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.

Differential adhesion and actomyosin cable collaborate to drive Echinoid-mediated cell sorting.

Cell sorting involves the segregation of two cell populations into `immiscible' adjacent tissues with smooth borders. Echinoid (Ed), a nectin ortholog, is an adherens junction protein in Drosophila, and cells mutant for ed sort out from the surrounding wild-type cells. However, it remains unknown which factors trigger cell sorting. Here, we dissect the sequence of this process and find that cell sorting occurs when differential expression of Ed triggers the assembly of actomyosin cable. Conversely, Ed-mediated cell sorting can be rescued by recruitment of Ed, via homophilic or heterophilic interactions, to the wild-type cell side of the clonal interface, even when differential Ed expression persists. We found, unexpectedly, that when actomyosin cable was largely absent, differential adhesion was sufficient to cause limited cell segregation but with a jagged tissue border (imperfect sorting). We propose that Ed-mediated cell sorting is driven both by differential Ed adhesion that induces cell segregation with a jagged border and by actomyosin cable assembly at the interface that smoothens this border.

Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye.

Planar cell polarity (PCP) refers to a second polarity axis orthogonal to the apicobasal axis in the plane of the epithelium. The molecular link between apicobasal polarity and PCP is largely unknown. During Drosophila eye development, differentiated photoreceptors form clusters that rotate independently of the surrounding interommatidial cells (ICs). Here, we demonstrate that both Echinoid (Ed), an adherens junction-associated cell adhesion molecule, and Flamingo (Fmi), a PCP determinant, are endocytosed via a clathrin-mediated pathway in ICs. Interestingly, we found that Ed binds the AP-2 adaptor and is required for the internalization of Fmi into ICs. Loss of ed led to increased amounts of Fmi on the cell membrane of non-rotating ICs and also to the misrotation of photoreceptor clusters. Importantly, overexpression of fmi in ICs alone was sufficient to cause misrotation of the adjacent photoreceptor clusters. Together, we propose that Ed, when internalized by AP-2, undergoes co-endocytosis with, and thereby decreases, Fmi levels on non-rotating ICs to permit correct rotation of ommatidial clusters. Thus, co-endocytosis of Ed and Fmi provides a link between apicobasal polarity and PCP.

Cell adhesion molecule Echinoid associates with unconventional myosin VI/Jaguar motor to regulate cell morphology during dorsal closure in Drosophila.

Echinoid (Ed) is a homophilic immunoglobulin domain-containing cell adhesion molecule (CAM) that localizes to adherens junctions (AJs) and cooperates with Drosophila melanogaster epithelial (DE)-cadherin to mediate cell adhesion. Here we show that Ed takes part in many processes of dorsal closure, a morphogenetic movement driven by coordinated cell shape changes and migration of epidermal cells to cover the underlying amnioserosa. Ed is differentially expressed, appearing in epidermis but not in amnioserosa cells. Ed functions independently from the JNK signaling pathway and is required to regulate cell morphology, and for assembly of actomyosin cable, filopodial protrusion and coordinated cell migration in dorsal-most epidermal cells. The effect of Ed on cell morphology requires the presence of the intracellular domain (Ed(intra)). Interestingly, Ed forms homodimers in vivo and Ed(intra) monomer directly associates with unconventional myosin VI/Jaguar (Jar) motor protein. We further show that ed genetically interacts with jar to control cell morphology. It has previously been shown that myosin VI is monomeric in vitro and that its dimeric form can associate with and travel processively along actin filaments. Thus, we propose that Ed mediates the dimerization of myosin VI/Jar in vivo which in turn regulates the reorganization and/or contraction of actin filaments to control changes in cell shape. Consistent with this, we found that ectopic ed expression in the amnioserosa induces myosin VI/Jar-dependent apical constriction of this tissue.

Weckle is a zinc finger adaptor of the toll pathway in dorsoventral patterning of the Drosophila embryo.

BACKGROUND: The Drosophila Toll pathway takes part in both establishment of the embryonic dorsoventral axis and induction of the innate immune response in adults. Upon activation by the cytokine Spätzle, Toll interacts with the adaptor proteins DmMyD88 and Tube and the kinase Pelle and triggers degradation of the inhibitor Cactus, thus allowing the nuclear translocation of the transcription factor Dorsal/Dif. weckle (wek) was previously identified as a new dorsal group gene that encodes a putative zinc finger transcription factor. However, its role in the Toll pathway was unknown. RESULTS: Here, we isolated new wek alleles and demonstrated that cactus is epistatic to wek, which in turn is epistatic to Toll. Consistent with this, Wek localizes to the plasma membrane of embryos, independently of Toll signaling. Wek homodimerizes and associates with Toll. Moreover, Wek binds to and localizes DmMyD88 to the plasma membrane. Thus, Wek acts as an adaptor to assemble/stabilize a Toll/Wek/DmMyD88/Tube complex. Remarkably, unlike the DmMyD88/tube/pelle/cactus gene cassette of the Toll pathway, wek plays a minimal role, if any, in the immune defense against Gram-positive bacteria and fungi. CONCLUSIONS: We conclude that Wek is an adaptor to link Toll and DmMyD88 and is required for efficient recruitment of DmMyD88 to Toll. Unexpectedly, wek is dispensable for innate immune response, thus revealing differences in the Toll-mediated activation of Dorsal in the embryo and Dif in the fat body of adult flies.

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion.

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. We show that, in the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and beta-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid- and Echinoid+ cells, like those between DE-Cadherin- and DE-Cadherin+ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion.

Echinoid synergizes with the Notch signaling pathway in Drosophila mesothorax bristle patterning.

echinoid (ed) encodes an immunoglobulin domain-containing cell adhesion molecule that negatively regulates the Egfr signaling pathway during Drosophila photoreceptor development. We show a novel function of Ed, i.e. the restriction of the number of notum bristles that arise from a proneural cluster. Thus, loss-of-function conditions for ed give rise to the development of extra macrochaetae near the extant ones and increase the density of microchaetae. Analysis of ed mosaics indicates that extra sensory organ precursors (SOPs) arise from proneural clusters of achaete-scute expression in a cell-autonomous way. ed embryos also exhibit a neurogenic phenotype. These phenotypes suggest a functional relation between ed and the Notch (N) pathway. Indeed, loss-of-function of ed reduces the expression of the N pathway effector E(spl)m8 in proneural clusters. Moreover, combinations of moderate loss-of-function conditions for ed and for different components of the N pathway show clear synergistic interactions manifested as strong neurogenic bristle phenotypes. We conclude that Ed is not essential for, but it facilitates, N signaling. It is known that the N and Egfr pathways act antagonistically in bristle development. Consistently, we find that Ed also antagonizes the bristle-promoting activity of the Egfr pathway, either by the enhancement of N signalling or, similar to the eye, by a more direct action on the Egfr pathway.

Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway.

echinoid (ed) encodes an cell-adhesion molecule (CAM) that contains immunoglobulin domains and regulates the EGFR signaling pathway during Drosophila eye development. Based on our previous genetic mosaic and epistatic analysis, we proposed that Ed, via homotypic interactions, activates a novel, as yet unknown pathway that antagonizes EGFR signaling. In this report, we demonstrate that Ed functions as a homophilic adhesion molecule and also engages in a heterophilic trans-interaction with Drosophila Neuroglian (Nrg), an L1-type CAM. Co-expression of ed and nrg in the eye exhibits a strong genetic synergy in inhibiting EGFR signaling. This synergistic effect requires the intracellular domain of Ed, but not that of Nrg. In addition, Ed and Nrg colocalize in the Drosophila eye and are efficiently co-immunoprecipitated. Together, our results suggest a model in which Nrg acts as a heterophilic ligand and activator of Ed, which in turn antagonizes EGFR signaling.