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

Abstract

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60/DYNC2I1 and the DYNC2H1–DYNC2LI1 dimer from dynein-2, and IFT54 and IFT57 from IFT-B contribute to the dynein-2–IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.

Roles for CEP170 in cilia function and dynein-2 assembly

“…1A, Table S1). We also previously identified CEP170 binding in non-TMT proteomic methods (Table S1) (Hiyamizu et al, 2023b). We validated the binding of CEP170 to WDR34 by coimmunoprecipitation (Fig.…”

“…Previously, we have used proteomic data generated using tagged WDR60 and WDR34 (Hiyamizu et al, 2023b; Shak et al, 2023; Vuolo et al, 2018) to identify dynein-2 interacting proteins. The most consistent hit in these data sets, CEP170, was identified in 12 new and previously published tandem mass tag (TMT) datasets, 11 of which having a Log 2 abundance ratio greater than 1 (Fig.…”

“…Along with cilia defects following loss of CEP170, the main conclusion of this work is the identification of, CEP170, as an interactor of dynein-2. To date, the majority of published dynein-2 interactions have only been reported in the context of IFT-A and IFT-B (Hiyamizu et al, 2023a; Hiyamizu et al, 2023b; Shak et al, 2023; Toropova et al, 2019; Tsurumi et al, 2019; Vuolo et al, 2018; Zhu et al, 2021) these interactions have given us great insight into how dynein-2 interacts with IFT-A and IFT-B. Whilst we cannot say for certain which subunit of dynein-2 CEP170 binds to, CEP170 can be robustly detected using either WDR34 and WDR60 in immunoprecipitations.…”

“…Previous work has defined interactions of WDR60 and WDR34 with other dynein-2 components and IFT proteins (Hiyamizu et al, 2023b; Shak et al, 2023; Vuolo et al, 2018). In this study, we extend that work to define the sDAP protein CEP170 as a dynein-2 interactor.…”

Hot-wiring dynein-2 uncovers roles for IFT-A in retrograde train assembly and motility

“…Interestingly, our data also show that the DQR mutations that favor motor untrapping do not significantly interfere with dynein-2 loading onto anterograde trains. We speculate that the multiple links established between dynein-2 and anterograde IFT trains (Hiyamizu et al, 2023;Lacey et al, 2023;Pedersen et al, 2006;Toropova et al, 2019;Zhu et al, 2021) are capable of accommodating any conformational changes resulting from the DQR mutations.…”