Interaction of WDR60 intermediate chain with TCTEX1D2 light chain of the dynein-2 complex is crucial for ciliary protein trafficking

Abstract

The dynein-2 complex mediates trafficking of ciliary proteins by powering the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Although 11 subunits are known to constitute the dynein-2 complex, with several light-chain subunits shared by the dynein-1 complex, the overall architecture of the dynein-2 complex has not been fully clarified. Utilizing the visible immunoprecipitation assay, we demonstrated the interaction modes among the dynein-2 subunits, including previously undefined interactions, such as that between WDR60 and the TCTEX1D2–DYNLT1/DYNLT3 dimer. The dynein-2 complex can be divided into three subcomplexes, namely DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. We established cell lines lacking WDR60 or TCTEX1D2, both of which are dynein-2–specific subunits encoded by ciliopathy-causing genes, and found that both WDR60-knockout (KO) and TCTEX1D2-KO cells show defects in retrograde ciliary protein trafficking, with WDR60-KO cells demonstrating more severe defects probably due to failed assembly of the dynein-2 complex. The exogenous expression of a WDR60 mutant lacking TCTEX1D2 binding partially restored retrograde trafficking to a level comparable to that of TCTEX1D2-KO cells. Thus, our results demonstrated that WDR60 plays a major role and TCTEX1D2 plays an auxiliary role in the dynein-2 complex to mediate retrograde ciliary protein trafficking.

Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

“…Smo is thought to enter cilia continuously but then rapidly exits in the absence of ligand. Our data are consistent with this, with loss of WDR60 resulting in aberrant accumulation of Smo in cilia, likely leading to perturbed hedgehog signaling (also recently validated by others [ Hamada et al, 2018 ]). Interestingly, abnormal accumulation of Smo in the cilia has also been observed in mouse fibroblasts with mutations in DHC2/DYNC2H1.…”

“…Our data show that the structural asymmetry within the dynein-2 motor is matched by functional asymmetry. Perhaps most strikingly, WDR34 is essential for axoneme extension during the early steps of ciliogenesis, whereas WDR60 is not required for cilium extension (the latter finding being validated recently by others [ Hamada et al, 2018 ]). Depletion of WDR34 using RNAi is also associated with ciliary defects ( Asante et al, 2013 ), whilst patient fibroblasts have shorter cilia with a bulbous tip ( Huber et al, 2013 ) and fibroblasts from WDR34 knockout mice have stumpy cilia and defects in Shh signaling ( Wu et al, 2017 ).…”

“…These findings are all consistent with roles for WDR34 and WDR60 in IFT. Moreover, further recent data found that WDR60 plays a major role in retrograde ciliary protein trafficking ( Hamada et al, 2018 ).…”

Intraflagellar transport trains and motors: Insights from structure

“…The tail asymmetry is generated by an unusual stoichiometry sub-complex of intermediate and light chains [ 49 ]. The C-terminal β-propeller domains of the intermediate chains (WDR34 and WDR60) each bind a copy of the heavy chain, and are heterodimerized by their N-proximal regions and an array of light chains (one DYNLRB dimer, three DYNLL dimers, and a presumptive DYNLT-TCTEX1D2 heterodimer) [ 29 , 49 , [131] , [132] , [133] , [134] , [135] , [136] ]. This sub-complex of intermediate and light chains stabilizes straightening of one heavy chain in the tail and steers the other into a zigzag conformation, which tailors dynein-2′s structure to the repeat of the IFT-B polymer [ 49 ].…”

Emerging mechanisms of dynein transport in the cytoplasm versus the cilium

“…An analysis of dynein-2 subunit interactions via a visible immunoprecipitation (VIP) assay has been published, demonstrating interactions between WDR60 and TCTEX1/3-TCTEX1D2; WDR34 and LC8 and Roadblock; and the intermediate chain-light chain complex with the heavy chain and light-intermediate chain [ 148 ].…”