The long-term objective of the proposed work is to understand the cellular processes that take place within the hagfish slime gland thread cell that result in the production and maturation of a long, precisely packaged, tapered cytoplasmic thread comprised almost exclusively of intermediate filaments (IFs). It is anticipated that as a result of these studies much will be learned about the interaction of IFs with each other as well as with other cytoskeletal components, and as such the GTC should prove to be a useful model system for studying the biology of IFs and IF-cytoskeletal interactions. The specific aims include (1) determining whether non-IF cytoskeletal components (e.g., microtubules and microfilaments) are essential for proper thread formation, (2) delineation of the dynamics of hagfish IF subunit incorporation into IFs and the developing thread in vivo, (3) determining whether maturational processes associated with thread aging (senescence) can be correlated with morphological and biochemical changes in the thread ('non-conventional') and cytoplasmic ('conventional') cytoskeletal components, and (4) evaluation of the transient forms of thread IF type I and type II keratin polypeptides that lead to the assembly of keratin IFs in vitro. The importance of studying IFs, and IF-IF and IF-cytoskeletal interactions is becoming increasingly apparent. In addition to contributing to the cytoskeletal architecture of most cells, IFs have been implicated in a number of other important cellular functions, including intracellular transport, organelle movement, and cell locomotion. In addition, antibodies to specific IFs provide useful information regarding cell origin, normal differentiation, and evidence of pathology, tumor origin, and neoplastic progression. The rather ubiquitous distribution of IFs, their apparent role in cytoskeletal functions and other important cellular processes, their potential role in pathological processes and their potential usefulness in pathological diagnoses clearly make IFs important structures to study. By employing morphological, biochemical, and immunocytochemical procedures, the proposed research outlines a comprehensive, multidisciplinary approach to the study of a cell (the GTC) that comes about as close to being an IF machine as nature probably intended to provide, and by accomplishing the specific aims it is anticipated that the usefulness of the GTC as a model system for studying IF-IF interactions and the interactions of IFs with other cytoskeletal components will become apparent.