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

ABSTRACT: 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.

SUBMITTER: Dymek EE

PROVIDER: S-EPMC6727744 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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

Dymek Erin E EE Lin Jianfeng J Lin Jianfeng J Fu Gang G Porter Mary E ME Nicastro Daniela D Smith Elizabeth F EF

Molecular biology of the cell 20190522 15

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 <i>Chlamydomonas pacrg</i> 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 lac ...[more]

PMID: 31116684

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Project description:UnlabelledBackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.ResultsPACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer's vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.

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

Publications

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

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