Cilia and cilia-related disease
Cilia and flagella are microtubule-based cell extensions. They are widely distributed in the mammalian body and most cell types possess motile or non-motile cilia. Loss of ciliary motility results in chronic airway infections and male infertility. Cilia also function in cellular sensing and signaling: The receptors for light and odors, for example, are located in modified cilia. Defects in the assembly or function of cilia are responsible for numerous diseases and developmental disorders including polydactyly (extra fingers and toes), blindness, anosmia (loss of the sense of smell) and polycystic kidney disease. The latter is one of the most common inherited disorders in humans. The goal of our research is to understand how cells assemble and maintain cilia, and to identify the molecular mechanisms underlying ciliary diseases.
Cilia and flagella are exceptionally well conserved in evolution allowing us to analyze their biology in model organisms. For most of our studies we use the unicellular green alga Chlamydomonas reinhardtii. Many genes that when defective cause ciliopathies in mammals are conserved in the Chlamydomonas genome. Chlamydomonas possess two typical 9+2 cilia and allows for 1) the isolation of cilia for biochemical analysis, 2) the genetic and molecular genetic manipulation of cilia, and 3) high resolution in vivo microscopy of cilia. This combination of features makes Chlamydomonas an ideal model to study ciliary biology.
Cargo transport by IFT
Cilia are devoid of ribosomes and therefore all proteins required to build a cilium need to be transported from the cell body into the organelle. The major mechanism of protein transport in cilia is intraflagellar transport (IFT). This bidirectional motility of protein particles along cilia was first observed in 1993 by Keith Kozminski while working on Chlamydomonas in the lab of Joel Rosenbaum at Yale University. The IFT pathway is well conserved in almost all eukaryotic cells with cilia. It is required for ciliary assembly, maintenance, and signaling and defects in IFT and ciliary protein transport have been linked to many human diseases including the obesity syndrome Bardet-Biedl syndrome (BBS). IFT particles (consisting of ~2 dozen distinct IFT particle proteins) travel from the cell body along the ciliary microtubules to the ciliary tip powered by kinesin-2 (anterograde IFT); they return to the cell body driven by cytoplasmic dynein (retrograde IFT). The particles are believed to function as carriers to move proteins from the cell body to their assembly sites in the cilium and probably to return signals received by the cilium to the cell body.
Cells expressing GFP-tagged KAP, a subunit of the anterograde IFT motor, imaged by DIC and TIRF microscopy.
Simultaneous TIRF imaging of IFT20-mCherry (red) and the axonemal protein DRC4-GFP (green). Transport of the DRC4 cargo by IFT can be observed in the bottom cilium on the right side.
It is currently unclear how the IFT machinery transports and delivers hundreds of different proteins required in the cilium. One current research goal is to understand how IFT selects and delivers proteins for transport into the cilium. Specific questions include: How do cells ensure that the right proteins are transported into cilia? How do IFT particles function as carriers for such a large variety of distinct proteins? How are IFT particles loaded and unloaded and is the amount of cargo transported by IFT regulated? Do all ciliary proteins utilize IFT to get into the cilium or are there alternative, yet to be discovered, transport systems?
Using total internal reflection fluorescence micrscopy (TIRFM) we are able to image protein transport inside cilia of living cells directly. TIRFM has a high signal-to-noise ratio allowing us to visualize single proteins tagged with green fluorescent protein (GFP) at video rates. Using two-color TIRFM, we can image IFT carriers together with various cargo proteins and determine, e.g., how often such transport events occur and where in the cilium cargoes are unloaded. This approach provides a direct read-out for IFT function. We use existing and newly generated Chlamydomonas mutants to identify proteins which regulate cargo trafficking by IFT. Targets include various CDK-like and MAP protein kinases; mutants in these kinases cause a long flagella-phenotype with cilia up to three times the length of wild-type cells. One aim is to understand how this network of protein kinases senses ciliary length and transmits this information to the IFT system in order to adjust the delivery and removal of material from the organelle. A second focus is placed on BBS proteins, which appear to function in exporting certain signaling proteins from cilia. In human defects in BBS proteins cause obesity and blindness; Chlamydomonas BBS mutants lack phototactic behavior, the ability to move toward or away from a light source. Our working hypothesis is that the BBS proteins function in cell body-to-cilium communication and that in bbs mutants the IFT system has lost its ability to carry information in the form of signaling proteins. The goal is to identify the principles which govern protein transport in eukaryotic cilia and determine how defects in this transport cause disease.
Ciliary Motility - outside and inside. (Presentation at the Mathematical and Computational Challenges in Cilia- and Flagella-Induced Fluid Dynamics meeting, Columbus, OH, October 2012)
Lechtreck, K.-F. (2016). Methods for Studying Movement of Molecules Within Cilia. Methods of Molecular Biology: Cilia. Eds.: S.T. Christensen & P. Satir. Volume 1454:83-96
Harris, J.A., Liu, Y., Yang, P., Kner, P., and Lechtreck, K.-F. (2016). Single particle imaging reveals IFT-independent transport and accumulation of EB1 in Chlamydomonas flagella. Molecular Biology of the Cell 27: 295-307.
Kubo T, Brown JM, Bellve K, Craige B, Craft JM, Fogarty K, Lechtreck KF, Witman GB. (2016). Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin. Journal of Cell Science 129:2106-19.
Tran, P.V. and Lechtreck, K.-F. (2016). An age of enlightenment for cilia: The FASEB Summer Research Conference on the “Biology of Cilia and Flagella”. Meeting Report. Developmental Biology 409:319-28
Lechtreck, K.-F. (2015) IFT-cargo interactions and protein transport in cilia. Trends in Biochemical Science 40:765-778.
Jiang, Y.-Y., Lechtreck, K.-F. and Gaertig, J. (2015). Total Internal Reflection Fluorescence Microscopy of Intraflagellar Transport in Tetrahymena thermophila. In: Methods in Cilia & Flagella, W.F. Marshall (Ed.) Elsevier Science and Technology. Vol 127; p. 223-237.
Vasudevan, K.K., Jiang Y.-Y., Lechtreck, K.-F., Kushida, Y., Alford, L.M., Sale, W.S., Hennessey, T. and Gaertig, J. (2015). Kinesin-13 regulates the quantity and quality of tubulin inside cilia. Mol. Biol Cell 26:478-94
Craft, J.M., Harris, J.A., Hyman, S., Kner, P., and Lechtreck, K.-F. (2015). Tubulin Transport by IFT is Upregulated during Ciliary Growth by a Cilium-autonomous Mechanism. J. Cell Biol. 208:223-237. (see also JCB biosights video)
Awata, J.,Takada, S., Standley, C., Lechtreck, K.-F., Bellvé, K.D., Pazour, G.J., Fogarty, K.E., and Witman, G.B. (2014). Nephrocystin-4 controls ciliary trafficking of membrane and large soluble proteins at the transition zone. J. Cell Sci. 127:4714–4727.
Bhogaraju, S., Weber, K., Engel, B.D., Lechtreck, K.-F. and Lorentzen, E. (2014) Getting tubulin to the tip of the cilium: One IFT train, many different tubulin cargo-binding sites? Bioassays.
Lechtreck, K.F. (2014) Chlamydomonas reinhardtii as a model for flagellar assembly. Perspectives in Phycology 1: 41 - 51 (Invited review for inaugural issue). (Request reprint)
Wren, K., Craft, J.M., Tritschler, D., Schauer, A., Patel,, D.K., Smith E.F., Porter, M.E., Kner, P., and Lechtreck, K.-F. (2013). A differential cargo loading model of ciliary length regulation by IFT. Current Biology 23, 2463–2471.
Lechtreck, K.-F., Gould, T.J., and Witman, G.B. (2013). Repair of the Flagellar Central Pair in Chlamydomonas reinhardtii. Cilia 2,15.
Lechtreck, K.-F., Brown, J.M., Sampaio, J.L., Shevchenko, A., Evans, J.E. and Witman, G.B. (2013). Cycling of the signaling protein Phospholipase D through cilia requires the BBSome only for the export phase. Journal of Cell Biology 201, 249-61.
Ludington, W.B., Wemmer, K.A., Lechtreck, K.-F., Witman, G.B., and Marshall, W.F. (2013). Avalanche-like behavior in ciliary import. PNAS 110, 3925-3930.
Lechtreck, K.-F. (2013). Visualizing IFT in Chlamydomonas flagella. Methods in Enzymology, 524, 265-284.
Craige, B., Tsao, C.-C., Diener, D.R., Hou, Y., Lechtreck, K.-F., Rosenbaum J.L., and Witman, G.B. (2010). CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. Journal of Cell Biology 190, 927-940.
Lechtreck, K.-F., Johnson, E.C., Sakai, T., Cochran, D., Ballif, B.A., Rush, J., Pazour, G.J., Ikebe, M., Witman, G.B. (2009). The Chlamydomonas BBSome is an IFT cargo required for export of specific signaling proteins from flagella. Journal of Cell Biology 187, 1117-1132.
Lechtreck , K.-F., Luro, S., and Witman, G.B. (2009). HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B. Cell Motil Cytoskel. 66, 469-82.
Lechtreck, K.-F., Delmotte, P., Robinson, M.L., Sanderson, M.J., and Witman, G.B. (2008). Mutations in Hydin impair ciliary motility in mice. Journal of Cell Biology 180, 633-643.
Lechtreck, K.-F. and Witman, G.B. (2007). Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility. Journal of Cell Biology 176, 473-482.