Given the broad range of mutant strains with deficits in behaviors relevant to an undergraduate course in physiology, we sought to develop two discovery-based experiments with C. elegans that would both amplify concepts being learned in class and permit students to participate in experimental design. The first experiment challenges students to characterize quantitatively the deficits in muscular function arising from selected missense mutations of
unc-54 (myosin heavy chain B). Cyclic interaction of myosin with actin is the basis of tension development in muscle cells, as well as eukaryotic cells generally, and the mechanical events are coupled to the hydrolysis of MgATP by myosin. The experiment exploits the availability of
unc-54 mutations ( e.g. ,
s74 and
s95 alleles) that have salient functional deficits without disruption of muscle structure. Following discussion of viscosity and Reynolds number, students develop swimming assays in which tail-beat frequency is examined as a function of bulk solution viscosity. The concepts that the students consider when designing their experiments include the following: osmolarity and its measurement; bulk solution viscosity and its measurement; muscular fatigue and endurance; and force-velocity relations and their interpretation in the context of muscular efficiency. Because of the linkage between mutations of human myosin isoforms and a range of muscle and non-muscle diseases, including lethal heart disease and hereditary deafness, students have found the experiment engaging. Moreover, the accessibility of the crystalline structure of the myosin head (containing the catalytic domain and the actin-binding regions) permits students to correlate their results with the emerging structure-function relationship of myosin. The experiment can also be extended to the analysis of other mutations affecting muscular function ( e.g. ,
unc-27 /Troponin-I-2; please see abstract presented at this meeting by Priest et al. , "Structural and Functional Disruption of Muscle Caused by Troponin I Mutation"), as well as to evaluation of muscle structure in wild-type and mutant muscles with rhodamine-phalloidin staining of filamentous actin. The second experiment, which spans portions of 3-4 laboratory sessions, aims: (1) to increase familiarity of students with computational tools used by physiologists to glean information from genomic databases, and (2) to illustrate how genomic information can be used to design physiological experiments probing human diseases with a model organism. Students "adopt" a disease linked to a mutant transporter, channel, or contractile protein and then use computational tools to identify a candidate homologue in C. elegans , to analyze protein domain architecture, and to formulate an hypothesis concerning the physiological role of the protein. Subsequently, students test their hypothesis by eliminating expression of the candidate homologue through RNA interference. Use of forward and reverse PCR primers incorporating the T7 RNA polymerase promoter sequence allows conservation of time and money when cDNA is transcribed, since double-stranded RNA can be prepared in a single reaction. Following an overnight soak in phosphate-buffered saline containing 100-1000 ng/microliter dsRNA, worms and progeny are examined for deficits in assays of chemotaxis, osmoregulation, egg-laying, and swimming, as well as in structural analyses of actin organization with rhodamine-phalloidin staining. Examples of adopted diseases include Bartter's Syndrome (linked to mutation of Na-K-2Cl cotransporter), affecting kidney function in humans, and Familial Hypertrophic Cardiomyopathy (linked to mutation of myosin, tropomyosin, troponin I, and troponin T), affecting human heart muscle. Two qualities of the experiment are its practical relevance, because of consideration of human diseases, and its integration of molecular, cellular, and organismal biology.