9722822 Lindemann The aim of this project is to understand the mechanism that is responsible for the rhythmic beating of eukaryotic cilia and flagella. The working hypothesis was developed in the previous grant period. It is called the Geometric Clutch model of axonemic functioning and will provide the framework for the proposed experimental investigations. This hypothesis contends that forces (strains) produced within the axoneme, transverse to the axis of the outer doublets (termed t-forces), are responsible for squeezing the doublets together or pulling them apart and thereby acting to engage and disengage the dynein motor proteins. When dynein motors are engaged, they generate motive force between the doublets. Stretched elastic elements between the doublets also contribute force to the doublets. The accumulated strain on each doublet multiplied by the local curvature of the flagellum is the major determinant of the t- force. This concept has been formulated into a computer model and the model has been remarkably successful at simulating the movements of living cilia and flagella. In the current proposal, key predictions of the Geometric Clutch hypothesis will be compared to the behavior of real cilia and flagella in controlled experiments. Tests are planned using bull sperm, mouse sperm, respiratory cilia from newt lung and Chlamydomonas reinhardtii. Dr. Lindemann has developed versions of the computer model which mimic the behavior of Chlamydomonas and newt lung cilia. A special version has also been produced which incorporates the unique feature of a bull sperm flagellum and can produce good simulations of bull sperm beating. Experimental tests will assess the validity of the t-force concept and the role it plays in the beat cycle. The role of calcium ions in biasing the flagellar beat will be examined, and also the roles of cAMP and ADP in regulating switching and power output of the beat cycle. Then, specific modification of the Geometri c Clutch working mechanism will be determined that must be implemented to produced calcium ion-, cAMP- and ADP- like changes in the beat of the computer simulated flagella. Structural mutants of Chlamydomonas reinhardtii will be used to explore the role of specific structures in the functioning of the beat cycle. Outer arm deficient mutants will be used to better define the role of the inner and outer row dynein arms. Spoke deficient mutants will be used to explore the role of the spokes and dynein regulatory complex in dynein bridge switching. Information gained from experiments will be used to improve the theoretical model and refine the concepts of the Geometric Clutch hypothesis. The ability of a cell to move relative to its environment is a critical attribute for essentially all living species. The specialized eukaryotic structures known as flagella and/or cilia endow cells with such motile ability in aqueous environments. These long slender structures, fully enclosed by the cell membrane, extend from the cell surface and move back and forth with a characteristic and cell type-specific wave form and beat frequency, resulting in the movement of the surrounding fluid relative to the cell. If the cells are unencumbered, as in the case of protozoa or sperm, the result is that the cell swims; if the cells are attached to a substratum, the result is a sweeping of the fluid over the cell surface, as in the case of epithelial cells that line the respiratory tract. The organelle at the core of the flagellum that is responsible for this beating is a highly organized complex of microtubules and motor proteins (dyneins) termed the axoneme. It is known that flagellar beating is the result of some of the microtubules of the axoneme sliding past the others within the confines of the membrane, and that this sliding is catalyzed by the dyneins using the energy derived from ATP hydrolysis. There is a large (although still incomplete) body of knowledge available co ncerning the protein composition and arrangement within the axoneme, as a result of electron microscopic observations, analysis of genetic mutants, and biochemical analyses. However, exactly how all these proteins interact in real time to result in the complex behavior of beating remains unknown. This project represents a unique opportunity to test a well defined theoretical construct which may explain the inner workings of the eukaryotic flagellum. If this project is successful, the working mechanism of the eukaryotic axoneme will be understood for the first time at a level of detail sufficient to explain most of the behaviors seen in nature. ***
