Structural organization of the C1b projection within the ciliary central apparatus

‘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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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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Proteome of the central apparatus of a ciliary axoneme

The Journal of Cell Biology ◽

10.1083/jcb.201902017 ◽

2019 ◽

Vol 218 (6) ◽

pp. 2051-2070 ◽

Cited By ~ 21

Author(s):

Lei Zhao ◽

Yuqing Hou ◽

Tyler Picariello ◽

Branch Craige ◽

George B. Witman

Keyword(s):

Mass Spectrometry ◽

Knowledge Base ◽

Genetic Screening ◽

Primary Ciliary Dyskinesia ◽

The Other ◽

Wild Type ◽

Central Apparatus ◽

Motile Cilia ◽

Ciliary Dyskinesia

Nearly all motile cilia have a “9+2” axoneme containing a central apparatus (CA), consisting of two central microtubules with projections, that is essential for motility. To date, only 22 proteins are known to be CA components. To identify new candidate CA proteins, we used mass spectrometry to compare axonemes of wild-type Chlamydomonas and a CA-less mutant. We identified 44 novel candidate CA proteins, of which 13 are conserved in humans. Five of the latter were studied more closely, and all five localized to the CA; therefore, most of the other candidates are likely to also be CA components. Our results reveal that the CA is far more compositionally complex than previously recognized and provide a greatly expanded knowledge base for studies to understand the architecture of the CA and how it functions. The discovery of the new conserved CA proteins will facilitate genetic screening to identify patients with a form of primary ciliary dyskinesia that has been difficult to diagnose.

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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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The Pcdp1 complex coordinates the activity of dynein isoforms to produce wild-type ciliary motility

Molecular Biology of the Cell ◽

10.1091/mbc.e11-08-0739 ◽

2011 ◽

Vol 22 (23) ◽

pp. 4527-4538 ◽

Cited By ~ 16

Author(s):

Christen G. DiPetrillo ◽

Elizabeth F. Smith

Keyword(s):

Primary Ciliary Dyskinesia ◽

Beat Frequency ◽

Structural Approach ◽

Wild Type ◽

Unique Role ◽

Ciliary Beating ◽

Ciliary Motility ◽

Central Apparatus ◽

Dynein Arms ◽

Ciliary Dyskinesia

Generating the complex waveforms characteristic of beating cilia requires the coordinated activity of multiple dynein isoforms anchored to the axoneme. We previously identified a complex associated with the C1d projection of the central apparatus that includes primary ciliary dyskinesia protein 1 (Pcdp1). Reduced expression of complex members results in severe motility defects, indicating that C1d is essential for wild-type ciliary beating. To define a mechanism for Pcdp1/C1d regulation of motility, we took a functional and structural approach combined with mutants lacking C1d and distinct subsets of dynein arms. Unlike mutants completely lacking the central apparatus, dynein-driven microtubule sliding velocities are wild type in C1d- defective mutants. However, coordination of dynein activity among microtubule doublets is severely disrupted. Remarkably, mutations in either outer or inner dynein arm restore motility to mutants lacking C1d, although waveforms and beat frequency differ depending on which isoform is mutated. These results define a unique role for C1d in coordinating the activity of specific dynein isoforms to control 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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Central Apparatus, the Molecular Kickstarter of Ciliary and Flagellar Nanomachines

International Journal of Molecular Sciences ◽

10.3390/ijms22063013 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3013

Author(s):

Zuzanna Samsel ◽

Justyna Sekretarska ◽

Anna Osinka ◽

Dorota Wloga ◽

Ewa Joachimiak

Keyword(s):

Protein Composition ◽

Eukaryotic Cells ◽

Two Phase ◽

Ciliary Beating ◽

Immotile Cilia ◽

Growing Body ◽

Central Apparatus ◽

Motile Cilia

Motile cilia and homologous organelles, the flagella, are an early evolutionarily invention, enabling primitive eukaryotic cells to survive and reproduce. In animals, cilia have undergone functional and structural speciation giving raise to typical motile cilia, motile nodal cilia, and sensory immotile cilia. In contrast to other cilia types, typical motile cilia are able to beat in complex, two-phase movements. Moreover, they contain many additional structures, including central apparatus, composed of two single microtubules connected by a bridge-like structure and assembling numerous complexes called projections. A growing body of evidence supports the important role of the central apparatus in the generation and regulation of the motile cilia movement. Here we review data concerning the central apparatus structure, protein composition, and the significance of its components in ciliary beating regulation.

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PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

The Journal of Cell Biology ◽

10.1083/jcb.132.3.359 ◽

1996 ◽

Vol 132 (3) ◽

pp. 359-370 ◽

Cited By ~ 115

Author(s):

E F Smith ◽

P A Lefebvre

Keyword(s):

Protein Interactions ◽

Insertional Mutagenesis ◽

Flagellar Motility ◽

Wild Type ◽

Protein Protein Interactions ◽

Cellular Functions ◽

Central Pair ◽

Central Apparatus ◽

Immunofluorescence Labeling ◽

Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

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Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer

Biochemistry and Cell Biology ◽

10.1139/o90-162 ◽

1990 ◽

Vol 68 (9) ◽

pp. 1083-1089 ◽

Cited By ~ 15

Author(s):

John J. Robinson

Keyword(s):

Embryonic Development ◽

Sea Urchin ◽

Organization Development ◽

Structural Organization ◽

Protein Composition ◽

Ruthenium Red ◽

Proteinase K ◽

Polypeptide Composition ◽

Layer Composition ◽

Precipitable Protein

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2+-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2+-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2+-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.Key words: hyaline layer, composition, organization, development.

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Murein components rescue developmental sporulation of Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.152.1.462-470.1982 ◽

1982 ◽

Vol 152 (1) ◽

pp. 462-470 ◽

Cited By ~ 1

Author(s):

L J Shimkets ◽

D Kaiser

Keyword(s):

Fruiting Body ◽

High Cell Density ◽

Myxococcus Xanthus ◽

Sodium Dodecyl ◽

Protein Composition ◽

Diaminopimelic Acid ◽

Nutrient Level ◽

High Cell ◽

Wild Type ◽

Nutrient Rich Medium

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.

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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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