Stuart Mudd Professor of Biology and Chair, Department of Biology Olin Building, Room 1257 1250 N. Dartmouth Ave. Claremont, CA 91711 (909) 607-2257 David_Asai@hmc.edu

Education & Professional Experience

• B.S., Stanford University
• M.S., Stanford University
• Ph.D., California Institute of Technology
• Postdoc, University of California, Santa Barbara
• Professor, Purdue University (1985-2003)
• Department Head, Purdue University (2000-2003)

Teaching

• Bio 52: Introduction to Biology
• Bio 189: Topics in Biochemistry and Molecular Biology
• Chem 25: Freshman Chemistry Laboratory

Asailum Inmates, 2006-07

• HMC students:
             Clarence Chan
             Yu-Long Chang
             Elizabeth Corpuz
             Jon Hetzel
             Erin Heyer
             Mark Hubenschmidt
             Hallie Kuhn
             Jon Litz
             Andrea Sand
             Oksana Sergeeva
             Hadley Watson
• Other inmates:
             David Wilkes, postdoctoral scientist
             Vidya Rajagopalan, postdoctoral scientist

Research Interests

The focus of our laboratory is to understand the structural basis for dynein functions. We use the tools of genetics, molecular biology, fluorescence light microscopy, and protein biochemistry.

Dynein is the molecular motor protein complex that transduces the free energy derived from the hydrolysis of ATP into mechanical work, resulting in dynein "walking" along the surface of a microtubule track. All eukaryotes express dynein — some (yeast, slime mold, filamentous fungi) have one dynein, whereas organisms with cilia/flagella express a family of approximately 15 different dyneins. Dynein is responsible for many cellular activities, including the retrograde transport of membrane-bounded organelles and vesicles, the positioning of organelles such as the Golgi apparatus and the mitochondria, the movement of nuclei and the establishment of the bipolar mitotic apparatus, and the beating of cilia and eukaryotic flagella. An important problem is to understand the structural specialization, regulation, and coordination of the different dyneins that work together in the living cell, testing the "multi-dynein hypothesis."

Dynein in situ is a large complex of several proteins, including heavy chains, intermediate chains, light-intermediate chains, and light chains. For example, ciliary outer arm dynein is a complex of approximately 10 proteins with a total molecular weight of ca. 2M. The heavy chains contain the motor activity of the complex, including the sites of ATP hydrolysis and microtubule track-binding. The smaller subunits mediate the tethering of the dynein to its molecular cargo and regulate the activity of the dynein. An important problem is to understand how each of the subunits contributes to dynein function in the living cell.

The ciliated protozoan, Tetrahymena thermophila, presents a powerful experimental system in which to dissect the structure-function relationships of dynein subunits. Tetrahymena possesses the biological complexity of metazoans in a single cell that is easy to grow in the laboratory. Tetrahymena expresses a family of dynein heavy chains that is similar in complexity and sequences to the family of dynein heavy chains expressed in "higher" eukaryotes including mammals. It is straightforward to modify any gene in Tetrahymena; modifications include changing the sequence of the gene, completely eliminating the gene, over-expressing the gene, and tagging the protein product of the gene. Gene replacement occurs exclusively by homologous recombination, and the phenomenon of phenotypic assortment permits the investigator to evaluate the effect of the modification of any gene (including an essential gene) in a living cell. The Tetrahymena genome sequencing project enables a strategy that combines bioinformatics and targeted gene replacement to study any protein in a living cell.

• Are the individual isoforms functionally specialized? The limited evidence is that the individual isoforms cannot substitute for one another; we are testing this hypothesis by utilizing homologous gene replacement/disruption methods in Tetrahymena.
• Although there are as many as 23 axonemal heavy chains in Tetrahymena., there are only two non-axonemal or “cytoplasmic” dyneins. We are focusing on the organization and cellular function of cytoplasmic dynein-2 in Tetrahymena. This includes our studies of the DYH2 heavy chain and the dynein-2 light-intermediate chain (D2LIC).

Confocal immunofluorescence image of conjugating Tetrahymena cells. When two Tetrahymena cells of different mating types couple, the germline micronucleus in each cell undergoes meiosis. This results in two haploid gametic micronuclei in each partner cell; the cells exchange gametic pronuclei which then fuse and give rise to a new somatic macronucleus in each progeny cell. Shown is an immunofluorescence image of a pair of mating cells in prophase I. In green are the microtubules; in blue are the nuclei. This is a projection of optical sections captured with our Zeiss LSM510 confocal laser scanning microscope.

Selected Publications (click here for full list)

Wilkes, D. E., H. E. Watson, D. R. Mitchell, and D. J. Asai. 2008. Twenty-five dyneins in Tetrahymena: A re-examination of the multidynein hypothesis. Cell Motil. Cytoskel. 65 (4): 342-351.

Wilkes, D. E., V. Rajagopalan, C. W. C. Chan, E. Kniazeva, A. Wiedeman, and D. J. Asai. 2007. The dynein light chain family in Tetrahymena thermophila. Cell Motil. Cytoskel. 64 (2): 82-96.

Haushalter, K. and D. J. Asai. 2006. Beyond Bio2010: If we build it, will they come? CUR Quarterly 26: 160-163.

Eisen, J., R. S. Coyne, M. Wu, D. Wu, M. Thiagarajan et al. 2006. Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biology 4: e286.

Adhiambo, C., J. D. Forney, D. J. Asai, and J. H. LeBowitz. 2005. The two cytoplasmic dynein-2 isoforms in Leishmania mexicana perform separate functions. Mol. Biochem. Parasitol. 143: 216-225.

Toba, S., T. M. Gibson, K. Shiroguchi, Y. Y. Toyoshima, and D. J. Asai. 2004. Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment. Cell Motility and the Cytoskeleton 58: 30-38.

Asai, D. J., and D. E. Wilkes. 2004. The dynein heavy chain family. J. Eukar. Microbiol. 51: 23-29.

Asai, D. J., and M. Koonce. 2001. The dynein heavy chain: structure, mechanics, and evolution. Trends Cell Biol. 11: 196-202.

Faruki, S., R. H. Geahlen, and D. J. Asai. 2000. Syk-dependent phosphorylation of microtubules in activated B-lymphocytes. J. Cell Sci. 113: 2557-2565.

Asai, D. J. 2000. Analysis of dynein structure and function in ciliated protozoa. Japan. J. Protozool. 33: 15-27.

Asai, D. J., and J. D. Forney, Editors. 2000. Tetrahymena thermophila. Methods in Cell Biology, Vol. 62, Academic Press, 580 pages.

Gibson, T. M., and D. J. Asai. 2000. Isolation and characterization of 22S outer arm dynein from Tetrahymena cilia. Meth. Cell Biol. 62: 433-440.

Lee, S.-W., J. C. Wisniewski, W. L. Dentler, and D. J. Asai. 1999. Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila. Mol. Biol. Cell 10: 771-784.

Criswell, P. S., and D. J. Asai. 1998. Evidence for four cytoplasmic dynein heavy chain isoforms in rat testis. Mol. Biol. Cell 9: 237-247.

Criswell, P. S., L. E. Ostrowski, and D. J. Asai. 1996. A novel cytoplasmic dynein heavy chain: expression of DHC1b in mammalian ciliated epithelial cells. J. Cell Sci. 109: 1891-1898.

Asai, D. J. 1996. Functional and molecular diversity of dynein heavy chains. Sems. Cell Biol. 7: 311-320.