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Abstract
Cilia are thin microtubule-based organelles, which act in cell sensing and movement. Cilia are gated from the cell body and devoid of ribosomes; thus, they rely on large bi-directional protein complexes, termed intraflagellar transport (IFT) trains to move material from the cell body into cilia. IFT trains are composed of repeats of IFT-A, IFT-B, kinesin-II (anterograde motor), and IFT-dynein (retrograde motor). Anterograde trains emerge from a pool of IFT proteins at the cilia base, traffic to the ciliary tip, remodel into retrograde trains and then return to the base. How these trains assemble at the base and remodel at the tip is poorly understood. Here, we use single-particle microscopy on fluorescently-tagged IFT proteins, representing different parts of the train, to reveal how these processes occur. Using different photobleaching approaches at the cilia base, we found that trains assemble in a stepwise fashion with IFT-A recruiting to the base first followed by IFT-B, kinesin-II, and lastly dynein and other cargos. Additionally, while IFT trains continuously cycle through cilia, only a portion of IFT-B is immediately reused on trains; IFT-A, dynein, and kinesin enter the cell body pool after cycling through cilia. Next, we utilized similar photobleaching techniques with single- and two-color imaging on fluorescent protein-tagged IFT proteins, and focused on the ciliary tip. We found that IFT-A, IFT-B, and IFT-dynein remain associated during train fragmentation. We postulate that fragmentation of anterograde trains at the tip releases IFT dynein from inhibitory contacts with IFT-B leading to its activation on the smaller fragments and initiating retrograde IFT.
Lastly, we analyzed how tubulin binds to IFT for import into cilia. We identified that when a strain lacking the CH domain of IFT54 (ΔCH-IFT54) was combined with strains defective in the establish IFT81/74 tubulin binding site (IFT81-5E and ΔN-IFT74), the moderate ciliogenesis defects of the single mutants are aggravated with many cells failing to assemble cilia. Analysis of the ift54-2 ΔCH-IFT54 ift81-1 CH5E-IFT81 double mutant revealed normal IFT but reduced frequency of tubulin transport. These observations suggest that the CH-domain of IFT54 participates in ciliary tubulin transport, likely via and a tripartite domain.
Lastly, we analyzed how tubulin binds to IFT for import into cilia. We identified that when a strain lacking the CH domain of IFT54 (ΔCH-IFT54) was combined with strains defective in the establish IFT81/74 tubulin binding site (IFT81-5E and ΔN-IFT74), the moderate ciliogenesis defects of the single mutants are aggravated with many cells failing to assemble cilia. Analysis of the ift54-2 ΔCH-IFT54 ift81-1 CH5E-IFT81 double mutant revealed normal IFT but reduced frequency of tubulin transport. These observations suggest that the CH-domain of IFT54 participates in ciliary tubulin transport, likely via and a tripartite domain.