A Research Blog

Dr Gerald Liew received his PhD in Biochemistry from Stanford University, and is currently a first year MD student at Duke-NUS Medical School. His primary research interest investigates the signalling pathways involved in ensuring proper primary cilium function. Today, Gerald shares with us more about this topic from his PhD Thesis.

At the primary cilium Proper cilia function is essential to life. As you read this paragraph, the cilia lining your windpipe are constantly beating to sweep mucus and dirt out of your lungs. But did you know that non-motile (primary) cilia can be found on the surface of almost every cell in the human body? For over a century, scientists believed primary cilia to be an unimportant evolutionary vestige. Only recently did we appreciate that the primary cilium functions as a cellular “antennae” to convert extracellular signals into intracellular responses, allowing a cell to sense its environment and respond accordingly1.  Within the cilium, intraflagellar transport (IFT) protein complexes move bidirectionally along its length, in a process termed intraflagellar transport (IFT). IFT is essential for the trafficking of ciliary cargoes needed for ciliogenesis1-5. Notably, mutations inindividual IFT proteins have also been linked to perturbations in developmental pathways (e.g. vertebrate Hedgehog signaling6) as well as disease (e.g. polycystic kidney disease7).

Figure 1: What are the molecular mechanisms that govern the trafficking of BBSome and associated cargoes into and out of the primary cilium? In particular, how is the nucleotide state of ARL6 regulated within the primary cilium? (Image credit: Max Nachury)

Research on “ciliopathies” such as Bardet-Biedl syndrome (BBS) has also yielded rich insights into the function of the primary cilium. BBS is a disease characterized by pleiotropic symptoms including blindness, deafness, obesity, kidney failure, and polydactyly8. To date, 21 genes have been implicated in causing BBS, but much remains to be learned about the function of BBS genes. Prior work by my host lab had shown that eight distinct BBS gene products assemble into a coat-like complex termed the BBSome9, which directly recognizes signaling receptors and is recruited to membranes by the ARF-like GTPase, ARL6/BBS310. Furthermore, the BBSome co-moves with IFT complexes along the cilium11,12, suggesting a role for the BBSome in the trafficking of ciliary cargoes. But the precise function of the BBSome and how ARL6 is regulated remained unclear (Fig. 1).

To understand how the trafficking of BBSome and signaling receptors might be perturbed in BBS patients, we focused on a Rab-like GTPase and IFT complex-B subunit (IFT27), which was recently found mutated in a BBS family13. Specifically, we investigated the role of IFT27 in regulating ciliary traffic with other GTPases. Strikingly, ARL6, BBSome and a candidate BBSome cargo, GPR161, accumulated in IFT27-deficient cilia. By measuring BBSome entry and exit rates into and out of the cilia, we determined that BBSome entry rates remained unaffected, while BBSome exit rates were reduced ~3-fold in the absence of IFT27. This indicated that IFT27 operates inside cilia to promote ciliary exit of the BBSome/ARL6 coat.

We next sought to determine how IFT27 promotes BBSome/ARL6 exit from cilia. Remarkably, a proteomics screen uncovered an interaction between ARL6 and IFT27. We were further able to demonstrate using pure recombinant proteins that ARL6 interacted with IFT27 only when ARL6 was stripped of GTP or GDP. Since the specific recognition of the nucleotide-empty form of a GTPase is a signature feature of guanine nucleotide exchange factors (GEFs), we hypothesized that IFT27 participates in GTP loading onto ARL6 to promote assembly of exit-bound and cargo-laden BBSome coats, and my host lab is currently pursuing this hypothesis.

Our work on dissecting the regulation of BBSome trafficking within cilia provides insights on modes of intracellular protein transport beyond the canonical coat complexes, and illuminates the molecular choreography leading the retrieval of signaling molecules from cilia back into the cell.

What is this paper: The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3 (http://www.cell.com/developmental-cell/abstract/S1534-5807(14)00590-5).

Where published: Developmental Cell (Vol 31, pg 265-278, 2014)

Who published: First author: Gerald Liew; Senior author: Maxence Nachury 

Where are the authors from: Gerald Liew is a first-year MD student at Duke-NUS Medical School. The work described in this blog post was performed when he was a PhD student at Stanford University working in the Nachury lab in the Department of Molecular and Cellular Physiology.

Funding information: Gerald Liew was supported by a National Science Scholarship from the Agency for Science, Technology and Research, Singapore.

Sources for further reading:

  1. 1. Nachury, M. V. How do cilia organize signalling cascades? Philos Trans R Soc Lond B Biol. 369,20130465 (2014).
  2. 2. Piperno, G. & Mead, K. Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella. Proc Natl Acad Sci USA. 94,4457–4462 (1997).
  3. 3. Cole, D. G. et al. Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol. 141, 993–1008 (1998).
  4. 4. Rosenbaum, J. L. & Witman, G. B. Intraflagellar transport. Nat. Rev. Mol. Cell Biol. 3,813–825 (2002).
  5. 5. Wren, K. N. et al. A differential cargo-loading model of ciliary length regulation by IFT. Curr. Biol. 23,2463–2471 (2013).
  6. 6. Huangfu, D. et al.Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature. 426, 83–87 (2003).
  7. 7. Pazour, G. J. et al. Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151,709–718 (2000).
  8. 8. Fliegauf, M., Benzing, T. & Omran, H. When cilia go bad: cilia defects and ciliopathies. Nat. Rev. Mol. Cell Biol. 8,880–893 (2007).
  9. 9. Nachury, M. V. et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 129, 1201–1213 (2007).
  10. 10. Jin, H. et al. The conserved Bardet-Biedl syndrome proteins assemble a coat that traffics membrane proteins to cilia. Cell. 141, 1208–1219 (2010).
  11. 11. Ou, G., Blacque, O. E., Snow, J. J., Leroux, M. R. & Scholey, J. M. Functional coordination of intraflagellar transport motors. Nature. 436, 583–587 (2005).
  12. 12. Lechtreck, K.-F. et al. The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella. J Cell Biol. 187, 1117–1132 (2009).
  13. 13. Aldahmesh, M. A. et al. IFT27, encoding a small GTPase component of IFT particles, is mutated in a consanguineous family with Bardet-Biedl syndrome. Hum. Mol. Genet. 23, 3307–3315 (2014).

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