Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy
Abstract
Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. In this work, we have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.
- Authors:
-
- Baylor College of Medicine, Houston, TX (United States)
- McGovern Medical School, Houston, TX (United States); Yale School of Medicine, New Haven, CT (United States)
- Publication Date:
- Research Org.:
- SLAC National Accelerator Lab., Menlo Park, CA (United States)
- Sponsoring Org.:
- USDOE; National Institutes of Health (NIH)
- OSTI Identifier:
- 1596756
- Grant/Contract Number:
- AC02-76SF00515; R01-EY007981; R01-EY026545; P30-EY002520; F32-EY027171; F31-EY028025; P41-GM103832; Q0035; AU-1714
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Proceedings of the National Academy of Sciences of the United States of America
- Additional Journal Information:
- Journal Volume: 116; Journal Issue: 47; Journal ID: ISSN 0027-8424
- Publisher:
- National Academy of Sciences
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; primary cilia; photoreceptors; ciliopathies; superresolution; cryoelectron tomography
Citation Formats
Robichaux, Michael A., Potter, Valencia L., Zhang, Zhixian, He, Feng, Liu, Jun, Schmid, Michael F., and Wensel, Theodore G. Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy. United States: N. p., 2019.
Web. doi:10.1073/pnas.1902003116.
Robichaux, Michael A., Potter, Valencia L., Zhang, Zhixian, He, Feng, Liu, Jun, Schmid, Michael F., & Wensel, Theodore G. Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy. United States. https://doi.org/10.1073/pnas.1902003116
Robichaux, Michael A., Potter, Valencia L., Zhang, Zhixian, He, Feng, Liu, Jun, Schmid, Michael F., and Wensel, Theodore G. Tue .
"Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy". United States. https://doi.org/10.1073/pnas.1902003116. https://www.osti.gov/servlets/purl/1596756.
@article{osti_1596756,
title = {Defining the layers of a sensory cilium with STORM and cryoelectron nanoscopy},
author = {Robichaux, Michael A. and Potter, Valencia L. and Zhang, Zhixian and He, Feng and Liu, Jun and Schmid, Michael F. and Wensel, Theodore G.},
abstractNote = {Primary cilia carry out numerous signaling and sensory functions, and defects in them, “ciliopathies,” cause a range of symptoms, including blindness. Understanding of their nanometer-scale ciliary substructures and their disruptions in ciliopathies has been hindered by limitations of conventional microscopic techniques. In this work, we have combined cryoelectron tomography, enhanced by subtomogram averaging, with superresolution stochastic optical reconstruction microscopy (STORM) to define subdomains within the light-sensing rod sensory cilium of mouse retinas and reveal previously unknown substructures formed by resident proteins. Domains are demarcated by structural features such as the axoneme and its connections to the ciliary membrane, and are correlated with molecular markers of subcompartments, including the lumen and walls of the axoneme, the membrane glycocalyx, and the intervening cytoplasm. Within this framework, we report spatial distributions of key proteins in wild-type (WT) mice and the effects on them of genetic deficiencies in 3 models of Bardet–Biedl syndrome.},
doi = {10.1073/pnas.1902003116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 47,
volume = 116,
place = {United States},
year = {Tue Nov 05 00:00:00 EST 2019},
month = {Tue Nov 05 00:00:00 EST 2019}
}
Web of Science
Works referenced in this record:
Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods
journal, January 2015
- Chuang, Jen-Zen; Hsu, Ya-Chu; Sung, Ching-Hwa
- Cilia, Vol. 4, Issue 1
Automated tilt series alignment and tomographic reconstruction in IMOD
journal, February 2017
- Mastronarde, David N.; Held, Susannah R.
- Journal of Structural Biology, Vol. 197, Issue 2
Structural and molecular bases of rod photoreceptor morphogenesis and disease
journal, November 2016
- Wensel, Theodore G.; Zhang, Zhixian; Anastassov, Ivan A.
- Progress in Retinal and Eye Research, Vol. 55
A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells
journal, September 2007
- Maerker, Tina; van Wijk, Erwin; Overlack, Nora
- Human Molecular Genetics, Vol. 17, Issue 1
Centrins in retinal photoreceptor cells: Regulators in the connecting cilium
journal, May 2008
- Trojan, Philipp; Krauss, Norbert; Choe, Hui-Woog
- Progress in Retinal and Eye Research, Vol. 27, Issue 3
Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans
journal, April 2010
- Cevik, Sebiha; Hori, Yuji; Kaplan, Oktay I.
- Journal of Cell Biology, Vol. 189, Issue 1
A G-protein activation cascade from Arl13B to Arl3 and implications for ciliary targeting of lipidated proteins
journal, November 2015
- Gotthardt, Katja; Lokaj, Mandy; Koerner, Carolin
- eLife, Vol. 4
A BBSome Subunit Links Ciliogenesis, Microtubule Stability, and Acetylation
journal, December 2008
- Loktev, Alexander V.; Zhang, Qihong; Beck, John S.
- Developmental Cell, Vol. 15, Issue 6
Practical limits of resolution in confocal and non-linear microscopy
journal, January 2003
- Cox, Guy; Sheppard, Colin J. R.
- Microscopy Research and Technique, Vol. 63, Issue 1
Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells
journal, December 2002
- Mikami, A.
- Journal of Cell Science, Vol. 115, Issue 24
The Proteome of the Mouse Photoreceptor Sensory Cilium Complex
journal, May 2007
- Liu, Qin; Tan, Glenn; Levenkova, Natasha
- Molecular & Cellular Proteomics, Vol. 6, Issue 8
Bardet–Biedl syndrome-8 (BBS8) protein is crucial for the development of outer segments in photoreceptor neurons
journal, November 2017
- Dilan, Tanya L.; Singh, Ratnesh K.; Saravanan, Thamaraiselvi
- Human Molecular Genetics, Vol. 27, Issue 2
Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM
journal, May 2013
- Li, Xueming; Mooney, Paul; Zheng, Shawn
- Nature Methods, Vol. 10, Issue 6
Bardet-Biedl syndrome type 4 (BBS4)-null mice implicate Bbs4 in flagella formation but not global cilia assembly
journal, June 2004
- Mykytyn, K.; Mullins, R. F.; Andrews, M.
- Proceedings of the National Academy of Sciences, Vol. 101, Issue 23
Super-resolution architecture of mammalian centriole distal appendages reveals distinct blade and matrix functional components
journal, May 2018
- Yang, T. Tony; Chong, Weng Man; Wang, Won-Jing
- Nature Communications, Vol. 9, Issue 1
Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARL13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain
journal, December 2013
- Cevik, Sebiha; Sanders, Anna A. W. M.; Van Wijk, Erwin
- PLoS Genetics, Vol. 9, Issue 12
Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy
journal, January 2019
- Yoon, Joshua; Comerci, Colin J.; Weiss, Lucien E.
- Biophysical Journal, Vol. 116, Issue 2
MosaicIA: an ImageJ/Fiji plugin for spatial pattern and interaction analysis
text, January 2013
- Shivanandan, Arun; Radenovic, Aleksandra; Sbalzarini, Ivo F.
- ETH Zurich
Integrative subcellular proteomic analysis allows accurate prediction of human disease-causing genes
journal, February 2016
- Zhao, Li; Chen, Yiyun; Bajaj, Amol Onkar
- Genome Research, Vol. 26, Issue 5
MosaicIA: an ImageJ/Fiji plugin for spatial pattern and interaction analysis
journal, January 2013
- Shivanandan, Arun; Radenovic, Aleksandra; Sbalzarini, Ivo F.
- BMC Bioinformatics, Vol. 14, Issue 1
BBSome function is required for both the morphogenesis and maintenance of the photoreceptor outer segment
journal, October 2017
- Hsu, Ying; Garrison, Janelle E.; Kim, Gunhee
- PLOS Genetics, Vol. 13, Issue 10
A Core Complex of BBS Proteins Cooperates with the GTPase Rab8 to Promote Ciliary Membrane Biogenesis
journal, June 2007
- Nachury, Maxence V.; Loktev, Alexander V.; Zhang, Qihong
- Cell, Vol. 129, Issue 6
The roles of evolutionarily conserved functional modules in cilia-related trafficking
journal, December 2013
- Sung, Ching-Hwa; Leroux, Michel R.
- Nature Cell Biology, Vol. 15, Issue 12
Super-Resolution Imaging Reveals TCTN2 Depletion-Induced IFT88 Lumen Leakage and Ciliary Weakening
journal, July 2018
- Weng, Rueyhung Roc; Yang, T. Tony; Huang, Chia-En
- Biophysical Journal, Vol. 115, Issue 2
The Renewal of Protein in Retinal rods and Cones
journal, October 1968
- Young, Richard W.; Droz, Bernard
- The Journal of Cell Biology, Vol. 39, Issue 1
Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)
journal, August 2006
- Rust, Michael J.; Bates, Mark; Zhuang, Xiaowei
- Nature Methods, Vol. 3, Issue 10
Ultra-High Resolution 3D Imaging of Whole Cells
journal, August 2016
- Huang, Fang; Sirinakis, George; Allgeyer, Edward S.
- Cell, Vol. 166, Issue 4
Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans
journal, March 2010
- Cevik, Sebiha; Hori, Yuji; Kaplan, Oktay I.
- The Journal of Cell Biology, Vol. 188, Issue 6
Genes and molecular pathways underpinning ciliopathies
journal, July 2017
- Reiter, Jeremy F.; Leroux, Michel R.
- Nature Reviews Molecular Cell Biology, Vol. 18, Issue 9
The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella
journal, December 2009
- Lechtreck, Karl-Ferdinand; Johnson, Eric C.; Sakai, Tsuyoshi
- The Journal of Cell Biology, Vol. 187, Issue 7
Cryo-electron tomography reveals conserved features of doublet microtubules in flagella
journal, September 2011
- Nicastro, D.; Fu, X.; Heuser, T.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 42
Three-dimensional structure of basal body triplet revealed by electron cryo-tomography: Electron cryo-tomography of basal body triplet
journal, December 2011
- Li, Sam; Fernandez, Jose-Jesus; Marshall, Wallace F.
- The EMBO Journal, Vol. 31, Issue 3
SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium
journal, June 2018
- Dharmat, Rachayata; Eblimit, Aiden; Robichaux, Michael A.
- The Journal of Cell Biology, Vol. 217, Issue 8
UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004
- Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
- Journal of Computational Chemistry, Vol. 25, Issue 13
Light-dependent phosphorylation of Bardet–Biedl syndrome 5 in photoreceptor cells modulates its interaction with arrestin1
journal, July 2013
- Smith, Tyler S.; Spitzbarth, Benjamin; Li, Jian
- Cellular and Molecular Life Sciences, Vol. 70, Issue 23
Motile cilia genetics and cell biology: big results from little mice
journal, September 2020
- Lee, Lance; Ostrowski, Lawrence E.
- Cellular and Molecular Life Sciences, Vol. 78, Issue 3
The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking
journal, June 2012
- Liem, Karel F.; Ashe, Alyson; He, Mu
- The Journal of Cell Biology, Vol. 197, Issue 6
Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81
journal, August 2013
- Bhogaraju, S.; Cajanek, L.; Fort, C.
- Science, Vol. 341, Issue 6149
Electron-tomographic analysis of intraflagellar transport particle trains in situ
journal, October 2009
- Pigino, Gaia; Geimer, Stefan; Lanzavecchia, Salvatore
- The Journal of Cell Biology, Vol. 187, Issue 1
Structure of Trypanosoma brucei flagellum accounts for its bihelical motion
journal, June 2011
- Koyfman, A. Y.; Schmid, M. F.; Gheiratmand, L.
- Proceedings of the National Academy of Sciences, Vol. 108, Issue 27
Spectrum of Clinical Diseases Caused By Disorders of Primary Cilia
journal, September 2011
- Ware, S. M.; Aygun, M. G. -; Hildebrandt, F.
- Proceedings of the American Thoracic Society, Vol. 8, Issue 5
Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.
journal, December 1987
- Horst, C. J.; Forestner, D. M.; Besharse, J. C.
- The Journal of Cell Biology, Vol. 105, Issue 6
The Conserved Bardet-Biedl Syndrome Proteins Assemble a Coat that Traffics Membrane Proteins to Cilia
journal, June 2010
- Jin, Hua; White, Susan Roehl; Shida, Toshinobu
- Cell, Vol. 141, Issue 7
Intraflagellar Transport: The Eyes Have It
journal, February 1999
- Rosenbaum, Joel L.; Cole, Douglas G.; Diener, Dennis R.
- The Journal of Cell Biology, Vol. 144, Issue 3
BBS7 is required for BBSome formation and its absence in mice results in Bardet-Biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking
journal, April 2013
- Zhang, Q.; Nishimura, D.; Vogel, T.
- Journal of Cell Science, Vol. 126, Issue 11
Mapping Synaptic Input Fields of Neurons with Super-Resolution Imaging
journal, October 2015
- Sigal, Yaron M.; Speer, Colenso M.; Babcock, Hazen P.
- Cell, Vol. 163, Issue 2
EMAN2: An extensible image processing suite for electron microscopy
journal, January 2007
- Tang, Guang; Peng, Liwei; Baldwin, Philip R.
- Journal of Structural Biology, Vol. 157, Issue 1
Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy
journal, April 2015
- Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.
- PLOS ONE, Vol. 10, Issue 4
Intrinsic Protein-Protein Interaction-mediated and Chaperonin-assisted Sequential Assembly of Stable Bardet-Biedl Syndrome Protein Complex, the BBSome
journal, April 2012
- Zhang, Qihong; Yu, Dahai; Seo, Seongjin
- Journal of Biological Chemistry, Vol. 287, Issue 24
Intraflagellar transport molecules in ciliary and nonciliary cells of the retina
journal, April 2010
- Sedmak, Tina; Wolfrum, Uwe
- The Journal of Cell Biology, Vol. 189, Issue 1
Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet–Biedl syndrome
journal, July 2015
- Datta, Poppy; Allamargot, Chantal; Hudson, Joseph S.
- Proceedings of the National Academy of Sciences, Vol. 112, Issue 32
Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen
journal, February 2020
- Ou, Li; Kong, Wing-Pui; Chuang, Gwo-Yu
- Scientific Reports, Vol. 10, Issue 1
The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance
journal, March 2002
- Pazour, Gregory J.; Baker, Sheila A.; Deane, James A.
- The Journal of Cell Biology, Vol. 157, Issue 1
The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia
journal, October 2018
- Jordan, Mareike A.; Diener, Dennis R.; Stepanek, Ludek
- Nature Cell Biology, Vol. 20, Issue 11
IFT20 Links Kinesin II with a Mammalian Intraflagellar Transport Complex That Is Conserved in Motile Flagella and Sensory Cilia
journal, June 2003
- Baker, Sheila A.; Freeman, Katie; Luby-Phelps, Katherine
- Journal of Biological Chemistry, Vol. 278, Issue 36
The Retinal Ciliopathies
journal, January 2007
- Adams, N. A.; Awadein, Ahmed; Toma, Hassanain S.
- Ophthalmic Genetics, Vol. 28, Issue 3
SARA-Regulated Vesicular Targeting Underlies Formation of the Light-Sensing Organelle in Mammalian Rods
journal, August 2007
- Chuang, Jen-Zen; Zhao, Yu; Sung, Ching-Hwa
- Cell, Vol. 130, Issue 3
Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography
journal, May 2007
- Nickell, Stephan; Park, Paul S. -H.; Baumeister, Wolfgang
- The Journal of Cell Biology, Vol. 177, Issue 5
Three-Dimensional Architecture of the Rod Sensory Cilium and Its Disruption in Retinal Neurodegeneration
journal, November 2012
- Gilliam, Jared C.; Chang, Juan T.; Sandoval, Ivette M.
- Cell, Vol. 151, Issue 5
ARL13B, PDE6D, and CEP164 form a functional network for INPP5E ciliary targeting
journal, November 2012
- Humbert, M. C.; Weihbrecht, K.; Searby, C. C.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 48
Super-resolution microscopy reveals that disruption of ciliary transition-zone architecture causes Joubert syndrome
journal, August 2017
- Shi, Xiaoyu; Garcia, Galo; Van De Weghe, Julie C.
- Nature Cell Biology, Vol. 19, Issue 10
Expansion Stimulated Emission Depletion Microscopy (ExSTED)
journal, April 2018
- Gao, Mengfei; Maraspini, Riccardo; Beutel, Oliver
- ACS Nano, Vol. 12, Issue 5
Computer Visualization of Three-Dimensional Image Data Using IMOD
journal, January 1996
- Kremer, James R.; Mastronarde, David N.; McIntosh, J. Richard
- Journal of Structural Biology, Vol. 116, Issue 1
Ablation of Whirlin Long Isoform Disrupts the USH2 Protein Complex and Causes Vision and Hearing Loss
journal, May 2010
- Yang, Jun; Liu, Xiaoqing; Zhao, Yun
- PLoS Genetics, Vol. 6, Issue 5
Structure of Trypanosoma Brucei Flagellum Accounts for its Bihelical Motion
journal, January 2012
- Schmid, Michael F.; Koyfman, Alexey Y.; Gheiratmand, Ladan
- Biophysical Journal, Vol. 102, Issue 3
Arbuscular cell invasion coincides with extracellular vesicles and membrane tubules.
text, January 2019
- Roth, Ronelle; Hillmer, Stefan; Funaya, Charlotta
- Apollo - University of Cambridge Repository
Ultra-High Resolution 3D Imaging of Whole Cells.
text, January 2016
- Huang, Fang; Sirinakis, George; Allgeyer, Edward
- Apollo - University of Cambridge Repository
Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins
journal, March 2015
- Johnson, Errin; Seiradake, Elena; Jones, E. Yvonne
- Scientific Reports, Vol. 5, Issue 1
Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors
journal, April 1996
- Beech, P. L.; Pagh-Roehl, K.; Noda, Y.
- Journal of Cell Science, Vol. 109, Issue 4
Fine structure of basal bodies (kinetosomes) and associated components of Tetrahymena
journal, January 1970
- Munn, E. A.
- Tissue and Cell, Vol. 2, Issue 4