scholarly journals FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

2019 ◽  
Vol 30 (21) ◽  
pp. 2659-2680 ◽  
Author(s):  
Jianfeng Lin ◽  
Thuc Vy Le ◽  
Katherine Augspurger ◽  
Douglas Tritschler ◽  
Raqual Bower ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Spatial Organization ◽  
Electron Tomography ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Extrinsic Factors ◽  
Ciliary Motility ◽  
Genes Encoding ◽  
Radial Spokes ◽  
Cytoplasmic Assembly

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.

scholarly journals FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

2019 ◽  
Author(s):  
Jianfeng Lin ◽  
Thuc Vy Le ◽  
Katherine Augspurger ◽  
Douglas Tritschler ◽  
Raqual Bower ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Spatial Organization ◽  
Electron Tomography ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Extrinsic Factors ◽  
Ciliary Motility ◽  
Genes Encoding ◽  
Radial Spokes ◽  
Dynein Arms

AbstractCiliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat, and highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for pre-assembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus,IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms to a specific location in the 96 nm repeat.IDA8encodes FAP57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple inner dynein arms and regulatory complexes.

scholarly journals FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c

2015 ◽  
Vol 26 (4) ◽  
pp. 696-710 ◽  
Author(s):  
Krishna Kumar Vasudevan ◽  
Kangkang Song ◽  
Lea M. Alford ◽  
Winfield S. Sale ◽  
Erin E. Dymek ◽  
...  
Keyword(s):  
Cell Motility ◽  
Electron Tomography ◽  
Macromolecular Complexes ◽  
Radial Spoke ◽  
Ciliary Motility ◽  
Cryo Electron Tomography ◽  
Ciliary Protein ◽  
Radial Spokes ◽  
Dynein Arms

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

scholarly journals Structural organization of the C1b projection within the ciliary central apparatus

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.

Molecular basis of ciliary defects caused by compound heterozygous IFT144/WDR19 mutations found in cranioectodermal dysplasia

2021 ◽  
Author(s):  
Yamato Ishida ◽  
Takuya Kobayashi ◽  
Shuhei Chiba ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Protein Trafficking ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Missense Variant ◽  
Cellular Level ◽  
Compound Heterozygous ◽  
Hypomorphic Allele ◽  
Genes Encoding ◽  
Specific Proteins ◽  
Compound Heterozygous Mutations

Abstract Primary cilia contain specific proteins to achieve their functions as cellular antennae. Ciliary protein trafficking is mediated by the intraflagellar transport (IFT) machinery containing the IFT-A and IFT-B complexes. Mutations in genes encoding the IFT-A subunits (IFT43, IFT121/WDR35, IFT122, IFT139/TTC21B, IFT140, and IFT144/WDR19) often result in skeletal ciliopathies, including cranioectodermal dysplasia (CED). We here characterized the molecular and cellular defects of CED caused by compound heterozygous mutations in IFT144 [the missense variant IFT144(L710S) and the nonsense variant IFT144(R1103*)]. These two variants were distinct with regard to their interactions with other IFT-A subunits and with the IFT-B complex. When exogenously expressed in IFT144-knockout (KO) cells, IFT144(L710S) as well as IFT144(WT) rescued both moderately compromised ciliogenesis and the abnormal localization of ciliary proteins. As the homozygous IFT144(L710S) mutation was found to cause autosomal recessive retinitis pigmentosa, IFT144(L710S) is likely to be hypomorphic at the cellular level. In striking contrast, the exogenous expression of IFT144(R1103*) in IFT144-KO cells exacerbated the ciliogenesis defects. The expression of IFT144(R1103*) together with IFT144(WT) restored the abnormal phenotypes of IFT144-KO cells. However, the coexpression of IFT144(R1103*) with the hypomorphic IFT144(L710S) variant in IFT144-KO cells, which mimics the genotype of compound heterozygous CED patients, resulted in severe ciliogenesis defects. Taken together, these observations demonstrate that compound heterozygous mutations in IFT144 cause severe ciliary defects via a complicated mechanism, where one allele can cause severe ciliary defects when combined with a hypomorphic allele.

scholarly journals Dependency relationships between IFT-dependent flagellum elongation and cell morphogenesis in Leishmania

Open Biology ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 180124 ◽  
Author(s):  
Jack Daniel Sunter ◽  
Flavia Moreira-Leite ◽  
Keith Gull
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Shape ◽  
Electron Tomography ◽  
Intraflagellar Transport ◽  
Flagellar Pocket ◽  
Cell Morphogenesis ◽  
Multiple Functions ◽  
Cell Shape Changes ◽  
Shape Changes

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation.

Abstract 13296: Risk Loci of Hypertrophic Cardiomyopathy Identified via the UK Biobank

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Alex Gyftopoulos ◽  
Yi-Ju Chen ◽  
Libin Wang ◽  
Charles H Williams ◽  
Young Wook Chun ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Large Population ◽  
Phenotypic Expression ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Genetic Profile ◽  
Uk Biobank ◽  
Disease Manifestation ◽  
Pathogenic Variants ◽  
The Uk

Introduction: Hypertrophic cardiomyopathy (HCM) is the most commonly inherited cardiac disease affecting 1:500 to 1:200 individuals worldwide. HCM has a heterogeneous genetic profile and phenotypic expression. More than 1400 known pathogenic variants have been identified in 11 sarcomere genes. In about 40% of HCM patients, the genetic cause may not be identified. The same mutation may lead to different phenotypes and severity in different individuals. Identification of novel HCM genes and modifiers will expand our understanding of the signaling pathways that are responsible for phenotypic expression of HCM. Methods: The UK Biobank comprises clinical and genetic data for greater than 500,000 individuals. We used OASIS, an information system for analyzing, searching, and visualizing associations between phenotype and genotype data to analyze this data. We compared control individuals to HCM individuals identified by ICD-10 code (I42.1 and I42.2) in a 20-to-1 fashion. Related individuals and those with confounding diagnoses were excluded. Results: The analysis was performed with Plink’s GLM option, and we identified 84 variants with a minor allele frequency of 0.5% or greater in 65 genes associated with HCM with a p < 1x10 -6 , including 4 with p < 5x10 -8 . The identified genes encode lncRNAs, miRNAs, and membrane proteins. Variants with high significance were identified in the genes encoding putative ciliary components DNAL4 (dynein axonemal light chain 4; p = 2.9x10 -8 ), MYO1D (unconventional myosin 1D; p = 3.1x10 -8 ), ITFAP (intraflagellar transport associated protein; p = 9.5x10 -8 ), CABCOCO1 (ciliary associated calcium biding coiled-coil 1; p = 3.7x 10 -7 ), EVL (Enah-Vasp-like; p = 4.4x 10 -7 ) and IFT122 (intraflagellar transport 122; p = 8.0 x10 -7 ). Conclusion: While none of these have previously associated with HCM, our findings suggest ciliary structure and function may play a role in disease manifestation. Our method is unique by pooling individuals in a large population set to identify potential causative or contributing mutations. Bioinformatic tools, such as OASIS, allow for the identification of previously unrecognized variants that may play a role in the development of HCM. This approach has identified numerous novel genes as possible risk loci.

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

2019 ◽  
Vol 30 (15) ◽  
pp. 1805-1816 ◽  
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.

scholarly journals Centrioles initiate cilia assembly but are dispensable for maturation and maintenance in C. elegans

2017 ◽  
Vol 216 (6) ◽  
pp. 1659-1671 ◽  
Author(s):  
Daniel Serwas ◽  
Tiffany Y. Su ◽  
Max Roessler ◽  
Shaohe Wang ◽  
Alexander Dammermann
Keyword(s):  
Fluorescence Microscopy ◽  
Transition Zone ◽  
Electron Tomography ◽  
Intraflagellar Transport ◽  
Basal Bodies ◽  
C Elegans ◽  
Cilia Formation ◽  
Syndrome Protein

Cilia are cellular projections that assemble on centriole-derived basal bodies. While cilia assembly is absolutely dependent on centrioles, it is not known to what extent they contribute to downstream events. The nematode C. elegans provides a unique opportunity to address this question, as centrioles do not persist at the base of mature cilia. Using fluorescence microscopy and electron tomography, we find that centrioles degenerate early during ciliogenesis. The transition zone and axoneme are not completely formed at this time, indicating that cilia maturation does not depend on intact centrioles. The hydrolethalus syndrome protein HYLS-1 is the only centriolar protein known to remain at the base of mature cilia and is required for intraflagellar transport trafficking. Surprisingly, targeted degradation of HYLS-1 after initiation of ciliogenesis does not affect ciliary structures. Taken together, our results indicate that while centrioles are essential to initiate cilia formation, they are dispensable for cilia maturation and maintenance.

scholarly journals Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum

2019 ◽  
Vol 116 (35) ◽  
pp. 17316-17322 ◽  
Author(s):  
Orly Levitan ◽  
Muyuan Chen ◽  
Xuyuan Kuang ◽  
Kuan Yu Cheong ◽  
Jennifer Jiang ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Spatial Organization ◽  
Higher Plants ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Spatial Differentiation ◽  
Thylakoid Membranes ◽  
Marine Diatom ◽  
Red Algal ◽  
Antenna Proteins

A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

scholarly journals The CSC connects three major axonemal complexes involved in dynein regulation

2012 ◽  
Vol 23 (16) ◽  
pp. 3143-3155 ◽  
Author(s):  
Thomas Heuser ◽  
Erin E. Dymek ◽  
Jianfeng Lin ◽  
Elizabeth F. Smith ◽  
Daniela Nicastro
Keyword(s):  
Electron Tomography ◽  
Structural Heterogeneity ◽  
Flagellar Motility ◽  
Wild Type ◽  
Localization Model ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
Health And Development ◽  
Cilia And Flagella

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ­ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

Sign in / Sign up

Export Citation Format

Share Document