imaged with spinning-disk confocal microscopy (SDCM). Etiolated seedlings were treated with inhibitors for 15 min, followed by time-lapse imaging using an 80-ms interval for 41 frames. Bar, 5 µm. E, Kymographs of the yellow lines depicted in D show fast movement of the vesicles over a 3.2-s time span. Bar, 5 µm. F, Quantification of XIK-YFP velocity from analysis of multiple kymographs. The motility of YFP-XIK was markedly decreased in PBP-treated cells and slightly reduced in BDM or MyoVin-1-treated cells compared to mock treatment. Values given are means ± se (n > 500 tracks from 10 hypocotyls per treatment; one-way ANOVA with Tukey’s post-hoc test, letters [a–c] denote samples/groups that show statistically signifcant differences from other groups, P < 0.001).