Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Purification and crystal structure of human ODA16: Implications for ciliary import of outer dynein arms by the intraflagellar transport machinery.

ABSTRACT: Motile cilia protrude from cell surfaces and are necessary to create movement of cells and fluids in the body. At the molecular level, cilia contain several dynein molecular motor complexes including outer dynein arms (ODAs) that are attached periodically to the ciliary axoneme, where they hydrolyse ATP to create the force required for bending and motility of the cilium. ODAs are preassembled in the cytoplasm and subsequently trafficked into the cilium by the intraflagellar transport (IFT) system. In the case of the green alga Chlamydomonas reinhardtii, the adaptor protein ODA16 binds to ODAs and directly to the IFT complex component IFT46 to facilitate the ciliary import of ODAs. Here, we purified recombinant human IFT46 and ODA16, determined the high-resolution crystal structure of the ODA16 protein, and carried out direct interaction studies of IFT46 and ODA16. The human ODA16 C-terminal 320 residues adopt the fold of an eight-bladed β-propeller with high overall structural similarity to the Chlamydomonas ODA16. However, the small 80 residue N-terminal domain, which in Chlamydomonas ODA16 is located on top of the β-propeller and is required to form the binding cleft for IFT46, has no visible electron density in case of the human ODA16 structure. Furthermore, size exclusion chromatography and pull-down experiments failed to detect a direct interaction between human ODA16 and IFT46. These data suggest that additional factors may be required for the ciliary import of ODAs in human cells with motile cilia.

SUBMITTER: Wang J

PROVIDER: S-EPMC7255517 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery.

Project description:Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

| S-EPMC2568026 | biostudies-literature

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46.

Project description:Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed ?-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal ?-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

| S-EPMC5418046 | biostudies-literature

The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16.

Project description:Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that the IFT46 N-terminus, ODA16, and outer arm dynein interact for IFT of the latter.

| S-EPMC5576905 | biostudies-literature

Shulin packages axonemal outer dynein arms for ciliary targeting.

Project description:The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo-electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

| S-EPMC7116892 | biostudies-literature

Trafficking of ciliary membrane proteins by the intraflagellar transport/BBSome machinery.

Project description:Bardet-Biedl syndrome (BBS) is a rare inherited disease caused by defects in the BBSome, an octameric complex of BBS proteins. The BBSome is conserved in most organisms with cilia, which are microtubule (MT)-based cell organelles that protrude from the cell surface and function in motility and sensing. Cilia assembly, maintenance, and function require intraflagellar transport (IFT), a bidirectional motility of multi-megadalton IFT trains propelled by molecular motors along the ciliary MTs. IFT has been shown to transport structural proteins, including tubulin, into growing cilia. The BBSome is an adapter for the transport of ciliary membrane proteins and cycles through cilia via IFT. While both the loss and the abnormal accumulation of ciliary membrane proteins have been observed in bbs mutants, recent data converge on a model where the BBSome mainly functions as a cargo adapter for the removal of certain transmembrane and peripheral membrane proteins from cilia. Here, we review recent data on the ultrastructure of the BBSome and how the BBSome recognizes its cargoes and mediates their removal from cilia.

| S-EPMC6737936 | biostudies-literature

Cutting off ciliary protein import: intraflagellar transport after dendritic femtosecond-laser ablation.

Project description:Primary cilia, organelles protruding from the surface of eukaryotic cells, act as cellular antennae to detect and transmit signals from the extracellular environment. They are built and maintained by continuous cycles of intraflagellar transport (IFT), where ciliary proteins are transported between the ciliary base and tip. These proteins originate from the cell body because cilia lack protein synthesis machinery. How input from the cell body affects IFT and ciliary function is not well understood. Here, we use femtosecond-laser ablation to perturb the dendritic input of proteins to chemosensory cilia in living Caenorhabditis elegans. Using fluorescence microscopy, we visualize and quantify the real-time response of ciliary proteins to dendritic ablation. We find that the response occurs in three distinct stages. First, IFT dynein is activated within seconds, redistributing IFT components toward the ciliary base; second, the ciliary axoneme shortens and motors slow down; and third, motors leave the cilium. Depletion of ATP by adding azide also results in IFT slowdown and IFT components leaving the cilium, but not in activation of retrograde IFT. These results indicate that laser ablation triggers a specific mechanism important for IFT regulation that allows the cilium to rapidly adapt to changes in the outside environment.

| S-EPMC7183794 | biostudies-literature

In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties.

Project description:Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6. IC2-NG moved by IFT; the transport was of low processivity and increased in frequency during flagellar growth. As expected, IFT of IC2-NG was diminished in oda16, lacking an ODA-specific IFT adapter, and in ift46 IFT46ΔN lacking the ODA16-interacting portion of IFT46. IFT loading appears to involve ODA16-dependent recruitment of ODAs to basal bodies followed by handover to IFT. Upon unloading from IFT, ODAs rapidly docked to the axoneme. Transient docking still occurred in the docking complex mutant oda3 indicating that the docking complex stabilizes rather than initiates ODA-microtubule interactions. In full-length flagella, ODAs continued to enter and move inside cilia by short-term bidirectional IFT and diffusion and the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA-IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella.

| S-EPMC6254574 | biostudies-literature

Shulin packages axonemal outer dynein arms for ciliary targeting

Project description:The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During cilio-genesis, these ∼1.8 MDa complexes are assembled in the cytoplasm and targeted to cilia via an unknown mechanism. Here we use the ciliate Tetrahymena to identify two novel factors (Q22YU3 and Q22MS1) which bind ODAs in the cytoplasm and are required for their delivery to cilia. We show that Q22YU3, which we name Shulin, locks the ODA motor domains into a closed conformation and inhibits motor activity. Cryo-EM reveals how Shulin stabilizes this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

2021-09-09 | PXD022396 | Pride

Intraflagellar Transport and Ciliary Dynamics.

Project description:Cilia and flagella are microtubule-based organelles whose assembly requires a motile process, known as intraflagellar transport (IFT), to bring tubulin and other components to the distal tip of the growing structure. The IFT system uses a multiprotein complex with components that appear to be specialized for the transport of different sets of cargo proteins. The mechanisms by which cargo is selected for ciliary import and transport by IFT remain an area of active research. The complex dynamics of cilia and flagella are under constant regulation to ensure proper length control, and this regulation appears to involve regulation at the stage of IFT injection into the flagellum, as well as regulation of flagellar disassembly and, possibly, of cargo binding. Cilia and flagella thus represent a convenient model system to study how multiple motile and signaling pathways cooperate to control the assembly and dynamics of a complex cellular structure.

| S-EPMC5334256 | biostudies-literature

Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.

Project description:Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ?65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.

| S-EPMC3791252 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data