Flagellar Growth: Boarder control on the IFT train
Cilia and flagella are cylindrical organelles that are present at the surface of many eukaryotic cells, where they detect changes in the local environment and—when they beat—help the cells to move. An individual cilium or flagellum grows by adding new protein subunits to its tip, using a special mechanism to move proteins from the body of the cell to the tip of the organelle.
Intraflagellar transport, or IFT, was discovered by Joel Rosenbaum and co-workers at Yale University twenty years ago while they were studying the green alga Chlamydomonas, which is a classic model for flagellum studies (Kozminski et al., 1993). In this form of transport, complexes containing about 20 IFT proteins are moved from the base to the tip of the flagella, and are then recycled back towards the base. This movement can be compared to trains travelling on microtubule tracks. At the time, it was proposed that the cargoes, or passengers, on the IFT ‘train’ are the precursors of the axoneme that forms the core of the flagellum (Figure 1A). This very reasonable hypothesis is supported by the observation that construction of the flagellum is inhibited if a single IFT protein is missing (Pazour et al., 2000).
However, demonstrating the presence of passengers on IFT trains turned out to be very tricky. IFT trains were purified from different organisms, confirming that IFT proteins were tightly bound together, but the presence of cargoes could not be shown convincingly. This suggests that association is transitory and cannot survive biochemical purification. So are the passengers hiding?
Now, in eLife, Hiroaki Ishikawa and Wallace Marshall, both from the University of California, San Francisco, and co-workers in the US, Japan and Germany report on a new IFT protein called IFT56 (also known as DYF13, TTC26B or PIFTC3) that could deliver a specific set of proteins that power the movement of flagella (Ishikawa et al., 2014). IFT56 would therefore function as a train conductor selecting particular proteins to board the train (Figure 1B). This exciting proposal is based on exhaustive analysis of cilia and flagella in zebrafish and Chlamydomonas when IFT56 expression was prevented. A mutation leading to the production of a severely truncated IFT56 protein did not interfere with train speed or frequency, but resulted in the formation of slightly shorter flagella with reduced motility. Proteomic analyses revealed that these flagella contained reduced amounts of several proteins associated with the generation or control of flagellum beating. Although the model is based on indirect evidence, the recent report that cargo proteins can finally be visualised (Wren et al., 2013) means that it is now possible to test this hypothesis: in other words, we will be able to unmask the passengers.
If IFT56 really acts as a conductor, how does it function? Recent data indicate that only a minority of the trains transport cargoes (Wren et al., 2013), despite the presence of IFT56 on all of them. So what controls loading? Is it simply the availability of cargoes or are some proteins marked in some way to indicate that they should be sent to the flagellum? In other words, do passengers need tickets to gain access to the train? This ticket could be a single post-translational modification such as phosphorylation.
The absence of IFT56 affects different organisms in different ways. In protozoa called trypanosomes, an absence of IFT56 causes flagella to go missing (Absalon et al., 2008; Franklin and Ullu, 2010), but in zebrafish (Zhang et al., 2012) and the green alga it only results in shorter flagella. This absence could impact the stability or movement of the IFT train. Perhaps in some species, the conductor is also an engineer, assisting train formation and function.
Alternatively, such a difference could reflect how the stability of the flagellum depends on the elements that power flagellar beating. For example, in Leishmania, the cousins of trypanosomes, a modification to the molecular motor results in the construction of much shorter flagella (Harder et al., 2010). This same modification in Chlamydomonas does not have this effect (Kamiya, 1988). In this case, the different phenotypes would be due to the nature of the flagellum itself–IFT56 and the IFT train would not play a direct role in determining them.
Intriguingly, IFT56 is also associated with IFT trains in immotile cilia that do not possess the motility elements discussed above (Blacque et al., 2005). This may seem to contradict the model proposed by Ishikawa et al. but could be explained by the conductor specialising to detect and transport any cargoes that possess the same ticket. In these conditions, IFT56 could ship very different protein complexes providing a single recognition element is shared between them.
Twenty years after the discovery of intraflagellar transport, we are now getting the first insights about putative cargoes. In the future, progress in live imaging, functional genomics and better understanding of the structure of IFT trains should illuminate the mechanisms by which cargoes are recognised, loaded and delivered to their destination.
Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomesMolecular Biology of the Cell 19:929–944.https://doi.org/10.1091/mbc.E07-08-0749
Functional genomics of the cilium, a sensory organelleCurrent Biology 15:935–941.https://doi.org/10.1016/j.cub.2005.04.059
Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtiiThe Journal of Cell Biology 107:2253–2258.https://doi.org/10.1083/jcb.107.6.2253
A motility in the eukaryotic flagellum unrelated to flagellar beatingProceedings of the National Academy of Sciences of the United States of America 90:5519–5523.https://doi.org/10.1073/pnas.90.12.5519
A differential cargo-loading model of ciliary length regulation by IFTCurrent Biology 23:2463–2471.https://doi.org/10.1016/j.cub.2013.10.044
Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafishMolecular Biology of the Cell 23:3069–3078.https://doi.org/10.1091/mbc.E12-01-0019
Article and author information
- Version of Record published: March 18, 2014 (version 1)
© 2014, Fort and Bastin
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
- Page views
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
- Cell Biology
Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.
- Cell Biology
Full-length mRNAs transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the 'transferome') is unknown. Here, we analyzed the transferome in an in vitro human-mouse cell co-culture model using RNA-sequencing. We found that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount of transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells. Typically, <1% of endogenous mRNAs undergo transfer. Non-selective, expression-dependent RNA transfer was further validated using synthetic reporters. RNA transfer appears contact-dependent via TNTs, as exemplified for several mRNAs. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.