Abstract
A wealth of knowledge regarding the functions of Extracellular Matrix (ECM) macromolecules from in vitro or disease models strives for validation in intact animals. In particular, the progression of cardiomyopathies is tightly linked to pathological changes in the heart ECM. To address this in the Drosophila model, we developed a novel adaptation of fluorescence recovery after photobleaching (FRAP), which allows us to assess ECM protein incorporation during growth in living, intact larvae. Recovery of fluorescently tagged protein is a proxy for addition or relocation of ECM protein. We focus on Collagen IVα (Viking), a conserved protein thought to be a stable component of the basement membrane (BM). We established a time course for Vkg-GFP fluorescence accretion in three different BMs through larval development, under normal conditions and when Matrix Metalloprotease or its inhibitor, TIMP is overexpressed. We demonstrate that the gain and loss of Collagen trimers from the basement membrane changes over developmental time and between tissues. High variability in measured fluorescence reduced the sensitivity of this approach. During growth, a strong phasic wave of Vkg accumulation was detected at the second to third instar ecdysis, potentially supporting growth of the new instar. Between organs, flux of Vkg was high in somatic muscle, intermediate in the heart and low in trachea. Heart-specific overexpression of mmp2 and its inhibitor timp, modified the dynamics of Vkg-GFP flux. We find that MMPs are positive regulators of Vkg/Col IV turnover in the ECM, in alignment with current models of ECM regulation.