Fig 1: Active Rab10 rescues defects in primary cilia formation but not length.(A) RPE-1 cells expressing GFP or GFP-Rab10 QL (active mutant) were starved and stained with Ac-tubulin antibody to mark primary cilia. Scale bars, 20 and 10 μm for low- and high-magnification images, respectively. (B and C) Quantification of experiments as in (A); means ± SEM; one-way analysis of variance (ANOVA), with Tukey’s posttest (***P < 0.001). n > 250 cells for (B) and 50 cells for (C). (D) Unstarved A459 WT cells were stained with Rab10 and CEP164 antibodies to reveal the mother centriole. Scale bar, 1.2 μm. (E) Intensity profiles along the straight blue line in (D). (F) Starved A549 cells were stained with Rab10 and Ac-tubulin antibodies for primary cilia (red). Scale bars, 5.5 μm. The yellow arrows in (F) represent the base of cilia (stained with anti–Ac-tubulin antibody). (G) Quantification of experiments as in (F). Means ± SEM; unpaired t test (***P < 0.001; n = 5). (H) Quantification of experiments as in (F); means ± SEM; Mann-Whitney U test (**P < 0.01; n > 50).
Fig 2: Model of DENND2B regulating primary cilia.(A) Schematic representation of DENND2B regulating primary cilia via activation of Rab10. (B) DENND2B activating RhoA and controlling primary cilia length.
Fig 3: Schematic model for the biogenesis of tubular recycling endosomes. EHBP1 helps link PI(4,5)P2-containing endosomes to Rab10 and KIF13 motor proteins, which may serve as the driving force to pull the endosomal membranes along microtubules and thus generate tubular recycling endosomes.
Fig 4: Analysis of LRRK2, LRRK2 Ser935, Rab10 and Rab10 Thr73 phosphorylation in LCLs from control, G2019S LRRK2-PD and sporadic PD patients.(A) Example of three control and three G2019S LRRK2 LCL lines (left), or two distinct control and five sporadic PD LCL lines (right), treated with or without 10 nM MLi2 for 2 h. Cells were subsequently lysed and extracts subjected to quantitative immunoblot analysis with the indicated antibodies, and membranes developed using Odyssey CLx scan Western Blot imaging system. Note that ‘sporadic 2’ and ‘sporadic 4' are two out of the three sporadic PD LCLs which display a centrosomal cohesion deficit. (B) Control, G2019S LRRK2 and sporadic PD LCL extracts were analyzed as described in (A), and immunoblots quantified for full-length LRRK2/tubulin ratio. (C) Immunoblots of the type depticted in (A) were quantified for LRRK2 Ser935/tublin ratio. * P < 0.05. (D) Immunoblots were quantified for LRRK2 Ser935/LRRK2 ratio. * P < 0.05 (E) Immunoblots were quantified for Rab10/tubulin ratio. (F) Immunoblots were quantified for Rab10 Thr73/tubulin ratio. * P < 0.05. (G) Immunoblots were quantified for Rab10 Thr73/Rab10 ratio. * P < 0.05. Statistical analysis was performed with Kruskal–Wallis test with Dunn's multiple comparison. All data are presented as whisker plots.
Fig 5: MICAL-L1 and Rab10 are residents on the same tubular recycling endosome.A–C, HeLa cells were cultured on cover slides, fixed, and immunostained with antibodies against endogenous MICAL-L1 (A, green) and Rab10 (B, red). A series of serial sections were obtained and the representative image is a snapshot from a 3D reconstitution. C, depiction of the merged image from A and B, showing the overlap between MICAL-L1 and Rab10 in yellow. D–F, the dashed boxes from A–C are shown as magnified insets in the zoomed regions depicted in D–F. G, the graph represents three independent experiments with at least 10 images each that were subjected to imaging and quantification of surface volume overlap. The scale bars represent 10 μm. Error bars denote standard deviation.
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