NEURONAL CYTOSKELETAL REMODELLING

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At the tip of a process, neurite elongation involves the conversion of the cytoplasm from a growth cone type organization to that of a neurite shaft. This transition involves a highly dynamic interaction between microtubules and actin filaments in which a pool of highly dynamic microtubules penetrate the dense actin network in the growth cone lamella. Later microtubules stabilize to form the neurite shaft. We hypothesize that microtubule-associated proteins (MAPs) mediate this transition by initially associating with the actin filaments, to organize the growth cone lamellae, and then with those microtubules that will ultimately form the shaft. We suggest that regulation of MAPs in this actin/microtubule binding is mediated by the phosphorylation state of the MAP. This model is based on observations from the Kosik lab that a malignant melanoma cell line (M2) lacking a major actin filament gelation protein, actin binding protein (ABP-280) can be rescued by MAP2c microinjections. These cells show defects in cortical gelation that are visibly corrected by microinjection of actin gelation proteins and by microinjection of MAP2c. MAP2c also induces the formation of two distinct structures--a flattened lamella rich in actin and multiple processes rich in microtubules. In comparison to mature MAP2 and tau, MAP2c most strongly induces morphologic changes, and is expressed very early in neuronal development. Here we will study the role of signal transduction pathways in regulating the organizational properties of MAP2c. Recently the Kosik lab has shown the growth factor NT-3, which can markedly enhance growth cone formation and spreading in cultured neurons, also increased the association between MAP2 and actin. This regulation will be studied by the co-injection of MAP isoforms and a variety of rac and rho proteins. The effects on the interaction of tau and MAP2 with actin in these experiments will be determined by direct immunolabeling, electron microscopy, and co- immunoprecipitations in the primary cultured neurons. Another aim of this proposal is to obtain training as a clinician scientist. We have set-up several vehicles via which I may obtain additional laboratory training and continue spending a small proportion of my time with clinically related matters.