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An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

ABSTRACT: Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.

SUBMITTER: McCafferty CL

PROVIDER: S-EPMC10541116 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

McCafferty Caitlyn L CL Papoulas Ophelia O Lee Chanjae C Bui Khanh Huy KH Taylor David W DW Marcotte Edward M EM Wallingford John B JB

bioRxiv : the preprint server for biology 20230921

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in <i>Tetrahymena thermophila</i>. From over 19, ...[more]

PMID: 37781579

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Project description:Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Project description:Background: Recessive mutation of the X-linked gene, PIH1 domain-containing protein 3 (PIH1D3), causes familial ciliopathy. PIH1D3 deficiency is associated with the defects of dynein arms in cilia, but how PIH1D3 specifically affects the structure and function of dynein arms is not understood yet. To gain insights into the underlying mechanisms of the disease, it is crucial to create a reliable animal model. In humans, rats, and mice, one copy of the PIH1D3 gene is located on the X chromosome. Interestingly, mice have an additional, intronless copy of the Pih1d3 gene on chromosome 1. To develop an accurate disease model, it is best to manipulate the X-linked PIH1D3 gene, which contains essential regulatory sequences within the introns for precise gene expression. This study aimed to develop a tailored rat model for PIH1D3-associated ciliopathy with the ultimate goal of uncovering the intricate molecular mechanisms responsible for ciliary defects in the disease. Methods: Novel Pih1d3-knockout (KO) rats were created by using TALEN-mediated non-homologous DNA recombination within fertilized rat eggs and, subsequently, underwent a comprehensive characterization through a battery of behavioral and pathological assays. A series of biochemical and histological analyses were conducted to elucidate the identity of protein partners that interact with PIH1D3, thus shedding light on the intricate molecular mechanisms involved in this context. Results: PIH1D3-KO rats reproduced the cardinal features of ciliopathy including situs inversus, defects in spermatocyte survival and mucociliary clearance, and perinatal hydrocephalus. We revealed the novel function of PIH1D3 in cerebrospinal fluid circulation and elucidated the mechanism by which PIH1D3 deficiency caused communicating hydrocephalus. PIH1D3 interacted with the proteins required for the pre-assembly and uploading of outer (ODA) and inner dynein arms (IDA), regulating the integrity of dynein arm structure and function in cilia. Conclusion: PIH1D3-KO rats faithfully reproduced the cardinal features of ciliopathy associated with PIH1D3 deficiency. PIH1D3 interacted with the proteins responsible for the pre-assembly and uploading of dynein arms in cilia, and its deficiency led to dysfunctional cilia and, thus, to ciliopathy by affecting the pre-assembly and uploading of dynein arms. The resultant rat model is a valuable tool for the mechanistic study of PIH1D3-caused diseases.

Dataset Information

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

Publications

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

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