Adherens junction-associated pores mediate the intercellular transport of endosomes and cytoplasmic proteins.

Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. Here, we demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, we observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, we present a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact.

Smurf Downregulates Echinoid in the Amnioserosa To Regulate Drosophila Dorsal Closure.

Drosophila dorsal closure is a morphogenetic movement that involves flanking epidermal cells, assembling actomyosin cables, and migrating dorsally over the underlying amnioserosa to seal at the dorsal midline. Echinoid (Ed)-a cell adhesion molecule of adherens junctions (AJs)-participates in several developmental processes. The disappearance of Ed from the amnioserosa is required to define the epidermal leading edge for actomyosin cable assembly and coordinated cell migration. However, the mechanism by which Ed is cleared from amnioserosa is unknown. Here, we show that Ed is cleared in amnioserosa by both transcriptional and post-translational mechanisms. First, Ed mRNA transcription was repressed in amnioserosa prior to the onset of dorsal closure. Second, the ubiquitin ligase Smurf downregulated pretranslated Ed by binding to the PPXY motif of Ed. During dorsal closure, Smurf colocalized with Ed at AJs, and Smurf overexpression prematurely degraded Ed in the amnioserosa. Conversely, Ed persisted in the amnioserosa of Smurf mutant embryos, which, in turn, affected actomyosin cable formation. Together, our results demonstrate that transcriptional repression of Ed followed by Smurf-mediated downregulation of pretranslated Ed in amnioserosa regulates the establishment of a taut leading edge during dorsal closure.

Novel transport function of adherens junction revealed by live imaging in Drosophila.

Adherens junctions are known for their role in mediating cell-cell adhesion. DE-cadherin and Echinoid are the principle adhesion molecules of adherens junctions in Drosophila epithelia. Here, using live imaging to trace the movement of endocytosed Echinoid vesicles in the epithelial cells of Drosophila embryos, we demonstrate that Echinoid vesicles co-localize and move with Rab5-or Rab11-positive endosomes. Surprisingly, these Echinoid-containing endosomes undergo directional cell-to-cell movement, through adherens junctions. Consistent with this, cell-to-cell movement of Echinoid vesicles requires the presence of DE-cadherin at adherens junctions. Live imaging further revealed that Echinoid vesicles move along adherens junction-associated microtubules into adjacent cells, a process requiring a kinesin motor. Importantly, DE-cadherin- and EGFR-containing vesicles also exhibit intercellular movement. Together, our results unveil a transport function of adherens junctions. Furthermore, this adherens junctions-based intercellular transport provides a platform for the exchange of junctional proteins and signaling receptors between neighboring cells.

Capulet and Slingshot share overlapping functions during Drosophila eye morphogenesis.

BACKGROUND: CAP/Capulet (Capt), Slingshot (Ssh) and Cofilin/Twinstar (Tsr) are actin-binding proteins that restrict actin polymerization. Previously, it was shown that low resolution analyses of loss-of-function mutations in capt, ssh and tsr all show ectopic F-actin accumulation in various Drosophila tissues. In contrast, RNAi depletion of capt, tsr and ssh in Drosophila S2 cells all affect actin-based lamella formation differently. Whether loss of these three related genes might cause the same effect in the same tissue remains unclear. METHODS: Loss-of-function mutant clones were generated using the MARCM or EGUF system whereas overexpression clones were generated using the Flip-out system. Immunostaining were then performed in eye imaginal discs with clones. FRAP was performed in cultured eye discs. RESULTS: Here, we compared their loss-of-function phenotype at single-cell resolution, using a sheet of epithelial cells in the Drosophila eye imaginal disc as a model system. Surprisingly, we found that capt and ssh, but not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. The capt/ssh mutant cells possessed: (1) hexagonal cell packing with discontinuous adherens junctions; and (2) largely complementary accumulation of excessive phosphorylated myosin light chain (p-MLC) and F-actin rings at the apical cortex. We further showed that the capt/ssh mutant phenotypes depended on the inactivation of protein kinase A (PKA) and activation of Rho. CONCLUSIONS: Although Capt, Ssh and Tsr were reported to negatively regulate actin polymerization, we found that Capt and Ssh, but not Tsr, share overlapping functions during eye morphogenesis.