One of the earliest steps in neuronal polarity is the rapid elongation of the axon, a process in which the cytoplasm converts 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 dynamic microtubules penetrate the dense actin network in the growth cone lamella and then stabilize to form the neurite shaft. The applicants 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 the microtubules that will ultimately form the shaft. The hypothesis includes some more speculative ideas that the regulation of MAPs in this actin/microtubule association is mediated by the phosphorylation state of the MAP. They base this model on observations of a malignant melanoma cell line (M2) that lacks a major actin filament gelation protein, actin binding protein (ABP-280) and the effects of MAP2c microinjections. These cells show defects in cortical gelation that are visibly corrected by the microinjection of actin gelation proteins and by microinjection of MAP2c. MAP2c also induces the formation of a flattened lamella and the elaboration of multiple processes. The contributions of each of the two filament systems to these effects can be separated by disruption of actin filaments with cytochalasin, resulting in loss of the lamella, but not the process formation; and disruption of microtubules with colchicine resulting in loss of process formation, but not the lamella. In comparison to mature MAP2 and tau, MAP2c most strongly induces morphologic changes, and is expressed very early in neuronal development. Here the applicants propose the following studies: investigate those sites on MAP2 and tau that are important for binding to, and organizing, actin filaments. They will construct truncated MAP2 and tau cDNAs, and express the resultant proteins for in vitro rheometry and in vivo microinjection studies. Similar studies will be carried out for intact dephosphorylated MAPs, and MAPs mutated at one or more phosphorylation sites. These studies will identify sequences and phosphorylation sites important to the organizational properties of a MAP. Second, they will look at the role of signal transduction pathways in regulating these organizational properties of MAPs. They have found that the growth factor NT-3, which can markedly enhance growth cone 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 modified rac and rho proteins. The effects on the interaction of tau and MAP2 with actin in these experiments will be determined by direct immunolabeling or co-immunoprecipitations in the primary cultured neurons. Finally, they will study the effects of other actin associated proteins on MAP organizational properties in ABP expressing; melanoma cells and in gelsolin deficient neuronal cultures.
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