The transition of plastic neurites to stabilized axons is of utmost importance in proper development and maintenance of synaptic connections. An hierarchical expression of neuronal intermediate filaments (IFs) accompanies these events, in which neurons rely on the generic vimentin IF system to initiate the outgrowth of highly-plastic, putative axonal neurites, then subsequently deposit neurofilaments (NFs) to stabilize these structures during synaptogenesis and maintain these synapses throughout life. Although it is clear that expression of Vm declines and that of NFs increases during this period, how the neuron effects this transition in the presence of an existing Vm network remains unclear. Our data indicates that, while the bulk of Vm and NFs form discrete IF networks, a portion of newly-expressed NF subunits (in particular, NF-H) undergoes transient association with Vm prior to its accumulation in NFs. These findings suggest that more rapid turnover of Vm filaments as opposed to NFs may contribute to the developmental transition between these networks. Studies in this pilot proposal will investigate (1) the relative turnover rate of Vm and NFs and (2) the subcellular localization of putative Vm-NF heteropolymers in differentiating NB2a/d1 neuroblastoma cells and chick dorsal root ganglion neurons in culture, using our established techniques of microinjection, immunofluorescence, confocal microscopy, immuno-electron microscopy, microdensitometry, radiolabeling, immunoprecipitation, electrophoresis and autoradiography. These analyses will confirm or deny our working hypotheses that (1) neurons have no means to regulate the formation of hetero- or homopolymers, and the accumulation of NF homopolymers is mediated by subunit trapping within relatively long-lived NFs coupled with a developmental decline in Vm, and (2) any such putative Vm and NF co- polymers represent transient assembly intermediates that are largely confined to the perikaryon and axon hillock region rather than undergoing axonal transport. Should these hypotheses be proven valid, they will demonstrate the method by which neurons effect the developmental switch between the that provides support to mature axons throughout life. Should they prove false, the studies of this pilot proposal will point the way towards subsequent investigations of neurofilament subunit exchange during development and establishment of the mature axonal cytoskeleton.
