Final Report Summary - BB: DICJI (Breaking barriers: Investigating the junctional and mechanobiological changes underlying the ability of Drosophila immune cells to invade an epithelium.) The ability of cells to penetrate into tissues underlies many pathological conditions such as metastasis and inflammation. Invasion requires coordination of changes in the biology of the invading cell with alterations in the adhesive behavior and integrity of the penetrated barrier. Recent work has shown that Drosophila hemocytes move through an epithelial barrier during embryonic development and require the small GTPase RhoL to alter Cadherin properties. The project aimed to identify morphological and biophysical changes in the epithelia during this immune cell transmigration, and to examine potential changes in junctional DE-Cadherin expression during hemocyte invasion, using immunofluorescence analysis and live imaging of junctional DE-Cadherin and the Actomyosin network. These experiments were planned to provide clear insight into whether changes in DE-Cadherin integrity precede hemocyte invasion. Cell-cell junctions are known sites of mechanotransduction and hence this project also planned to evaluate possible changes in the mechanobiology of Adherens junctions during transmigration. I aimed to use a FRET based tension sensor as well as laser nanoablation of cell junctions to assess cortical tension during hemocyte invasion. Analysis of morphological changes in the germband during hemocyte migration using immunofluorescent imaging of fixed embryos as well as 2-photon imaging of live ones revealed that hemocytes migrate into the tail by squeezing between two epithelial tissues, namely the caudal ectoderm and the hindgut visceral mesoderm. I was able to show that this migration of hemocytes into the tail brings about changes in DE- Cadherin localization in the neighboring cells, at least in part through apoptosis of these cells. Due to the depth of the tissue we were examining, FRET based approaches proved intractable, and we needed to modify our equipment to use laser nanoablation of cell junctions. However, by modifying the junctional tension in the ectodermal cells of the tail using various genetic tools I showed that changes in junctional tension accompany hemocyte invasion into the tail. Our studies should have relevance for future studies of pathologies like inflammation and cancer metastasis and prompt an examination of how cortical tension is altered in these systems.