Figure 3.
Figure 3. KHC knockdown blocked movement of Dsg2 but not Dsc2. (A) First frames show areas (boxes) analyzed by live-cell imaging in control or KHC-deficient cells 3 d after transfection (Video 4). The three magnified columns highlight selected vesicles (arrows) at three time points. Vesicle trajectories taken from the analyzed area are shown on the right, where colored tracks represent the movement of selected vesicles. Bars, 20 µm. (B) Bar graphs show quantification of distribution of particle movements (equal to instantaneous velocity; for each set of bar P < 0.05) and the ratio of rapid movements to the average number of analyzed particles (15–20 particles per cell) in each cell (three to four cells were analyzed per experiment) for Dsg2 in control and knockdown (KD) conditions (*, P = 0.03 for the ratio of rapid movements). In kinesin-1–deficient cells, the number of particle movements and the ratio of rapid movements for Dsg2 were decreased compared with controls. shNT, shRNA nontargeting. (C) Distribution of particle movements and the ratio of rapid movements to the average amount of analyzed particles for Dsc2 in KHC knockdown cells are similar to the control (P = 0.9). (D) Immunoblot showing level of KHC knockdown (∼50%, n = 3; *, P < 0.05). α-Tubulin (Tub) is the loading control. (E) Maximum projection of time-lapse analysis of cells into which cDNAs encoding Dsg2-GFP or Dsc2-GFP (not depicted) were coinjected with or without KHC cDNA into the nuclei of nontargeting (NT) siRNA (siNT) or siKHC-silenced cells (Video 6). Representative trajectories of Dsg2-GFP movements are shown in red. Bar, 5 µm. Bar graph on right is a population analysis showing particles engaging in long-range, short-range, or stationary movements as a percentage of Dsg2-GFP vesicles. The percentage of long-range movements is partially restored by the KHC-mCherry rescue construct (*, P < 0.05). n above the bars represents the number of vesicle trajectories that have been analyzed. Number of cells that been analyzed: siRNA nontargeting treated = 7, siKHC treated = 9, and siKHC with KHC-mCherry coinjection = 7. Error bars represent SEM.

KHC knockdown blocked movement of Dsg2 but not Dsc2. (A) First frames show areas (boxes) analyzed by live-cell imaging in control or KHC-deficient cells 3 d after transfection (Video 4). The three magnified columns highlight selected vesicles (arrows) at three time points. Vesicle trajectories taken from the analyzed area are shown on the right, where colored tracks represent the movement of selected vesicles. Bars, 20 µm. (B) Bar graphs show quantification of distribution of particle movements (equal to instantaneous velocity; for each set of bar P < 0.05) and the ratio of rapid movements to the average number of analyzed particles (15–20 particles per cell) in each cell (three to four cells were analyzed per experiment) for Dsg2 in control and knockdown (KD) conditions (*, P = 0.03 for the ratio of rapid movements). In kinesin-1–deficient cells, the number of particle movements and the ratio of rapid movements for Dsg2 were decreased compared with controls. shNT, shRNA nontargeting. (C) Distribution of particle movements and the ratio of rapid movements to the average amount of analyzed particles for Dsc2 in KHC knockdown cells are similar to the control (P = 0.9). (D) Immunoblot showing level of KHC knockdown (∼50%, n = 3; *, P < 0.05). α-Tubulin (Tub) is the loading control. (E) Maximum projection of time-lapse analysis of cells into which cDNAs encoding Dsg2-GFP or Dsc2-GFP (not depicted) were coinjected with or without KHC cDNA into the nuclei of nontargeting (NT) siRNA (siNT) or siKHC-silenced cells (Video 6). Representative trajectories of Dsg2-GFP movements are shown in red. Bar, 5 µm. Bar graph on right is a population analysis showing particles engaging in long-range, short-range, or stationary movements as a percentage of Dsg2-GFP vesicles. The percentage of long-range movements is partially restored by the KHC-mCherry rescue construct (*, P < 0.05). n above the bars represents the number of vesicle trajectories that have been analyzed. Number of cells that been analyzed: siRNA nontargeting treated = 7, siKHC treated = 9, and siKHC with KHC-mCherry coinjection = 7. Error bars represent SEM.

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