Meeting Abstract
We are interested in structural and functional changes to muscle resulting from RNAi against the sallimus gene, which encodes a giant sarcomere associated protein (gSAP). Sallimus (sls) is an ortholog of vertebrate titin and like most animal gSAPs, sls possesses both compliant (Ig) and stiffer elastic domains (PEVK). There are ten identified sls isoforms in fruit fly that express different proportions of compliant and stiff domains, potentially offering diverse mechanical tissue properties. Our previous experiments show that RNAi against sls in Drosophila larvae does not knock down all sls isoforms equally. Namely, the expression of isoforms most rich in PEVK domains is dramatically reduced. Additionally, sls RNAi yields a significant increase in gSAP length and in the expression of isoforms rich in compliant domains rather than those isoforms consisting of more elastic domains. Experiments described here are aimed at determining how sarcomere length increases. It’s plausible that (1) post-RNAi sls has been cut during the process of alternative splicing, or (2) that the reduced expression of PEVK domains yields a more easily elongated (i.e. compliant) gSAP. We used stimulated emission depletion microscopy (STED) and dual labelling immunohistochemistry (IHC) to measure the distance between known regions of the sls protein. Commercially available anti-kettin binds ~175 nm from the Z-disk and the custom made Klg3/4 antibody (gift of B. Bullard) binds ~0.5 mm from the Z-disk in control tissue. We predict that if the sls protein is somehow cut during splicing (post-RNAi), then the distance between these two points will decrease. Alternatively, the distance between antibodies would increase if the post-RNAi sls proteins, that are rich with compliant domains, were simply elongated.
