Combinations of deletion and missense variations of the dynein-2 DYNC2LI1 subunit found in skeletal ciliopathies cause ciliary defects

Abstract

Cilia play crucial roles in sensing and transducing extracellular signals. Bidirectional protein trafficking within cilia is mediated by the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes, with the aid of kinesin-2 and dynein-2 motors. The dynein-2 complex drives retrograde trafficking of the IFT machinery after its transportation to the ciliary tip as an IFT cargo. Mutations in genes encoding the dynein-2-specific subunits (DYNC2H1, WDR60, WDR34, DYNC2LI1, and TCTEX1D2) are known to cause skeletal ciliopathies. We here demonstrate that several pathogenic variants of DYNC2LI1 are compromised regarding their ability to interact with DYNC2H1 and WDR60. When expressed in DYNC2LI1-knockout cells, deletion variants of DYNC2LI1 were unable to rescue the ciliary defects of these cells, whereas missense variants, as well as wild-type DYNC2LI1, restored the normal phenotype. DYNC2LI1-knockout cells coexpressing one pathogenic deletion variant together with wild-type DYNC2LI1 demonstrated a normal phenotype. In striking contrast, DYNC2LI1-knockout cells coexpressing the deletion variant in combination with a missense variant, which mimics the situation of cells of compound heterozygous ciliopathy individuals, demonstrated ciliary defects. Thus, DYNC2LI1 deletion variants found in individuals with skeletal ciliopathies cause ciliary defects when combined with a missense variant, which expressed on its own does not cause substantial defects.

Multiple interactions of the dynein-2 complex with the IFT-B complex are required for effective intraflagellar transport

“…It is noteworthy that mutations in all of the dynein-2-specific subunits and in all of the IFT-A subunits are known to cause skeletal ciliopathies of varying clinical severity ( McInerney-Leo et al, 2015 ; Reiter and Leroux, 2017 ; Schmidts, 2014 ; Zhang et al, 2018 ). We recently revealed the relationships between defects in protein–protein interactions and ciliary defects caused by mutations of DYNC2LI1 and IFT-A subunits (IFT122 and IFT144) found in skeletal ciliopathies ( Ishida et al, 2021 ; Qiu et al, 2022 ; Takahara et al, 2018 ).…”

Molecular basis underlying the ciliary defects caused by IFT52 variations found in skeletal ciliopathies

“…Mutations in subunits of the IFT-A and dynein-2 complexes are known to cause skeletal ciliopathies (Schmidts, 2014;Schmidts et al, 2015;McInerney-Leo et al, 2015;Zhang et al, 2018), and we recently clarified the molecular basis underlying these ciliopathies caused by mutations in the IFT-A and dynein-2 subunits (IFT122, IFT144, and DYNC2LI1) (Takahara et al, 2018;Ishida et al, 2021;Qiu et al, 2022). In the present study, we investigated the molecular and ciliary defects caused by skeletal ciliopathy-associated mutations of IFT52, which is a subunit of the IFT-B complex and constitutes, together with other subunits, the binding site for heterotrimeric kinesin-II.…”

“…Quantification of fluorescence intensities and statistical analyses were performed as described previously ( Qiu et al. , 2022 ; Zhou et al.…”

“…Quantification of fluorescence intensities and statistical analyses were performed as described previously (Qiu et al, 2022;Zhou et al, 2022). Briefly, all images acquired under the same setting were analyzed using ZEN 3.1 software (Carl Zeiss).…”

“…In particular, mutations in subunits of the IFT-A complex and the dynein-2 complex result in skeletal ciliopathies. We recently demonstrated the molecular mechanisms underlying ciliary defects caused by mutations in the IFT-A subunits IFT122 and IFT144 and a dynein-2 subunit, DYNC2LI1, which are found in skeletal ciliopathies (Takahara et al, 2018;Ishida et al, 2021;Qiu et al, 2022).…”