, 1X Phosphate-buffered saline (PBS)
, 1X Phosphate-buffered saline with Ca 2+ and Mg 2+ (PBS(Ca/Mg))
, Fixation solution: 4% Paraformaldehyde (PFA)
, Permeabilization buffer for HeLa and RPE1 cells: 0.1% Triton X-100, 1 mg/ml BSA in PBS. Buffer should be passed through a sterile filter (0.2µm) to avoid cellular contamination for long-term storage
, Permeabilization buffer for IMCD cells: 0.3% Triton X-100, 10 mg/ml BSA, 4% donkey serum and 0.02% sodium azide in PBS (see Note 2)
, Blocking buffer for HeLa and RPE1 cells: 1 mg/ml BSA in PBS
, Blocking buffer for IMCD cells: 10 mg/ml BSA, 4% donkey serum and 0.02% sodium azide in PBS
, Quenching solution: 50 mM NH 4 Cl in PBS
, Mounting media for HeLa and RPE1 cells: PBS-glycerol mix (1:1) using the SlowFade Light Antifade Kit containing DAPI from Molecular Probes
, Mounting media for IMCD cells: Immu-Mount
, Microtubule depolymerizing solutions: Nocodazole (Sigma). 10µM for immediate depolymerization (1h). 1µM for cell-cycle synchronization (24h)
, Microtubule stabilizing solution: 10µM Taxol (Paclitaxel)
, Somatostatin-14 (Phoenix Pharmaceuticals)
, 3 Equipment: 1. Cells are grown in an incubator under standard growth conditions (37°C and 5% CO 2 )
, Cells are seeded on 12 mm glass coverslips
, For HeLa and RPE1 cells, coverslips are mounted on plain glass microscope slides (1mm, Pearl). For IMCD cells, coverslips are sandwiched between microscope cover glass, ThermoFisher Scientific) and a microscope slide
, 8-well µ-Slide, Ibidi
, 35 mm, high Glass Bottom, Ibidi. Neurons were plated on 35 mm glassbottomed (No. 1.5) dishes (MatTek) that had been coated with 40 µg
, Electroporation is performed in 0.4 cm gap Gene Pulser/MicroPulser cuvettes
Ciliogenesis: building the cell's antenna, Nat. Rev. Mol. Cell Biol, vol.12, pp.222-234, 2011. ,
Motile and non-motile cilia in human pathology: from function to phenotypes, J. Pathol, vol.241, pp.294-309, 2016. ,
The centrosome in cells and organisms, Science, vol.335, pp.422-426, 2012. ,
The ciliary pocket, Curr. Opin. Cell Biol, vol.25, pp.78-84, 2013. ,
URL : https://hal.archives-ouvertes.fr/inserm-02263810
Gated entry into the ciliary compartment, Cell. Mol. Life Sci. CMLS, vol.73, pp.119-127, 2016. ,
IFT-Cargo Interactions and Protein Transport in Cilia, Trends Biochem. Sci, vol.40, pp.765-778, 2015. ,
Ins and outs of GPCR signaling in primary cilia, EMBO Rep, vol.16, pp.1099-1113, 2015. ,
G-Protein-Coupled Receptor Signaling in Cilia, Cold Spring Harb. Perspect. Biol, 2017. ,
Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization, Science, vol.259, pp.825-829, 1993. ,
Targeting of betaarrestin2 to the centrosome and primary cilium: role in cell proliferation control, PloS One, vol.3, p.3728, 2008. ,
Beta-arrestin-mediated localization of smoothened to the primary cilium, Science, vol.320, pp.1777-1781, 2008. ,
, Proteomics of Primary Cilia by Proximity Labeling, vol.35, pp.497-512, 2015.
Smoothened determines ?-arrestin-mediated removal of the G protein-coupled receptor Gpr161 from the primary cilium, J. Cell Biol, vol.212, pp.861-875, 2016. ,
Recruitment of ?-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization, Mol. Cell. Biol, vol.36, pp.223-235, 2016. ,
An Actin Network Dispatches Ciliary GPCRs into Extracellular Vesicles to Modulate Signaling, Cell, vol.168, pp.252-263, 2017. ,
Role of ?-arrestins and arrestin domaincontaining proteins in G protein-coupled receptor trafficking, Curr. Opin. Cell Biol, vol.27, pp.63-71, 2014. ,
Non-visual arrestins are constitutively associated with the centrosome and regulate centrosome function, J. Biol. Chem, vol.285, pp.8316-8329, 2010. ,
The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells, J. Cell Biol, vol.149, pp.317-330, 2000. ,
Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine, Nature, vol.406, pp.1005-1009, 2000. ,