Figure 6: MYO5B interaction with Rab10 determines fission of PPVs at the TGN.

(a) Rat fibroblasts were transfected with TD-Rab10 together with vehicle or exon D plasmids. Time-lapse imaging shows a fission event in control cells (red arrowhead) and the persistence of a tubule-like structure that was finally collapsed in exon D cells. Scale bar=20 μm. (b,c) Quantification for the number of tubules (b) and vesicles (c). Data are shown as mean±s.e.m. At least 15 cells at each group were analyzed. *P<0.05, ***P<0.001, Student’s t-test. (d–f) Fibroblasts transfected with TD-Rab10 were treated for 1.5 h with indicated amounts of TAT-exon D or scrambled peptides. The static number of tubules (e) and vesicles (f) were quantified. Data are shown as mean±s.e.m. At least 15 cells at each group were analyzed. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test. Scale bar=20 μm. (g) Fibroblast transfected with vehicle (control) or plasmids encoding exon D or siMYO5B were subject to EM analysis. Note the normal Golgi structure in control cell and the appearance of many tubule-like structures with heterogeneous shapes and sizes (blue arrows) in exon D or siMYO5B cells. Cross sections of tubules exhibit vesicle-like morphology (yellow arrowheads). Scale bars=500 nm. (h) Quantification for the number of abnormal membrane structures, including tubules, cross sections of tubules or accumulated vesicles per 9 μm² perinuclear region. Data are shown as mean±s.e.m. ***P<0.001, Student’s t-test, at least 20 cells at each group were analyzed.