Multicellular organisms possess complex mechanisms that coordinate the activity of their diverse cells and organs, entailing the release of signaling molecules by some types of cells and the ability to bind and respond appropriately to these molecules by other types. Chicken ciliary neurotrophic factor (chCNTF) is a protein required for proper development of the avian nervous system. Produced by cells innervated by ciliary ganglion neurons, chCNTF must be released by them in order to bind to, and mediate development of, the neurons. CNTF lacks an N-terminal sequence that targets proteins into a well-characterized secretory pathway in cells; prior studies indicate that it is not secreted through this """"""""classical"""""""" pathway. The goal of this proposal is thus to characterize its means of secretion. These results will be significant in clarifying the basic methods used for communication by eukaryotic cells and, because CNTF and related proteins are essential for proper embryonic development, may identify sources of developmental defects and/or potential manipulations that enhance intercellular communication. The cellular compartments involved in CNTF export will be identified by: 1) metabolic labeling of CNTF in cultured cells followed by fractionation of cells and characterization of the compartments in which it resides; 2) attaching green fluorescent protein to CNTF to allow it to be followed through living cells; and 3) isolating organelles and assessing their ability to take up CNTF in vitro. The region of chCNTF necessary to target it to an export pathway will also be identified by testing the ability of various hybrids of chCNTF and rat CNTF (a closely related protein that is not secreted) to be exported by cells.
