Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie01 - Supplemental Movie 1. Effects of BICD2-N and p50 overexpression on DIC2-GFP distribution and dynamics. HeLa cells stably expressing DIC2-GFP were transfected with constructs expressing mCherry-α-tubulin and either HA-p50 or HA-BICD-N, and imaged using TIRF microscopy with a high penetration depth with a 500 ms exposure (2 frames/s) for 57.5 s. 5 consecutive frames were averaged. Color panels show overlays of DIC2-GFP (green) and mCherry-α-tubulin (red); the panels next to them represent the inverted DIC-GFP signal. Left panels, control, middle -- cotransfection with HA-p50, right - cotransfection with HA-BICD-N. Movie is sped up 28.75 times.
- Movie02 - Supplemental Movie 2. DHC-GFP dynamics in control, dynactin and LIS1-depleted cells. HeLa cells stably expressing DHC-GFP were transfected with different siRNAs and imaged three days later using TIRF microscopy with a high penetration depth with a 500 ms exposure (2 frames/s) for 50 s. 5 consecutive frames were averaged. Left – control siRNA, middle - p150Glued siRNA, right – LIS1 siRNA. Movie is sped up 25 times.
- Movie03 - Supplemental Movie 3. p50-GFP dynamics in control, dynein and LIS1-depleted cells. HeLa cells stably expressing p50-GFP were transfected with different siRNAs and imaged using TIRF microscopy with a high penetration depth with a 500 ms exposure (2 frames/s) for 50 s. 5 consecutive frames were averaged. Left – control siRNA, middle – DHC siRNA, right – LIS1 siRNA. Movie is sped up 25 times.
- Movie04 - Supplemental Movie 4. Rab6 vesicle movement in an MRC5-SV cell transiently expressing FKBP2-GFP-Rab6A. MRC5-SV cells transiently expressing FKBP2-GFP-Rab6A were imaged using wide field microscopy with the time interval of 5 s (exposure time 500 ms); total duration is 11 min. Images were processed by subtraction of the same image subjected to Low Pass filtration (MetaMorph) to allow simultaneous visualization of small vesicles and the Golgi complex; contrast is inverted. Movie is sped up 153.5 times.
- Movie05 - Supplemental Movie 5. Rab6 vesicle movement in an MRC5-SV cell transiently expressing FKBP2-GFP-Rab6A and HA-BICD2-N-FRB before and after rapalog addition. MRC5-SV cells transiently expressing FKBP2-GFP-Rab6A were imaged using wide field microscopy with the time interval of 5 s (exposure time 100 ms), total duration is 27 min. Rapalog was added at 1.6 min after beginning of the movie (indicated). Images were processed by subtraction of the same image subjected to Low Pass filtration (MetaMorph) to allow simultaneous visualization of small vesicles and the Golgi complex; contrast is inverted. Movie is sped up 149.6 times.
- Movie06 - Supplemental Movie 6. DIC2-GFP and Rab6A dynamics in cells co-expressing FKBP2-BICD2-N-FRB. HeLa cells stably expressing DIC2-GFP were transiently transfected with FKBP2-Rab6A, mStrawberry-Rab6A and HA-BICD-N-FRB, and imaged using wide field microscopy with a 500 ms exposure (2 frames/s) for 100s. Left panel – DIC2-GFP (signal inverted), middle – mStrawberry-Rab6A (signal inverted), right – overlay with DIC2-GFP in green and mStrawberry-Rab6A in red. Movie is sped up 15 times.
- Movie07 - Supplemental Movie 7. DIC2-GFP recruitment to Rab6A-positive membranes in cells co-expressing FKBP2-BICD2-N-FRB and FKBP2-Rab6A. HeLa cells stably expressing DIC2-GFP were transiently transfected with FKBP2-Rab6A, mStrawberry-Rab6A and HA-BICD-N-FRB, and imaged using wide field microscopy with a 500 ms exposure (2 frames/s) for 20 s. Images were collected at 39 min after rapalog addition. Left panel – DIC2-GFP (signal inverted), middle – mStrawberry-Rab6A (signal inverted), right – overlay with DIC2-GFP in green and mStrawberry-Rab6A in red. Movie is sped up 3 times.
- Movie08 - Supplemental Movie 8. Rab6 vesicle movement along MTs in an MRC5-SV cell transiently expressing FKBP2-GFP-Rab6A and mCherry-α-tubulin. MRC5-SV cells transiently expressing FKBP2-GFP-Rab6A (green) and mCherry-α-tubulin (red) were imaged using TIRF microscopy with a high penetration depth with a 100 ms exposure (10 frames/s) for 50 s. Time interval between frames is 500 ms (5 consecutive frames made with 100 ms exposure were averaged). Images were processed by applying Blur filter (Photoshop). Movie is sped up 15 times.
- Movie09 - Supplemental Movie 9. Rab6 vesicle movement in an MRC5-SV cell transiently expressing FKBP2-GFP-Rab6A and HA-BICD2-N-FRB after rapalog addition. MRC5-SV cells transiently expressing FKBP2-GFP-Rab6A (green) and mCherry-α-tubulin (red) were imaged using TIRF microscopy with a high penetration depth with a 100 ms exposure (10 frames/s) for 50 s. Time interval between frames is 500 ms (5 consecutive frames made with 100 ms exposure were averaged). Images were processed by applying Blur filter (Photoshop). The first frame of the movie corresponds to 47.5 s after rapalog addition. Movie is sped up 15 times.