Carolyn D. Silflow - US grants

Work from our laboratory has shown that the tubulin gene family in the unicellular eukaryote Chlamydomonas reinhardtii consists of 2 beta tubulin genes coding for identical protein products, and 2 alpha tubulin genes coding for slightly different gene products. Why this haploid organism maintains 23 genes for each of the tubulin types, and what roles are played by each of the genes during the life cycle, are questions we can now address. Three approaches using molecular biology and genetics will be used to understand the functions of the tubulin genes. 1) To determine the degree of coordination in the expression of the genes, specific DNA probes will be used to quantitate the level of transcripts from each of the genes under a variety of conditions in which tubulin protein assembly and tubulin gene expression is regulated. These conditions include passage through the cell cycle, which triggers tubulin gene expression at a specific stage; amputation of the flagella, which triggers a massive increase in the expression of the tubulin (and other flagellar genes); and induced depolymerization of flagellar or cytoplasmic microtubules, which decreases expression of the tubulin genes. 2) To locate regions of the tubulin genes important for regulation of expression, transformation of modified tubulin-lacZ fusion genes into, Chlamydomonas will be used to examine their expression under the conditions described above. These transformation experiments will make it possible to examine possible control regions of the genes both 3' and 5' to the coding sequence. Of particular interest as a possible control region will be the third intervening sequence of the beta tubulin genes, which is 89% conserved between the 2 genes. 3) Resistance to anti-microtubule drugs will be used to select mutants in the tubulin genes. Examination of mutants will allow us to determine the function of each of the tubulin genes by analyzing the cell process disrupted in individual mutants. Comparison of altered and wild type genes should help identify regions of the tubulins required for drug binding. A number of tubulin-binding drugs, such as colcemid and vinblastine, have found widespread use as chemotherapeutic agents. The proposed research should provide significant insight into the action of anti-tubulin drugs, as well as the more generally important question of the regulation of gene expression in a eukaryotic system.

The unicellular green alga Chlamydomonas reinhardtii has several unique characteristics that make it the model system of choice for molecular genetic studies of genes involved in eukaryotic flagella, chloroplasts, cell-cell interactions, and a number of biochemical pathways. We will develop a molecular map of the Chlamydomonas genome to complement the extensive genetic map that currently exists. Restriction fragment length polymorphism that distinguish the DNA from two interfertile strains will be used to follow the segregation of molecular markers in genetic crosses. Molecular markers will be mapped with respect to phenotypic markers on each of the nineteen linkage groups. Sets of linked molecular markers will be ordered on each linkage group. The ends of linkage groups will be mapped using molecular markers associated with telomere. Precise correlation between the molecular map and the genetic map will be sought by carrying out a "chromosome walk" in one region of the genome. This project will greatly facilitate molecular and genetic analysis of Chlamydomonas nuclear genes currently underway in many laboratories including our own. The single-celled green alga Chlamydomonas has become an important model organism for the study of a number of problems of fundamental biological interest. This is a proposal to develop a detailed and comprehensive map of the DNA and genes of this organism. Such a map will be a valuable resource to workers in the field, and will greatly facilitate their work.

The long-term goal of the proposed research is to understand the genetic control of growth and development of higher plants. The investigation focuses on the genetic control of morphogenesis in the model plant Arabidopsis thaliana. The major objective of the present study is to determine the specific roles of individual tubulin genes and tubulin isoforms in the microtubule complexes centrally involved in the processes of cell division and cell elongation that govern the morphogenesis of plant tissues and organs. The specific aims of the proposed investigations are 1) to characterize the spatial and temporal patterns of expression of individual tubulin genes by northern blot and in situ hybridization experiments, 2) to localize, spatially and temporally, the individual tubulin isoforms in developing plant tissues and organs by western blot analyses and immunofluorescence microscopy carried out with isoform-specific antibodies, 3) to use immunofluorescence microscopy and isoform- specific antibodies to determine which isoforms are present in microtubule containing structures such as preprophase bands, mitotic spindles, phragmoplasts, and the cortical arrays, 4) to identify the cis-acting sequences and trans-acting proteins and genes that control the tissue specific expression of Arabidopsis tubulin genes and 5) to utilize "overexpressed" antisense gene specific sequences in transgenic plants produced by Agrobacterium tumefaciens Ti plasmid mediated transformation to probe the function of individual tubulin genes. %%% Microtubules are filamentous polymers that play central roles in several basic processes of the cells of higher organisms. In plant cells they are involved in cell division and cell expansion, processes key to plant morphogenesis. This project is represents a detailed molecular study of the components of microtubules in plants.

The long-term goal of the proposed research is to understand the roles of microtubule-based components of the cytoskeleton in plant development. Morphogenesis in plants is controlled primarily by processes that establish the planes of cell divisions and the axes of cell elongations, and three microtubule arrays unique to plant cells play central roles in these morphogenetic processes. Furthermore, the absence of centrioles in plant cells makes the potential role of gamma-tubulin in the nucleation of microtubules of particular interest in plants. These investigations focus on the genetic control of microtubule-based processes in the model plant Arabidopsis thaliana, which contains 15 characterized alpha and beta-tubulin genes and at least one gamma-tubulin gene. The specific objectives of the proposed investigations are (1) to use immunofluorescence or immunogold microscopy and gamma-tubulin- specific antibodies to localize gamma tubulin within cells throughout the cell cycle, (2) to characterize spatial and temporal patterns of expression of the gamma tubulin gene(s) and selected beta-tubulin genes, and (3) to investigate the utilization and function of the pollen-localized alpha 1-isoform by means of immunolocalization with our alpha 1-tubulin-specific antibody and by analysis of transgenic plants harboring "antisense RNA" chimeric genes.

Cilia and flagella, and the basal bodies from which they grow, are highly conserved throughout the evolution of eukaryotic cells. from unicellular protists to various types of differentiated cells in mammalian systems. Basal bodies, which are analogous to centrioles in structure, serve as the organizing site for a complex set of cytoskeletal structures including microtubule rootlets and various striated fibers. These cytoskeletal elements appear to be involved in segregating basal bodies properly during the cell cycle, in determining or maintaining cellular polarity, in positioning of other cellular organelles, and in anchoring cilia and flagella in the cell. The unicellular biflagellate green alga Chlamydomonas has long been used as a model genetic system to study the genes involved in flagellar function. Previous genetic studies of mutants with abnormal flagellar number have shown that this phenotype results from defects in the replication, maturation, and segregation of basal bodies during the cell cycle. The recent development of methods for insertional mutagenesis in Chlamydomonas has greatly facilitated the cloning of genes identified by mutation. These new methods will be used to clone genes involved in basal body function. The specific aims are: 1) to use insertional mutagenesis to identify genes involved in basal body replication, segregation, and localization by isolating mutants with abnormal flagellar number; 2) to clone the genes identified by mutation and to characterize them by DNA sequencing; 3) to examine the function of each gene through detailed cytological analysis of the mutant phenotype; and 4) to examine the spatial and temporal localization of the gene products using immunocytochemistry. It is likely that many of the proteins involved in basal body function identified in this project will have homologs in mammalian cells such as ciliated epithelial cells, sperm cells, and sensory cells. Results from this project will provide a better understanding of the cytoskeleton in these cells.

Basal bodies and centrioles are microtubule organelles with both longitudinal and nine-fold rotational asymmetry. In addition to their role in ciliary assembly, basal bodies are important for the rotational positioning of motile cilia to properly orient the direction of ciliary beat. Little is known about the molecular components that establish or recognize the asymmetry to assemble appendages onto basal bodies at specific locations. Using the model organism Chlamydomonas reinhardtii, a unicellular, biflagellate green alga, the PI has isolated a series of mutants with a variable flagellar number (vfl) and with defects in rotational positioning of basal bodies, in orientation of probasal bodies with respect to mature basal bodies, and in segregation of basal bodies during cell division. Two VFL genes have been cloned. This new project continues the PI's study of the vfl mutants. The specific aims of the project are to 1) Clone and characterize the VFL4, VFL5, and VFL6 genes and localize the VFL5 and VFL6 gene products using light and electron microscopy; 2) Identify additional genes involved in basal body segregation and positioning using genetic approaches including insertional mutagenesis and chemical mutagenesis; 3) Examine interactions among vfl genes and identify interacting proteins using genetic approaches and yeast two-hybrid technology. The experimental approach will identify evolutionarily conserved molecules that specify rotational asymmetry in basal bodies as well as molecular components of fibers that bind to specific sites on the walls of basal bodies. The study will advance the long-term goal of understanding the molecular mechanisms that underlie the structure of basal bodies, the attachment of appendages to specific sites on the wall of the basal body, and the initiation of basal body duplication at a specified rotational position. The project will provide research training at three levels: undergraduate, graduate and postdoctoral.

Project Summary

This award renews support for a collection of 2300 strains of the unicellular green alga consisting of mutants of C. reinhardtii and closely related species. Chlamydomonas is an excellent model system for studies of photosynthesis, flagellar biogenesis and function, signal transduction, and other aspects of cell biology. The recent completion of the genome sequence of Chlamydomonas will likely accelerate the adoption of this organism as an experimental tool. The collection, now called the Chlamydomonas Resource Center (formerly, the Chlamydomonas Genetics Center at Duke University), serves as the central repository to receive, catalogue, preserve and distribute wild type and mutant cultures of C. reinhardtii and other Chlamydomonas species in which extensive genetic analysis has been done. It also serves as the resource center to disseminate information on this organism to the international scientific community. Given its ease of culture and striking flagellar motility, Chlamydomonas is also an ideal organism for laboratory exercises for high school students and college undergraduates. Thus, the Center also provides materials and advice for students and teachers interested in adopting Chlamydomonas for classroom and individual science projects. In addition to live cultures, the Center also maintains and distributes molecular reagents useful for research on Chlamydomonas, including genomic and cDNA clones of Chlamydomonas nuclear, chloroplast and mitochondrial genes, and cDNA libraries. A web site [http://www.chlamy.org] provides descriptions of all cultures in the collection, historical information and reference material on genetic loci and mutant alleles, genetic and molecular maps, an address directory, and an extensive bibliography.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests support for an EMBO Workshop on the Cell and Molecular Biology of Chlamydomonas, to be held in May 27, 2008 to June 1, 2008 in Hyeres, France. The conference is the biennial meeting for all researchers studying the green algal system Chlamydomonas. Chlamydomonas is an excellent model organism for research in cell biology, genetics, biochemistry, biophysics, plant physiology, evolution, and genomics. The conference brings together investigators from different fields and disciplines, allowing them to integrate their specialized vision of these organisms into a broader perspective. Three overarching themes for the conference are Chlamydomonas and Medicine, Chlamydomonas and Energy, and Chlamydomonas Biology. The research community will have the opportunity to present and discuss new results. Poster presentations, platform sessions, and workshops provide the structure for exchange of information and will provide an opportunity to discuss the development of technology and of experimental resources. The conference will facilitate discussion and planning of joint efforts to exploit information from the recently completed genome sequence. The conference promotes participation by all members of the research community. Graduate students, postdoctoral fellows, and assistant professors are encouraged to present their work during platform and poster sessions. Women and minorities are encouraged to give talks and presentations. The conference will provide excellent opportunities for researchers to experience personal contacts with other workers from the geographically diverse Chlamydomonas community which includes persons from North America, Europe, Asia, and Australia. [unreadable] [unreadable] [unreadable]

Scientific research

Cilia are highly conserved organelles present in all major phylogenetic groups of eukaryotic organisms. They have been adapted for motility of single cells and for fluid transport over cell layers. In multicellular animals, they play specialized roles in embryo development and in signaling pathways in diverse cell types. The nine doublet microtubules in typical cilia grow from a template of nine triplet microtubules that compose the wall of the basal body (centriole). Basal body docking with the plasma membrane provides a site for assembly of a cilium. Components of intraflagellar transport, required for ciliary assembly and maintenance, converge at the basal body. Initiation of ciliary assembly depends on the conversion of triplet microtubules to doublet microtubules at the distal end of the basal body. Using as a model system the unicellular, biflagellate green alga Chlamydomonas reinhardtii, this project investigates a class of "uni" mutants with defects in docking of basal bodies with the plasma membrane and in the conversion of triplet microtubules to doublet microtubules required for ciliary assembly. The molecular basis of these processes is investigated through biochemical characterization of the products of the UNI1 and UNI2 genes. The role of phosphorylation in the function of the Uni2 protein is being studied. The functions of the Uni1 and Uni2 proteins are being examined through analysis of mutant phenotypes, subcellular localization of the proteins, and protein interactions.

Broader Impacts
This project integrates research and education by providing mentored research experiences for undergraduate students. Students receive training in cell biology, molecular biology, and genetics disciplines while carrying out research. The projects help students to develop the ability for critical thinking and the skills for written and oral science communication.

Chlamydomonas reinhardtii has been a long standing model system in the biological sciences for studies of photosynthesis, flagellar development and other aspects of cell biology. Recently, as a fast-growing, photosynthetic eukaryote, Chlamydomonas has attracted a great deal of interest as a source of biomass and renewable energy. University of Minnesota-Twin Cities houses the Chlamydomonas Resource Center (CRC) and its utility is enhanced by the availability of the sequence of the nuclear, chloroplast and mitochondrial genomes. CRC has a large collection of mutants and a complete genetic map linked to the molecular map. The PI serves as the new Director of CRC and, in its 30th year of operation, this stock collection center will continue to act as the central repository to receive, catalogue, preserve and distribute wild type and mutant cultures of C. reinhardtii. The Center will continue to maintain and distribute molecular tools for Chlamydomonas research, including genomic and cDNA clones of Chlamydomonas nuclear, chloroplast and mitochondrial genes, and cDNA libraries. The Center will continue to distribute teaching kits, with reagents, strains and instructions for high school and beginning college biology class experiments. A web site [http://www.chlamy.org] will provide descriptions of all cultures in the collection, historical information and reference material on genetic loci and mutant alleles, genetic and molecular maps, and an extensive bibliography.

The unicellular green alga Chlamydomonas reinhardtii serves as a unique model system for studies of photosynthesis, metabolic pathways, flagellar biogenesis and function, signal transduction, and other aspects of cell biology. Chlamydomonas allows researchers to apply the powerful genetic techniques available in a simple microbial system to an organism with chloroplasts and flagella. The system also provides a platform for applied research in biofuels and in protein engineering. Uniquely among algal systems, over 50 years of experimentation by hundreds of laboratories has generated a collection of more than 4000 mutants affecting many cell processes. The Chlamydomonas Resource Center, in its 36th year of operation, serves as the central repository to receive, catalogue, preserve and distribute wild type and mutant cultures of C. reinhardtii. It also maintains and disseminates molecular reagents such as plasmids and provides information on this organism to the international scientific community. The Center prepares and distributes teaching kits, with simple reagents, strains and instructions to allow high school and college biology students and classes to perform sophisticated experiments, producing hydrogen, for example, without needing expensive laboratory facilities. By maintaining and distributing mutant strains that can be used to illuminate the functioning of flagella and chloroplasts, and by encouraging the interest of a new generation of researchers, the Chlamydomonas Resource Center plays a critical role in ensuring that this valuable model system continues to provide unique insights into many areas of biology.

The aims of this project are to: 1) improve the security of the collection by cryopreservation of all living strains in liquid nitrogen, in duplicate; 2) make the mutants in the collection more useful to users by preparing and distributing custom collections of strains using a recently acquired replica-plating robot; and 3) enhance the educational use of the collection by recruiting undergraduate students to test and refine our current teaching kits, and to help the co-directors of the Center design new laboratory experiments to be distributed to teachers through the website. These enhancements of Center activities will insure that users have access to the information and strains needed to make optimal use of this increasingly important model organism for research and teaching. A frequently updated web site (chlamy.org) provides descriptions of all cultures and plasmids in the collection, historical information and reference material on genetic loci and mutant alleles, a library of useful laboratory protocols and laboratory exercises, as well as genetic and molecular maps, and links to other relevant information.