Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

AbstractNearly all motile cilia contain a central apparatus (CA) composed of two connected singlet-microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild type Chlamydomonas with CA mutants. We identified a large (>2 MDa) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold-labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and three-dimensional (3D) structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.SummaryFu et al. use a wild-type vs. mutant comparison and cryo-electron tomography of Chlamydomonas flagella to identify central apparatus (CA) subunits and visualize their location in the native 3D CA structure. The study provides a better understanding of the CA and how it regulates ciliary motility.

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Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

The Journal of Cell Biology ◽

10.1083/jcb.201906006 ◽

2019 ◽

Vol 218 (12) ◽

pp. 4236-4251 ◽

Cited By ~ 8

Author(s):

Gang Fu ◽

Lei Zhao ◽

Erin Dymek ◽

Yuqing Hou ◽

Kangkang Song ◽

...

Keyword(s):

Molecular Mechanisms ◽

Electron Tomography ◽

3D Structure ◽

Protein Composition ◽

Ciliary Beating ◽

Ciliary Motility ◽

Cryo Electron Tomography ◽

Central Apparatus ◽

Subunit Organization ◽

Insight Into

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

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Structural organization of the C1b projection within the ciliary central apparatus

10.1101/2021.06.16.448709 ◽

2021 ◽

Author(s):

Kai Cai ◽

Yanhe Zhao ◽

Lei Zhao ◽

Nhan Phan ◽

George Witman ◽

...

Keyword(s):

Electron Tomography ◽

3D Structure ◽

Protein Composition ◽

Ciliary Motility ◽

Cryo Electron Tomography ◽

Radial Spokes ◽

Candidate Protein ◽

Central Apparatus ◽

Subtomogram Averaging ◽

Subunit Organization

'9+2' motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA projections remain largely unknown. Here, we combined genetic approaches and quantitative proteomics with cryo-electron tomography and subtomogram averaging to compare the CA of wild-type Chlamydomonas with those of two CA mutants. Our results show that two conserved proteins, FAP42 and FAP246, are localized to the L-shaped C1b projection of the CA. We also identified another novel CA candidate protein, FAP413, which interacts with both FAP42 and FAP246. FAP42 is a large protein that forms the peripheral 'beam' of the C1b projection, and the FAP246-FAP413 subcomplex serves as the 'bracket' between the beam (FAP42) and the C1b 'pillar' that attaches the projection to the C1 microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of both the C1b and C1f projections, and loss of any of these proteins leads to ciliary motility defects. Our results provide insight into the subunit organization and 3D structure of the C1b projection, suggesting that the FAP246-FAP413-FAP42 subcomplex is part of a large interconnected CA-network that provides mechanical support and may play a role in mechano-signaling between the CA and radial spokes to regulate dynein activity and ciliary beating.

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Structural organization of the C1b projection within the ciliary central apparatus

Journal of Cell Science ◽

10.1242/jcs.254227 ◽

2021 ◽

Author(s):

Kai Cai ◽

Yanhe Zhao ◽

Lei Zhao ◽

Nhan Phan ◽

Yuqing Hou ◽

...

Keyword(s):

Structural Organization ◽

Protein Composition ◽

Wild Type ◽

Large Protein ◽

Ciliary Motility ◽

Central Apparatus ◽

Motile Cilia ◽

Ca Protein

‘9+2’ motile cilia contain 9 doublet microtubules and a central apparatus (CA) composed of two singlet microtubules with associated projections. The CA plays crucial roles in regulating ciliary motility. Defects in CA assembly or function usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of most CA-projections remain largely unknown. Here, we combined genetic, proteomic, and cryo-electron tomographic approaches to compare the CA of wild-type Chlamydomonas with those of three CA-mutants. Our results show that two proteins, FAP42 and FAP246, are localized to the L-shaped C1b-projection of the CA, where they interact with the candidate CA-protein FAP413. FAP42 is a large protein that forms the peripheral ‘beam’ of the C1b-projection, and the FAP246-FAP413 subcomplex serves as the ‘bracket’ between the beam (FAP42) and the C1b ‘pillar’ that attaches the projection to the C1-microtubule. The FAP246-FAP413-FAP42 complex is essential for stable assembly of the C1b, C1f and C2b-projections, and loss of these proteins leads to ciliary motility defects.

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In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

Scientific Reports ◽

10.1038/s41598-020-74104-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Swetha Vijayakrishnan ◽

Marion McElwee ◽

Colin Loney ◽

Frazer Rixon ◽

David Bhella

Keyword(s):

Electron Microscopy ◽

Structure Determination ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Cell Nuclei ◽

Nuclear Egress ◽

Virus Capsids ◽

Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

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In situ structure determination of virus capsids imaged within cell nuclei by correlative light and cryo-electron tomography

10.1101/2020.02.06.936948 ◽

2020 ◽

Author(s):

Swetha Vijayakrishnan ◽

Marion McElwee ◽

Colin Loney ◽

Frazer Rixon ◽

David Bhella

Keyword(s):

Electron Microscopy ◽

Structure Determination ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Cell Nuclei ◽

Nuclear Egress ◽

Virus Capsids ◽

Cryo Electron Tomography

AbstractCryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures at atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (>500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised equipment of limited availability. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions reveal that the capsid associated tegument complex is present on capsids prior to nuclear egress. We show that this approach to cryogenic imaging of cells is suited to both correlative light/electron microscopy and 3D structure determination.

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Structure and function of outer dynein arm intermediate and light chain complex

Molecular Biology of the Cell ◽

10.1091/mbc.e15-10-0723 ◽

2016 ◽

Vol 27 (7) ◽

pp. 1051-1059 ◽

Cited By ~ 18

Author(s):

Toshiyuki Oda ◽

Tatsuki Abe ◽

Haruaki Yanagisawa ◽

Masahide Kikkawa

Keyword(s):

Molecular Mechanisms ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Coiled Coil ◽

Chain Complex ◽

Flagellar Motility ◽

Chemically Induced Dimerization ◽

And Function

The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs.

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The Morphology and Assembly of Respiratory Syncytial Virus Revealed by Cryo-Electron Tomography

Viruses ◽

10.3390/v10080446 ◽

2018 ◽

Vol 10 (8) ◽

pp. 446 ◽

Cited By ~ 21

Author(s):

Zunlong Ke ◽

Rebecca Dillard ◽

Tatiana Chirkova ◽

Fredrick Leon ◽

Christopher Stobart ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Electron Tomography ◽

Rna Virus ◽

3D Structure ◽

Three Dimensional ◽

The Elderly ◽

Cryo Electron Tomography ◽

Infected Cells ◽

Syncytial Virus ◽

Tract Disease

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. With repeat infections throughout life, it can also cause substantial disease in the elderly and in adults with compromised cardiac, pulmonary and immune systems. RSV is a pleomorphic enveloped RNA virus in the Pneumoviridae family. Recently, the three-dimensional (3D) structure of purified RSV particles has been elucidated, revealing three distinct morphological categories: spherical, asymmetric, and filamentous. However, the native 3D structure of RSV particles associated with or released from infected cells has yet to be investigated. In this study, we have established an optimized system for studying RSV structure by imaging RSV-infected cells on transmission electron microscopy (TEM) grids by cryo-electron tomography (cryo-ET). Our results demonstrate that RSV is filamentous across several virus strains and cell lines by cryo-ET, cryo-immuno EM, and thin section TEM techniques. The viral filament length varies from 0.5 to 12 μm and the average filament diameter is approximately 130 nm. Taking advantage of the whole cell tomography technique, we have resolved various stages of RSV assembly. Collectively, our results can facilitate the understanding of viral morphogenesis in RSV and other pleomorphic enveloped viruses.

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A cryo–electron tomography workflow reveals protrusion-mediated shedding on injured plasma membrane

Science Advances ◽

10.1126/sciadv.abc6345 ◽

2021 ◽

Vol 7 (13) ◽

pp. eabc6345

Author(s):

Shrawan Kumar Mageswaran ◽

Wei Yuan Yang ◽

Yogaditya Chakrabarty ◽

Catherine M. Oikonomou ◽

Grant J. Jensen

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Electron Tomography ◽

Membrane Damage ◽

Light And Electron Microscopy ◽

Actin Dynamics ◽

Endosomal Sorting ◽

Plasma Membrane Damage ◽

Cryo Electron Tomography ◽

Vacuolar Protein

Cryo–electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the ‘endosomal sorting complex required for transport’ machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.

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Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations

Communications Biology ◽

10.1038/s42003-019-0748-0 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 8

Author(s):

C. Keith Cassidy ◽

Benjamin A. Himes ◽

Dapeng Sun ◽

Jun Ma ◽

Gongpu Zhao ◽

...

Keyword(s):

Molecular Mechanisms ◽

Electron Tomography ◽

Conformational Dynamics ◽

Molecular Simulations ◽

Adaptor Protein ◽

Signaling Mechanism ◽

E Coli ◽

Signaling Complex ◽

Chemical Gradients ◽

Cryo Electron Tomography

AbstractTo enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.

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