Abstract
ABSTRACTIntraflagellar transport (IFT) particles are composed of polyprotein complexes IFT-A and IFT-B as well as cargo adaptors such as the BBSome. Two IFT-B subunits, IFT25 and IFT27 were found to form a heterodimer, which is essential in exporting the BBSome out of the cilium but not involved in flagellar assembly and cytokinesis in vertebrates. Controversial results were, however, recorded to show that defects in IFT, flagellar assembly and even cytokinesis were caused by IFT27 knockdown in Chlamydomonas reinhardtii. Using C. reinhardtii as a model organism, we report that depletion of IFT25 has no effect on flagellar assembly and does not affect the entry of the BBSome into the flagellum, but IFT25 depletion did impair BBSome movement out of the flagellum, clarifying the evolutionally conserved role of IFT25 in regulating the exit of the BBSome from the flagellum cross species. Interestingly, depletion of IFT25 causes dramatic reduction of IFT27 as expected, which does not cause defects in flagellar assembly and cytokinesis in C. reinhardtii. Our data thus support that Chlamydomonas IFT27, like its vertebrate homologues, is not involved in flagellar assembly and cytokinesis.
Highlights
Cilia and flagella project from the surface of most eukaryotic cells in interphase and share the same organelle structure that consists of a microtubule extension called axoneme surrounded by a specialized plasma membrane (Dutcher, 2003; Rosenbaum and Witman, 2002)
The underlying molecular mechanisms seem different, both cases gained a common outcome that loss of IFT27 causes defects in intraflagellar transport (IFT) and flagellar assembly. These results suggest that IFT25 and IFT27 probably play a role in IFT and flagellar assembly in a species-dependent manner (Eguether et al, 2014; Huet et al, 2014; Keady et al, 2012; Liew et al, 2014; Qin et al, 2007)
The mean frequencies of anterograde and retrograde IFT25::HA::GFP were measured to be 0.89±0.06 particle/sec (n=100) and 1.04±0.10 particle/sec (n=100), respectively, similar to the frequencies observed for IFT56-GFP (Ishikawa et al, 2014) (Fig. 1E). These results show that IFT25 distribution and movement in flagella are similar to the other IFT proteins studied to date in C. reinhardtii, and the N-terminal GFP-tagged IFT25 was functional in IFT and flagellar assembly and can be used to replace the native IFT25
Summary
Cilia and flagella project from the surface of most eukaryotic cells in interphase and share the same organelle structure that consists of a microtubule extension called axoneme surrounded by a specialized plasma membrane (Dutcher, 2003; Rosenbaum and Witman, 2002). These organelles differ in performing signaling and motility-based functions they are assembled by an evolutionally conserved process called intraflagellar transport (IFT), where the linear IFT trains of large protein complexes undergo robust bidirectional motility along the axoneme (Kozminski et al, 1993; Pigino et al, 2009). How IFT-A, IFT-B and the BBSome interact to assemble functional IFT trains remains largely unknown, recent studies have shown that the IFT-B subunit IFT74 is required for the coupling between IFT-A and IFT-B, at least in C. reinhardtii (Brown et al, 2015), and a second IFT-B subunit, the small GTPase IFT27, plays a role in linking the BBSome to IFT-B as found in the mouse model (Eguether et al, 2014; Liew et al, 2014)
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