The existence of intermediate filament (IF) protein insoforms with characteristic cell-type distributions suggests either a cellular function or mechanism of regulation that is specific for each protein. Studies of IF protein function and regulation have been limited, however, by the resistance of IF to depolymerization and biochemical manipulation. Recent advances in recombinant DNA technology have shown that the introduction, into the nucleus or cytoplasm, of nucleic acid sequences coding for the gene (sense message), or for a sequence complementary to the gene (anti-sense message), can either enhance or inhibit, respectively, the amount of translation competent mRNA available to the cell, thereby simulating a dominant """"""""mutant"""""""" phenotype that can be used to analyze gene or protein function.
Our aims are (i) to use recombinant DNA techniques to enhance or inhibit the synthesis of individual IF proteins in single cells and cell populations, and (ii) to use these genetically engineered cells to study the function and regulation of IF proteins in nerve and muscle. PBR322 plasmids engineered to direct IF protein transcription, or to direct anti-sense transcripts of IF oligonucleotides will be introduced into chondrocytes, presumptive myoblasts, and neurons either by microinjection or transfection. These cells will then be used for biochemical and immunohistochemical analysis of cytoskeletal protein synthesis and distribution, as well as morphological and ultrastructural analysis. Areas of particular interest will be the contribution of desmin to myofibril integrity; the ability of mature neurons to transport and degrade vimentin; the role of neurofilaments in neurite stability; and the role of IF in cell viability and differentiation. Many studies have reported the abnormal accumulation of IF proteins in human pathology: e.g., desmin in inherited myopathies, neurofilament proteins and vimentin in the neurofibrillary tangles of Alzheimer's disease, and neurofilaments in motor neuron disease; however, the current ignorance of normal IF regulation and function makes these findings difficult to interpret. For this reason, the emphasis of this proposal on the function and regulation of IF proteins in neurogenesis and myogenesis should prove valuable for future studies of development and degenerative neurologic disorders that may relate to IF protein dysfunction.
