Structure of the Decorated Ciliary Doublet Microtubule

Ma, Mingyu; Stoyanova, Mihaela; Rademacher, Griffin; Dutcher, Susan K.; Brown, Alan; Zhang, Rui

doi:10.1016/j.cell.2019.09.030

Cited by 215 publications

(426 citation statements)

References 138 publications

(155 reference statements)

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“…Some of these microtubule internal proteins (MIPs) were associated with the internal wall of the microtubules (Supplementary Figure 1D,F), while others seemed to be floating in the microtubule lumen (Supplementary Figure 1C,E). In both cases, their arrangement differed from the periodic MIPs decoration typical of motile cilia 8,13,35,36 . The lack of an obvious periodical decoration by MIPs and large axonemal complexes, raises the question about how the stability of the primary cilium axoneme is maintained.…”

Section: An Eb1-like Protein Decorates A-tubules In Primary Ciliamentioning

confidence: 86%

“…The periodical arrangement of a large number of MIPs in motile cilia has been shown to substantially contribute to microtubule doublet stabilization 42 . However, microtubules in motile and primary cilia strongly differ in number and type of MIPs 11,13 .…”

Section: Discussionmentioning

confidence: 99%

“…This was certainly facilitated by the ease of use of several model organisms such as Chlamydomonas 8,9,10 , sea urchin 8,11 , and Tetrahymena 8 , which generously provide large amounts of motile cilia with simple deciliation protocols 12 . Structural studies of motile cilia have been revolutionized particularly by the advent of cryo-electron microscopy (cryo-EM) 13 and subtomogram averaging (STA). Cryo-electron tomography (cryo-ET) has enabled the unambiguous localization of protein complexes within motile cilia and the visualization of their structure and conformational changes required for ciliary activity.…”

Section: Introductionmentioning

confidence: 99%

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The molecular structure of primary cilia revealed by cryo-electron tomography

Kiesel

Viar

Tsoy

et al. 2020

Preprint

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Primary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known "9+0" arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.We would like to thank the Electron Microscopy Facility (in particular Tobias Fürstenhaupt, Weihua Leng, Michaela Wilsch-Bräuninger) and the Light Microscope Facility from the Services and Facilities of the MPI-CBG for their support. We are thankful to Helin Rägel and Cécilie Martin-Lemaitre for their tips on MDCKII cell culture, Noreen Walker for the Imaris tutorial, and Tim-Oliver Buchholz for denoising cryo-tomography data. We thank Pavel Tomancak, Florian Jug, Dennis Diener, and Jan Brugues for the fruitful discussions and suggestions to the manuscript. We thank Oscar Gonzales for IT support. . developed the cryo-peel-off method, prepared samples for FM and EM imaging, acquired and reconstructed room temperature and cryo-tomograms, contributed to FM data acquisition, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. G.A.V. prepared samples and contributed to data acquisition of the room temperature tomography, analyzed cryo-EM data with subtomogram averaging and tomogram segmentation, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. N.T. analyzed cryo-ET data to average the microtubule singlets, contributed to supplementary figure preparation, contributed to the interpretation of data, and contributed to writing and revising the manuscript. R.M. pre...

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Section: An Eb1-like Protein Decorates A-tubules In Primary Ciliamentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The molecular structure of primary cilia revealed by cryo-electron tomography

Kiesel

Viar

Tsoy

et al. 2020

Preprint

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“…This is consistent with the observation that immunofluorescence detection of CrCCDC103 in isolated axonemes was weak in regions where outer arms had assembled, presumably due to limited antibody accessibility, but much stronger at both the axonemal base and tip where these motors were absent. To date, no cryo-EM densities assigned to CCDC103 structures have been reported (e.g., Ma et al, 2019), and so whether these CCDC103 oligomers are arrayed on the microtubule surface or embedded within the doublets as are many other axonemal microtubule-associated proteins remains to be determined.…”

Section: Potential Modes Of Ccdc103 Polymerization and Predictions mentioning

confidence: 99%

“…Building ciliary axonemes which contain many hundreds of different proteins presents a daunting problem in macromolecular assembly (Ma et al, 2019;Nicastro et al, 2006;Pazour, Agrin, Leszyk, & Witman, 2005;Pazour & King, 2019). This issue is readily exemplified by the complexity of axonemal inner and outer dynein arm incorporation onto the nine doublet microtubules.…”

Section: Introductionmentioning

confidence: 99%

The outer dynein arm assembly factor CCDC103 forms molecular scaffolds through multiple self‐interaction sites

King

Patel‐King

2019

Cytoskeleton

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CCDC103 is a small protein with unusual biophysical properties that is required for outer dynein arm assembly on ciliary axonemes. Mutations in both human and zebrafish CCDC103 proteins lead to primary ciliary dyskinesia. Previous studies revealed that this protein can oligomerize and appears to be arrayed along the entire length of the ciliary axoneme. CCDC103 also binds purified microtubules directly and indeed stabilizes them. Here we use biochemical approaches to identify two regions of CCDC103 that mediate self-interaction. In both cases, these associations are stable to heating in the presence of detergent and are not disrupted by strong reducing agents. One interaction region consists of a 27-residue inherently disorder segment that can mediate heat/detergent-resistant dimerization when attached to unrelated monomeric proteins. The second interface includes the C-terminal RPAP3_C alpha helical domain. Our data suggest that CCDC103 can form an unconventional polymer and we propose models for how the monomers might be organized. We also use molecular modeling of the RPAP3_C domain to determine the structural consequences of the pathogenic H154P mutation found in human PCD patients. K E Y W O R D S Axoneme, Chlamydomonas, cilia, dynein, flagella, microtubule ABBREVIATIONS: CCDC, coiled coil domain containing; FAP, flagella-associated protein; MBP, maltose-binding protein; ODA-DC, outer dynein arm docking complex; RPAP3, RNA polymerase II-associated protein 3; TCEP, Tris(2-carboxyethyl)phosphine.

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Algal Ciliary Motility

Hunter

Penny

Dutcher

2021

Encyclopedia of Life Sciences

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The microtubule‐based cilium (also known as a flagellum) extends from the surface of most eukaryotic cells. This long, slender organelle is biochemically complex and is conserved in many eukaryotic organisms. The unicellular alga, Chlamydomonas reinhardtii, serves as a tractable organism for studying the assembly and function of cilia. Cilia provide locomotion and environmental sensing. To produce motility, specialised molecular motors called outer and inner dynein arms generate force to produce waveform. These motors are regulated by other macromolecular complexes in the cilium. The ease of genetic and biochemical approaches combined with electron microscopy has provided the ability to perform in‐depth studies of the regulation of ciliary assembly and function. Results from Chlamydomonas researchers have broadened our knowledge of motile cilia diseases in humans. There are still many outstanding questions to be addressed. Key Concepts Chlamydomonas reinhardtii is a haploid, motile, single‐celled alga that is useful for the study of cilia. The Chlamydomonas cilium is a complex structure that generates asymmetric and symmetric waveforms to allow forward and backward swimming, respectively, and orientation to environmental signals. During ciliary motility, dynein arms pull microtubule doublets past each other to generate sliding, which is converted into axonemal bending, although the mechanism for how this occurs is still controversial. Outer dynein arms control the ciliary beat frequency by changing the velocity of doublet sliding. Inner dynein arms in conjunction with the radial spokes and the central pair apparatus, control the bend amplitude and the shape of the waveform. The axoneme is divided into 96 nm repeating segments that contain regulatory, enzymatic and structural components essential for motility; these include the inner and outer dynein arms, N‐DRC, MIA complex and radial spokes as well as two central microtubules. IFT (intraflagellar transport) is essential for building the cilium by transporting cargo into (anterograde) and out of (retrograde) the cilium. Cell biological research about ciliary components, including the central pair apparatus, radial spokes and N‐DRC, provides key information about how ciliary motility is regulated. Genetic, biochemical and microscopic methods used in Chlamydomonas have allowed molecular resolution of interactions between regulatory complexes. Studies of Chlamydomonas cilia have provided additional insights to the structure, function and regulation of cilia, and have helped with the understanding of human cilia‐related diseases called ciliopathies.

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Structure of the Decorated Ciliary Doublet Microtubule

Cited by 215 publications

References 138 publications

The molecular structure of primary cilia revealed by cryo-electron tomography

The molecular structure of primary cilia revealed by cryo-electron tomography

The outer dynein arm assembly factor CCDC103 forms molecular scaffolds through multiple self‐interaction sites

Algal Ciliary Motility

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