Intraflagellar transport (IFT) particles or trains are composed of IFT-A and IFT-B complexes. To assess the working mechanism of the IFT-A complex in IFT and ciliogenesis, we have analyzed ift43 mutants of Chlamydomnonas in conjunction with mutants of the other IFT-A subunits. An ift43 null mutant or a mutant with a partial deletion of the IFT43 conserved domain has no or short flagella. The mutants accumulate not only IFT-B but also IFT-Ain the short flagella, which is in contrast to an ift140 null mutant. The IFT43 conserved domain is necessary and sufficient for the function of IFT43. IFT43 directly interacts with IFT121 and loss of IFT43 results in instability of IFT-A. A construct with a partial deletion of the IFT43 conserved domain is sufficient to rescue the instability phenotype of IFT-A, but results in diminishing of IFT-A at the peri-basal body region. We have further provided evidence for the direct interactions within the IFT-A complex and shown that the integrity of IFT-A is important for its stability and cellular localization. Finally, we show that both IFT43 and IFT140 are involved in mobilizing ciliary precursors from the cytoplasmic pool during flagellar regeneration, suggesting a novel role of IFT-A in transporting ciliary components in the cytoplasm to the peri-basal body region.

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.

The length of cilia is robustly regulated [1]. Previous data suggest that cells possess a sensing system to control ciliary length [2-5]. However, the details of the mechanism are currently not known [6, 7]. Such a system requires a mechanism that responds to ciliary length, and consequently, disruption of that response system should alter ciliary length [1]. The assembly rate of cilium mediated by intraflagellar transport (IFT) gradually decreases as the cilium elongates and eventually is balanced by the constant rate of disassembly, at which point cilium elongation stops [8, 9]. Because the rate of IFT entry into the cilium also decreases as the cilium elongates [10], regulation of IFT entry could provide the mechanism for length control. Previously, we showed that phosphorylation of the FLA8/KIF3B subunit of the anterograde kinesin-II IFT motor blocks IFT entry and flagellar assembly in Chlamydomonas [11]. Here, we show in Chlamydomonas that cellular signaling in response to alteration of flagellar length regulates phosphorylation of FLA8/KIF3B, which restricts IFT entry and, thus, flagellar assembly to control flagellar length. Cellular levels of phosphorylated FLA8 (pFLA8) are tightly linked to flagellar length: FLA8 phosphorylation is reduced in cells with short flagella and elevated in cells with long flagella. Depletion of the phosphatases CrPP1 and CrPP6 increases the level of cellular pFLA8, leading to short flagella due to decreased IFT entry. The results demonstrate that ciliary length control is achieved by a cellular sensing system that controls IFT entry through phosphorylation of the anterograde IFT motor.