9316975 Ayme-Southgate Projectin is a giant protein present in Drosophila muscle, which has been shown to share structural homologies with two other giant muscle proteins, titin (from vertebrates) and twitchin (from a nematode, C. elegans). Their similarity is found at the level of two amino acid domains present in all three proteins and repeated many times in a regular pattern. The functions of these proteins are under intense investigation and titin is, at this time, the best understood. The different isoforms of projectin have been implicated in several possible functions which are related to the functions proposed for either titin or twitchin. However, no specific role has been definitely demonstrated. Partial information is available on the structure of projectin but the complete amino acid sequence is still to be determined. The long term objective of this project is to understand the structure and functions of projectin and how projectin participates in the assembly and maintenance of the myofibrils. The specific aims are to: 1, determine the projectin gene structure and splicing pattern for the indirect flight muscle (IFM) isoform; 2, determine exons specific for the IFM isoform; 3, determine the orientation of the IFM isoform in the sarcomere (i.e., whether the N- terminus is towards the Z band or the A/I junction). To achieve this last aim, two monoclonal antibodies will be exploited which, by immunoelectron microscopy, have been shown to specifically recognize epitopes at either the Z band or the A/I junction. %%% Muscle cells possess a highly-organized subcellular structure, with cytoskeletal elements and "motors" exquisitely arranged to subserve the specific function of contraction, and internal membranes arranged to subserve the function of regulation of the contractile activity. Although a great deal is already known about the organization and structure of muscle cells, some unsolved problems remain. One of these unsolved problems conce rns the role(s) of certain very large proteins (600,000 to 2,000,000 daltons), recently discovered, which are situated within the A and I bands of the sarcomeres. These large proteins are arranged in periodic arrays in conjunction with periodic arrays of proteins whose roles have been well characterized in muscle function, myosin (the "motor") and alpha actinin. Circumstantial evidence suggests that, for one or another of these very large proteins, they may confer elasticity to the muscle cell, or be involved in regulation of contraction, or be involved in the general assembly of myofibrils (the subcellular units of cytoskeletal organization in muscle cells). In the model insect, Drosophila, there are at least two isoforms of projectin, one found in synchronous muscle and the other found in asynchronous muscle. These muscle types have significantly different contractile properties: synchronous muscle is "regular" muscle, and contracts once per nerve impulse, whereas asynchronous muscle (also known as "indirect flight muscle") contracts several times per nerve impulse, allowing for the high frequency wing beat necessary for insect flight. The two different projectin isoforms are distinguished by different molecular sizes and they have somewhat different subcellular localizations in their respective muscle tissues. The experiments to be carried out in this project will address key aspects of the structure and function of these very large muscle proteins. The information obtained from these studies should be helpful in understanding the functions of these large muscle proteins, not only in the very interesting case of insect flight vs. regular muscles, but in all animals, including humans. ***
