MUTATIONAL ANALYSIS OF AMOEBOID CHEMOTAXIS

Amoeboid chemotaxis is an important phenomenon in which signal transduction and cell motility interact. It may be critical to movement of cancer cells away from the original in metastasis as well as the ability of white blood cells such as to respond rapidly to infection. Dictyostelium discoideum amoebae provide a powerful model system for analyzing amoeboid motility and chemotaxis using a genetic approach. They have a habloid genome which allbws expression of be analyzed genetically, and are easily cultivited. The ultimate goal of this study is a dissection of amoeboid motility and chemotaxis by analyzing the biochemical and behavioral properties of mutants. Mutants will be generated by chemical mutagenesis. Dictyostelium mutants will be selected using an established selection chamber and screening assay. The mutants that are selected will be analyzed in terms of their biochemistry and behavior. Chemoattractant binding, production of intracellular messengers (such as IP3, calcium, and cGMP), and actin/myosin association with the cytoskeleton will assayed. Computer-controlled image analysis will be used to determine single cell speed, turning, orientation and shape in response to both stable chemoattractant gradients and rapid changes in concentration. Genetic studies will determine complementation groups and insure that the biochemical defect is associated with the behavioral alteration. The wild-type copies of the mutant genes will be cloned by complementing selected mutants with a library of genomic DNA in a Dictyostelium transformation vector. Chemotactic transformants will be selected on the basis of morphology or using the selection chamber. Recovery of the transformation vector from chemotactic transformants will lead to the identification of wild-type sequences that can complement the mutation. Tbe long term objective is to identify the biochemical processes responsible for particular behavioral responses.