Diverse functions for intern associated proteins in Drosophila adult muscle

The ability to adhere to the extracellular matrix (ECM) is critical for numerous cell types and tissues including epithelia and muscle. Cell-ECM adhesion is primarily mediated by integrins which provide a direct link between the ECM and the actin cytoskeleton. Integrin adhesions are frequently associated with a core of 60 different proteins (integrin-associated proteins, IAPs). Integrins are required for muscle attachment and in Drosophila, loss of integrins and several IAPs results in embryonic lethality and muscle detachment. However, the IAPs FAK, RSU1, tensin, vinculin and zyxin are not required for viability or embryonic muscle attachment. Furthermore, FAK, RSU1, tensin and vinculin have been observed to localise to muscle attachment sites in Drosophila, indicating that they have some function in muscle attachment. Unlike FAK, RSU1, tensin and vinculin, it was not previously known whether zyxin is expressed in Drosophila muscles. To test this, I generated a genomic zyxin-GFP construct that should contain most of the endogenous zyxin promotor. The genomic zyxin-GFP construct was not observed at muscle attachment sites, suggesting that it is not normally expressed in muscle. I wished to know whether FAK, RSU1, tensin and vinculin are required for muscle function. Various behavioural assays were employed to test for muscle function in larvae and adult flies. The results suggest that larval muscle function was normal in flies lacking these IAPs, but that adult muscle function might be impaired, although it proved difficult to demonstrate a clear functional defect. I then tested whether the IAPs FAK, RSU1, tensin and vinculin are required for normal morphology of adult muscles, focusing on the adult indirect flight muscles (IFMs). The IFMs are fibrillar muscles which attach to the cuticle via specialised epithelial cells known as tendon cells. At the end of the myofibril, where the myofibril attaches to the tendon cell, is a dense region of actin and IAPs known as the modified terminal Z-band (MTZ). I have found that the MTZ is not a homogenous zone of proteins, but is instead organised into at least three distinct layers. Because of the similarity between the structure of the MTZ with that of a hand, I refer to the layers as ‘fingers’, ‘palm’ and ‘wrist’. I discovered that the IAPs FAK, RSU1, tensin and vinculin are each required for the proper structure of the MTZ in unique ways. The fingers were elongated in IFMs lacking FAK, RSU1, tensin or vinculin, while the palm was disrupted in IFMs lacking RSU1, tensin or vinculin. Finally, I was intrigued by the enrichment of the actin-binding protein filamin/Cheerio in the palm and wished to know if it is required for palm function. Deletion of the C-terminus of filamin/Cheerio resulted in a reduction in palm length. Filamin/Cheerio is a mechanosensitive protein which exists in a closed and open conformation. I found that filamin/Cheerio must be open in order to help form a normal palm. Furthermore, vinculin is required to convert filamin/Cheerio from and closed to an open filamin/Cheerio state so that it can perform its function in the palm.Wellcome Trust Studentshi