The goal of these investigations is to understand the regulation of cytoskeletal biosynthesis and function during mammalian cell development. The system utilized is the differentiation of mouse 3T3-adipocytes, where large and specific decreases in mRNA levels for actin and tubulin appear to play an important regulatory role in the differentiation process. The proposed experiments, which emphasize the regulation of cytoskeletal gene expression, cell structure and lipogenesis, are particularly relevant to human diseases involving fat cell number and function, such as obesity, diabetes and liposarcoma. Experiments underway use cloned cDNA probes for Beta-actin and Beta-tubulin to assay transcription during differentiation in both isolated nuclei and whole cells. If transcriptional changes cannot quantitatively account for changes in these mRNAs, we will study mRNA turnover and processing. The build-up and processing of nuclear RNAs will get particular attention in light of evidence suggesting the build-up of a putative actin mRNA precursor during differentiation. The actin gene active in adipocytes will be isolated and used to define the primary transcription unit and to ask if this putative actin precursor has a structure consistent with such a role. Further studies involve construction of in vitro systems to study relevant control mechanisms which are operating. Experiments will be performed to understand the normal physiological role of the cytoskeletal and morphological regulation of lipogenic gene expression shown to exist in adipocytes. In particular, the role of cyclic AMP's effects on cell adhesion and the cytoskeleton in the suppression of lipogenic gene expression by this key physiological agent will be examined by exposing differentiating cells to a variety of cyclic AMP agents while quantitatively varying substrate adhesiveness or treating with anticytoskeletal drugs. The subsequent expression of lipogenic protein and RNA will be studied with antibodies and cDNA clones previously constructed. Finally, the reversibility of cellular and molecular differentiation-dependent changes will be studied. This is now experimentally approachable by replating differentiated cells which have had lipid accumulation blocked and hence, retain firm attachment to the sub-stratum. Subsequent cell growth and cytoskeleton synthesis will be studied at the protein and RNA levels.