Figure 6.
Figure 6. Intracellular trafficking of Dsc2, but not Dsg2, was affected by blocking kinesin-2 function with DN KIF3A. (A) Representative videos show behavior of Dsg2- and Dsc2-GFP in cells imaged 12 h after double transfection with motorless DN KIF3A–RFP constructs. First frames show the areas (boxes) analyzed (Video 7), and the three magnified columns show selected vesicles (colored arrows) at three time points. Vesicle trajectories taken from the analyzed area are shown on the left, where colored tracks represent the movement of selected vesicles. Bars, 20 µm. (B) Distribution of particle movements (equal to instantaneous velocity; *, P < 0.05 for each set of bars) and the ratio of rapid movements to the average amount of analyzed particles (15–20 particles per cell) in each cell (three cells were analyzed per experiment) for Dsc2 in control cells and cells expressing the DN mutant (*, P = 0.05 for the ratio of rapid movements). Particle movements and the ratio of rapid movements for Dsc2 were decreased in cells with inhibited kinesin-2 function compared with control cells. (C) Distribution of long vectors and the ratio of long vectors to the average amount of analyzed particles (15–20 particles in each cell) for Dsg2 in cells expressing DN KIF3A are similar to that observed in control cells (P = 0.8). (D). Immunoblot showing level of DN KIF3A–RFP expression and total level of endogenous KIF3A for experiments in A and B. Cntrl, control. (E) Maximum projection of time-lapse analysis of cells into which cDNAs encoding Dsc2-GFP or Dsg2-GFP (not depicted) were coinjected with or without KIF3A cDNA into the nuclei of nontargeting (NT) siRNA (siNT) or siKIF3A-silenced cells (Video 8). Bar, 5 µm. Representative trajectories of Dsc2-GFP movements are shown in red. Bar graph on the right is a population analysis showing particles engaging in long-range, short-range, or stationary movements as a percentage of Dsc2-GFP vesicles. The percentage of long-range movements is partially restored by the KHC-mCherry rescue construct (P = 0.001). n above the bars represents the number of vesicles trajectories that have been analyzed. Number of cells: nontargeting siRNA = 8, siKIF3A = 10, and siKIF3A + KIF3A-RFP = 8.

Intracellular trafficking of Dsc2, but not Dsg2, was affected by blocking kinesin-2 function with DN KIF3A. (A) Representative videos show behavior of Dsg2- and Dsc2-GFP in cells imaged 12 h after double transfection with motorless DN KIF3A–RFP constructs. First frames show the areas (boxes) analyzed (Video 7), and the three magnified columns show selected vesicles (colored arrows) at three time points. Vesicle trajectories taken from the analyzed area are shown on the left, where colored tracks represent the movement of selected vesicles. Bars, 20 µm. (B) Distribution of particle movements (equal to instantaneous velocity; *, P < 0.05 for each set of bars) and the ratio of rapid movements to the average amount of analyzed particles (15–20 particles per cell) in each cell (three cells were analyzed per experiment) for Dsc2 in control cells and cells expressing the DN mutant (*, P = 0.05 for the ratio of rapid movements). Particle movements and the ratio of rapid movements for Dsc2 were decreased in cells with inhibited kinesin-2 function compared with control cells. (C) Distribution of long vectors and the ratio of long vectors to the average amount of analyzed particles (15–20 particles in each cell) for Dsg2 in cells expressing DN KIF3A are similar to that observed in control cells (P = 0.8). (D). Immunoblot showing level of DN KIF3A–RFP expression and total level of endogenous KIF3A for experiments in A and B. Cntrl, control. (E) Maximum projection of time-lapse analysis of cells into which cDNAs encoding Dsc2-GFP or Dsg2-GFP (not depicted) were coinjected with or without KIF3A cDNA into the nuclei of nontargeting (NT) siRNA (siNT) or siKIF3A-silenced cells (Video 8). Bar, 5 µm. Representative trajectories of Dsc2-GFP movements are shown in red. Bar graph on the right is a population analysis showing particles engaging in long-range, short-range, or stationary movements as a percentage of Dsc2-GFP vesicles. The percentage of long-range movements is partially restored by the KHC-mCherry rescue construct (P = 0.001). n above the bars represents the number of vesicles trajectories that have been analyzed. Number of cells: nontargeting siRNA = 8, siKIF3A = 10, and siKIF3A + KIF3A-RFP = 8.

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