ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of Septate Junction integrity in Drosophila epithelial cells

Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterized. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum, alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-Actin and non-muscle Myosin II in the plane of adherens junctions. On the other hand, β-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation, and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the septate junction, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.

Methods

LIve imaging acquisition: LSM Zeiss 880 AiryScan equipped with a 63X N.A.1.4. objective and controlled by ZEN software.

Fixed specimens: LSM Zeiss 880 AiryScan equipped with a 63X N.A.1.4. objective and controlled by ZEN software or LEICA SPE equipped with a 63X N.A. 1.4

Laser ablations: were performed on live pupae aged for 16h to 19h APF using a Leica SP5 confocal microscope equipped with a 63X N.A. 1.4 objective or a LSM Zeiss 880 AiryScan equipped with a 63X N.A. 1.4 objective. Ablation was carried out on epithelial cell membranes at AJ level with a two-photon laser-type Mai-Tai HP from Spectra Physics set to 800 nm and a laser power of 2.9W.

Images were then analyzed and quantitated using Fiji
Statistical analyses: All information concerning the statistical details is provided in the main text and in figure legends, including the number of samples analyzed for each experiment. Prism 8 software and R 4.2.1 were used to perform the analyses.