Eg5 is required for migration of CARTS. (A and B) HeLa cells cotransfected with plasmids for GFP-Eg5 T112N and PAUF-mRFP were imaged at 1-s interval for ∼3 min. The cells expressing PAUF-mRFP alone were observed as a control. (A) Tracking of CARTS was performed using Imaris software. The tracks are colorized as shown in the time scale at the bottom. The images of the indicated time points are shown. Bars, 5 µm. See also Videos 2–4. (B) Histogram of velocity distribution of CARTS migration in control cells (n = 546 punctate elements in 7 cells) and GFP-Eg5 T112N–expressing cells (n = 829 punctate elements in 7 cells). The migration rate is shown according to the speed mean values. A χ2 test shows that both speed distributions are significantly different (χ2 = 129.8; P < 0.001). (C and D) The reaction mixture for the biogenesis of CARTS was incubated with DMSO (control), 100 µM monastrol (Mona), or 20 µM nocodazole (Noc) at 4 or 32°C. The high-speed pellet containing CARTS was Western blotted with anti-TGN46 antibody. The input refers to permeabilized HeLa cells used as starting material in these experiments. (D) Quantification of CARTS formation. The average values of three independent experiments are shown (mean ± SD).
