The study we propose will explore the functions of the various domains of the NF(M) subunit by using recombinant DNA technology to produce truncated forms of the neurofilament protein. We will express these proteins first in cultured fibroblasts lacking other intermediate filament proteins, secondly, in transgenic mice and in mice in which the endogenous NF(M) gene has been""""""""knocked out"""""""" by homologous recombination. The participation of the truncated proteins in assembly, processing and transport will be assessed by immunofluorescence, immuno-election microscopy and biochemical techniques. It is expected that some truncated forms will show abnormal assembly, phosphorylation or transport, and some may interfere with the processing of the native NFs or of other cytoskeletal elements as well. Ultimately, this work will allow the description of functional domains of NF(M) and yield insight into the biological functions of the NFs. Specifically, we will: 1. Alter copies of the human NF(M) gene so that they encode proteins containing a heterologous eleven amino acid """"""""tag"""""""" at either the N terminus or at an internal position so that they can be immunocytochemically identified. Because of this tag, further alterations can be made, in principle, in any part of the NF(M) sequence without abrogating the immunodetection. We will verify the functional neutrality of the """"""""tag"""""""" by shoeing that the tagged NF(M)'s behave the same as the unaltered human (NF(M) in transgenic mice. 2. Prepare and analyze the behavior of """"""""tagged"""""""" NF(M) proteins carrying deletions in either their amino terminus, carboxy terminus, acidic domain, or the tandemly repeated polyphosphorylation site. Genes encoding these altered forms of the NF(M) will be introduced into fibroblast cells and the patterns of expression studied using immunomicroscopy. We will examine the character and localization of the immunofluorescence to assess filament formation, association with organelles, lysosomes, vacuoles and nuclear or cytoplasmic membranes. Altered NF(M)'s showing interesting phenotypes in fibroblast cells will be studied in detail in the neurons of transgenic mice or mice in which the NF(M) gene has been inactivated by a homologous recombination """"""""knock out"""""""" event. These latter mice are being created by us in a research program funded elsewhere. We anticipate that the expression of the altered (NF(M) proteins in normal transgenic mice and in mice carrying an NF(M) null mutation will offer us an unobstructed view of the contributions of each protein domain to the assembly, processing, transport and function of neurofilaments in their normal cellular context, the neuron.
Showing the most recent 10 out of 555 publications
