The Interactive Fly

Zygotically transcribed genes

Genes coding for junctional proteins

  • Neuromuscular junction
  • Development of junctions and distribution of proteins in junctions
  • Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis
  • The tumor suppressor CYLD controls epithelial morphogenesis and homeostasis by regulating mitotic spindle behavior and adherens junction assembly
  • Myosin II controls junction fluctuations to guide epithelial tissue ordering
  • Interface contractility between differently fated cells drives cell elimination and cyst formation
  • Wunen, a Drosophila lipid phosphate phosphatase, is required for junction-mediated barrier function
  • Pasiflora proteins are novel core components of the septate junction
  • A tetraspanin regulates septate junction formation in Drosophila midgut
  • Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila
  • Occluding junctions maintain stem cell niche homeostasis in the fly testes
  • Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila, muscle attachment sites and scolopale cells
  • A tendon cell specific RNAi screen reveals novel candidates essential for muscle tendon interaction
  • Diverse integrin adhesion stoichiometries caused by varied actomyosin activity
  • Adherens junction distribution mechanisms during cell-cell contact elongation in Drosophila
  • Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye
  • Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex
  • Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly
  • The triple-repeat protein Anakonda controls epithelial tricellular junction formation in Drosophila
  • Local and tissue-scale forces drive oriented junction growth during tissue extension
  • A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion
  • Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium
  • Wave propagation of junctional remodeling in collective cell movement of epithelial tissue: Numerical simulation study
    Genes coding for components of the subapical complex

    Genes coding for adherens junction proteins

    Genes coding for proteins of the basal junction of cleavage furrow

    Genes coding for septate junction proteins

    Integrin adhesion (focal adhesion) junctions

    Non-junctional proteins that indirectly effect junctions and cell polarity

    Development of junctions and distribution of proteins in junctions

    Analysis has been carried out on the pattern and development of cellular junctions in the different tissues of the Drosophila embryo from the blastoderm stage until hatching. The cellular junctions found include: gap junctions, two types of septate junctions, and several types of cell-cell and cell-substrate adherens junctions. During early and mid embryogenesis (stages 4 to 13) only spot adherens junctions, gap junctions, and zonulae adherentes (circumferential adherens junctions) prevail. Scattered spot adherens junctions are already formed at the blastoderm stage. During and shortly after gastrulation, spot adherens junctions become concentrated at the apical pole and fuse into continuous zonulae adherentes in the posterior endoderm and the ectoderm. In addition to the zonulae adherentes, ectodermally derived epithelia possess scattered gap junctions and form pleated septate junctions and hemiadherens junctions during late embryogenesis (stages 14 to 17). Hemiadherens junctions (HAJ) are junctions connecting muscles directly to epidermal cells and are characterized either by the presence of an intervening extracellular electron-dense material (connecting HAJ) or by the presence of an intervening tendon (tendon HAJ) (Tepass, 1994 and Prokop, 1998).

    Mesenchymal tissues (i.e., all nonepithelial tissues of the embryo, including the neural primordium and, transiently, the mesoderm and endoderm) possess both spot adherens junctions and gap junctions at a low frequency. Initially, the midgut epithelium does not establish a junctional complex and possesses only gap junctions and spot adherens junctions. Only late in development does a circumferential smooth septate junction develop; zonulae adherentes are missing. The various derivatives of the mesoderm express spot adherens junctions, hemiadherens junctions, and gap junctions, but never zonulae adherentes or septate junctions. After organogenesis, several different types of tissue-specific adherens junctions are formed, among them connecting hemiadherens junctions (between gut epithelium and visceral muscle and early during the formation of the muscle tendon junction); muscle tendon junctions (between somatic muscle and tendon cells); fasciae adherentes (between the cells of both the visceral muscle and the dorsal vessel), and autocellular nephrocyte junctions (in nephrocytes). Interesting exceptions to the general pattern of junctional development are provided by the outer epithelial layer of the proventriculus and the Malpighian tubules. Both tissues develop as typical ectodermal epithelia and possess zonulae adherentes. During late embryogenesis, both epithelia lose the zonulae adherentes and form smooth rather than pleated septate junctions, thereby expressing a junctional complex similar to that of the endodermally derived midgut epithelium (Tepass, 1994).

    The distribution of proteins in the apico-lateral cell junctions has been examined in Drosophila imaginal discs. Antibodies to phosphotyrosine (PY), Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin (which marks filamentous actin) labels the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin 3 (Fas3) and Coracle (Cor) label the more basal septate junction. The junctional proteins labeled by these antibodies undergo specific changes in distribution during the cell cycle. A loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to loss of the septate junctions and the formation of what appear to be ectopic adherens junctions. Based on staining with PY and Dlg antibodies, the apico-lateral junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat facets, discs overgrown, lethal (2) giant discs and warts. However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal giant larvae show abnormal distribution of the junctional markers, including a complete loss of apico-basal polarity in loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).

    The role of integrins was examined in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). At the ultrastructural level two types of muscle attachments can be distinguished: direct and indirect. At the direct muscle attachments, single muscles (such as the transverse muscles) attach to epidermal cells directly such that the hemiadherens junctions (HAJs) in opposing cells are separated by only 30-40nm, with a thin line of extracellular electron-dense material in between. These closed paired HAJs are referred to as connecting HAJs. Indirect muscle attachments occur at the segmental border, where the ends of multiple muscles attach at the same epidermal site, and contain extensive extracellular matrix consisting of fuzzy electron dense fibers, separated by up to several micrometers. This is referred to as tendon matrix because, like the vertebrate tendons, it is an extracellular matrix used to attach the muscles. Since HAJs at indirect muscle attachments are not closely paired but connected to the tendon matrix, they are referred to as tendon HAJs. Both types of muscle attachments have a common molecular basis: both contain PS integrins; both are sites were large secreted proteins Tiggrin and Masquerade accumulate; the intracellular appearance of connecting HAJs and tendon HAJs looks similar; connecting HAJs and tendon HAJs can appear together at the same site; they both appear to arise from short connecting HAJs; and both HAJs are separated from the extracellular electron dense matrix by a translucent gap of a few nanometers (Prokop, 1998).

    Muscle attachments and neuromuscular junctions were examined ultrastructurally in single or double mutant Drosophila embryos lacking PS1 integrin (alphaPS1betaPS), PS2 integrin (alphaPS2betaPS), and/or their potential extracellular ligand Laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions to extracellular matrix, but not for their intracellular link to the cytoskeleton. The intracellular electron-dense material of connecting HAJs and tendon HAJs connects to microfilaments in the muscles, and to microtubules in the epidermis. The epidermal microtubules are anchored at the other end to apical focal HAJs that connect to the cuticle (Prokop, 1998).

    The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscle and epidermal HAJs to electron dense extracellular matrix. PS2 integrin is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component Laminin A, while adhesion mediated by PS2 integrin does not (Prokop, 1998).

    At neuromuscular junctions (NMJs) the formation of functional synapses occurs normally in embryos lacking PS integrins and/or Laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly in embryos lacking laminin A. It is suggested that neuromuscular contact does not require laminin A directly at its point of contact, but requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of Laminin A. In contrast, loss of PS integrin function causes the boutons to make a more extensive contact with the muscle surface. Since no PS integrins are found at neuromuscular contacts it seems likely that the boutons can adhere to more muscle area because the muscle surfaces are more relaxed (allowing them to bend around the bouton) in the severely detached muscles of embryos lacking both PS integrins functions. Adhesion molecules expressed at Drosophila NMJs, like Fasciclin II, Fasciclin III or Connectin, are unlikely to mediate adhesion at the mature embryonic NMJ because they either fade during stage 16 or show no phenotype when mutated. Instead, mutant analysis reveals the existence of yet unknown embryonic adhesion factors downstream of mef2 regulation. Such factors might include laminin receptors that promote adhesion, or other receptors that displace the basement synaptic cell junction. Identification of mef2-dependent receptors might be aided by the use of lamA mutation as a sensitized background (Prokop, 1998).

    Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis

    Morphogenesis of epithelial tissues relies on the interplay between cell division, differentiation and regulated changes in cell shape, intercalation and sorting. These processes are often studied individually in relatively simple epithelia that lack the complexity found during organogenesis when these processes might all coexist simultaneously. To address this issue, this study makes use of the developing fly retinal neuroepithelium. Retinal morphogenesis relies on a coordinated sequence of interdependent morphogenetic events that includes apical cell constriction, localized alignment of groups of cells and ommatidia morphogenesis coupled to neurogenesis. Live imaging was used to document the sequence of adherens junction (AJ) remodelling events required to generate the fly ommatidium. In this context, it was demonstrated that the kinases Rok and Drak function redundantly during Myosin II-dependent cell constriction, subsequent multicellular alignment and AJ remodelling. In addition, it was shown that early multicellular patterning characterized by cell alignment is promoted by the conserved transcription factor Atonal (Ato). Further ommatidium patterning requires the epidermal growth factor receptor (EGFR) signalling pathway, which transcriptionally governs Rho-kinase (rok) and Death-associated protein kinase related (Drak)-dependent AJ remodelling while also promoting neurogenesis. In conclusion, this work reveals an important role for Drak in regulating AJ remodelling during retinal morphogenesis. It also sheds new light on the interplay between Ato, EGFR-dependent transcription and AJ remodelling in a system in which neurogenesis is coupled with cell shape changes and regulated steps of cell intercalation (Robertson, 2013).

    In Drosophila, Rok seems to be the main kinase responsible for phosphorylating the Myosin regulatory light chain (Sqh) during epithelial patterning and apical cell constriction. This is the case for the activation of MyoII during intercalation as germband extension proceeds, but also during various instances of compartment boundary formation and cell sorting situations in the embryo and in the wing imaginal disc. The current work reveals that in the constricting cells of the MF, Rok functions redundantly with Drak, a kinase recently shown to phosphorylate Sqh both in vitro and in vivo (Neubueser, 2010). It is noteworthy that previous work has shown that RhoGEF2 is not required for cell constriction in the MF, suggesting that perhaps another guanine exchange factor (GEF) might function redundantly with RhoGEF2 to promote cell constriction. These data on Drak reinforce the idea that redundancies exist in this context. Because the RhoA (Rho1 -- FlyBase) loss of function abolishes this cell response entirely, it would be expected that Drak function is regulated by RhoA. In addition, the current data indicate that Drak acts redundantly with Rok during MyoII-dependent multicellular alignment and AJ remodelling during ommatidia patterning. It will be interesting to test whether Drak functions in other instances of epithelial cell constriction or MyoII-dependent steps of AJ remodelling in other developmental contexts in Drosophila (Robertson, 2013).

    This study demonstrates a two-tiered mechanism regulating the planar polarization of MyoII and Baz. In the constricting cells in the posterior compartment, MyoII and Baz are segregated from one another and this is exacerbated by the wave of cell constriction in the MF. Upon Ato-dependent transcription in the MF cells, this segregated pattern of expression is harnessed and these factors become planar polarized at the posterior margin of the MF. This is independent of the core planar polarity pathway including the Fz receptor and is accompanied by a striking step of multicellular alignment. Previous work has demonstrated that Ato upregulates E-Cad transcription at the posterior boundary of the MF. In addition, apical constriction leads to an increase in E-Cad density at the ZA. The current data are therefore consistent with both hh-dependent constriction and ato-dependent transcriptional upregulation of E-Cad promoting differential adhesion, thus leading to a situation in which the ato+ cells maximize AJ contacts between themselves and minimize contact with the flanking cells that express much less E-Cad at their ZA. This typically leads to a preferential accumulation of cortical MyoII at the corresponding interface. Such actomyosin cables are correlated with increased interfacial tension, and it is proposed that this is in turn responsible for promoting cell alignment. Unfortunately, the very small diameter of these constricted cells precludes direct measurements of the AJ-associated tension using laser ablation experiments (Robertson, 2013).

    Supra-cellular cables of MyoII have been previously associated with cell alignment in various epithelia and have also been observed at the boundary of sorted clones, whereby cells align at a MyoII-enriched interface. Interestingly, this study found that the actomyosin cable defining the posterior boundary of the MF is also preferentially enriched for Rok, a component of the T1, MyoII-positive AJ in the ventral epidermis (Simoes Sde, 2010). This indicates an important commonality between actomyosin cable formation during cell sorting and the process of cell intercalation. However, unlike during intercalation, this study found that in the developing retina baz is largely dispensable for directing the pattern of E-Cad and actomyosin planar polarization. Further work will therefore be required to understand better the relationship between Baz and E-Cad at the ZA during ommatidia morphogenesis. It is speculated that the creation of a high E-Cad versus low E-Cad boundary in the wake of the MF might be sufficient to promote Rok and MyoII enrichment at the posterior AJs. This posterior Rok and MyoII enrichment might perhaps prevent E-Cad accumulation by promoting E-Cad endocytosis, as has been recently shown in the fly embryo (Robertson, 2013).

    This study has used live imaging to define a conserved step of ommatidia patterning that consists of the coalescence of the ommatidial cells' AJs into a central vertex to form a 6-cell rosette. The corresponding steps of AJ remodelling require Rok, Drak, Baz and MyoII, a situation compatible with mechanisms previously identified during cell intercalation in the developing fly embryo. The steps of AJ remodelling required to transform lines of cells into 5-cell pre-clusters are transcriptionally regulated downstream of EGFR in a ligand-dependent manner. Interestingly, in the eye EGFR signalling is activated in the cells that form lines and type1-arcs in the wake of the MF and, thus, are undergoing AJ remodelling. Previous work examining tracheal morphogenesis in the fly has demonstrated that interfaces between cells with low levels versus high levels of EGFR signalling correlate with MyoII-dependent AJ remodelling in the tracheal placode. This situation resembles that which is described in this study in the wake of the MF. In the eye, however, it was found that EGFR signalling is not required to initiate cell alignment. Nevertheless, taken together with work in the tracheal placode and previous studies related to multicellular patterning in the developing eye, this work indicates a conserved function for the EGFR signalling pathway in promoting MyoII-dependent AJ remodelling. This leaves open several interesting questions; for example, it is not presently clear how EGFR signalling can promote discrete AJ suppression and elongation. It is, however, tempting to speculate that previously described links between EGFR signalling and the expression of E-Cad or Rho1 might play a role during this process (Robertson, 2013).

    The tumor suppressor CYLD controls epithelial morphogenesis and homeostasis by regulating mitotic spindle behavior and adherens junction assembly

    The molecular mechanisms that contribute to the morphogenesis and homeostasis of the epithelium remain elusive. This study reports a novel role for the cylindromatosis (CYLD) tumor suppressor in these events. The results show that CYLD depletion disrupts epithelial organization in both Drosophila egg chambers and mouse skin and intestinal epithelia. Microscopic analysis of proliferating cells in mouse epithelial tissues and cultured organoids reveals that loss of CYLD synergizes with tumor-promoting agents to cause the misorientation of the mitotic spindle. Mechanistic studies show that CYLD accumulates at the cell cortex in epithelial tissues and cultured cells, where it promotes the formation of epithelial adherens junctions through the modulation of microtubule dynamics. These data suggest that CYLD controls epithelial morphogenesis and homeostasis by modulating the assembly of adherens junctions and ensuring proper orientation of the mitotic spindle. These findings thus provide novel insight into the role of CYLD in development, tissue homeostasis, and tumorigenesis (Xie, 2017).

    Myosin II controls junction fluctuations to guide epithelial tissue ordering

    Under conditions of homeostasis, dynamic changes in the length of individual adherens junctions (AJs) provide epithelia with the fluidity required to maintain tissue integrity in the face of intrinsic and extrinsic forces. While the contribution of AJ remodeling to developmental morphogenesis has been intensively studied, less is known about AJ dynamics in other circumstances. AJ dynamics were studied in an epithelium that undergoes a gradual increase in packing order, without concomitant large-scale changes in tissue size or shape. Neighbor exchange events were found to be driven by stochastic fluctuations in junction length, regulated in part by junctional actomyosin. In this context, the developmental increase of isotropic junctional actomyosin reduces the rate of neighbor exchange, contributing to tissue order. A model is proposed in which the local variance in tension between junctions determines whether actomyosin-based forces will inhibit or drive the topological transitions that either refine or deform a tissue (Curran, 2017).

    Interface contractility between differently fated cells drives cell elimination and cyst formation

    This study finds that ectopic expression of transcription factors that specify cell fates causes abnormal epithelial cysts in Drosophila imaginal discs. Cysts do not form cell autonomously but result from the juxtaposition of two cell populations with divergent fates. Juxtaposition of wild-type and aberrantly specified cells induces enrichment of actomyosin at their entire shared interface, both at adherens junctions as well as along basolateral interfaces. Experimental validation of 3D vertex model simulations demonstrates that enhanced interface contractility is sufficient to explain many morphogenetic behaviors, which depend on cell cluster size. These range from cyst formation by intermediate-sized clusters to segregation of large cell populations by formation of smooth boundaries or apical constriction in small groups of cells. In addition, single cells experiencing lateral interface contractility are eliminated from tissues by apoptosis. Cysts, which disrupt epithelial continuity, form when elimination of single, aberrantly specified cells fails and cells proliferate to intermediate cell cluster sizes. Thus, increased interface contractility functions as error correction mechanism eliminating single aberrant cells from tissues, but failure leads to the formation of large, potentially disease-promoting cysts. These results provide a novel perspective on morphogenetic mechanisms, which arise from cell-fate heterogeneities within tissues and maintain or disrupt epithelial homeostasis (Bielmeier, 2016).

    Wunen, a Drosophila lipid phosphate phosphatase, is required for septate junction-mediated barrier function

    Lipid phosphate phosphatases (LPPs) are integral membrane enzymes that regulate the levels of bioactive lipids such as sphingosine 1-phosphate and lysophosphatidic acid. The Drosophila LPPs Wunen (Wun) and Wunen-2 (Wun2) have a well-established role in regulating the survival and migration of germ cells. This study now shows that wun has an essential tissue-autonomous role in development of the trachea: the catalytic activity of Wun is required to maintain septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate crucial luminal components, suggesting a role for phospholipids in SJ function. The integrity of the blood-brain barrier is also lost in wun mutants, indicating that loss of SJ function is not restricted to the tracheal system. Furthermore, by comparing the rescue ability of different LPP homologs it was shown that wun function in the trachea is distinct from its role in germ cell migration (Ile, 2012).

    This study demonstrates a role for an LPP in development of the trachea. In the absence of wun function, the trachea suffers from breaks in the dorsal trunk (DT), non-uniform lumen diameter, and loss of luminal components resulting from ineffective paracellular barrier function. wun functions tissue autonomously and Wun activity can be replaced by that of the close paralog Wun2 and two mouse homologs, but not by a catalytically dead LPP (Ile, 2012).

    Defects are only seen in the trachea when wun is removed both maternally and zygotically and this is likely to explain why wun has not been previously uncovered in screens performed to identify genes required for tracheal development. The genetic data suggest that maternally provided Wun protein or the product of the reaction it catalyses lasts at least until the start of tracheal system formation and that zygotically expressed Wun in tracheal cells is sufficient to provide this activity. (Ile, 2012).

    In germ cells the Wunens function redundantly: the germ cell death caused by loss of both proteins from germ cells can be rescued by expression of either protein alone, indicating that the two proteins have overlapping substrate specificities. In the trachea the situation is similar in that overexpression of wun2 in the trachea is able to substitute for loss of wun. To determine whether the roles of Wunens in germ cell migration and tracheal development are identical two mammalian LPPs with different activities were used. mLPP3 is able to substitute for Wunens in germ cell migration and survival assays, whereas mLPP2 is not, in spite of both proteins being highly expressed and localizing to the cell surface. As expected, it was found that mLPP3-GFP is able to rescue the tracheal phenotypes of wun wun2 M-Z− embryos; however, mLPP2-GFP is also able to do so. Thus, mLPP2 lacks an activity required for germ cell migration but possesses activity sufficient for tracheal development. It is concluded that the crucial LPP substrate or substrates for germ cell and tracheal development are different. Wunens and mLPP3 show relatively little substrate specificity and can dephosphorylate the lipid essential for both germ cell and tracheal development. mLPP2, by contrast, shows more restrictive specificity and can only dephosphorylate the lipid that is crucial for tracheal development. (Ile, 2012).

    The localization of Wun-GFP to particular regions of the tracheal cell plasma membrane is intriguing. Mammalian LPPs have been demonstrated to localize to specific plasma membrane domains. Human (h) LPP1 (PPAP2A -- Human Genome Nomenclature Committee) sorts apically, whereas hLPP3 colocalizes with E-cadherin in Madin-Darby canine kidney (MDCK) cells and the C-terminal domain of hLPP3 has been shown to bind the AJ protein p120 catenin (catenin δ1). However, it remains to be confirmed whether specific Wun-GFP localization is crucial for activity. Wun2-myc can also rescue and, although the protein shows no specific localization, there might be sufficient present at SJs or apical membranes to fulfill the requirement for LPP activity. What is clear is that the LPPs, just as in germ cell migration, are playing more than a structural role because Wun2-H326K showed no rescue ability (Ile, 2012).

    How does loss of Wun affect the tracheal epithelial cells? Overall, the polarity of these cells is unaffected, but AJ proteins are weaker at the apical surface. In this respect the tracheal phenotype is reminiscent of weak alleles of shotgun, which encodes Drosophila E-cadherin. Such mutants exhibit incomplete fusion of the DT and uneven luminal diameter. However, no genetic interaction between wun wun2 and shotgun is seen, suggesting that reduced DE-cadherin is not the critical factor in causing the tracheal defects in wun wun2 mutants. (Ile, 2012).

    SJs were also affected in the wun wun2 M-Z− mutants: SJ components were not confined to the subapical region, as in wild type, and paracellular barrier function was lost. However, mutants for all essential SJ components reported to date display an abnormally elongated and convoluted DT. This phenotype is not seen in wun wun2 M-Z− mutants, indicating that although barrier activity may be lost, SJs must still be present and indeed they are seen by EM. This situation is similar to that of yrt M-Z− embryos, which also show compromised paracellular barrier function despite a normal complement of septa when examined ultrastructurally (Ile, 2012).

    The lack of luminal accumulation of Serp and Verm in the wun wun2 M-Z animals is striking. Embryos mutant for serp and verm also display trachea with an abnormally elongated and convoluted DT. As this is not seen in wun wun2 M-Z animals, it is suspected that the loss of luminal Serp and Verm is not absolute. Indeed, extremely weak luminal staining id occasionally seen, but mostly Serp and Verm are detected in the hemolymph of late embryos. Although the possibility cannot be excluded that Serp and Verm are incorrectly secreted at the basolateral surface, the possibility is favored that Serp and Verm are apically secreted but owing to defects in the SJ-mediated paracellular barrier they diffuse from the lumen into the hemolymph. (Ile, 2012).

    The differential accumulation of Serp and Verm versus the 2A12 antigen in the tracheal lumen in various mutant backgrounds has been interpreted to suggest that multiple secretory pathways exist. The first is actin dependent and is based on the observation that in dia mutants the 2A12 antigen is not present in the tracheal lumen whereas Verm is. The second is SJ dependent and is based on the fact that in mutants for the α subunit of the Na/K-ATPase, the 2A12 antigen accumulates in the lumen but Verm, which at stage 15 can been seen both in the lumen and in the tracheal cells, is undetectable in the lumen and tracheal cells by stage 16. Similar results have been obtained with mutants for other SJ components, including those encoded by sinuous, Lachesin, varicose, coracle and kune-kune. Based on these data, it is likely that the failure in Serp and Verm accumulation results, at least in part, from their diffusion out of the trachea. The difference in behavior between Serp, Verm and ANF-GFP versus the 2A12 antigen might depend more on the strength of their interaction with luminal components than on differences in their secretion. (Ile, 2012).

    A model is proposed in which Wun expression at the cell surface leads to changes in intracellular lipid levels, which affects both AJs and SJs. These changes result in paracellular barrier defects and prevent particular luminal components from accumulating. Although the identification of which lipid or lipids are being affected and how changes in their levels and/or localization result in defects in specific tissues is ongoing, one potential Wun substrate, S1P, is known to increase barrier function in HUVEC cells via an S1P1-dependent pathway (Ile, 2012).

    What is particularly striking is that the role of Wun in barrier function for the trachea and ventral nerve cord in Drosophila appears to be representative of a more conserved aspect of LPP function. mLPP3 has an essential embryonic role in establishing vascular endothelial cell interactions during early development. In addition, mice with postnatal inactivation of Lpp3 specifically in the vascular endothelium are viable but have impaired vascular endothelial barrier function leading to vascular leakage, particularly in the lungs . Thus, it appears that mLPP3 is required in both the establishment and maintenance of vascular integrity in a tissue-autonomous fashion (Ile, 2012).

    Recent studies have demonstrated crucial roles for lipids in establishing or maintaining epithelial cell plasma membrane identity. For example, phosphoinositides are central to establishing the apical surface during lumen formation in MDCK cells and glycosphingolipids are needed to maintain apicobasal domain identity in C. elegans intestinal cells. Modulation of lipid levels coupled with cell biological analyses in a developmental context will be invaluable in exploring this fascinating field further (Ile, 2012).

    Pasiflora proteins are novel core components of the septate junction

    Epithelial sheets play essential roles as selective barriers insulating the body from the environment and establishing distinct chemical compartments within it. In invertebrate epithelia, septate junctions (SJs) consist of large multi-protein complexes that localize at the apicolateral membrane and mediate barrier function. This study reports the identification of two novel SJ components, Pasiflora1 (CG7713) and Pasiflora2 (CG8121), through a genome-wide glial RNAi screen in Drosophila. Pasiflora mutants show permeable blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins. Consistent with the observed phenotypes, the genes are co-expressed in embryonic epithelia and glia and are required cell-autonomously to exert their function. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetraspan membrane proteins conserved across the protostome-deuterostome divide. Both proteins localize at SJs and their apicolateral membrane accumulation depends on other complex components. In fluorescence recovery after photobleaching experiments, pasiflora proteins were found to be core SJ components as they are required for complex formation and exhibit restricted mobility within the membrane of wild-type epithelial cells, but rapid diffusion in cells with disrupted SJs. Taken together, these results show that Pasiflora1 and Pasiflora2 are novel integral components of the SJ and implicate a new family of tetraspan proteins in the function of these ancient and crucial cell junctions (Deligiannaki, 2015).

    The generation of distinct chemical milieus within the body is essential for metazoan development. This compartmentalization is accomplished by epithelia that impede paracellular diffusion and selectively transport substances via membrane channels and transporters. To provide a barrier, epithelia have a narrow intercellular space, which is sealed by specialized junctions, including tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates. SJs are the ancestral sealing junctions and are found in all invertebrates from sponges to arthropods but are also present in vertebrates. In electron micrographs, SJs appear as an array of regularly spaced septa, which operate by extending the travel distance for solutes through the paracellular route. SJs are found in both primary epithelia, such as epidermis, trachea and hindgut, and secondary epithelia, which develop through mesenchymal-epithelial transition, such as the blood-brain barrier (BBB) and midgut. The BBB ensheaths the nervous system and is required to maintain its homeostasis. Owing to the high potassium content of the hemolymph, animals with a defective BBB die of paralysis. In Drosophila, the BBB is a squamous epithelium established late in embryogenesis by SJ-forming subperineurial glia (SPG). In addition to providing a paracellular barrier, SJs also serve as a fence for the diffusion of proteins across the lateral membrane. Molecularly and functionally homologous SJs are found in vertebrates at the node of Ranvier, where they form the paranodal junction between axons and myelinating glia (Deligiannaki, 2015).

    The SJ consists of a large multi-protein complex. In Drosophila, more than 20 proteins have been characterized that when missing lead to disruption of SJs and loss of barrier integrity. Most of these are transmembrane (TM) and lipid-anchored proteins that localize at the SJ, such as the claudins Sinuous (Sinu), Megatrachea (Mega) and Kune-kune (Kune), the cell adhesion molecules Neurexin IV (Nrx-IV), Contactin (Cont), Neuroglian (Nrg), Lachesin (Lac) and Fasciclin III, the sodium pump with its two subunits ATP&alpha and Nervana 2 (Nrv2), Melanotransferrin (Transferrin 2) and Macroglobulin complement-related (Mcr). The complex also includes the intracellular scaffold proteins Coracle (Cora) and Varicose (Vari) that interact with the cytoplasmic tails of membrane proteins and connect them to the actin cytoskeleton. A hallmark of SJ proteins is that they are interdependent for localization, and removal of one component is sufficient to destabilize the whole complex. In addition, half of the known SJ proteins can be co-immunoprecipitated from tissue extracts and detected by mass spectrometry (MS), further suggesting that they function together in a multi-protein complex. Fluorescence recovery after photobleaching (FRAP) experiments have been instrumental in classifying most SJ proteins as core components based on their limited mobility after photobleaching and the observation that upon loss of function other SJ proteins diffuse rapidly into the bleached region due to impaired complex formation (Deligiannaki, 2015).

    Accompanying epithelial morphogenesis, SJs are remodeled into mature junctions. At embryonic stage 12, SJ proteins accumulate along the lateral membrane of columnar epithelial cells. Subsequently, they gradually localize at more apical compartments and by stage 15 are restricted to the apicolateral membrane, basal to adherens junctions. The Ly-6 proteins Crooked (Crok), Crimpled (Crim) and Coiled (Cold) are required for SJ formation; however, they do not reside at SJs and instead localize to cytoplasmic puncta. In Ly-6 mutants, the FRAP kinetics of SJ proteins mirrors that of core complex mutants and therefore Ly-6 proteins are thought to be involved in the assembly of SJ (sub)complexes in an intracellular compartment. The subsequent relocalization of SJs requires endocytosis from the basolateral membrane and recycling to the apicolateral compartment. Gliotactin (Gli) and Discs-large (Dlg) localize at SJs but, in contrast to core components and Ly-6 proteins, upon their loss of function the complex is properly formed and SJ proteins, although mislocalized, retain their restricted mobility. Together with a lack of physical interactions with SJ components, this result suggests that Gli and Dlg are required for complex localization rather than its assembly (Deligiannaki, 2015).

    In contrast to SJs, TJs localize apically of the zonula adherens and in electron microscopy appear as a series of fusions of adjacent membranes. Although the set of proteins that composes the TJ is different from that of the SJ, the two complexes share a key molecular component, the claudins. Claudins are a tetraspan membrane family of 20-34kDa proteins with intracellular N- and C-termini and constitute a main component of TJs. The larger first extracellular loop contains a claudin family signature motif and bears critical residues that define TJ charge and size selectivity in a tissue-specific manner. Claudins are part of a large protein clan, comprising the PMP22/EMP/MP20/Claudin, MARVEL, tetraspanin, connexin and innexin families, which share the same overall topology but differ in size and motif composition of extracellular and intracellular domains. Many members of this clan can form homo- and heterotypic oligomers on the same and neighboring membranes and play essential roles in junctional complexes, including TJs, gap junctions and the casparian strip of plants, as well as in membrane traffic and fusion events. Claudins have been shown to interact with other tetraspan proteins such as occludins, tetraspanins and MARVEL, as well as cell adhesion proteins and receptors. Similarly, tetraspanins form microdomains in the plasma membrane, in which cell adhesion proteins, TM receptors and their signaling components are enriched and, thereby, are thought to be modulated in their activity (Deligiannaki, 2015).

    This study identifies and characterizes two new core components of the SJ, Pasiflora1 and Pasiflora2, which are part of a novel tetraspan protein family that is conserved across the prostostome-deuterostome divide and is characterized by specific sequence features. Both proteins localize at SJs, show interdependence for localization and restricted mobility with known SJ members and are required for the integrity of epithelial barriers. This work provides new insight into the composition of the SJ and implicates a second family of tetraspan proteins in the development of these crucial cell junctions (Deligiannaki, 2015).

    This study has identified two previously uncharacterized proteins, Pasiflora1 and Pasiflora2, as novel components of the Drosophila SJ. Several lines of evidence support this notion. First, pasiflora1 and pasiflora2 mutants exhibit all the characteristic phenotypes associated with disrupted SJs: breakdown of blood-brain and tracheal barriers, overelongated dorsal trunks, and SJ mislocalization in a variety of tissues. In the BBB, SJs appear severely disorganized and in columnar epithelia SJ proteins fail to localize at the apicolateral membrane and instead spread basolaterally. Second, the genes are co-expressed in embryonic epithelia that rely on SJs for their function and the proteins overlap with Cora at the apicolateral membrane. Similar to known SJ proteins, pasiflora localization depends on other complex members, as they spread basolaterally in SJ mutant backgrounds. Finally, using FRAP it was demonstrated that pasiflora proteins are core SJ components. In stage 15 epidermal cells, Nrg-GFP displays limited lateral mobility after photobleaching owing to its incorporation in the large multi-protein complex. By contrast, in pasiflora mutants, Nrg-GFP diffuses rapidly, indicating that SJ complex formation is compromised. Overexpressed pasiflora proteins also move slowly within the membrane of wt cells, but diffuse rapidly in cells with disrupted SJs, showing that they are themselves associated with the SJ complex (Deligiannaki, 2015).

    An emerging idea is that not all SJ proteins are as interdependent as previously thought and that distinct subcomplexes exist within the large, highly ordered, multi-protein complex. The current observations and those of others indicating that in SJ mutants the localization of other complex members is differentially affected and that the fluorescence of GFP-tagged SJ proteins does not fully recover after photobleaching support this notion (Deligiannaki, 2015).

    Pasiflora proteins are conserved in arthropods and beyond and share the global topological features of the tetraspan superfamily, with short conserved sequence motifs. The ability of different tetraspan families to form ribbons based on homo- and heterotypic interactions in cis within the plasma membrane suggests that pasifloras, together with claudins, are involved in forming the highly regularly spaced septa of the SJ. Freeze-fracture experiments have shown that SJs form ribbons, with an apparent size of a single septum of 10 nm and a regular spacing of 15-20 nm. Depending on the tissue, these ribbons are either highly aligned with each other (mature ectoderm) or meandering (developing wing disc). In the SJ, the plasma membranes of neighboring cells are not fused but closely juxtaposed at a distance of 15 nm and there is no evidence in invertebrates that different tissues have distinct paracellular permeability. Claudins and pasifloras are therefore unlikely to create pores in trans with specific size and charge selectivity. This suggests that the small claudins and pasifloras act only in cis to form ribbons, while the single-pass membrane proteins of the complex mediate the trans interaction with the neighboring cell via their large extracellular adhesive domains. To date, the structural basis for the intermolecular interaction between tetraspan proteins has not been resolved. The pasiflora proteins belong to a larger family with nine members in Drosophila. Pasiflora1 and Pasiflora2 are expressed in embryonic epithelia and glia and act non-redundantly during SJ formation. Little is known about the other family members: Fire exit is expressed in exit and peripheral glia, which also form SJs; CG15098 is expressed in the midgut, which forms structurally different, smooth SJs (Deligiannaki, 2015).

    This study reveals that the composition of the SJ complex strongly resembles that of other junctional and TM protein complexes, where adhesive or signaling receptors are embedded in a complex environment of hydrophobic tetraspan proteins of different types, in this case three different claudins and two different members of the novel pasiflora family. Membrane complexes such as the SJ are particularly refractory to biochemical and structural analysis owing to their hydrophobicity and large size. However, due to their crucial function in all invertebrates and the vertebrate paranode, it is possible, by genetic means, to identify and study the structural core components as well as the biogenesis of the complex. Given the medical importance of the paranodal SJ in particular and of tetraspan proteins in general, this discovery of pasiflora proteins opens the possibility of studying these proteins and their interactions in a highly accessible and sensitive paradigm (Deligiannaki, 2015).

    A tetraspanin regulates septate junction formation in Drosophila midgut

    Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes via the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of SJs are observed: pleated SJs (pSJs) and smooth SJs (sSJs), which are present in ectodermally- and endodermally-derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Snakeskin (Ssk), in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh, identified a tetraspanin family protein, Tetraspanin 2A (Tsp2A), as a novel protein involved in sSJ formation in Drosophila. Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised (Tsp2A) expression caused by RNAi or the CRISPR/Cas9 system is associated with defects in the ultrastructure of sSJs, changes localization of other sSJ proteins, and impairs barrier function of the midgut. In most Tsp2A-mutant cells, Mesh fails to localize to sSJs and is distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

    Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, specialized intercellular junctions, designated as occluding junctions, regulate the free diffusion of solutes through the paracellular pathway. In vertebrates, tight junctions act as occluding junctions, whereas, in invertebrates, septate junctions (SJs) are the functional counterparts of tight junctions. SJs form circumferential belts around the apicolateral regions of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. SJs are subdivided into several morphological types that differ among different animal phyla, and several phyla possess multiple types of SJs that vary among different types of epithelia (Izumi, 2016).

    In arthropods, two types of SJs exist: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectodermally-derived epithelia and surface glia surrounding the nerve cord, while sSJs are found mainly in endodermally-derived epithelia, such as the midgut and the gastric caeca. The outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs, although these epithelia are ectodermal derivatives. The criteria distinguishing these two types of SJs are the arrangement of the septa. In oblique sections of lanthanum-treated preparations, the septa of pSJs are visualized as regular undulating rows but those in sSJs are observed as regularly spaced parallel lines. In freeze-fracture images, the rows of intramembrane particles in pSJs are separated from one another, whereas those in sSJs are fused into ridges. To date, more than 20 pSJ-related proteins, including pSJ components and regulatory proteins involved in pSJ assembly, have been identified and characterized in Drosophila. In contrast, few genetic and molecular analyses have been carried out on sSJs. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, have been identified and characterized. Ssk consists of 162 amino acids and has four membrane-spanning domains, two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop. Mesh has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain, and a sushi domain. Mesh transcripts are predicted to be translated into three isoforms of which the longest isoform consists of 1,454 amino acids. In Western blot studies, Mesh is detected as a main ~90 kDa band and a minor ~200 kDa band. Compromised expression of ssk or mesh causes defects in the ultrastructure of sSJs and in the barrier function of the midgut against a 10-kDa fluorescent tracer. Ssk and Mesh physically interact with each other and are mutually dependent for their sSJ localization. Thus, Mesh and Ssk play crucial roles in the formation and barrier function of sSJs (Izumi, 2016).

    Tetraspanins are a family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. Among several protein families with four transmembrane domains, tetraspanins are characterized especially by the structure of the second extracellular loop. It contains a highly conserved cysteine-cysteine-glycine (CCG) motif and 2 to 4 other cysteine residues. These cysteines form 2 or 3 disulfide bonds within the loop. Tetraspanins are believed to play a role in membrane compartmentalization and are involved in many biological processes, including cell migration, cell fusion and lymphocyte activation, as well as viral and parasitic infections. Several tetraspanins regulate cell-cell adhesion but none are known to be involved in the formation of epithelial occluding junctions. In the Drosophila genome, there are 37 tetraspanin family members, and some have been characterized by genetic analyses. Lbm, CG10106 and CG12143 participate in synapse formation. Sun associates with light-dependent retinal degeneration. TspanC8 subfamily members, including Tsp3A, Tsp86D and Tsp26D, are involved in the Notch-dependent developmental processes via the regulation of a transmembrane metalloprotease, ADAM10. However, the functions of most other Drosophila tetraspanins remain obscure (Izumi, 2016).

    This study identified a tetraspanin family protein, Tsp2A, as a novel molecular component of sSJs in Drosophila. Tsp2A is required for sSJ formation and for the barrier function of Drosophila midgut. Tsp2A and two other sSJ-specific membrane proteins Mesh and Ssk show mutually dependent localizations at sSJs and form a complex with each other. Therefore, it is concluded that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

    Of the sSJ-specific components, Mesh is a membrane-spanning protein and has an ability to induce cell-cell adhesion, implying that it is a cell adhesion molecule and may be one of the components of the electron-dense ladder-like structures in sSJs. In contrast, both Ssk and Tsp2A are unlikely to act as cell adhesion molecules in sSJs because each of the two extracellular loops of Ssk (25 and 22 amino acids, respectively) appear to be too short to bridge the 15-20-nm intercellular space of sSJs. Furthermore, overexpression of EGFP-Tsp2A in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2016).

    Several observations in Tsp2A-mutants may provide clues for understanding the role of Tsp2A in sSJ formation. In most Tsp2A-mutant midgut epithelial cells, Mesh fails to localize to the apicolateral membranes but was distributed in the cytoplasm, possibly to specific intracellular membrane compartments. To further examine where Mesh was localized in Tsp2A-mutant cells, the midgut was doublestained with the anti-Mesh antibody and the antibodies against typical markers of various intracellular membrane compartments, including the Golgi apparatus (anti-GM130), early endosomes (anti-Rab5), recycling endosomes (anti-Rab11) and lysosomes (anti-LAMP1). However, it was not possible to detect any overlap between staining by these markers and that of Mesh. The staining pattern in Tsp2A-mutant midgut epithelial cells produced with the anti-KDEL antibody, which labels endoplasmic reticulum, was similar, although not identical with that produced by the anti-Mesh antibody (Izumi, 2016).

    Interestingly, some tetraspanins are known to control the intracellular trafficking of their partners. For instance, a mammalian tetraspanin, CD81 is necessary for normal trafficking or for surface membrane stability of a phosphoglycoprotein, CD19, in lymphoid B cells. The TspanC8 subgroup proteins, which all possess eight cysteine residues in their large extracellular domain, regulate the exit of a metalloproteinase, ADAM10, from the ER and differentially control its targeting to either late endosomes or to the plasma membrane. Consequently, TspanC8 proteins regulate Notch signaling via the activation of ADAM10 in mammals, Drosophila and Caenorhabditis elegans. If Mesh is retained in the trafficking pathway from endoplasmic reticulum to plasma membrane in Tsp2A-mutant cells, Tsp2A may have an ability to promote the intracellular trafficking of Mesh in the secretory pathway. To clarify the role of Tsp2A in sSJ formation, it will be necessary to determine the intracellular membrane compartment where Mesh was localized in Tsp2A-mutant cells (Izumi, 2016).

    Tsp2A, Mesh and Ssk are mutually dependent for their localization at sSJs. Consistent with this intimate relationship, the co-immunoprecipitation experiment revealed that Tsp2A physically interacts with Mesh and Ssk in vivo. However, the amount of Ssk observed in the co-immunoprecipitation with EGFP-Tsp2A was barely enriched relative to that in the extracts of embryos expressing EGFP-Tsp2A. This was particularly striking in comparison to the degree of enrichment of Mesh in the co-immunoprecipitation with EGFP-Tsp2A. To interpret these results, the detailed manner of the interaction between Tsp2A, Mesh and Ssk proteins needs to be further clarified. Many tetraspanin family proteins are known to interact with one another and with other integral membrane proteins to form a dynamic network of proteins in cellular membranes. Tetraspanins are also believed to have a role in membrane compartmentalization. Given such functional properties of tetraspanins, Tsp2A may determine the localization of sSJs at the apicolateral membrane region by membrane domain formation (Izumi, 2016).

    In the Tsp2A-mutant midgut epithelial cells, Lgl was distributed throughout the basolateral membrane region, whereas it was localized in the apicolateral membrane region in the wild-type. In view of the role of Lgl in the formation of the apical-basal polarity of ectodermally-derived epithelial cells, it is of interest to consider whether this abnormal localization of Lgl in the Tsp2A-mutant affects epithelial polarity. However, in the Tsp2A-mutant midgut epithelial cells, Dlg still showed polarized concentration into the apicolateral membrane region and the Lgl never leaked into the apical membrane domain. These observations suggest that the lack of Tsp2A does not affect the gross apical-basal polarity of the midgut epithelial cells (Izumi, 2016).

    Some tetraspanins have been reported to be involved in the regulation of cell-cell adhesion. A mammalian tetraspanin, CD151, regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin reorganization, or through complex formation with α3γ1 integrin. A mammalian tetraspanin, CD9, is concentrated in the axoglial paranodal region in the brain and in the peripheral nervous system, and CD9 knockout mice display defects in the formation of paranodal septate junctions and in the localization of paranodal proteins. Paranodal septate junctions have electron-dense ladder-like structures and their molecular organization is similar to that of pSJs but tetraspanins involved in pSJ formation have not been reported in Drosophila (Izumi, 2016).

    Interactions between several tetraspanins and claudins, the key integral membrane proteins involved in the organization and function of tight junctions, are also known. Claudin-11 forms a complex with OAP-1/Tspan-3 and chemical crosslinking reveals a direct association between claudin-1 and CD9. Furthermore, the interaction between claudin-1 and CD81 is shown to be required for hepatitis C virus infectivity. To date, no tight junction defect has been reported in CD9 knockout mice, CD81 knockout mice, or CD9/CD81 double knockout mice. Further investigation is necessary to clarify whether the interactions between tetraspanins and tight junction proteins are involved in the formation and function of tight junctions (Izumi, 2016).

    Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila

    During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). This study examined the major stages and underlying mechanisms of MET during blood-brain barrier (BBB) formation in Drosophila. Contact with the basal lamina was shown to be essential for the growth of the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the paracellular space, are not required for MET, but are necessary for the establishment of polarized SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signalling pathway regulates SPG cell growth and shape, with different levels of signalling causing distinct phenotypes. Timely, well-coordinated SPG growth is essential for the uniform insertion of SJs and thus the insulating function of the barrier. This is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium and of the key role trimeric G protein signalling plays in this important morphogenetic process (Schwabe, 2017).

    This study of Drosophila BBB development represents the first dynamic in vivo study of MET and secondary epithelium formation. The data shed particular light on the roles of the basal lamina and of the insulating SJs, as well as on the function of GPCR signaling in this important morphogenetic process. Once SPG reach the CNS surface, contact with the basal lamina is essential for the extensive growth of the SPG during epithelium formation. Previous in vitro studies have shown that adhesion to basal lamina components is necessary for cell spreading and proliferation, however this study is the first to demonstrate in vivo that attachment to the basal lamina is essential for non-proliferative cell growth and ensheathment. Attachment to the ECM occurs primarily through focal adhesions and integrins, which in turn can activate MAPK signaling, triggering cell proliferation and growth. In addition, adhesion to the ECM has been shown to provide traction, which facilitates cell spreading. Contact to the ECM may thus provide the SPG with both growth signals and attachment sites. Being highly expressed on the basal lamina facing side of SPG, Dystroglycan (Dg) is an excellent candidate for mediating ECM attachment. However, zygotic mutants of Dg show no BBB defects and germline clones could not be analyzed due to Dg's role in oogenesis (Schwabe, 2017).

    Beyond supporting SPG growth, contact with the basal lamina likely provides an important cue for polarizing the cells, as judged by their strong enrichment of Dg at the basal lamina facing (basal) membrane compartment. Previous studies have shown that Dg and its ligand Pcan/Trol are required for the establishment of polarity in follicle cells. However, when the basal lamina and thus its ligand Pcan are depleted, Dg is still expressed and polarized in the SPG, suggesting that glial polarity can be supported by the residual basal lamina or that additional polarizing signals exist (Schwabe, 2017).

    Once SJs have formed, the GPCR Moody and the Mdr65 transporter are asymmetrically distributed within the SPG, further demonstrating that these cells possess distinct apical and basal membrane compartments. This polarized distribution is coincident with and dependent on the presence of SJs, demonstrating for the first time that SJs serve a function in cell polarity. By acting as a fence and preventing diffusion of membrane proteins across the lateral compartment, the SJs maintain asymmetric protein distributions, which could result from polarized exocytosis or endocytosis. Intriguingly, a separate study has identified PKA as a crucial antagonistic effector of Moody signaling. PKA has been shown to regulate polarized exocytosis at the trans-Golgi network in different types of epithelia. Apical-basal polarity plays an important morphogenetic role in the continued growth of the SPG epithelium during larval stages and in the function of the BBB (Schwabe, 2017).

    Signaling by the GPCR Moody plays a critical role both in regulating the growth of individual SPG and in synchronizing this process across the entire SPG cell population. In Moody pathway mutants, glial growth behavior is more erratic, and more variable between cells. This increased variability of glial cell shape, size, and growth causes a significant delay of epithelial closure of up to 1.5 hrs. This delay is not caused by an earlier delay in glial migration or by a delay in SJ formation (Schwabe, 2017).

    The detailed dynamic analysis reveals that, in moody and loco mutants, the spatio-temporal coordination of cell spreading is impaired. Spreading cells, like other motile cells, show fluctuating exploratory motions of the leading edge visible as cycles of protrusion and retraction. This complex process can be broken down into discrete steps: actin protrusion of the leading edge, adhesion to the ECM, and myosin-driven contraction against adhesions. Time-lapse recordings indicate that Moody signaling has its most pronounced effect on the stabilization of protrusions, as evidenced by an increase in the ratio of retractions to extensions, and the marked shift of cell contours over time. The destabilization of protrusions might be due to weaker integrin-mediated interaction of focal adhesions with the ECM, but also due to impaired stress-mediated maturation of focal adhesions. The fact that both under- and overactivity of the Moody pathway impair protrusion stabilization may be due to the feedback between actin-myosin and focal adhesion, which also causes the well-known biphasic response of migration speed to adhesion strength of migrating cells. While the loss of moody has no significant effects on the other parameters that were measured, the loss of loco also reduces the frequency and size of protrusions, suggesting that actin polymerization may be specifically affected by increased GPCR signaling activity. Cumulatively, these impairments in protrusion/retraction behavior lead to retarded, non-isometric growth of SPG and to the irregular cell shapes observed in moody and loco mutants (Schwabe, 2017).

    Interestingly, PKA, Rho1 and MLCK have been identified as important downstream effectors of Moody signaling. All three factors are well known to control actin-myosin contraction; Rho1 and MLCK as positive regulators, PKA as a negative regulator. More recently, Rho1 activity has been shown to also drive actin polymerization at the leading edge, and a PKA-RhoGDI-Rho1 regulators feedback loop has been suggested to act as a pacemaker of protrusion- retraction cycles (Schwabe, 2017).

    The role of Moody pathway signaling in directed and well-coordinated cell growth is strikingly similar to the function of trimeric G protein signaling in other contexts. In Dictyostelium, G protein signaling is essential for directed cell migration: When all G protein signaling is abolished, cells are still mobile and actively generate protrusions, however these protrusions form in random directions, with the result that the cells lose their directionality. During gastrulation in Drosophila, signaling by the G12 ortholog Concertina and the putative GPCR ligand Folded Gastrulation synchronizes apical actin-myosin constrictions of mesodermal precursor cells, and thereby effects their concerted invagination. Thus, a major role of G protein signaling during development may be to modulate basic cellular behaviors such as cell growth, protrusion, or contraction, and reduce variability within cells and between neighboring cells, with the goal of generating uniform patterns and behaviors (Schwabe, 2017).

    Synchronized growth behavior of SPG is not only important for rapid epithelial closure but, ultimately, also for generating an evenly sealed BBB. All the evidence supports the notion that the defects in SJ organization that are responsible for the BBB failure are a secondary consequence of the morphogenetic function of the GPCR pathway. Cell contacts precede and are necessary for SJ formation, and the growth of cell contacts and SJ accumulation are strongly correlated. Delayed and more erratic cell- cell contact formation, as is the case in Moody pathway mutants, is likely to result in uneven circumferential distribution of SJ material later on; conversely, the timing of SJ formation per se is not affected by the pathway, arguing against a direct effect. Since the insulating function of SJs depends on their length, a decrease in the length in some local areas will result in insulation defects. Moreover, since SJs are known to form very static complexes, any irregularity in SJ distribution may be retained for long periods of time (Schwabe, 2017).

    Although under- and overactivity of the Moody pathway lead to globally similar outcomes, impaired epithelium formation and failure of BBB insulation, the data point to subtly different subcellular effects of the two types of pathway modulation. During MET, loco mutants (which this study confirms as inducing pathway overactivity) show predominantly retarded growth, presumably as a result of curtailed protrusive activity, while moody mutants show severe fluctuation and variability in growth. It will be very interesting to investigate these distinct outcomes of Moody pathway misregulation in greater detail (Schwabe, 2017).

    Occluding junctions maintain stem cell niche homeostasis in the fly testes

    Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. This study shows that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously express markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis (Fairchild, 2016).

    The hub regulates stem cell behavior in multiple ways. First, the hub physically anchors the stem cells by forming an adhesive contact with both germline stem cells (GSCs) and cyst stem cells (CySCs). The hub thus provides a physical cue that orients centrosomes such that stem cells predominantly divide asymmetrically, perpendicular to the hub. Following asymmetric stem cell division, one daughter cell remains attached to the hub and retains stem cell identity while the other is displaced from the hub and differentiates. Second, hub cells produce signals, including the STAT ligand Unpaired-1 (Upd), Hedgehog (Hh), and the BMP ligands Decapentaplegic (Dpp) and Glass-bottomed boat (Gbb), that signal to the adjacent stem cells to maintain their identity. As germ cells leave the stem cell niche, two somatic cyst cells surround and encapsulate them to form a spermatocyst. As spermatocysts move from the apical to the basal end of the testis, both somatic cyst cells and germ cells undergo a coordinated program of differentiation. Previous studies have shown that differentiation of encapsulated germ cells requires their isolation behind a somatic occluding junction-based permeability barrier. Specifically, a role was identified for septate junctions, which are functionally equivalent to vertebrate tight junctions, in establishing and maintaining a permeability barrier for each individual spermatocyst (Fairchild, 2016).

    During analysis of septate junction protein localization, it was observed that some, notably Coracle, were expressed in both the hub and the differentiating cyst cells. Moreover, knockdown of septate junction components in the somatic cells of the gonad resulted in enlarged hubs. Based on these results, the role of septate junction components in regulating the number of hub cells was explored in detail. To this end, RNAi was used to knock down the expression of the core septate junction components Neurexin-IV (Nrx-IV) and Coracle (Cora) in both the hub and cyst cell populations and the number of hub cells counted in testes from newly eclosed and 7-day-old adults. RNAi was expressed using three tissue-specific drivers: upd-Gal4, expressed in hub cells; tj-Gal4, expressed weakly in hub cells and strongly in both CySCs and early differentiating cyst cells; and eyaA3-Gal4, expressed strongly in all differentiating cyst cells, weakly in CySCs, and at negligible levels in the hub. To visualize hub cells, multiple established hub markers, including upd-Gal4, upd-lacZ, Fasciclin-III (FasIII), and DN-cadherin (DNcad) were used. Surprisingly, it was found that knockdown of Nrx-IV or cora driven by upd-Gal4 gave rise to normal hubs. In comparison, knockdown of Nrx-IV or cora using tj-Gal4 or eyaA3-Gal4 led to large increases in the number of the hub cells. Hub growth was not uniform and varied between testes, but median hub cells numbers in Nrx-IV and cora knockdown testes grew by 30% and 55%, respectively, between 1 and 7 days post-eclosion (DPEs). However, in extreme cases, hubs contained up to five times the number of cells found in age-matched control testes. This result was confirmed using a series of controls that discounted the possibility that hub overgrowth was due to temperature or leaky expression of the RNAi lines. These results suggested that hub growth occurred as a result of knockdown of septate junction proteins in cyst cells rather than the hub. This was further supported using another somatic driver that is not thought to be expressed in the hub, c587-Gal4. However, analysis of c587-Gal4 was complicated by the fact this driver severely impacted fly viability when combined with Nrx-IV or cora RNAi lines (Fairchild, 2016).

    Intriguingly, hub growth largely occurred after adult flies eclosed and not in earlier developmental stages. For example, when the driver eyaA3-Gal4 was used to knock down Nrx-IV or cora, hubs from 1-day-old adults were not larger than controls, but hubs from 7-day-old adults were significantly larger. Moreover, overgrowth phenotypes were recapitulated when temperature-sensitive Gal80 was used to delay induction of eyaA3-Gal4-mediated Nrx-IV knockdown until after eclosion. Hub growth manifested both in a higher mean number of hub cells per testis and by a shift in the distribution of hub cells per testis upward, toward larger hubs sizes. This distribution suggested a gradual, stochastic process of hub growth, resulting in a population of testes containing a range of hub sizes. These results reveal progressive hub growth in adults upon knockdown of septate junction components in cyst cells and suggest that this growth is not driven by events occurring in the hub itself but rather by events occurring in cyst cells (Fairchild, 2016).

    Niche size has been shown in various tissues, including vertebrate hematopoietic stem cells and somatic stem cells in the fly ovary, to be an important factor in regulating the number of stem cells that the niche can support. In the fly testes, it has been shown that mutants having few hub cells could nonetheless maintain a large population of GSCs. To determine how a larger hub, containing more cells, affects niche function, the number of GSCs and CySCs was monitored after knockdown of septate junction components in cyst cells. Overall, the average number of germ cells contacting the hub grew substantially in Nrx-IV or cora knockdown testes between 1 and 7 DPEs. To confirm that the germ cells contacting the hub were indeed GSCs, spectrosome morphology was studied, and it was found to be to be consistent with that seen in wild-type GSCs. Moreover, in individual testes, there was a positive correlation between the number of hub cells and the number of GSCs. Similar growth was also observed in the number of CySCs, defined as cyst cells expressing Zfh1, but not the hub cell marker DNcad. Control testes (from tj-Gal4 x w1118 progeny) had on average 34.3 CySCs whereas Nrx-IV and cora knockdown testes had 53.4 and 50.2 CySCs, respectively. These results show the importance of maintaining a stable stem cell niche size, as enlarged hubs were active and could support additional stem cells, which may result in the excess production of both germ cells and cyst cells (Fairchild, 2016).

    Next, it was of interest to determine the mechanism driving hub growth in adult flies upon knockdown of septate junction components in cyst cells. One possible mechanism that can explain this growth is hub cell proliferation. However, a defining feature of hub cells is that they are not mitotically active. Consistent with this, a large number of testes were stained for the mitotic marker phospho-histone H3 (pH3), and cells were never observed where upd-LacZ and pH3 were detected simultaneously. These results argue that division of hub cells is unlikely to explain hub growth in the adult Nrx-IV and cora knockdown testes. To determine the origin of the extra hub cells, the lineage of eyaA3-expressing cells was traced using G-TRACE (Evans, 2009). eyaA3 was chosed as both the expression pattern of septate junctions, and Nrx-IV or cora knockdown results suggested that hub growth involved differentiating eyaA3-positive cyst cells. The eyaA3-Gal4 driver utilizes a promoter region of the eya gene, which is required for somatic cyst cell differentiation and is expressed at very low levels in CySCs and at high levels in differentiating cyst cells. Using G-TRACE allows identification of both cells that previously expressed eyaA3-Gal4 (marked with GFP) and cells currently expressing eyaA3-Gal4 (marked with a red fluorescent protein [RFP]); additionally, the hub was identified using expression of upd-LacZ and FasIII. In control experiments at both 1 and 7 DPEs, few GFP-positive cells were observed in the hub. Those few GFP-positive cells could be explained by the transient expression of eya in the embryonic somatic gonadal precursor cells that form both hub and cyst cell lineages or extremely low levels of expression in adult hub cells. When G-TRACE was combined with knockdown of Nrx-IV, the results were strikingly different. Initially, 1 DPE, hubs were only slightly larger than controls and few GFP-positive hub cells were observed. In comparison, 7-DPE hubs contained on average more than twice as many cells compared to controls. Importantly, hub growth in Nrx-IV knockdowns was largely attributable to the incorporation of GFP-positive cells. Moreover, a population of upd-LacZ-labeled cells that were also RFP-positive was observed consistent with ongoing or recent expression of eyaA3-Gal4 in hub cells. These results suggest that knockdown of Nrx-IV or cora leads cyst cells to adopt hallmarks of hub cell identity and express hub-cell-specific genes (Fairchild, 2016).

    To learn more about the differentiation state of non-endogenous hub cells in Nrx-IV and cora knockdown testes, various markers were used to label the stem cell niche. This analysis showed normal expression of hub cell markers, such as Upd, FasIII, DNcad, as well as Hedgehog (hh-LacZ), Armadillo (Arm), and DE-Cadherin (DEcad). It was asked how cells that were previously, and in some instances were still, eyaA3 positive could express multiple hub-cell fate markers. To answer this question, the signaling mechanisms that determine hub fate were investigated in Nrx-IV and cora knockdown testes. Hub growth phenotypes similar to those produced by Nrx-IV and cora knockdown have been described previously, most notably in agametic testes that lack germ cells, suggesting that the germline regulates the formation of hub cells. One specific germline-derived signal shown to regulate hub fate is the epidermal growth factor (EGF) ligand Spitz. In embryonic testes, somatic cells express the EGF receptor (EGFR), which, when activated, represses hub formation. EGFR-induced mitogen-activated protein kinase (MAPK) signaling, visualized by staining for di-phosphorylated-ERK (dpERK), was active in CySCs and spermatogonial-stage cyst cells. Quantifying dpERK-staining intensity in cyst cell nuclei showed that MAPK activity was lower in CySCs following knockdown of Nrx-IV or cora, suggesting reduced EGFR signaling. Moreover, the effect of Nrx-IV or cora knockdown on MAPK signaling was not restricted to CySCs, as lower dpERK staining was observed at a distance from the hub. To see whether disruption of EGFR signaling could underlie hub defects in Nrx-IV and cora knockdown testes, attempts were made to rescue these phenotypes by increasing EGF signaling. When a constitutively activated EGF receptor (EGFR-CA) was co-expressed in cyst cells along with Nrx-IV RNAi, hub growth was attenuated, resulting in a reduction in the average number of hub cells compared to expressing only Nrx-IV RNAi. Similar results were also observed in the growth of the GSC population, suggesting that reduced EGFR activation in cyst cells contributes to the overall growth of the stem cell niche caused by the knockdown of Nrx-IV or cora. Surprisingly, analysis of testes with loss-of-function mutations in the EGFR/MAPK pathway reveals different phenotypes than those observed: encapsulation is disrupted and CySCs are lost, but hub size is largely unaffected. This result shows that the partial reduction in EGFR/MAPK signaling seen in Nrx-IV and cora knockdown testes results in distinct phenotypes and highlights the complexity of EGFR signaling in the fly testis (Fairchild, 2016).

    Another pathway that is documented to regulate hub cell fate is Notch signaling. Notch plays important roles in hub specification in embryos. The Notch ligand Delta is produced by the embryonic endoderm and acts to promote hub cell specification in the anterior-most somatic gonadal precursor cells. Whereas it has been suggested that Notch acts in the adult to regulate hub fate, such a role has not been clearly demonstrated. A reporter for the Notch ligand Delta (Dl-lacZ) was observed in hub cells of both control and Nrx-IV knockdown testes. Intriguingly, reducing Notch signaling efficiently rescued the hub overgrowth seen in adult Nrx-IV knockdown testes. When a dominant-negative Notch (Notch-DN) was co-expressed in the somatic cells, along with Nrx-IV RNAi, the growth of the hub was reduced compared to the expression of Nrx-IV RNAi alone. Growth in the GSC population was not significantly reduced by co-expression of Notch-DN, suggesting that the Notch pathways may modulate hub growth through a different mechanism compared to the EGFR pathway. Because Notch is well established to regulate hub growth in the embryo, temperature-sensitive Gal80 was used to delay expression of Notch-DN and confirm that the reduction in hub cells was due to disruption of post-embryonic Notch signaling. These results suggest that Notch signaling in cyst cells may contribute to the hub overgrowth phenotypes caused by septate junction knockdown in the adult testes (Fairchild, 2016).

    In addition to Notch and EGFR, other signaling pathways that regulate hub size may contribute to the hub growth seen upon somatic knockdown of septate junction components. For example, it has been previously shown that the range of BMP signaling is expanded following Nrx-IV or cora knockdown in cyst cells. Constitutive activation of BMP signaling in the germline was shown to increase the size of the hub and the number of GSCs. Additionally, the relative expression levels of the genes drm, lines, and bowl regulate hub size in the adult. In particular, it is known that lines maintains a “steady state” in the testes by repressing expression of a subset of hub genes in the cyst cell population. Unlike lines mutants, Nrx-IV or cora knockdowns generally lack ectopic hubs. This may reflect the more gradual hub growth seen in septate junction knockdowns or, alternatively, highlight key mechanistic differences in how hub growth is achieved in each respective genetic background. The current work is consistent with the model whereby occluding junctions are required for proper soma-germline signaling in the fly testes. This signaling maintains stem cell niche homeostasis by preventing somatic cyst cells from adopting hub cell fate, which would lead to niche overgrowth. It is well established that, in embryonic testes, hub fate is both positively and negatively regulated by signals from the germline and the endoderm.The results, and recent findings about the genes lines and traffic jam, argue that, in the adult testes, hub fate is actively repressed in the cyst cell lineage. Failure to repress hub fate allows cyst cells to exhibit features of hub cells and act as a functional stem cell niche. However, these cyst-cell-derived hub cells are distinct from the true endogenous hub cells in that they show non-hub-cell features, including expression of the differentiating cyst cell markers eyaA3-Gal4 and β3-tubulin. The data suggest that, following disruption of septate junctions proteins, the signaling environment surrounding the somatic cells is altered such that cyst cells gradually begin expressing hub cell markers (Fairchild, 2016).

    One major outstanding question is how eyaA3-Gal4-expressing cyst cells become incorporated into the endogenous hub. Previously, it was shown that a septate-junction-mediated permeability barrier forms by the four-cell spermatogonial-stage spermatocyst. The hub growth phenotypes induced by Nrx-IV and cora knockdowns may occur due to defects in cell-cell signaling, possibly involving EGFR and Notch, that manifest in these later spermatocysts. However, this model requires an explanation for how these cyst cells translocate back to and join the hub. Alternatively, signaling defects in these later spermatocysts are somehow instructing earlier cyst cells, such as CySCs, to join the hub. It is easier to envisage the latter model, as early cyst cells are spatially much closer to the hub, but the sequence of signaling events in such a case will be complex and require further elucidation. The ability of CySCs to convert into hub cells in wild-type testes is a controversial subject. However, the incorporation of CySCs into the hub does not necessitate complete conversion into hub cells but could rather involve simple de-repression or activation of genes that confer hub cell function, including regulators of the cell-cycle- and hub-cell-specific signaling ligands. Notably, the transition between CySC and hub cell fate is linked to the cell cycle (Fairchild, 2016).

    Why would loss of the septate-junction-mediated somatic permeability barrier result in disruption of signaling between the soma and germline? There are many possible answers, but it is possible to speculate about two such mechanisms that explain hub overgrowth. One possibility is that germline differentiation, which is dependent on the permeability barrier, is required for the release of signals that maintain stem cell niche homeostasis. Another possibility is that the permeability barrier locally concentrates germline-derived signals that repress hub cell fate by trapping them in the luminal space between the encapsulating cyst cells and the germline. The latter scenario could explain the observation that activated EGFR signaling partially rescues hub overgrowth. In this model, septate junctions allow localized buildup of the EGF ligand Spitz, ensuring that sufficient signaling is available to repress hub fate. It is more difficult to draw strong conclusions about how Notch signaling is altered when septate junctions are disrupted, particularly as the Notch ligand Delta appears restricted to the hub. Overall, an unexpected role was found for an occluding-junction-based permeability barrier in mediating stem cell niche homeostasis. This work highlights how the architecture of the stem cell niche system in the fly testes, which is highly regular and contains a reproducible number of stem cells and niche cells, is in fact the result of an active and dynamic signaling environment (Fairchild, 2016).

    Cbl-associated protein regulates assembly and function of two tension-sensing structures in Drosophila, muscle attachment sites and scolopale cells

    Cbl-associated protein (CAP) localizes to focal adhesions and associates with numerous cytoskeletal proteins; however, its physiological roles remain unknown. This study demonstrates that Drosophila CAP regulates the organization of two actin-rich structures in Drosophila: muscle attachment sites (MASs), which connect somatic muscles to the body wall; and scolopale cells, which form an integral component of the fly chordotonal organs and mediate mechanosensation. Drosophila CAP mutants exhibit aberrant junctional invaginations and perturbation of the cytoskeletal organization at the MAS. CAP depletion also results in collapse of scolopale cells within chordotonal organs, leading to deficits in larval vibration sensation and adult hearing. This study investigated the roles of different CAP protein domains in its recruitment to, and function at, various muscle subcellular compartments. Depletion of the CAP-interacting protein Vinculin results in a marked reduction in CAP levels at MASs, and vinculin mutants partially phenocopy Drosophila CAP mutants. These results show that CAP regulates junctional membrane and cytoskeletal organization at the membrane-cytoskeletal interface of stretch-sensitive structures, and they implicate integrin signaling through a CAP/Vinculin protein complex in stretch-sensitive organ assembly and function (Bharadwaj, 2013).

    Interactions between cells and the extracellular matrix (ECM) are crucial for many biological processes. These include cell migration, directed process outgrowth, basement membrane-mediated support of tissues and maintenance of cell shape. Communication between cells and ECM proteins often occurs through the action of α/β-integrin heterodimers, a receptor complex that forms adhesive contacts, including focal adhesions, hemiadherens junctions, costameres and myotendinous junctions. In response to extracellular forces, focal adhesions undergo structural changes and initiate signaling events that allow adaptation to tensile stress. Vinculin is thought to be the primary force sensor in the integrin complex, mediating homeostatic adaptation to external forces (Bharadwaj, 2013 and references therein).

    Vinculin-binding partners include proteins belonging to the CAP (Cbl-associated protein) protein family. However, the physiological significance of this association is unknown. Mammalian CAP proteins are components of focal adhesions in cell culture. In myocytes, CAP localizes to integrin-containing complexes called costameres that anchor sarcomeres to muscle cell membranes. There are three mammalian CAP protein family members: CAP, Vinexin and ArgBP2. CAP associates in vitro with many proteins, including the cytoskeletal regulators Paxillin, Afadin and Filamin, vesicle trafficking regulators such as Dynamin and Cbl, and the lipid raft protein Flotillin. In vitro studies demonstrate that CAP regulates the reassembly of focal adhesions following nocodazole dissolution. However, despite extensive studies on CAP, little is known about its functions in vivo. Cap (Sorbs1) mutant mice are defective in fat metabolism, and targeted deletion of the vinexin gene results in wound-healing defects. Drosophila CAP binds to axin and is implicated in glucose metabolism . Analysis of CAP function in mammals is complicated by potential functional redundancy of the three related CAP proteins. Therefore, the function of Drosophila CAP, the single CAP family member in Drosophila, was examined in vivo (Bharadwaj, 2013 and references therein).

    The Drosophila muscle attachment site (MAS) is an excellent system for studying integrin signaling. Somatic muscles in each segment of the fly embryo and larva are connected to the body wall through integrin-mediated hemiadherens junctions. Somatic muscles in flies lacking integrins lose their connection to the body wall. Surprisingly, flies lacking Vinculin, a major component of cytosolic integrin signaling complexes, are viable and show no muscle defects. Thus, unlike its mammalian counterpart, Drosophila Vinculin is apparently dispensable for the initial assembly of integrin-mediated adhesion complexes at somatic MASs (Bharadwaj, 2013).

    The fly MAS is structurally analogous to the fly chordotonal organ. These organs transduce sensations from various stimuli, including vibration, sound, gravity, airflow and body wall movements. The chordotonal organ is composed of individual subunits called scolopidia, each containing six cell types: neuron, scolopale, cap, ligament, cap attachment and ligament attachment cells. Chordotonal neurons are monodendritic, and their dendrites are located in the scolopale space, a lymph-filled extracellular space completely enveloped by the scolopale cell. Within the scolopale cell, a cage composed of actin bars, called scolopale rods, facilitates scolopale cell envelopment of the scolopale space. Thus, like the MAS, the actin cytoskeleton plays a specialized role in defining chordotonal organ morphology. Similarities between MASs and chordotonal organs include the requirement during development in both tendon and cap cells for the transcription factor Stripe. Furthermore, both of these cell types maintain structural integrity under force and so are likely to share common molecular components dedicated to this function (Bharadwaj, 2013 and references therein).

    This study shows that the Drosophila CAP protein is selectively localized to both muscle attachment sites and chordotonal organs. In Drosophila CAP mutants morphological defects are observed that are indicative of actin disorganization in both larval MASs and the scolopale cells of Johnston's organ in the adult. The morphological defects in scolopale cells result in vibration sensation defects in larvae and hearing deficits in adults. It was also found that, like its mammalian homologues, Drosophila CAP interacts with Vinculin both in vitro and in vivo. These results reveal novel CAP functions required for actin-mediated organization of cellular morphology, lending insight into how CAP mediates muscle and sensory organ development and function (Bharadwaj, 2013).

    Integrin-based adhesion complexes are crucial for cell attachment to the extracellular matrix. These complexes change their composition and architecture in response to extracellular forces, initiating downstream signaling events that regulate cytoskeletal organization. This study has investigated the role played by the CAP protein in two stretch-sensitive structures in Drosophila: the MAS and the chordotonal organ. CAP mutants exhibit aberrant junctional invaginations at the MAS and collapse of scolopale cells in chordotonal organs. This study highlights a crucial integrin signaling function during development: the maintenance of membrane morphology in stretch-sensitive structures (Bharadwaj, 2013).

    The morphological defects observed in CAP mutants could result from an excessive integrin signaling, or possibly accumulation of additional membranous components related to integrin signaling, in CAP mutants, owing to defects in endocytosis at the MAS. This is consistent with known interactions between CAP family members and vesicle trafficking regulators, including Dynamin and Synaptojanin, which are required for internalization of transmembrane proteins. Alternatively, CAP may be required for proper organization of the actin cytoskeleton at MASs, and the aberrant membrane invaginations that were observe are a secondary consequence of these cytoskeletal defects. This idea garners support from known interactions between CAP and various actin-binding proteins, including Vinculin, Paxillin, Actinin, Filamin and WAVE2. A third possibility is that CAP and Vinculin are regulators of membrane stiffness at the MAS, and aberrant junctional infoldings observed in CAP and vinculin mutants derive from diminished membrane rigidity in the presence of persistent myofilament contractile forces. Biophysical studies demonstrate that Vinculin-deficient mammalian cells in vitro show reduced membrane stiffness. Interestingly, the CAP protein ArgBP2 interacts with Spectrin, a protein important for cell membrane rigidity maintenance. These models for CAP function at MASs, however, are not mutually exclusive. Interestingly, disruption of the ECM protein Tiggrin leads to MAS phenotypes similar to CAP. Future studies on CAP interaction with Tiggrin and other CAP-interacting proteins will shed light on mechanisms underlying CAP function. Nevertheless, this study demonstrates in vivo the importance of CAP in stretch-sensitive organ morphogenesis, and it will be interesting to determine whether this function is phylogenetically conserved (Bharadwaj, 2013).

    Apart from the MAS, CAP is also expressed at high levels in chordotonal organ scolopale cells, and this study has found that CAP mutants are defective in vibration sensation, a hallmark of chordotonal organ dysfunction. However, only the initial fast hunching response to vibration is disrupted in CAP mutant larvae. This may result from a partial loss of chordotonal function in these organs in the absence of CAP. A functional defect was also observed in the adult Johnston's organ; CAP mutant flies show diminished sound-evoked potentials. Importantly, the scolopale cells in CAP mutants appear partially collapsed. The extracellular space within the scolopale cell is lined by an actin cage, and CAP may influence the proper assembly of this actin cage or its association with the scolopale cell membrane. Ch organs are mechanosensory detectors and are constantly exposed to tensile forces. Thus, CAP apparently influences cytoskeletal integrity in two actin-rich structures: the MAS and the chordotonal organ, both of which are involved in force transduction (Bharadwaj, 2013).

    Mammalian and Drosophila CAP bind to Vinculin. Vinculin is required for the recruitment of the mammalian CAP protein vinexin to focal adhesions in NIH3T3 cells in vitro. Consistent with this observation, a dramatic decrease was seen in CAP levels at MASs in vinculin mutants, but residual levels of CAP protein remain. Furthermore, CAP localization at the muscle fiber Z-lines is completely unaltered in vinculin mutants. These observations indicate that Vinculin is not the sole upstream regulator of CAP localization. vinculin mutants show some of the phenotypic defects observed in CAP mutants; however, these defects are less pronounced. Therefore, the residual CAP pool that is recruited to MASs in a Vinculin-independent manner is apparently sufficient for partial CAP function. Assessment of CAP and Vinculin function at the larval MAS shows that these proteins are required for maintaining the integrity of junctional membranes in the face of tensile forces. CAP proteins may serve as scaffolding proteins at membrane-cytoskeleton interfaces and facilitate the assembly of protein complexes involved in cytoskeletal regulation and membrane turnover (Bharadwaj, 2013).

    Mutations in the CAP-binding protein filamin cause myofibrillar myopathy. This, in combination with data showing a crucial role for CAP in regulation of muscle morphology, sets the stage for investigating how loss of CAP protein function might influence the etiology of myopathies (Bharadwaj, 2013).

    A tendon cell specific RNAi screen reveals novel candidates essential for muscle tendon interaction

    Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. This study used dorsal longitudinal muscles of Drosophila and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. A genetic screen using RNAi-mediated knockdown in tendon cells was performed to find out molecular players involved in the formation and maintenance of myotendinous junction; 21 candidates were found out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating the screen. Candidates were found with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells (Tiwari, 2015).

    Diverse integrin adhesion stoichiometries caused by varied actomyosin activity

    Cells in an organism are subjected to numerous sources of external and internal forces, and are able to sense and respond to these forces. Integrin-mediated adhesion links the extracellular matrix outside cells to the cytoskeleton inside, and participates in sensing, transmitting and responding to forces. While integrin adhesion rapidly adapts to changes in forces in isolated migrating cells, it is not known whether similar or more complex responses occur within intact, developing tissues. Changes in integrin adhesion composition were studied upon different contractility conditions in Drosophila embryonic muscles. All integrin adhesion components tested were still present at muscle attachment sites (MASs) when either cytoplasmic or muscle myosin II was genetically removed, suggesting a primary role of a developmental programme in the initial assembly of integrin adhesions. Contractility does, however, increase the levels of integrin adhesion components, suggesting a mechanism to balance the strength of muscle attachment to the force of muscle contraction. Perturbing contractility in distinct ways, by genetic removal of either cytoplasmic or muscle myosin II or eliminating muscle innervation, each caused unique alterations to the stoichiometry at MASs. This suggests that different integrin-associated proteins are added to counteract different kinds of force increase (Bulgakova, 2017).

    Adherens junction distribution mechanisms during cell-cell contact elongation in Drosophila

    During Drosophila gastrulation, amnioserosa (AS) cells flatten and spread as an epithelial sheet. This study used AS morphogenesis as a model to investigate how adherens junctions (AJs) distribute along elongating cell-cell contacts in vivo. As the contacts elongated, total AJ protein levels increased along their length. However, genetically blocking this AJ addition indicated that it was not essential for maintaining AJ continuity. Implicating other remodeling mechanisms, AJ photobleaching revealed non-directional lateral mobility of AJs along the elongating contacts, as well as local AJ removal from the membranes. Actin stabilization with jasplakinolide reduced AJ redistribution, and live imaging of myosin II along elongating contacts revealed fragmented, expanding and contracting actomyosin networks, suggesting a mechanism for lateral AJ mobility. Actin stabilization also increased total AJ levels, suggesting an inhibition of AJ removal. Implicating AJ removal by endocytosis, clathrin endocytic machinery accumulated at AJs. However, dynamin disruption had no apparent effect on AJs, suggesting the involvement of redundant or dynamin-independent mechanisms. Overall, it is proposed that new synthesis, lateral diffusion, and endocytosis play overlapping roles to populate elongating cell-cell contacts with evenly distributed AJs in this in vivo system (Goldenberg, 2013).

    This study has documented three major behaviours of AJ proteins as AS cell-cell contacts elongate: new addition to the contacts, lateral movement along the contacts, and removal from the contacts. Once delivered to the contacts, AJ proteins appear to be redistributed by the expansion and contraction of fragmented actomyosin networks along the contacts, and by endocytosis from the contacts. However, the even distribution of AJ proteins could not be perturbed by singly disrupting any of these mechanisms, suggesting that they function redundantly for robust localization of AJs during AS morphogenesis (Goldenberg, 2013).

    A ~50% increase in total AJ proteins was quantified along elongating contacts from early to mid cell elongation. The exocyst complex closely colocalizes with AJs at all cell-cell contacts in the tissue, suggesting a role in DE-cad exocytosis, as evident in other Drosophila tissues (Langevin, 2005). However, eliminating this net new addition has no noticeable effect on the even distribution of DE-cad along elongating contacts. Notably, within 2h of gastrulation, these mutants begin the lose cell-cell adhesion in the ventral neurectoderm, as holes left from delaminating neuroblasts fail to reform. At this later stage, cell-cell adhesion is still maintained in the AS in the mutants, and the residual maternally supplied DE-cad remains remarkably continuous along the fully elongated contacts. Thus, despite the rapidly expanding circumferences of the cells, net new addition of AJ proteins is not essential, and a stable pool of AJ proteins can apparently be effectively redistributed to maintain AJ continuity and cell-cell adhesion (Goldenberg, 2013).

    The fact that total AJ protein levels increase by only ~1.5-fold while the elongating cell-cell contact lengths increase by ~2-fold, suggests that local AJ densities decrease, and thus that AJ proteins are locally lost either though lateral movement or removal from the membrane. Indeed, direct measurements of AJ proteins showed a ~50% decrease in local intensities, and the apparent heights of AJs decreased as well. Furthermore, iFRAP analyses revealed that local AJ patch lengths often increase, or decrease, at rates of 25% per min. Additionally, the local intensities of these patches can drop by up to 30% per min. By quantifying multiple parameters for the same AJ region over time, it was found that decreasing AJ protein levels did not correlate with the lateral spreading of the complexes, suggesting removal from the membrane. Notably, neither the lateral AJ movements nor the AJ losses correlated directly with the lengthening of the overall contacts, suggesting that the redistribution mechanisms are not directly triggered by contact elongation. Instead, it is proposed that local mechanisms result in constitutive expansions and condensations of cadherin-catenin clusters along the contacts, as well as constitutive removal, recycling and re-addition of AJ proteins along the contacts. These mechanisms would result in AJ plasticity that could allow pre-existing AJ proteins to populate elongating cell-cell contacts as they form (Goldenberg, 2013).

    A key element regulating AS cell shape change appears to be the loss of actomyosin networks from cell-cell contacts. It is evident that these actomyosin networks restrain protrusive microtubule bundles from initiating elongation of the apical domain and cell rotation [36]. Thus, their weakening appears to permit the cell shape change. This study found that experimentally stabilizing actin networks rigidifies the cell cortex and also inhibits AJ redistribution. Thus, the normal weakening of actomyosin networks may contribute to both AS cell shape change and AJ plasticity along elongating contacts (Goldenberg, 2013).

    Weakening actomyosin networks could promote AJ remodeling in two ways: by increasing lateral mobility or by increasing endocytosis. Actin is known to tether and restrain AJs in many systems. For example, in the ectoderm neighbouring the AS, actomyosin levels at AJs are normally maintained at higher levels than in the AS, but disrupting actin or α-catenin leads to the lateral mobility of AJ puncta with residual patches of actin attached. With the normal loss of myosin from AS cells, it was found that its networks fragment, and that these fragments expand and contract. It is proposed that these networks may expand and condense associated cadherin-catenin clusters. Also, the loss of actomyosin networks from the AS may contribute to AJ endocytosis, consistent with the observed increase in AJ protein levels at contacts with actin stabilization (Goldenberg, 2013).

    There are clear examples where single AJ remodeling mechanisms play a dominant role in controlling cell-cell adhesion. For example, perturbing dynamin activity has a dramatic effect on AJ localization in the pupal notum, but has no clear effect at elongating AS cell-cell contacts. Such differences may be due to the relative effectiveness of other AJ distribution mechanisms in the tissues. For example, new synthesis of AJ proteins is much lower in the pupal notum versus the early embryo. Tissues with multiple effective AJ distribution mechanisms may maintain adhesion more robustly. Defining such redundancies and their contributions to robust cell-cell adhesion is important for understanding different developmental and homeostatic processes, as well as different disease states, e.g., the propensity of cancerous tissues to metastasize (Goldenberg, 2013).

    Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye

    Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. This study shows that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. They uncovered a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and showed that apical Magi recruited the RASSF8-ASPP complex during AJ remodeling in IOCs. Further, this Magi complex was required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. They propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites (Zaessinger, 2015).

    As Magi is the sole Drosophila homolog of the three vertebrate MAGI scaffolds, it offers a powerful system with which to investigate the functions of these important proteins. Using newly generated null alleles, this study has shown that Magi coordinates the number and packing of IOCs in the developing Drosophila pupal eye by regulating AJ dynamics. Magi is necessary in the IOCs to localize the RASSF8-ASPP complex correctly during their junctional remodeling. This ensures the integrity of E-Cad-based junctions and the correct localization of Baz, α- and β-catenin. Based on these observations and on the growing evidence of a role for Baz in AJ remodeling, a model is proposed whereby, during AJ remodeling in IOCs, Magi recruits the RASSF8-ASPP complex, which helps to localize Baz at the membrane and regulates the sites of E-Cad accumulation (Zaessinger, 2015).

    Junction remodeling is a key step during morphogenesis, in which cells in a tissue change position and neighbors. For instance, in the developing pupal eye, IOCs found between ommatidia organize as a single row of cells. During this process existing contacts are eliminated and new ones are established by remodeling E-Cad-based junctions. In Magi mutants, rearrangement defects and some incorrect localization of IOCs were observed. At the same time, E-Cad-based AJs were interrupted in Magi mutant cells. It is proposed that this defect in AJ remodeling leads to IOCs remaining at the wrong place in the lattice. The most parsimonious model is that the defects in AJ remodeling trigger the defects in cell numbers seen in Magi mutants by preventing apoptosis, although it was not possible to fully substantiate this as the effect of Magi on apoptosis was not statistically significant. If the model is correct, it still remains unclear how disrupted junctions would lead to a failure in apoptosis. One possibility is that IOCs only receive the correct 'death signal' when they have rearranged to contact the correct cells. Thus, in Magi mutants, the defective AJs would lead to apoptosis failure because the IOCs did not attain their position in the 'death zone' to receive the killing signal (Zaessinger, 2015).

    These junctional defects are reminiscent of those seen for magi-1 mutants in the nematode C. elegans, in which magi-1 loss of function enhanced the defects caused by cadherin and catenin mutations and disrupted cell migration during enclosure (Lynch, 2012). MAGI scaffolds are thus implicated in the fine regulation of AJs in both flies and nematodes. A similar role has been suggested for MAGI proteins in mammalian epithelial cells. In overexpression studies, human MAGI1 reduced the Src-induced invasiveness of MDCK cells and stabilized E-Cad-mediated intercellular aggregation (Kotelevets, 2005). By analogy, the overexpression phenotype of Drosophila Magi could thus be due to stronger AJs, although this remains to be experimentally tested. The overexpression effects of MAGI-1b were sensitive to PTEN and AKT activities (Kotelevets, 2005) and mammalian MAGI scaffolds have also been implicated in PTEN activation through their direct binding to PTEN. However, this study did not detect any physical interaction between Drosophila Magi and Pten, and the overexpression phenotype of Magi, at least in the Drosophila eye, appeared insensitive to Pten. Although these are negative observations, they suggest that in Drosophila Magi and Pten do not form a complex to regulate AJs (Zaessinger, 2015).

    Despite its effects on eye development, Magi mutants exhibit slightly enlarged wings. Whether this is dependent on E-Cad belt integrity and AJ dynamics remains to be established. The fact that ASPP shows a very similar wing phenotype supports this model (Zaessinger, 2015).

    Rather than binding and modulating the activity of Pten, this analysis supports a model whereby Magi, by binding to the RASSF8-ASPP complex, recruits and stabilizes Baz at the membrane. Accumulation of Baz has been shown to specify and initiate the formation of new AJs both in cellularizing embryos and in photoreceptors. It is proposed that Baz recruited at the membrane of IOCs will in turn promote the stabilization or the proper distribution around the cell cortex of AJ material. Since biochemical and genetic experiments suggest that RASSF8 and Magi act together in a complex, it is proposed that the effects of Magi on AJs and on Baz membrane recruitment are mediated by RASSF8, and are thus likely to involve ASPP. Indeed, mammalian ASPP2 binds PAR3 and is required for PAR3 localization at junctions both in cell culture and in the mouse neuroepithelium. This suggests that Magi might control Baz localization through ASPP. However, Baz membrane recruitment is unlikely to be the only step to form correct AJs downstream of Magi/RASSF8/ASPP. Previous studies have implicated C-terminal Src kinase (Csk) and its action on Src kinase, and the relationships between Magi, Baz and Csk should be investigated in the future (Zaessinger, 2015).

    During IOC remodeling, Magi therefore appears to be a crucial upstream regulator of AJs. However, the mechanisms governing Magi membrane localization are still unknown. One hypothesis is that the membrane recruitments of different AJ components and regulators are dependent on each other in stabilization loops. However, this is unlikely to be the case for Magi as it is still perfectly localized at the membrane in ASPP, RASSF8 and baz mutants, and in ASPP; RASSF8 double mutants (Zaessinger, 2015).

    Another possibility is that Magi would require mature AJs with E-Cad to be at the membrane. No direct correlation was found between E-Cad accumulation around the apical membrane and Magi membrane localization. For instance, in ASPP; RASSF8 double-mutant cells, E-Cad belt interruptions were detected either without or with Magi, indicating that Magi localization does not require E-Cad directly. Furthermore, an extensive domain mapping of Magi failed to identify a single domain (WW or PDZ) that would be required for Magi recruitment, suggesting that it might be independent of these domains or that several redundant mechanisms may be at play. The nature of the signal required for Magi membrane localization thus remains to be uncovered (Zaessinger, 2015).

    Even though Magi binds to RASSF8 directly and both proteins function together during Drosophila eye morphogenesis, their mutant phenotypes are not identical. First, RASSF8 mutants have a wing rounding phenotype, which is absent in Magi mutants. Second, whereas RASSF8 has a significant role in the global developmental apoptosis rate in the pupal eye, no significant effect could be detected for Magi and ASPP. Taken together, this suggests that the assembly of a Magi-RASSF8-ASPP complex might be context dependent or that RASSF8 has Magi-independent functions (Zaessinger, 2015).

    Although the human N-terminal RASSF (RASSF7-10) proteins lack any PPxY motifs, one is present in ASPP2 and has been shown to bind to MAGI1. It is therefore possible that MAGI-ASPP complexes are formed in all organisms but the precise mode of interaction differs: mediated by RASSF8 in the fly, but direct in humans (Zaessinger, 2015).

    MAGI scaffolds have been suggested to play a role in tumorigenesis. First, they are bound and inactivated by several viral oncoproteins. Second, MAGI1 has been shown to exhibit tumor suppressor activity in colorectal cancer cell lines in xenograft model. Finally, mutations in MAGI2 and MAGI3 are reported in colon, prostate and breast cancers. Documented alterations include deletion of the second WW motif of MAGI2 and a MAGI3:AKT3 fusion leading to a disruption of MAGI3. Based on the current work, it is proposed that these are loss-of-function mutations. It would be interesting to investigate whether changes in AJ dynamics are associated with these MAGI mutations in human cancers and whether they contribute to tumorigenesis (Zaessinger, 2015).

    Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex

    The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).

    A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains (Dobrosotskaya, 1997). There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin23, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).

    Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).

    Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts (Gregorc, 2007). MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).

    There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).

    The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs (Zaessinger, 2015). Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).

    Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).

    PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).

    Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).

    Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).

    In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).

    Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).

    Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

    Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, the behavior of E-cadherin-rich junction clusters were tracked, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. These findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT (Weng, 2016).

    This study shows that during Drosophila gastrulation, subapical junctions are repositioned toward the apical surface and are strengthened as the cortical tension increases. Both these phenomena follow apical myosin activation and thus may reflect a mechanosensitive response of junctional complexes to the tension generated by this activation of myosin. The junctional responses occur on the time scale of individual myosin pulses and are temporally correlated with those pulses. Such junctional changes depend on myosin activity but do not require Sna, given that ectopic myosin activation recapitulates similar junctional responses in Sna-negative tissues. This phenomenon may not be restricted to Drosophila embryos. The increased contractile actomyosin on the apical cortex of human cell lines deficient for the cortex actin regulator Merlin is associated with a condensation of adherens junctions toward the apical surface, suggesting that the response of adherens junctions to cortical tension can be of general significance (Weng, 2016).

    The changes in junction mass and density suggest that, rather than being simple passive anchors for contractile actomyosin filaments, adherens junctions respond to the contractile actomyosin by restructuring and repositioning themselves, potentially involving aggregation and rearrangement of E-Cad molecules within the plasma membrane or vesicle-based redistribution of E-Cad. Indeed, actomyosin organization has been shown to be critical in the lateral clustering of E-Cad molecules. The change in E-Cad clustering is considered an active mechanosensitive mechanism to strengthen the adhesion. Alternatively, the adhesion can also be remodeled through the vesicle-based mechanisms, and endocytosis of E-Cad has been shown to be up-regulated when junctions are under actomyosin-generated stress. The repositioning could also arise through restructuring rather than passive dragging, if for example recycling and turnover rates in the basal regions of the junctions differ from apical regions. Overall, regardless of the underlying mechanism, this mechanosensitivity may be advantageous, providing a direct self-corrective mechanism that allows junctions to adjust their localization and intensity to match the mechanical force they experience (Weng, 2016).

    Although the molecular mechanism for the junction strengthening requires further investigation, the data suggest that it is resistant to the posttranscriptional disassembly of adherens junctions downstream of Sna. The phenotype of myosin knockdown in this study resembles that previously described for cta; T48 double mutants, in which apical actomyosin cannot be activated and junctions are lost only in the ventral mesodermal cells. In all scenarios in which Sna expression is associated with junction loss (ventral cells in cta; T48 mutants, ventral cells in myosin knockdown mutants, and ectodermal cells with ectopic Sna expression), Sna is expressed in cells in the absence of myosin contractility. Maintenance of adherens junctions ultimately relies on the balance between assembly and disassembly rates of junctional components. Thus mechanical force likely modulates the assembly/disassembly balance and therefore remains in a homeostatic relationship with the junctions bearing the force (Weng, 2016).

    In the early stages of embryogenesis analyzed in this study, E-Cad is maternally provided and thus not subject to direct transcriptional repression. The disassembly of junctions in the absence of myosin contraction must therefore reflect a posttranscriptional regulation on junctions, likely performed by one or several of Sna’s transcriptional targets. Much effort has been invested in identifying transcription targets of Sna, but it is not known which, if any, of its known targets might play such a role. One mesodermally expressed gene, Traf4, is required for fine-tuning junction morphology, but its expression appears to depend on the other mesodermal determinant, Twist, rather than Sna. One gene repressed by Sna in Drosophila mesoderm, bearded, is required for the subapical positioning of adherens junctions in cells not expressing Snail. It is not clear, however, whether Bearded plays a direct role in junction disassembly or a more general role in apical polarity or the apical myosin contractility that drives repositioning. The posttranscriptional regulation of adherens junction disassembly may allow more rapid and effective EMT than a disassembly relying on transcriptional down-regulation of junctional components such as E-Cad. Identifying and characterizing the relevant Sna targets in Drosophila may provide insights into the underlying mechanism for this disassembly, especially with respect to its apparent sensitivity to externally exerted tension. The force-dependent resistance to this Sna function may help in dissecting the underlying molecular functions. Further exploration of Sna’s posttranscriptional effect on junctions and how myosin contraction antagonizes Sna will shed light on understanding of EMT processes (Weng, 2016).

    The triple-repeat protein Anakonda controls epithelial tricellular junction formation in Drosophila

    In epithelia, specialized tricellular junctions (TCJs) mediate cell contacts at three-cell vertices. TCJs are fundamental to epithelial biology and disease, but only a few TCJ components are known, and how they assemble at tricellular vertices is not understood. This study describes a transmembrane protein, Anakonda (Aka), which localizes to TCJs and is essential for the formation of tricellular, but not bicellular, junctions in Drosophila. Loss of Aka caused epithelial barrier defects associated with irregular TCJ structure and geometry, suggesting that Aka organized cell corners. Aka is necessary and sufficient for accumulation of Gliotactin at TCJs, suggesting that Aka initiated TCJ assembly by recruiting other proteins to tricellular vertices. Aka's extracellular domain had an unusual tripartite repeat structure that might mediate self-assembly, directed by the geometry of tricellular vertices. Conversely, Aka's cytoplasmic tail is dispensable for TCJ localization. Thus, extracellular interactions, rather than TCJ-directed intracellular transport, appear to mediate TCJ assembly (Byri, 2015).

    Epithelial cells are linked via intercellular junctions that provide paracellular diffusion barriers, maintenance of polarity, and cell-to-cell communication. Bicellular junctions (BCJs) make up the most abundant intercellular contacts in epithelia. They connect two neighboring plasma membranes and are organized into distinct complexes along the apical-basal axis, collectively forming the apical junctional belt. However, at certain positions along the cell perimeter, three cell corners meet and the bicellular junctional complex is disjointed. Here, specialized tricellular junctions (TCJs) connect epithelial cells. TCJs play important roles in epithelial barrier functions and cytoskeletal organization, and are preferential sites for trans-endothelial migration of neutrophils and metastatic cancer cells, as well as for the spreading of intracellular pathogens (Byri, 2015).

    Due to the geometry of three-cell vertices, the sealing of the epithelium at these sites requires a dedicated junctional organization. Ultrastructural analyses using freeze-fracture electron microscopy (EM) showed that the zonula occludens, tight junctions (TJs) in vertebrates and septate junctions (SJs) in arthropods, changes characteristics when approaching a three-cell vertex. Instead of continuing parallel to the epithelial plane around the cell perimeter, the junctional strands extend basally, forming a bicellular seal along the apical-basal axis. In vertebrates, these structures are termed central sealing elements, and three such parallel TJ extensions enclose a narrow (approximately 10 nm) central canal at each TCJ. Similarly, in invertebrates the SJ strands turn by 90 degrees when approaching a tricellular corner, forming three parallel limiting strands that surround the tricellular juncture space, resembling the central sealing element in vertebrates. Within and perpendicular to the vertical juncture space are a series of diaphragms, which appear linked not only to three limiting septa, but also to the three cell corner membranes, thereby forming true tricellular contacts (Byri, 2015).

    Despite the fundamental biological importance of TCJs, only few of their components are known, and the mechanism of their localized assembly at three-cell vertices is not understood. In vertebrates, the Occludin family protein Tricellulin localizes to TCJs and is recruited there by lipolysis-stimulated lipoprotein receptor (LSR) and related proteins (ILDR1 and ILDR2). Additionally, the cytoplasmic PDZ-domain-containing protein Tjp2iso3 associates with Tricellulin in Sertoli cells. The Neuroligin-like transmembrane protein Gliotactin in Drosophila is the only TCJ protein characterized so far in invertebrates. It is not clear what kind of cues direct the accumulation of these proteins at tricellular vertices, and the features of known TCJ proteins do not explain the distinct structure of three-cell contacts observed by EM. This study describes a transmembrane protein, Anakonda (Aka), which accumulates at TCJs in Drosophila epithelia. Aka can initiate TCJ assembly and may do so through its large extracellular domain, which exhibits an unusual triple-repeat structure (Byri, 2015).

    Ultrastructural analyses revealed a unique junctional architecture at points in epithelia where three cells meet. Yet, only few TCJ-specific proteins are known, and how they assemble into a tightly localized complex exclusively at tricellular vertices is not understood. This study describes transmembrane protein Anakonda, which is shown to play a critical role in TCJ assembly and epithelial barrier formation. First, Aka was shown to localize to TCJs in ectodermal and endodermal epithelia, suggesting that Aka is a core TCJ component. Second, Aka was demonstrated to be specifically required for the assembly and correct geometry of tricellular, but not of bicellular SJs, and Aka was shown to localize to TCJs independently of bicellular SJs. Third, Aka is required for recruiting or maintaining Gli at tricellular vertices. Conversely, Gli is not required for TCJ localization of Aka. Fourth, Aka mis-expression causes premature accumulation of Gli at TCJs, indicating that Aka acts upstream of Gli in initiating TCJ formation (Byri, 2015).

    Aka protein contains a large extracellular domain with a conserved tripartite repeat structure. This unique structure distinguishes Aka from other known TCJ proteins. Tricellulin, a four-pass transmembrane protein in vertebrates, localizes to the central sealing elements, suggesting that it participates in the specialized bicellular contacts that surround each three-cell vertex. Tricellulin is recruited to TCJs by the immunoglobulin domain transmembrane protein LSR, but how LSR localizes to TCJs is still unclear. In Drosophila, Gli, a transmembrane protein with a cholinesterase domain lacking catalytic activity, accumulates at TCJs, but is also involved in bicellular SJ organization. Phosphorylation-dependent endocytic turnover and indirect association with Dlg are required for accumulation of Gli at TCJs. However, the mechanisms underlying TCJ-specific localization are not understood for Gli or any other TCJ protein (Byri, 2015).

    The results suggest that Aka acts at an early step during TCJ assembly by recruiting other TCJ components, including Gli, to tricellular vertices. TCJ localization of Aka and Gli does not require Aka’s C terminus, including the PDZ-binding motif. Similarly, Gli was found to localize to TCJs independently of its PDZ-binding motif. These findings suggest that Aka and Gli might be targeted to TCJs through their extracellular domains, rather than through a cytoplasmic localization machinery. Because mislocalized Aka protein is not sufficient to recruit Gli to ectopic locations, Aka might not interact directly with Gli, or only do so in the context of TCJs. Consistent with this notion, we were not able to detect co-immunoprecipitation of Aka and Gli. Interestingly, overexpression of Gli or absence of Aka causes Gli to spread from TCJs to BCJs. Thus, Gli does not have an intrinsic propensity to localize to TCJs, but by default localizes to the apicolateral membrane domain occupied by SJs along the cell perimeter. TCJ accumulation of Gli therefore requires (1) association with the apicolateral membrane and (2) Aka-dependent recruitment of Gli specifically to TCJs. The findings show that unlike Gli, Aka does not depend on bicellular SJs for its localization to TCJs, and suggesting that Aka protein might have intrinsic properties that lead to its accumulation at TCJs (Byri, 2015).

    Tricellular vertices display a unique geometry where three plasma membranes are in close proximity and at fixed angles. The exceptional curvature of the plasma membrane at these sites implies distinct physical properties compared to bicellular contacts. Intriguingly, it was found that TCJ assembly depends on Aka expression in all three cells adjoining a vertex, suggesting that extracellular interactions between Aka molecules from different cells are essential for TCJ formation. Considering the size of Aka's extracellular domain (303 kDa), it is likely that a single Aka protein spans the entire TCJ canal (25–30 nm), although shorter Aka isoforms may constitute different structures. Shorter Aka species might carry out functions that may or may not be related to the function of full-length Aka at TCJs. Alternatively, Aka fragments may reflect turnover of TCJ complexes, e.g., during junctional remodeling (Byri, 2015).

    It is tempting to speculate that the three repeat regions in full-length Aka protein, with apparent similar domain organization, could make equal contacts with the three cell corners in the plane perpendicular to the central TCJ canal. Possibly, interaction with membrane components in the tricellular region occurs via the three SR domains, since such domains are found in receptors that recognize a wide range of molecular patterns, including surface proteins, carbohydrates, lipids, lipopolysaccharides, and peptidoglycans associated with pathogens or apoptotic cells. Because such three-way contacts may, for steric reasons, only be possible at vertices, they might selectively stabilize Aka complexes at these sites. Computer simulations suggest that the enrichment of Aka at three-cell vertices could theoretically be enhanced by the reduction of dimensionality in the tricellular region, which may promote stacking interactions between Aka molecules within the central TCJ canal. This idea is consistent with the regular structure of the TCJ diaphragms and the equal dihedral angles near TCJs as observed with electron microscopy. Stacking might occur with Aka molecules rotated by 120 or 240 degrees within the stack, perhaps in a helical array, depending on the contributing cell. Notably, such a scenario would explain the finding that TCJ formation requires Aka protein production by all three cells adjoining a vertex. A high priority of future studies will be to investigate the arrangement of Aka proteins at TCJs. Together, the current results on Aka localization, the geometry of three-cell vertices, and the triple-repeat structure of Aka protein suggest a mechanism of TCJ formation, which is promoted by self-assembly of Aka at tricellular contacts. Such self-assembly might additionally involve interactions with other membrane-associated or extracellular components, and could cooperate with bicellular adhesion molecules that zip up bicellular contacts. It will be interesting to test whether the geometry of tricellular vertices and the specific properties of Aka protein are sufficient to direct its accumulation to TCJs. Conversely, perturbed TCJ geometry upon loss of Aka may have long-range effects, such as the loss of cell-cell adhesion that this study observed in late-stage aka embryos. Interestingly, depletion of Tricellulin from mammalian cells affects cell shape and the F-actin network, suggesting that TCJs may in fact have organizing activity on the entire cell (Byri, 2015).

    The existence of aka homologs in invertebrates and cephalochordates correlates with the presence of SJs in these groups. However, the proposed self-assembly model for TCJ formation in Drosophila may apply also to vertebrates, although the corresponding proteins remain to be discovered. A better understanding of TCJ assembly will be a key step toward elucidating how these poorly characterized cellular structures provide epithelial barrier function, while at the same time allowing the passage of migrating lymphocytes, metastatic cells, and intracellular pathogens (Byri, 2015).

    Local and tissue-scale forces drive oriented junction growth during tissue extension

    Convergence-extension is a widespread morphogenetic process driven by polarized cell intercalation. In the Drosophila germ band, epithelial intercalation comprises loss of junctions between anterior-posterior neighbours followed by growth of new junctions between dorsal-ventral neighbours. Much is known about how active stresses drive polarized junction shrinkage. However, it is unclear how tissue convergence-extension emerges from local junction remodelling and what the specific role, if any, of junction growth is. This study reports that tissue convergence and extension correlate mostly with new junction growth. Simulations and in vivo mechanical perturbations reveal that junction growth is due to local polarized stresses driven by medial actomyosin contractions. Moreover, it was found that tissue-scale pulling forces at the boundary with the invaginating posterior midgut actively participate in tissue extension by orienting junction growth. Thus, tissue extension is akin to a polarized fluid flow that requires parallel and concerted local and tissue-scale forces to drive junction growth and cell-cell displacement (Collinet, 2015).

    A Par-1-Par-3-centrosome cell polarity pathway and its tuning for isotropic cell adhesion

    To form regulated barriers between body compartments, epithelial cells polarize into apical and basolateral domains and assemble adherens junctions (AJs). Despite close links with polarity networks that generate single polarized domains, AJs distribute isotropically around the cell circumference for adhesion with all neighboring cells. How AJs avoid the influence of polarity networks to maintain their isotropy has been unclear. In established epithelia, trans cadherin interactions could maintain AJ isotropy, but AJs are dynamic during epithelial development and remodeling, and thus specific mechanisms may control their isotropy. In Drosophila, aPKC prevents hyper-polarization of junctions as epithelia develop from cellularization to gastrulation. This study shows that aPKC does so by inhibiting a positive feedback loop between Bazooka (Baz)/Par-3, a junctional organizer, and centrosomes. Without aPKC, Baz and centrosomes lose their isotropic distributions and recruit each other to single plasma membrane (PM) domains. Surprisingly, loss- and gain-of-function analyses show that the Baz-centrosome positive feedback loop is driven by Par-1, a kinase known to phosphorylate Baz and inhibit its basolateral localization. Par-1 was found to promote the positive feedback loop through both centrosome microtubule effects and Baz phosphorylation. Normally, aPKC attenuates the circuit by expelling Par-1 from the apical domain at gastrulation. The combination of local activation and global inhibition is a common polarization strategy. Par-1 seems to couple both effects for a potent Baz polarization mechanism that is regulated for the isotropy of Baz and AJs around the cell circumference (Jiang, 2015).

    The identification of Par-1 as an inducer of Baz-centrosome co-recruitment is surprising given its well-established role in inhibiting Baz complex formation in Drosophila, C. elegans, and mammalian systems. It is proposed that Par-1 contributes to both global inhibition and local promotion of Baz complex assembly, providing a simple and potent Baz polarization mechanism (Jiang, 2015).

    The Baz-centrosome positive feedback loop is evident from the specific accumulation of Baz next to cortical centrosomes, the MT requirement for Baz accumulation, the Baz requirement for centrosome recruitment, and the dynein role for drawing Baz and centrosomes together. Significantly, Par-1 is also necessary and sufficient for the loop and seems to have two direct roles. One is promotion of astral microtubules around the centrosome, an effect consistent with known effects of Par-1 on MT regulators, but requiring further elucidation in the Drosophila embryo. The other is the phosphorylation of Baz at Ser-151 and Ser-1085. These modifications have well-characterized inhibitory effects on Baz cortical association, but strikingly, they are also enriched where the Baz-centrosome positive feedback loop occurs and appear necessary for Baz entry into the loop. It is speculated that phospho-regulated Baz-14-3-3 protein interactions mediate further protein interactions, or induce conformational changes, important for Baz-MT association. Indeed, 14-3-3 proteins can bridge MT motors, a Par-3 conformational change induces direct MT binding, Par-3 directly binds a dynein subunit, and other links to MTs are known (Jiang, 2015).

    Although the Par-1-Par-3-centrosome pathway can be a potent Baz polarization mechanism, it is normally attenuated within a homeostatic system. During early cellularization, Par-1 localizes over the entire PM and presumably phosphorylates Baz and MT regulators. In response, it is proposed that Baz is continually displaced and diffuses over the PM but is additionally primed for MT interactions. Simultaneously, the two centrosomes found atop each nucleus would provide the positional information for localizing Baz around the apical circumference through dynein-mediated MT associations. As Baz accumulates, it recruits aPKC to the apical domain, from where aPKC then displaces Par-1. Normally, this Baz-aPKC-Par-1 negative feedback loop seems to keep the Par-1-Baz-centrosome pathway in check. In the absence of aPKC, the Par-1- Baz-centrosome pathway continues unabated, leading to excessive Baz and centrosome polarization, loss of AJ isotropy, and later epithelial dissociation (Jiang, 2015).

    Intriguingly, focused accumulations of Par-3 and AJs colocalize with cortical centrosomes during C. elegans intestinal development and during zebrafish collective cell migration. Moreover, Par-1 induces centrosomal MT interactions with AJs during human liver lumen formation in vitro and is needed for Baz-centrosome associations during the asymmetric division of Drosophila germline stem cells. Thus, induction of the Par- 1-Par-3-centrosome pathway, with regulated shifts to aPKC or Par-1 activities, may be generally relevant to developmental transitions of animal tissues (Jiang, 2015).

    Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium

    Apoptosis is a mechanism of eliminating damaged or unnecessary cells during development and tissue homeostasis. During apoptosis within a tissue, the adhesions between dying and neighboring non-dying cells need to be remodeled so that the apoptotic cell is expelled. In parallel, the contraction of actomyosin cables formed in apoptotic and neighboring cells drive cell extrusion. To date, the coordination between the dynamics of cell adhesion and the progressive changes in tissue tension around an apoptotic cell is not fully understood. Live imaging of histoblast expansion, which is a coordinated tissue replacement process during Drosophila metamorphosis, shows remodeling of adherens junctions (AJs) between apoptotic and non-dying cells, with a reduction in the levels of AJ components, including E-cadherin. Concurrently, surrounding tissue tension is transiently released. Contraction of a supra-cellular actomyosin cable, which forms in neighboring cells, brings neighboring cells together and further reshapes tissue tension toward the completion of extrusion. A model is proposed in which modulation of tissue tension represents a mechanism of apoptotic cell extrusion, and would further influence biochemical signals of neighboring non-apoptotic cells (Teng, 2016).

    This study reports the temporal sequence of events during apoptotic cell extrusion, with a focus on the remodeling of AJs, the cytoskeleton, and mechanical tension. After caspase-3 starts to be activated in the polyploid larval epithelial cells (LECs), those undergoing apoptosis initiate apical constriction. It was reasoned that the initiation of this constriction could be due to a combination of actomyosin cable formation in the dying cell and the activity of caspase-3, which assists in the upregulation of actomyosin contractility. Indeed, it has been shown in tissue culture that the cleavage of Rho associated kinase by caspase- 3 is involved in phosphorylation and activation of myosin light chain, which regulates actomyosin contractility. It is proposed that the actomyosin cable that forms in apoptotic LECs is responsible for the early stages of apoptotic cell extrusion. During apical constriction, the level of AJ components including E-cad strongly reduced in a caspase-3-dependent manner. In the neighboring non- dying cells, this reduction is found only at the interface between the apoptotic cell and its neighbors. Since caspase-3 is not activated in the neighboring cells, it is speculated that the reduction of E-cad is a consequence of a loss of trans-interactions between E-cad of the neighboring cell, and E-cad of the apoptotic cell, which undergoes caspase-3-dependent cleavage. This often, but not always, leads to plasma membrane separation, which is suggestive of a loosening of AJ-dependent adhesion. It has been reported that anillin organizes and stabilizes actomyosin contractile rings at AJs and its knock-down is associated with a reduction of E-cad and β-Catenin levels at AJs, leading to AJ disengagement. A gradual decrease in the level of E-cad, and a gradual increase in MyoII accumulation in apoptotic cells was observed prior to the strong reduction of E-cad levels. This lead to the hypothesis that mechanical tension exerted on the cell interface between apoptotic LECs and neighboring cells by the contraction of the actomyosin cable, which forms in the apoptotic cell, is large enough to rupture the weakened contacts between plasma membranes at AJs upon the strong reduction of E-cad levels (Teng, 2016).

    Interestingly, and by contrast, there are cases when AJs are not disengaged even after the level of E-cad is reduced. In these cases the cells exhibit a separation of actomyosin cables from the membrane. It is speculated that the state of cell-cell contacts at AJs, i.e., whether they will disengage or remain engaged during apoptosis, is dependent on which of the following links is weaker: The link between two plasma membranes, or the link between the plasma membrane and the actomyosin cable. Both of these links would be weakened by a strong, albeit incomplete, reduction of E-cad levels. When the former is weaker than the latter, the two plasma membranes could be detached. When the former is stronger than the latter, the two plasma membranes could remain in contact, and the actomyosin cable could be detached from the plasma membrane (Teng, 2016).

    In parallel with the reduction of E-cad levels and the associated release of tension, a supra-cellular actomyosin cable begins to form in neighboring cells. These observations prompted a speculation that the release of tissue tension triggers MyoII accumulation in neighboring cells. Subsequent contraction of this outer ring helps to reshape tissue tension, which is transiently released when E-cad is reduced. As a consequence, the neighboring cells are stretched. Upon completion of apical constriction, neighboring non-apoptotic cells form de novo AJs and the stretched cells undergo cell division and/or cell-cell contact rearrangement. These processes allow a relaxation of the high tension associated with the stretching of cells. Finally, measurements of caspase-3 activity, and the observations from caspase inhibition experiments, lead to a conclusion that the characteristics associated with apoptotic cell extrusion reported in this study are the consequences of the apoptotic process, rather than the cause (Teng, 2016).

    In addition to the progressive remodeling of AJs and modulation of tissue tension during apoptosis, the mechanical role was examined of apoptosis 'apoptotic force' in tissue morphogenesis, which has been proposed, demonstrated, and discussed. It was shown that the mechanical force generated by the contraction of actomyosin cables formed when LECs undergo apoptosis, especially boundary LECs, promotes tissue expansion, along with histoblast proliferation and migration. Nonetheless, it cannot be ruled out that this apical contraction is in part driven by a decrease in cell volume, which can be triggered by caspase activation. Intriguingly, it was found that apoptosis of non-boundary LECs did not affect tissue expansion. This raised the possibility that the mechanical influence of apoptosis in neighboring tissues is dependent not only on the physical connections between cells, but also on the mechanical properties of cells, including cell compliance. If a tissue is soft, for instance, the tensile forces generated by apoptotic process could be absorbed by nearest-neighbor cells and would not propagate to cells further than a single cell away. It is speculated that the apoptotic process could mechanically contribute to cell death-related morphogenesis, only when apoptosis takes place at optimal mechanical properties of a tissue (Teng, 2016).

    This study presents a framework for understanding how cell adhesions and tissue tension are progressively modulated during apoptosis in a developing epithelium. It is concluded that tissue tension reshaping, including the transient release of tension upon a reduction in the levels of AJ components, represents a mechanism of apoptotic cell extrusion. It would be important to explore how this transient modulation in mechanical tension would further influence the biochemical nature of neighboring non-apoptotic cells (Teng, 2016).

    Wave propagation of junctional remodeling in collective cell movement of epithelial tissue: Numerical simulation study

    During animal development, epithelial cells forming a monolayer sheet move collectively to achieve the morphogenesis of epithelial tissues. One driving mechanism of such collective cell movement is junctional remodeling, which is found in the process of clockwise rotation of Drosophila male terminalia during metamorphosis. However, it still remains unknown how the motions of cells are spatiotemporally organized for collective movement by this mechanism. Since these moving cells undergo elastic deformations, the influence of junctional remodeling may mechanically propagate among them, leading to spatiotemporal pattern formations. Using a numerical cellular vertex model, this study found that the junctional remodeling in collective cell movement exhibits spatiotemporal self-organization without requiring spatial patterns of molecular signaling activity. The junctional remodeling propagates as a wave in a specific direction with a much faster speed than that of cell movement. Such propagation occurs in both the absence and presence of fluctuations in the contraction of cell boundaries (Hiraiwa, 2017).


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    Zygotically transcribed genes

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