Arl8b-dependent MT plus end–directed transport of late endosomes regulates FAs. (A) MP1 endosomes are transported along MTs. Time-lapse images of HeLa cells expressing GFP-MP1 (green), mCherry-Paxillin (red), and mCherry-tubulin (red) show colocalization of MP1 and MTs (white arrowheads). Representative individual endosome moves along MTs toward two FAs (bottom panels, white arrowheads). See also Video 7 and 8. (B) Nocodazole treatment of a cell transfected as in A results in MT depolymerization and “trapping” of few GFP-MP1 endosomes in FAs (white arrows and arrowheads). Time-lapse images of the same cell show that positions of GFP-MP1 endosomes do not change in time due to abolished MP1 transport. (C) Arl8b knockdown in MEFs. IF: anti-LAMP1 (green), anti-tubulin (red) antibodies, and Hoechst. LAMP1-positive late endosomes collapse to the perinuclear region upon Arl8b knockdown (white arrow). WB: anti-Arl8b antibody, anti-tubulin used as loading control. (D) The p14^−/−;p14-GFP MEFs treated with control and Arl8b RNAi. The late p14-GFP endosomes cluster in the Arl8b RNAi-treated cells (red arrow). See also Video 9. (E) The graph on the left shows the quantification of average FA length in MEFs. Mean in percent ± SEM compared with control p14^f/− MEFs treated with control RNAi (mean of FA length in control p14^f/− MEFs treated with control RNAi was taken as 100%). See also Table S1. The graph on the right shows the migration speed of p14^−/−;p14-GFP MEFs transfected with control (n = 26) and Arl8b siRNA (n = 66) in wound-healing assay (µm/h, mean of cell migration speeds ± SD). (F) Colocalization of Paxillin and Rab7 in MEFs. Images from time-lapse series of MEF cells coexpressing GFP-Rab7 (green) and mCherry-Paxillin (red). White arrows indicate FAs targeted by GFP-Rab7. See also Video 10.