Figure 3.
Dynactin depletion reduced the number of minus end–directed dynein molecules on MT. (a) HILO image (left) and kymograph (right) of a cell expressing GFP-p150. Representative stationary (switching to stationary from minus [1] or plus end–directed [3]), minus end–directed (4), and plus end–directed events (2) are indicated with the white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (b) Summed intensity projection over time (∼40 s) of the GFP-p150 time-lapse in a, showing structures that resemble MTs, indicating the likely persistent association of p150 with MTs. (c) Plot of position versus time for the p150 molecules tracked, showing stationary events (gray), minus end–directed events (teal), and plus end–directed events (magenta). n = 213 spots from 35 cells across three independent experiments. (d) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP in cells treated with 25 nM negative control (NC) siRNA (left) and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph. (e) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP in cells treated with 25 nM siRNA against endogenous p150 and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph. (f) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 68% (n = 2 independent experiments). (g) Comparison of displacement versus time plots of single dynein molecules in cells treated with 25 nM NC siRNA (top) and 25 nM siRNA against p150 (bottom). Data for NC obtained from n = 274 binding events tracked across ∼60 cells from two independent experiments. Data for RNAi obtained from n = 299 binding events tracked ∼60 cells from two independent experiments. Fisher’s exact test yielded a significant difference in dynein behavior in NC and p150-RNAi (P < 0.05). (h) Histogram of velocities of mDHC-GFP in the background of NC RNAi (top) and p150 RNAi (bottom; for data from d). In a, b, d, and e, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData F3.

Dynactin depletion reduced the number of minus end–directed dynein molecules on MT. (a) HILO image (left) and kymograph (right) of a cell expressing GFP-p150. Representative stationary (switching to stationary from minus [1] or plus end–directed [3]), minus end–directed (4), and plus end–directed events (2) are indicated with the white, teal, and magenta arrowheads, respectively, in the kymograph and in the insets below. (b) Summed intensity projection over time (∼40 s) of the GFP-p150 time-lapse in a, showing structures that resemble MTs, indicating the likely persistent association of p150 with MTs. (c) Plot of position versus time for the p150 molecules tracked, showing stationary events (gray), minus end–directed events (teal), and plus end–directed events (magenta). n = 213 spots from 35 cells across three independent experiments. (d) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP in cells treated with 25 nM negative control (NC) siRNA (left) and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph. (e) HILO microscopy image (left) from a 10-s-long time-lapse video of mDHC-GFP in cells treated with 25 nM siRNA against endogenous p150 and the corresponding kymograph (right). Representative stationary, minus end–directed, and plus end–directed events are indicated with white, teal, and magenta arrowheads, respectively, in the kymograph. (f) Representative western blot to verify the knockdown of p150 by RNAi. Quantification of western blot confirmed that the RNAi successfully knocked down levels of p150 by an average of 68% (n = 2 independent experiments). (g) Comparison of displacement versus time plots of single dynein molecules in cells treated with 25 nM NC siRNA (top) and 25 nM siRNA against p150 (bottom). Data for NC obtained from n = 274 binding events tracked across ∼60 cells from two independent experiments. Data for RNAi obtained from n = 299 binding events tracked ∼60 cells from two independent experiments. Fisher’s exact test yielded a significant difference in dynein behavior in NC and p150-RNAi (P < 0.05). (h) Histogram of velocities of mDHC-GFP in the background of NC RNAi (top) and p150 RNAi (bottom; for data from d). In a, b, d, and e, “N” marks the location/direction of the nucleus. Source data are available for this figure: SourceData F3.

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