"Being responsible for my own research project has been a great incentive to step out of my scientific comfort zone and explore areas less familiar to me such as Molecular Biology. These bacteria were transformed to produce a plasmid containing a synthetic piece of double stranded DNA I designed." - Lisi Krainer
Regulation of ciliary dynein activity and assembly, and the role of the central pair complex in ciliary motility regulation.
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Dynein and kinesin ATPases are molecular motors for many types of microtubule-based cell motility, including mitosis, directed transport of vesicles, maintenance of the endoplasmic reticulum and Golgi apparatus, and motility of (eukaryotic) cilia and flagella. We study the role of dyneins in microtubule sliding and eukaryotic flagellar bend formation, and in the regulation of flagellar waveform and beat frequency, through a combination of genetic, biochemical, and molecular approaches. While the role of dyneins as microtubule-associated motors for both ciliary and cytoplasmic motility is well recognized, little is known about their regulation or their mode of attachment to the loads they carry (vesicles or chromosomes in the cytoplasm, doublet microtubules in cilia and flagella). Our experimental system is the flagellated unicellular alga Chlamydomonas reinhardtii, since dynein subunits in Chlamydomonas flagella are homologous to subunits in mammalian dyneins but are more easily approached through genetic and molecular analyses. Human conditions that inhibit motility of these organelles (Kartagener''s syndrome, Primary Ciliary Diskinesia) are known to cause upper respiratory illnesses and male infertility. We are using in vitro mutagenesis and transformation of cloned genes to test specific hypotheses regarding the function of selected dynein subunits, and to understand mechanisms that regulate dynein assembly and activity. We are also using in vivo mutagenesis and selecting mutations that alter normal dynein function, characterizing these mutations at the molecular level, and correlating specific motility defects (analyzed by light microscopy) with defects in subunit structure and assembly. These studies provide the framework for determining general mechanisms of dynein assembly and function in all types of cell motility. The recent identification of kinesins associated with flagellar microtubules suggests that these motor proteins also play an important role in flagellar motility, and we are seeking to understand this role.
Little is known of dynein regulation in any cellular motility system, but the limited information suggests that kinase-phosphatase cascades are involved. In the flagellum, regulation is used to turn flagellar beating on and off, change beat frequency or waveform, and activate of dyneins in the precise patterns needed to propagate bends. In Chlamydomonas, flagellar motility is regulated in part by changes in intracellular calcium concentrations and some aspects of waveform regulation are known to involve protein kinase A-like and protein phosphatase I-like activities, but the mechanisms that link these changes to regulation of dynein ATPase activity are not known. We are selecting mutations that alter dynein regulation, as manifested by changes in flagellar beat parameters, and testing each mutation to see which signal transduction cascade has been altered and which flagellar structures are involved. Mutations that prevent dynein assembly into flagella are also analyzed to characterize genes that are important for cytoplasmic assembly of dynein subunits, targeting to the flagellar compartment, and attachment of dynein motors to doublet microtubules. These studies will provide key information about mechanisms that regulate the intracellular distribution and activity of dyneins.
My favorite research organism,
Carbajal-Gonzalez, B., T. Heuser, X. Fu, J. Lin, D. R. Mitchell, B. Smith and D. Nicastro. (2013) Conserved structural motifs of the central pair complex of eukaryotic flagella. Cytoskeleton 70:101-120.
Mitchison,H.M., M. Schmidts, N.T. Loges, J. Freshour, A. Dritsoula, R.A. Hirst, C. O'Callaghan, H. Blau, M. Al Dabbagh, H. Olbrich, P.L. Beales, T. Yagi, H. Mussaffi, E.M.K. Chung, H. Omran, and D.R. Mitchell (2012) Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia. Nature Genetics 44:381-389.
Hom, E.F.Y., G.B. Witman, E.H.Harris, S.K. Dutcher, R. Kamiya, D.R. Mitchell, G.J. Pazour, M.E. Porter, W.S. Sale, M. Wirschell, T. Yagi and S.M. King (2011) A unified taxonomy for ciliary dyneins. Cytosk. 68:555-565.
Gao, C., G. Wang, J. Amack and D. R. Mitchell (2010) Oda16/Wdr69 is essential for axonemal dynein assembly and ciliary motility during zebrafish embryogenesis. Developmental Dynamics 239:2190-2197.
Wei, M., P. Sivadas, H. A. Owen, D. R. Mitchell and P. Yang (2010) Chlamydomonas mutants display reversible deficiencies in flagellar beating and axonemal assembly. Cytosk. 67:71-80.
Mitchell, D. R. (2010) Polyglutamylation, the GLU that makes microtubules sticky. Current Biology 20:R234-R236.
Mitchell, D. R., and B. Smith (2009). Analysis of the central pair microtubule complex in Chlamydomonas reinhardtii. Ch. 13 In Cilia and Flagella, Part A, S. King and G. Pazour, eds., Methods in Cell Biology 92:197-213.
Duquesnoy, P., E. Escudier, L. Vincensini, J. Freshour, A-M. Bridoux, A. Coste, A. Dechildre, J. de Blic, M. Legendre, G. Montantin, H. Tenreiro, A-M. Vojtek, C. Loussert, A. Clément, D. Escalier, P. Bastin, D. R. Mitchell and S. Amselem (2009) Loss-of function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia. Am. J. Hum. Genet. 85:890-896.
Satir, P., D. R. Mitchell and G. Jekely. (2008). How did the cilium evolve? Ch. 3 In: Curr. Top. Dev. Biol. Vol 85, Ciliary Function in Mammalian Development, Ed. B. K. Yoder, Elsevier, pp. 63-82.
Omran, H, D. Kobayashi, H. Olbrich, T. Tsukahara, N. T. Loges , H. Hagiwara, Q. Zhang, G. Leblond, E. O’Toole, C. Hara, H. Mizuno, H.i Kawano, M. Fliegauf, T. Yagi, S. Koshida, A. Miyawaki, H. Zentgraf, H. Seithe, R. Reinhardt, Y. Watanabe, R. Kamiya, D. R. Mitchell & H. Takeda. (2008) Ktu/PF13 is required for axonemal dynein arm formation. Nature 456:611-616.
Ahmed, N. T., C. Gao, B. J. Lucker, D. Cole, and D. R. Mitchell. (2008) ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. J. Cell Biology 183:313-322.
Wilkes, D. E., H. E. Watson, D. R. Mitchell and D. J. Asai.(2008) Twenty-five dyneins in Tetrahymena: A re-examination of the multi-dynein hypothesis. Cell Motil. Cytosk. 65:342-351.
Lindemann, C. B. and D. R. Mitchell. (2007) Evidence for axonemal distortion during the flagellar beat of Chlamydomonas. Cell Motil. Cytosk. 64:580-589.
Freshour, J., R. Yokoyama, and D. R. Mitchell.(2007) Chlamydomonas flagellar outer row dynein assembly protein Oda7 interacts with both outer row and I1 inner row dyneins. J. Biol. Chem. 282:5404-5412.
Mitchell, D. R. (2007). The evolution of eukaryotic cilia and flagella as motile and sensory organelles. Adv Exp Med Biol. 607:130-40.
Mitchell, B. F., L. B. Pedersen, M. Feely, J. L. Rosenbaum, and D. R. Mitchell. (2005) ATP production in Chlamydomonas flagella by glycolytic enzymes. Mol. Biol. Cell 16:4509-4518.
Ahmed, N. T., and D. R. Mitchell. (2005) Oda16, a highly conserved protein essential for flagellar dynein assembly and regulation. Mol. Biol. Cell 16:5004-5012.
Yokoyama, R., E. O’Toole, S. Ghosh, and D. R. Mitchell. (2004) Regulation of flagellar dynein activity by a central pair kinesin. PNAS 101:17398-17403.
Mitchell, D. R. (2004). Speculations on the evolution of 9+2 organelles and the role of central pair microtubules. Biol.Cell 96:691-696.
Mitchell, D. R., and M. Nakatsugawa. (2004) Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella. J. Cell Biol. 166:709-715.
Zhang, H., and D. R. Mitchell. (2004) CPC1, a Chlamydomonas central pair protein with an adenylate kinase domain. J. Cell Sci. 117:4179-4188.