Experiments were repeated independently three ( a, b) or four times ( c) with similar results. (Lower right, schematic) Proposed model for Rab8A- and Rab13-dependent conformational change of JRAB indicator. Differences were considered significant when p < 0.05. Differences among groups were tested by ANOVA with Tukey’s post-hoc multiple comparison test. Asterisks indicate statistical significance. (Lower left) Averaged emission ratios of Venus/CFP ( n = 4) for HA, HA-Rab13DA, HA-Rab8ADA, and HA-Rab8ADN. (upper panels) Emission spectra of lysates from cells expressing JRAB indicator with HA, HA-Rab8ADA, or HA-Rab13DA were recorded at 1-nm intervals during excitation at 433 nm. c JRAB indicator was expressed in HEK293 cells with HA, HA-Rab8ADA, HA-Rab8ADN, or HA-Rab13DA, and the level of FRET in cell lysates was measured using a spectrofluorometer. Total cell lysates (Input) were also analyzed with anti-Myc or anti-HA antibody. Pulled-down Myc-JRAB (Beads) was detected by western blotting (WB) with anti-Myc antibody. b HEK293 cell lysates containing Myc-JRAB with HA-Rab13DA, HA-Rab8ADA, or HA were subjected to pulldown assays using GST-JRAB-CC4 (aa 840–930), which binds to JRAB-CH+LIM, but not to either Rab8ADA or Rab13DA. Total cell lysates (Input) were also analyzed with anti-GFP or anti-HA antibody. Pulled-down GFP-JRAB-C (Beads) was detected by western blotting (WB) with anti-GFP antibody. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.Ī Lysates of HEK293 cells co-expressing the GFP-tagged JRAB C-terminal region with HA-Rab13DA, HA-Rab8ADA, or HA were subjected to pulldown assays using GST-JRAB-CH+LIM. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. tubular endosomes play essential roles in diverse cellular functions. 11 Department of Biochemistry, Tokushima University Graduate School of Medical Sciences, Tokushima, Japan.10 Department of Neuroscience, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.9 Department of Cell Biology, Tokushima University Graduate School of Medical Sciences, Tokushima, Japan.8 Department of Cancer Genetics, Nagoya University Graduate School of Medicine, Nagoya, Japan.7 Division of Molecular Genetics, Aichi Cancer Center Research Institute, Nagoya, Japan.6 Department of Optical Imaging, Advanced Research Promotion Center, Tokushima University, Tokushima, Japan.
5 Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Aichi, Japan.4 Department of Physics, Nagoya University, Nagoya, Japan.3 Department of Post-LED Photonics Research, Institute of Post-LED Photonics, Tokushima, Japan.
2 Department of Interdisciplinary Researches for Medicine and Photonics, Institute of Post-LED Photonics, Tokushima, Japan.
0 kommentar(er)
