News and Publications

Genetic Specification of the Structure and Function of Microtubule-Organizing Centers

B.P.-H. Huang, V. Lee

The temporal and spatial distribution of microtubules in eukaryotic cells is controlled by discrete organelles known as microtubule-organizing centers (MTOCs). MTOCs show remarkable structural variation among different organisms but have similar functions of organizing interphase microtubule arrays and determining the bipolarity of the mitotic spindle; as such, the centers are crucial for the fidelity of cellular reproduction and cytoplasmic organization. Our investigations are directed toward determining the genetic specification of the structure and function of MTOCs. We use the basal body complex in the unicellular green alga Chlamydomonas reinhardtii as a model system.

Current efforts are directed toward understanding the structure and function of centrin, an EF-hand calcium-binding protein that is a conserved component of MTOCs in divergent species. Centrin, also known as caltractin, has been cloned at the DNA level from a number of different organisms, including yeast, algae, frogs, mice, and humans. Genetic analyses indicate that Chlamydomonas centrin and its yeast homolog in Saccharomyces cerevisiae, CDC31p, are required for the normal duplication and segregation of the basal body complex and the spindle pole body, the major MTOCs in the respective cell types. As a conserved component of MTOCs in divergent species, centrin is a potential novel target for the development of new therapeutic approaches that influence critical functions of microtubules.

Relationships between the structure and function of centrin are being investigated by studying the consequences of expressing site-specific mutations in centrin in transient and stable transformants of Chlamydomonas cells. Genetic and biochemical approaches were used to detect other genes and gene products that affect the basal body complex in Chlamydomonas and proteins that interact with centrin. One protein that interacts with centrin in algae is the cytoskeletal protein actin. The structural, biochemical, and functional aspects of this interaction are being investigated.

It has now been established that at least 3 genes encode distinct centrin proteins in the human genome. The mouse homologs for 2 of these genes have been isolated. We determined that one of the mammalian genes is ubiquitously expressed in different tissues, whereas another appears to be specifically expressed only in the testis. The localization and developmental pattern of expression of the different centrin proteins are being investigated in mouse embryos and in cultured cells. The function of centrin as a component of the mammalian centrosome is being examined by studying the consequences of disrupting centrin expression in cultured cells; we are using antibody microinjection, antisense strategies, and expression of dominant negative mutations. Transgenic mice in which the testis-specific centrin gene has been disrupted are being produced to assess the role of the protein in the development and function of the testis.

PUBLICATIONS

Lee, V.D., Huang, B. Centrin. In: Guidebook to the Cytoskeletal and Motor Proteins. Kreis, T., Vale, R. (Eds.). Oxford University Press, New York, in press.