NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF CELLULAR COMMUNICATION

(Adjunct)

Professor:
Ritsu Kamiya
Associate Professor:
Katsushi Owaribe
Research Associate:
Takako Minoura
Institute Research Fellow:
Osamu Kagami
Graduate student:
Itsushi Minoura (University of Tokyo)


The research in this laboratory, started in November, 1996, is aimed at an understanding of the molecular mechanisms that regulate the assembly and function of cytoskeletal proteins. Current research effort is centered on the function of axonemal dyneins, microtubule-based motor proteins that produce force for flagellar beating. The organism employed is Chlamydomonas, a biflagellate green alga particularly suited for genetic and molecular biological studies.

A single flagellar axoneme contains at least eleven kinds of dynein heavy chains in inner and outer arms. To understand the specific function of each heavy chain, we have been isolating and characterizing mutants that lack different kinds of axonemal dyneins. Their motility phenotypes have indicated that different dynein species differ in function in a fundamental manner. For example, the outer-arm heavy chains are important for flagellar beating at high frequency, whereas the inner-arm heavy chains are important for producing proper waveforms. Indirect evidence also suggests that the force generation properties differ greatly among different heavy chains. We are currently trying to directly measure the force production by different dyneins using micro-physiological techniques.

The inner dynein arms are known to contain actin as a subunit. Hence the two independent motility systems of eukaryotes - the actin-based and microtubule-based motility systems - should somehow cooperate in the inner-arm dynein although the function of actin in dynein arms is totally unknown at present. Several years ago, we found that the mutant ida5, lacking four out of the seven subspecies of inner-arm dyneins, has a mutation in the actin-encoding gene. Intriguingly, Chlamydomonas has been known to have only a single gene of conventional actin, and the mutant ida5 was found to express no conventional actin at all. On close inspection, the cytoplasm and axonemes of this mutant were found to contain a novel actin-like protein (NAP) which displays exceptionally low homology (64% in amino acid identity) to conventional actin. The mutant ida5 is deficient in the formation of the fertilization tubule and thus has a low mating efficiency. However, it displays normal cell division and grows as rapidly as wild type, possibly because NAP can substitute for actin in important cellular functions. Thus conventional actin and NAP may overlap in some, but not all, cellular functions. It is interesting to note that NAP is expressed in significant amount only in the mutant lacking actin; i.e., the expression of NAP appears to depend on the presence of actin. We are currently investigating how such regulation takes place.

We have recently succeeded in transforming the mutant ida5 with cloned actin gene and found that inner dynein arms become restored upon transformation. Transformation with the NAP-encoding gene is under way. Studies with artificially mutated actin gene will enable us to determine what functions are carried out by actin and NAP and, in particular, whether actin or NAP is really essential for cytokinesis, assembly and function of inner dynein arms, or other fundamental processes in Chlamydomonas.

Fig. 1.
Fertilization tubules in wild-type(A) and ida5 (B) mt+ gametes produced in response to a 1 hour exposure to 10 mM dibutyryl-cAMP and 1 mM IBMX. Bar, 0.3 mm. Wild-type fertilization tubules have been shown to contain F-actin bundles.


Publication List:
Hayashi, M., Yagi, T., Yoshimura, K., and Kamiya, R. (1998). Real-time observation of Ca2+-induced basal body reorientation in Chlamydomonas. Cell Motil. Cytoskeleton 41, 49-56.
Kagami, O., Gotoh, M., Makino, Y., Mohri, H., Kamiya, R., and Ogawa, K. (1998). A dynein light chain of sea urchin sperm flagella is a homolog of mouse Tctex 1, which is encoded by a gene of the t complex sterility locus. Gene 211, 383-386.
Kato-Minoura, T., Uryu, S., Hirono, M., and Kamiya, R. (1998). Highly divergent actin expressed in a Chlamydomonas mutant lacking the conventional actin gene. Biochem. Biophys. Res. Comm. 251, 71-76.
Ohara, A., Kato-Minoura, T., R., Kamiya, R. and Hirono, M. (1998). Recovery of flagellar inner-arm dynein and the fertilization tubule in Chlamydomonas ida5 mutant by transformation with actin genes. Cell Struct. Funct., 23, 273-281.
Matsuda, A., Yoshimura, K., Sineshchekov, O. A., Hirono, M., and Kamiya, R. (1998). Isolation and characterization of novel Chlamydomonas mutants that display phototaxis but not photophobic response. Cell Motil. Cytoskeleton, 41, 353-362.
Okamoto, M., Ito, M., and Owaribe, K. (1998). Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster. Develop. Growth and Differ. 40, 11-18.
Hirako, Y., Usukura, J., Uematsu, J., Hashimoto, T., Kitajima, Y., and Owaribe, K. (1998). Cleavage of BP180, a 180 kDa bullous pemphigoid antigen, yields a 120 kDa collagenous extracellular polypeptide. J. Biol. Chem. 273, 9711-9717.
Hirako, Y., and Owaribe, K. (1998). Hemidesmosomes and their unique transmembrane protein BP180. Microscopy Research and Technique 43, 207-217.


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Last Modified: 12:00, May 28, 1999