Cell Biology of Cilia; Cilia-related diseases
Cilia and cilia-related diseases
Cilia and flagella are microtubule-based cell extensions. Most cell types possess in the mammalian body possess motile or non-motile cilia. Defects in cilia function cause a plethora of diseases and developmental disorders referred to as ciliopathies. Loss of ciliary motility, for example, results in chronic airway infections, situs anomalies including congenitial heart defects, and male infertility. Cilia also function in cellular sensing and signaling: The receptors for light and odors, for example, are located in modified cilia. To perform their motile and sensory functions, cilia must possess the right composition, structure, and size. Defects in ciliary sensing result in blindness, anosmia (loss of the sense of smell), polydactyly (extra fingers and toes), obesity, severe skeletal malformations, and polycystic kidney disease (PKD). PKD is one of the most common inherited disorders in humans with an incidence of approximately 600,000 people in the United States and over 12,000,000 worldwide.
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
Because cilia lack ribosomes all proteins required to build a cilium need to be transported from the cell body where they are synthesized into the organelle. The major mechanism of protein transport in cilia is intraflagellar transport (IFT). IFT is well conserved and required for ciliary assembly. In IFT, multimegadalton protein arrays (=IFT trains) travel from the cell body along the ciliary microtubules to the ciliary tip and back using the molecular motors kinesin-2 (to the tip; anterograde IFT) and IFT dynein (to the base; retrograde IFT). The trains functions as carriers to move proteins from the cell body into cilium and to return proteins back to the cell body. Our goal is to understand how proteins are selected for transpor tinto cilia, and how the amount and timing of protein entering the cilium are regulated.
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 in the right amount 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?
We use a combination of biochemistry, genetics & molecular biology, and in vivo imaging to study protein transport in cilia. Using total internal reflection fluorescence microscopy (TIRFM), we are able to image protein transport inside cilia of living cells at the single molecule level with video rates. Using this approach, we demonstrated that many structural ciliary proteins (tubulin etc.) are cargoes of IFT. The transport of these proteins is upregulated when cilia are too short. However, how cells measure the length of their cilia and how they regulate how much cargo is delivered by the IFT trains remains currently unknown. A second focus of our research is the analysis of IFT-cargo binding sites in particularly the binding of tubulin, the main structural protein of cilia. In our studies of the rare inherited disorder Bardet-Biedl syndrome (BBS), we are currently determining the molecular mechanism by which the BBSome (a complex of 8 BBS proteins) removes proteins that perturb cilia function from the organelle.
Videos on our research:
Paper at the 5th Internayional CAESAR conference (Bonn, Germany, 2015)
Ciliary Motility - outside and inside. Presentation at the Mathematical and Computational Challenges in Cilia- and Flagella-Induced Fluid Dynamics meeting. (Columbus, OH, October 2012)
Dai, J., Barbieri, F., Mitchell, D.R., and Lechtreck, K.-F. (2018). In vivo analysis of outer arm dynein transport reveals cargo-specific IFT properties. Mol. Biol. Cell 22 Aug 2018. E18-05-0291.
Wingfield, J.L. and Lechtreck, K.-F. (2018) Chlamydomonas basal bodies as flagella organizing centers. Invitated review to Special Issue "Comparative Biology of Centrosomal Structures in Eukaryotes". Cells 7(7), 79; doi: 10.3390/cells7070079
Lechtreck, K.-F., Mengoni, I., Okivie, B., and Hilderhoff, K.B. (2018). In vivo analyses of radial spoke transport, assembly, repair and maintenance. Cytoskeleton (in press).
Hunter, E.L., Lechtreck, K., Hwang, J., Fu, G., Alford L.M., Gokhale, A., Yamamoto, R, Kamiya, R., Lin, H., Yang, F., Nicastro, D., Dutcher, S.K., Wirschell, M., and Sale, W.S. (2018). The IDA3 adapter, required for IFT transport of I1 dynein, is regulated by ciliary length. MBoC 29:886-896.
Liu P. and Lechtreck, K.-F. (2018). The Bardet-Biedl syndrome protein complex is an adapter expanding the cargo range of intraflagellar transport trains for ciliary export. PNAS 01.15.2017. doi: 10.1073/pnas.1713226115
Liu, Y., Visetsouk, M., Mynlieff, M.,Qin, H., Lechtreck, K.-F., and Yang, P. (2017). H+- and Na+- elicited swift distinct changes of the microtubule system in heterotrophic Chlamydomonas. Elife Sep 6; doi: 10.7554/eLife.26002.
Snouffer, A., Brown, D., Le, H., Walsh, J., Lupu, F., Norman, R., Lechtreck, K.-F., Ko, H.W., and Eggenschwiler, J. (2017). Cell Cycle-Related Kinase (CCRK) regulates ciliogenesis and Hedgehog signaling in mice. PLOS Genetics 13:e1006912
Wingfield, J.L., Mengoni, I., Bomberger, H., Jiang, Y.-Y., Walsh, J.D., Brown, J.M., Picariello, T., Cochran, C.A., Zhu, B., Pan, J., Eggenschwiler, J., Gaertig, J., Witman, G.B., Kner, P., and Lechtreck, K.-F. (2017). IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium. Elife May 31. doi: 10.7554/eLife.26609.
Lechtreck K.-.F, Van De Weghe J.C., Harris J.A., Liu P. (2017). Protein transport in growing and steady-state cilia. Traffic 18: 277-286.
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.