Structure and Function of the Inner Dynein Arms

The long term goal of the proposed research is to understand the mechanisms that regulate the dynein ATPases and generate microtubule-based motility. The dynein motors provide the driving force for ciliary and flagellar motility and contribute to microtubule-directed transport inside cells. Although much is known about dynein polypeptide composition, little is understood about the mechanisms that target the dyneins to their appropriate cargoes or regulate their activity. This proposal will capitalize on the well-characterized structural organization of the flagellar axoneme and the numerous motility mutants available in Chlamydomonas to ask how a single cell controls the assembly and activity of its multiple dynein motors. Preliminary studies have identified a family of genes that are thought to encode several inner arm dynein isoforms. The size and expression of the corresponding transcripts will be analyzed to determine if these clones encode flagellar proteins or cytoplasmic variants. Gene specific probes will be used to identify dynein heavy chain mutations by RFLP mapping procedures. The biochemical and structural phenotypes o the dynein mutants will be examined to analyze the physical relationship between various dynein isoforms and toe determine their distributions within the axoneme. Specific antibody probes based on domains of unique sequence will be used to address similar questions. These studies will establish the organization of the dynein motors within the flagellar axoneme. %%% Chlamydomonas is a unicellular green alga which swims in its natural habitat by waving and rotating its flagella, which are long slender cellular extensions containing a highly organized system of microtubules and other proteins which are specialized for this purpose. Flagella (and their homologous shorter structures, cilia) are the cellular organelles responsible for swimming motility (and the movement of a cell's aqueous environment around the cell) in a broad variety of eukaryotic cell types, ranging from the Protists (unicellular organisms) to human cells (e.g., spermatozoa and pulmonary airway epithelia). As such, a detailed understanding of how flagella function at the molecular level is of fundamental importance to biology. In addition, these remarkable inventions of nature are of great interest in terms of potential commercial applications, either as biomolecular materials whose properties can be harnessed through biofabrication, or as model systems for biomimetic reverse engineering and nanofabrication. This project will advance our understanding of the motor molecules of flagella, through a combined approach using genetics, biochemistry, and morphological analysis in a well-defined and highly tractable model system.