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.
Highlights
Cilia play crucial roles in sensing and transducing extracellular signals
Bidirectional protein trafficking within cilia, and the entry and exit of proteins across the ciliary gate composed of the transition zone (TZ) is mediated by the intraflagellar transport (IFT) machinery 7,8, which was originally identified in flagella of the green alga Chlamydomonas and is often referred to as ‘IFT particles’ or ‘IFT trains’9–11
Lysates prepared from HEK293T cells coexpressing DYNC2H1(N)-mCherry and any of the DYNC2LI1 constructs fused to EGFP were subjected to immunoprecipitation with glutathione S-transferase (GST)-fused anti-mChe nanobodies (Nb) (LaM-2 version)33 prebound to glutathione-Sepharose beads, followed by SDS-PAGE and immunob
Summary
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. Cilia are microtubule-based projections from the surfaces of a variety of eukaryotic cells, and perceive and transduce mechanical signals, such as fluid flow, and biochemical signals, such as the Hedgehog (Hh) family of morphogens. To achieve these functions, specific proteins exist on the ciliary membrane, including G proteincoupled receptors (GPCRs) and ion channels. Within the IFT machinery, the IFT-B complex, which is composed of 16 subunits, mediates anterograde trafficking driven by the kinesin-II motor, and the export of ciliary membrane proteins across the TZ together with the BBSome. Recent studies in Caenorhabditis elegans suggested that the IFT-A complex and IFT dynein are required for the integrity and gating function of the T Z14,15
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