Kenneth W. Foster, PhD - US grants

We have established the visual system of the unicellular eukaryote, Clamydomonas reinhardtii, as a model for the study of vision. Its rhodopsin receptor appears to be homologous with that of bovine. We propose to use the ease of genetic, behavioral, and biochemical manipulation of Chlamydomonas to improve our knowledge of the molecular mechanisms of vision. The nature of the retinal environment in its opsin and the mechanism of transduction will be studied with a series of retinal analogs by behavior and in vivo differential spectroscopy. The receptor protein and its membrane will be isolated for related spectroscopic information. The components of Chlamydomonas' visual processing that shape, communicate and maintain its visual signal to its flagella are also likely to be homolgous to the similar components found in higher organism photoreceptors. As is true for other rhodoposin receptors the signal has steps in the form of ionic currents. Particularly suitable for cells of this size are the attached- and whole-cell clamp electrophysiological techniques. Several of the ionic pathway components of its visual signal processor will be identified by combining this approach with pharmacological, light, ionic and mutant modifications of conditions. An extensive search for mutants that effect any aspect of the visual process will be started. At the same time search for specific mutants of the opsin and the hypothesized retinal isomerase will be begun. The ease of obtaining mutants which enable isolation of each protein involved in the transduction process is a very attractive feature of this model system. The work proposed in this grant application should yield information relevant to the molecular details of transduction, signal processing, biosynthesis of chromophore, cell maintenance, effect of nutrition, influence of lipids, adapation, and cell homeostasis. Understanding these molecular mechanisms should make it possible to understand the effects of some genetic diseases, nutrition, light, and drugs on the transduction portion of the visual process.

Support is requested for a preparative ultracentrifuge, liquid scintillation counter and an, autoclave. This equipment is urgently needed for biochemical studies of photoresponses in two eukaryotic microorganisms, the fungus Phycomyces blakesleeanus and the alga Chlamydomonas reinhardtii. In Phycomyces, blue light controls several response, including phototropism, light growth responses, carotene synthesis, and sporangiophore development. The blue-light photoreceptors in Phycomyces and in many other plants and fungi remain to be identified. The availablity of well characterized mutants, provides a strategy for biochemical isolation of photoreceptors and other molecules involved in photosensory transduction processes. In Chlamydomonas, phototaxis and carotene synthesis employ a rhodopsin photoreceptor. This rhodopsin, however, has yet to be purified in sufficient quantity for spectroscopic analysis. The increasing level of biochemistry work in the two biophysics research groups requires that appropriate equipment be available within the physics building, instead of only in the biology building across campus. The ultracentrifuge is required for cell fractionation, in particular membrane fractionation. The liquid scintillation counter will be used for receptor binding studies and for tracing signaling pathways using radioactively labeled probes. The autoclave will be used for sterilization of rowth media and apparatus.

DESCRIPTION (Provided by Applicant): Sensory signal processing will be studied in the unicellular alga, Chlamydomonas reinhardtii. Its sensory network controls two cilia that propel and steer the cell through its aqueous environment, and allow it to track a light source (phototaxis). The specific aims are a) to identify components of the system that are important and predictive of the phototaxis behavior and b) to determine the nature and importance of the interactions among these components. These goals are of general importance in biology, for every cell faces the complex task of evaluating multiple, and sometimes contradictory, sensory inputs. Responses must be well regulated for optimal performance. Cells employ sophisticated strategies such as nonlinear processing, adaptive feedback, and potentially chaotic control. To investigate these processes, an established model of ciliary motility has been chosen with an extensive database of known components. Three preparations will be studied on the time scale of important interactions: the intact cell, the detergent-permeabilized cell, and the deciliated living cell. The last two preparations allow selective study of two nonoverlapping portions, cilia and cell body respectively, of the transduction network. The dynamic responses are to be measured by a) stereo-CCD ciliary images sampled at 900 Hz and b) net electrical current of the deciliated cells. Modulation of selected wavelengths of light and varied temperature, viscosity, external pH, extracellular ion concentrations, and pharmacological agents will affect the ciliary responses. Internal uncontrolled variables associated with phototaxis; namely intraciliary calcium, pH, and membrane potential will be measured. The dynamic response information will be analyzed by suitable quantitative methods, and predictive models will be developed and tested. Analysis of mutations of the ciliary structures in Chlamydomonas has identified structural and biochemical components that relate to features of the dynamic response. With this new information, our current model of three nonlinear parallel pathways controlling the ciliary stroke frequency, phase, and stroke velocity during phototaxis will be refined. Analysis of the nonlinear dynamical behavior of the system should lead to an understanding of the integrated components that generate this cellular behavior. Since many diseases involve intracellular signaling, understanding the complex and inherently nonlinear strategies employed by cells is of broad clinical significance.

In this project the PIs will determine the motor-generated shear forces and doublet sliding in the biciliated unicellular alga Chlamydomonas reinhardtii under wide range of conditions with the goal of establishing the correct mechanism for self-organizing beating structure. First the PIs will record long durations of high temporal and spatial resolution images of ciliary beating using electron-multiplying CCD camera. Next they will determine accurately the drag force exerted by the fluid along the cilium using a slender body theory for low Reynolds number flows. The force generated by motors along the length of a cilium will then be determined by balancing the hydrodynamic drag to internal forces due to sliding and bending. The precise images of the cilia will also allow the PIs to determine the relative sliding of the doublets. The force-sliding data will be used to test various models of self-organizing beating structure as well as to determine the dynamics of the basal body that connects the two cilia to the cell body. The proposed study will add a significant tool for determining the active internal forces generated during ciliary beating. This in-vivo technique will complement the in-vitro molecular genetic, electron microscopy, atomic force microscopy (AFM), and sliding assay techniques currently used to understand the physiology of ciliary beating. Several graduate students will be trained in interdisciplinary research and to work as a team with several advisers. They will participate in all phases including experiment development, data collection, data analysis, and modeling. Undergraduate students will significantly aid the project by characterizing individual ciliary mutants that have mechanical or control function defects and will learn to use AFM. High school students will assist with mutants. The physics of cilia will be incorporated in undergraduate and graduate courses.