Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for IFT of radial spokes

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Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

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  1. Evaluation Summary:

    This paper is of broad interest to readers interested in motile cilia and cargo transport mediated by intraflagellar transport (IFT). It examines how radial spokes are trafficked into cilia by IFT, which represents a key process in the assembly of motile cilia. The authors demonstrate that an adaptor protein (ARMC2) is needed for association of radial spokes with the IFT machinery. They also find that the interaction of ARMC2/radial spokes with individual IFT trains (or particles) occurs in a stochastic manner rather than there being a specialized subset of trains specifically designated for a particular cargo. The results support the key claims in the paper.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    Building cilia requires bidirectional intraflagellar transport (IFT) that moves components in and out of the growing organelles. This process involves multi-component IFT trains being driven by kinesin and dynein motors. Cargos are known to attach to these trains allowing them access to the distal cilium where offloading generally occurs. This process provides a mechanism by which axonemal components reach the site of assembly. Defects in this process have profound impact on mammalian physiology and development. Chlamydomonas is an extremely powerful model system in which to dissect this complex process as numerous genetic, biochemical and microscopic tools can be readily applied. Although numerous proteins have been shown to require IFT for their transport into cilia, how many different components interact with the IFT trains to achieve this and whether those associations are inter-dependent or even exclusionary remains poorly understood. In this manuscript, the authors take advantage of a Chlamydomonas mutant, pf27, which assembles normal looking radial spokes (RS) but only in the very proximal part of the organelle even though reconstitution experiments indicate that the assembly sites in the remainder of the axoneme are fully functional. Using a candidate gene approach, they identify the gene product PF27 as ARMC2 a protein containing multiple armadillo repeats and whose dysfunction in mammals is known to disrupt lung function and yield male infertility. They find that ARMC2 and RS are trafficked together by IFT, while in the absence of RS, ARMC2 is moved by itself. The conclusion is that ARMC2 is an adaptor linking RS to IFT trains. Assembly of RS in the proximal region of pf27 cilia is proposed to occur by diffusion of RS from the cell body. This would seem to imply a certain leakiness to the ciliary gate even for large mega-dalton sized complexes. The authors suggest that IFT-adaptor interactions are critical for control of cargo entry into cilia. While that certainly seems quite reasonable, control of the adaptor-cargo association is likely equally important for productive transport, off-loading and axonemal incorporation.

    Using fluorescent tags and TIRF microscopy, the authors observe that ARMC2 is moved by IFT to the ciliary tip but is there off-loaded and diffuses back to the cell body - this has been seen with several other ciliary proteins previously. They carefully characterize the co-transport and release of ARMC2 and a RS marker protein RSP3, and clearly demonstrate that RSP3 transport fails in the absence of ARMC2.

    One important issue is whether individual IFT trains are dedicated to a particular cargo or set of cargoes (i.e. are active), or if all trains have similar cargo binding capacity and associations of individual complexes with any given train is stochastic. The experimental results reported for ARMC2 and outer arm dynein closely fit values derived from calculated probabilities of chance interactions with any given train. This is an important result and is well supported by the numeric data.

  3. Reviewer #2 (Public Review):

    In this work, Lechtreck et al showed that the IFT-dependent transport of radial spokes in Chlamydomonas requires ARMC2/PF27. They first identified the pf27 mutant strain as an armc2 mutant strain and showed that radial spoke localization in this mutants is restricted to the proximal part oft he flagella. The authors showed by using tagged ARMC2 and the radial spoke protein RSP3 that ARMC2/PF27 is highly enriched in growing flagella and that ARMC2/PF27 and RSP3 comigrated on anterograde trains. Furthermore, after unloading at the flagellar tip, only ARMC2/PF27 diffused back to the cell body and RSP3 was attached to the flagellar axoneme, supporting the role of ARMC2/PF267 as an IFT cargo adapter specific for radial spoke proteins. The authors also show that ODA16 (an outer dynein arm adapter protein) and IDA3 (an inner dynein arm cargo protein) move independently of ARMC2/PF17 indicating that unrelated cargoes are distributed stochastically onto IFT trains. The conclusions of this paper are well supported by the results.