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

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 C1a-e-c supercomplex within the ciliary central apparatus

10.1101/773416 ◽

2019 ◽

Cited By ~ 2

Author(s):

Gang Fu ◽

Lei Zhao ◽

Erin Dymek ◽

Yuqing Hou ◽

Kangkang Song ◽

...

Keyword(s):

Molecular Mechanisms ◽

Electron Tomography ◽

3D Structure ◽

Three Dimensional ◽

Protein Composition ◽

Wild Type ◽

Ciliary Motility ◽

Cryo Electron Tomography ◽

Central Apparatus ◽

Subunit Organization

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 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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PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e19-01-0063 ◽

2019 ◽

Vol 30 (15) ◽

pp. 1805-1816 ◽

Cited By ~ 14

Author(s):

Erin E. Dymek ◽

Jianfeng Lin ◽

Gang Fu ◽

Mary E. Porter ◽

Daniela Nicastro ◽

...

Keyword(s):

Cell Motility ◽

Electron Tomography ◽

Structural Studies ◽

Ciliary Beating ◽

Ciliary Motility ◽

Functional Studies ◽

Cryo Electron Tomography ◽

Motile Cilia

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

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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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3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography

Journal of Structural Biology ◽

10.1016/j.jsb.2012.07.003 ◽

2012 ◽

Vol 180 (2) ◽

pp. 318-326 ◽

Cited By ~ 44

Author(s):

Ke Wang ◽

Korrinn Strunk ◽

Gongpu Zhao ◽

Jennifer L. Gray ◽

Peijun Zhang

Keyword(s):

Structure Determination ◽

Mammalian Cells ◽

Electron Tomography ◽

3D Structure ◽

Cryo Electron Tomography ◽

3D Structure Determination

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In SituStructures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography

Journal of Bacteriology ◽

10.1128/jb.00117-19 ◽

2019 ◽

Vol 201 (13) ◽

Cited By ~ 17

Author(s):

Shiwei Zhu ◽

Maren Schniederberend ◽

Daniel Zhitnitsky ◽

Ruchi Jain ◽

Jorge E. Galán ◽

...

Keyword(s):

Salmonella Enterica ◽

Electron Tomography ◽

Additional Insight ◽

Content Type ◽

Bacterial Flagellum ◽

Cryo Electron Tomography ◽

Serovar Typhimurium ◽

Flagellar Assembly ◽

Insight Into

ABSTRACTThe bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, includingPseudomonas aeruginosa,Vibriospp., andSalmonella enterica. The bacterial flagellum has been studied extensively in the model systemsEscherichia coliandSalmonella entericaserovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determinein situstructures of polar flagella inP. aeruginosaand peritrichous flagella inS. Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.IMPORTANCEThe bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolutionin situstructures of polar flagella inPseudomonas aeruginosaand peritrichous flagella inSalmonella entericaserovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in bothP. aeruginosaandS. Typhimurium.

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Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex

10.1101/684316 ◽

2019 ◽

Author(s):

Long Gui ◽

Kangkang Song ◽

Douglas Tristchler ◽

Raqual Bower ◽

Yan Si ◽

...

Keyword(s):

Electron Tomography ◽

Core Complex ◽

Ciliary Motility ◽

C Terminus ◽

Large N ◽

Cryo Electron Tomography ◽

N Terminus ◽

Regulatory Complex ◽

Cilia And Flagella ◽

Linker Domain

ABSTRACTThe nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the eleven (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a sub-complex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N-terminus of DRC4 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits” associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.Significance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1–11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.

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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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