This project is divided into four sections all of which share a common focus on the elements of the neuronal cytoskeleton and the molecules that are responsible for the interactions between these elements. The first series of studies focuses on the interactions between neurofilaments and microtubules in vitro with specific attention to the role of microtubule accessory proteins and the P150 and P200 proteins of the neurofilaments in these interactions. The second set uses the techniques of microinjection and video assisted image-intensified microscopy to study the dynamics of DTAF-tubulin in the living cell. Phenomena such as wall exchange, organizing centers and treadmilling will be examined by fluorescence photobleaching recovery. The effecs of microinjected proteins and antibodies on the cytoskeleton will also be assessed. The third section will focus on the separation of the individual MAP components in both the tau and HMW complexes, their comparison by peptide fingerprinting, determination of their function in promoting the assembly and stability of microtubules and the effects of phosphorylation of MAPs on modifying these effects. Antibodies will be raised against each of the MAPs and used for microinjection and localization studies. The final section of the project will address the function of the 94,000 dalton microtubule assembly inhibitory protein in vivo and in vitro. it will be compared to the 95,000 dalton intermediate filament-associated protein and the SV-40 T antigen.
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