High molecular weight MAPs from brain tissue will be characterized with regard to ultrastructure, phosphorylation, and function. The ultimate goal is to understand how microtubules function in the cell. Polyclonal and monoclonal antibodies to MAP 1A, MAP 1B, and MAP 2 were produced in the previous project period. One of the antibodies recognizes a phosphorylated epitope. Further hybridoma production will be carried out to obtain monoclonal antibodies to MAP 1C and to other phosphorylated or dephosphorylated epitopes of each of the MAPs. MAPs will be characterized in vitro by rapid-freeze, deep-etch electron microscopy. Individual molecules and molecules associated with microtubules will be examined. The position of the epitopes recognized by the anti-MAP antibodies will be determined directly by electron microscopy. Immunoelectron microscopy, using thin section, whole mount, and rapid-freeze, deep-etch electron microscopy, will be used to localize the MAPs in cells. This information will be compared with the results of the in vitro electron microscopic analysis to define the interactions mediated by the MAPs between microtubules and other cellular structures. Evaluation of changes in MAP composition, distribution, and phosphorylation state in brain and in cultured cells will be examined. PC 12 cell will be investigated to determine the effect of NGF and other effectors on MAP phosphorylation state and distribution. Antibodies recognizing phosphorylated epitopes will be used to identify and, ultimately, isolate MAP kinases. Several approaches will be taken to investigate MAP function directly. Antibodies to the MAPs will be injected into cultured cells to determine effects on neurite formation, mitosis, organelle transport, and other microtubule related functions. In addition, attempts will be made to develop a system for reconstituting organelle transport in crude and purified microtubule preparations. These are basic studies that should be of considerable importance for understanding the structure and functional properties of the neuronal cytoskeleton. Such information should be of profound importance for understanding Alzheimer's disease and other neuropathological conditions. In addition, because the MAP 1 polypeptides are components of the mitotic spindle in a wide variety of cells, the project should provide important new information of relevance to understanding both normal and abnormal cell division.
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