Single-molecule Imaging in the Primary Cilium

Overview

Journal Methods Cell Biol

Specialty Cell Biology

Date 2023 May 10

PMID 37164543

Authors

Lucien E Weiss

Julia F Love

Joshua Yoon

Colin J Comerci

Ljiljana Milenkovic

Tomoharu Kanie

Peter K Jackson

Tim Stearns

Anna-Karin Gustavsson

Affiliations

Soon will be listed here.

Abstract

The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level.

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References

Jia S, Vaughan J, Zhuang X . Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function. Nat Photonics. 2014; 8:302-306. PMC: 4224117. DOI: 10.1038/nphoton.2014.13. View

Gahlmann A, Ptacin J, Grover G, Quirin S, von Diezmann L, Lee M . Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. Nano Lett. 2013; 13(3):987-93. PMC: 3599789. DOI: 10.1021/nl304071h. View

Sharonov A, Hochstrasser R . Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc Natl Acad Sci U S A. 2006; 103(50):18911-6. PMC: 1748151. DOI: 10.1073/pnas.0609643104. View

Chen Z, Geffroy L, Biteen J . NOBIAS: Analyzing anomalous diffusion in single-molecule tracks with nonparametric Bayesian inference. Front Bioinform. 2022; 1. PMC: 9053523. DOI: 10.3389/fbinf.2021.742073. View

Oswald F, Prevo B, Acar S, Peterman E . Interplay between Ciliary Ultrastructure and IFT-Train Dynamics Revealed by Single-Molecule Super-resolution Imaging. Cell Rep. 2018; 25(1):224-235. DOI: 10.1016/j.celrep.2018.09.019. View

Ram S, Prabhat P, Chao J, Ward E, Ober R . High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells. Biophys J. 2008; 95(12):6025-43. PMC: 2599831. DOI: 10.1529/biophysj.108.140392. View

Weiss L, Milenkovic L, Yoon J, Stearns T, Moerner W . Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol. Proc Natl Acad Sci U S A. 2019; 116(12):5550-5557. PMC: 6431229. DOI: 10.1073/pnas.1816747116. View

Conduit S, Davies E, Fulcher A, Oorschot V, Mitchell C . Superresolution Microscopy Reveals Distinct Phosphoinositide Subdomains Within the Cilia Transition Zone. Front Cell Dev Biol. 2021; 9:634649. PMC: 8120242. DOI: 10.3389/fcell.2021.634649. View

Granik N, Weiss L, Nehme E, Levin M, Chein M, Perlson E . Single-Particle Diffusion Characterization by Deep Learning. Biophys J. 2019; 117(2):185-192. PMC: 6701009. DOI: 10.1016/j.bpj.2019.06.015. View

10.

Lukinavicius G, Reymond L, DEste E, Masharina A, Gottfert F, Ta H . Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods. 2014; 11(7):731-3. DOI: 10.1038/nmeth.2972. View

11.

Kiesel P, Alvarez Viar G, Tsoy N, Maraspini R, Gorilak P, Varga V . The molecular structure of mammalian primary cilia revealed by cryo-electron tomography. Nat Struct Mol Biol. 2020; 27(12):1115-1124. PMC: 7610599. DOI: 10.1038/s41594-020-0507-4. View

12.

Yoon J, Comerci C, Weiss L, Milenkovic L, Stearns T, Moerner W . Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy. Biophys J. 2019; 116(2):319-329. PMC: 6349968. DOI: 10.1016/j.bpj.2018.11.3136. View

13.

Briscoe J, Therond P . The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol. 2013; 14(7):416-29. DOI: 10.1038/nrm3598. View

14.

Waters A, Beales P . Ciliopathies: an expanding disease spectrum. Pediatr Nephrol. 2011; 26(7):1039-56. PMC: 3098370. DOI: 10.1007/s00467-010-1731-7. View

15.

Singla V, Reiter J . The primary cilium as the cell's antenna: signaling at a sensory organelle. Science. 2006; 313(5787):629-33. DOI: 10.1126/science.1124534. View

16.

Lau L, Lee Y, Sahl S, Stearns T, Moerner W . STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein. Biophys J. 2012; 102(12):2926-35. PMC: 3379620. DOI: 10.1016/j.bpj.2012.05.015. View

17.

Shi X, Garcia 3rd G, Van De Weghe J, McGorty R, Pazour G, Doherty D . Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome. Nat Cell Biol. 2017; 19(10):1178-1188. PMC: 5695680. DOI: 10.1038/ncb3599. View

18.

Lukinavicius G, Umezawa K, Olivier N, Honigmann A, Yang G, Plass T . A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat Chem. 2013; 5(2):132-9. DOI: 10.1038/nchem.1546. View

19.

Ye F, Breslow D, Koslover E, Spakowitz A, Nelson W, Nachury M . Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors. Elife. 2013; 2:e00654. PMC: 3736543. DOI: 10.7554/eLife.00654. View

20.

Juette M, Gould T, Lessard M, Mlodzianoski M, Nagpure B, Bennett B . Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat Methods. 2008; 5(6):527-9. DOI: 10.1038/nmeth.1211. View