The Interactive Fly

Zygotically transcribed genes

Genes coding for junctional proteins

  • Neuromuscular junction
  • Development of junctions and distribution of proteins in junctions
  • 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
  • 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
  • Diverse integrin adhesion stoichiometries caused by varied actomyosin activity
  • Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex
  • 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

    Adherens Junctions
  • The tumor suppressor CYLD controls epithelial morphogenesis and homeostasis by regulating mitotic spindle behavior and adherens junction assembly
  • Atonal and EGFR signalling orchestrate rok- and Drak-dependent adherens junction remodelling during ommatidia morphogenesis
  • Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye
  • Adherens junction distribution mechanisms during cell-cell contact elongation in Drosophila
  • Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly
  • Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor
  • The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis
  • Adherens junction-associated pores mediate the intercellular transport of endosomes and cytoplasmic proteins
  • Adherens junction length during tissue contraction is controlled by the mechanosensitive activity of actomyosin and junctional recycling
  • Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis
  • Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth

    Septate Junctions
  • Pasiflora proteins are novel core components of the septate junction
  • A tetraspanin regulates septate junction formation in Drosophila midgut
  • Bark beetle controls epithelial morphogenesis by septate junction maturation in Drosophila
  • Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues
  • The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions

    Myotendinous junctions
  • 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
  • Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila
    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, and myotendenous 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).

    Bark beetle controls epithelial morphogenesis by septate junction maturation in Drosophila

    Epithelial tissues separate body compartments with different compositions. Tight junctions (TJs) in vertebrates and septate junctions (SJs) in invertebrates control the paracellular flow of molecules between these compartments. This epithelial barrier function of TJs and SJs must be stably maintained in tissue morphogenesis during cell proliferation and cell movement. This study shows that Bark beetle (Bark), a putative transmembrane scavenger receptor-like protein, is essential for the maturation but not the establishment of SJs in Drosophila. Embryos that lack bark establish functional SJs, but due to rudimentary septae formation during subsequent embryonic development, these become non-functional. Furthermore, cell adhesion is impaired at the lateral cell membrane and the core protein complexes of SJs are mis-localised, but appear to form otherwise normally in such embryos. A model is proposed in which Bark acts as a scaffold protein that mediates cell adhesion and mounting of SJ core complexes during cell rearrangement in tissue morphogenesis (Hildebrandt, 2015).

    This study describes the characterisation and functional analysis of the putative scavenger receptor protein Bark during epithelial morphogenesis. Bark was found to be required for the maturation but not the establishment of SJs. Furthermore, Bark is involved in epithelial cell adhesion during SJ maturation (Hildebrandt, 2015).

    The initiation and establishment of SJ formation appear to be independent of bark activity. This conclusion is based on the observation that the ultrastructural analysis of developing wild-type and bark mutant SJs is similar during stage 15 and 16. At stage 15, single septae have already formed in tracheal and epithelial cells of wild-type and bark mutant embryos, even though the typical ladder-like septae structure of later stages is not yet established. At stage 16 electron-dense material appears in SJs of bark mutant embryos as found in wild-type embryos. Thus, the ultrastructural analysis suggests a normal formation of SJs in bark mutants until stage 16. In addition, integral SJ components are also normally localised in bark mutants at stage 15 suggesting correct assembly of the SJs. Furthermore, the exocytosis of Serpentine and Vermiform into the tracheal lumen mediated by SJs during stage 16 is not affected in bark mutants, suggesting normal SJ function independent of bark. Remarkable is the delay of tracheal dorsal trunk elongation in bark mutants. Embryos in which the initial SJ formation is affected as found in mega display dorsal trunk elongation about two hours earlier than observed in bark mutants. Thus, establishment of SJs seems to be normal in bark mutants, since dorsal trunk elongation is indicative for disrupted SJs. Taken together, these results suggest that Bark does not critically participate in the initial morphogenesis and functional properties of SJs (Hildebrandt, 2015).

    In contrast, subsequent SJ maturation (stage 17) strongly depends on Bark. Rhodamine-dextran injection experiments revealed that the transepithelial barrier function of SJs is compromised in bark mutants. In such embryos the wild-type ladder-like SJ structure is disrupted, only rudimentary septae are formed and cell adhesion is impaired. This phenotype is distinct from the archetypal SJ phenotype observed in mutants of the SJ core complexes, e.g., in mega or Nrg mutant embryos. In these mutants the septae are either reduced in number or are absent, while cell adhesion seems not to be affected, i.e., the uniform spacing between the plasma membranes of adjacent epithelial cells is maintained. Individual bark mutant embryos develop a great diversity of septae ranging from no detectable septae in places where septae would normally form to rudimentary septae and up to wild-type like septae. Furthermore, such mutant embryos show an erratic spacing of the epithelial plasma membranes; in extreme cases the plasma membranes detach from each other resulting in gaps between the cells. Such cell adhesion defects of bark epithelial cells have also been observed in mutants of Gliotactin, a marker for tricellular junctions, but so far in no other mutant that affects SJs (Hildebrandt, 2015).

    The phenotypic differences between bark and archetypal SJ lack-of-function mutant embryos are also observed in gain-of-function experiments. Overexpression of Bark does not interfere with normal development or the barrier function of SJs and rescues the bark mutant phenotype. In contrast, overexpression of other SJ components causes mis-localisation of the components and a disruption of the barrier function. Thus, it has been proposed that SJ components are functionally interdependent. The observation that the level of Bark is not critical for bark function supports the argument that bark mediates a distinct role during SJ maturation in late embryogenesis (Hildebrandt, 2015).

    The detachment of lateral cell membranes, which occasionally deteriorate and form gaps between the epithelial cells of bark mutant embryos, indicates that Bark plays also a role in epithelial cell adhesion in addition to its function in SJ integrity. As Bark represents a large transmembrane protein, it may mediate the cell-cell adhesion through homophilic interactions. This possibility was tested, and putative homophilic Bark binding was analyzed in a cell aggregation assay, but no homophilic Bark binding was detected. However, homophilic binding in the embryo might not been detected, since posttranslational modifications may not occur in the cell culture system. Such modifications could include the attachment of sugar moieties via several potential glycosylation sites noted in the extracellular Bark domain and/or binding of sugar moieties to Bark via its lectin domain. Heterophilic Bark binding with an already identified SJ component is also not very likely, since the lack of such components has no effect on cell adhesion in the region of SJs. Thus, it is speculated that Bark mediates its cell adhesion function by homophilic binding, which depends on specific posttranslational modifications, or in conjunction with an unknown interaction partner (Hildebrandt, 2015).

    The distinctive feature of SJs is that they must maintain their functional properties, in particular the control of the transepithelial barrier function, while cells rearrange during tissue morphogenesis. Thus, epithelial layers must be able to simultaneously alter cell-cell-contacts, shuffle SJ protein components and establish functional SJ structures, which create distinct fluid compartments. Given these features, the questions that come to mind are: What is the molecular basis of these distinct requirements and what is the role of Bark? (Hildebrandt, 2015).

    The recent finding of a stable SJ multiprotein core complex is an important step in understanding SJ protein dynamics. The SJ core complex is preassembled intracellularly before its incorporation into the SJs at the plasma membrane. Interestingly, the core complex seems to be stable even in cells actively rearranging their contacts (Oshima, 2011). FRAP experiments indicate that the core complex is also stable in bark mutants, but the morphology and function of SJs are severely affected. The mis-localisation of SJ core components along the basolateral cell membrane in stage 17 bark mutants suggest that the SJ core complexes do not properly coalesce to assemble SJs in the apicolateral membrane region. This observation is consistent with previous binding studies suggesting a direct interaction of Bark with Mega or another SJ core protein (Jaspers, 2012). Thus, it is speculated that Bark provides a scaffold-like matrix serving as a platform for the SJ core complexes, which assemble into functional SJs. The potential Bark matrix is not necessary for establishment of SJs, but becomes essential during tissue morphogenesis, i.e., during SJ maturation to ensure that SJ core complexes remain well-ordered and able to sustain the epithelial barrier function. This role of Bark becomes particularly apparent during morphogenesis of the embryonic tracheal system, which is established by extensive cell shape changes and cell-cell rearrangements. Such a possible role would explain why a lack of Bark leads not only to cell adhesion defects and SJ failure, but also to a severely mis-shapen, convoluted tracheal system (Hildebrandt, 2015).

    A scenario that would then allow for cell-cell rearrangement is that Bark is regulated in a way that it may detach from defined regions within the SJs and thereby reduce both cell adhesion and SJ integrity, which results in SJ core complex release. Such opened SJ sub-regions may in turn tolerate cell-cell rearrangements, while nearby sub-regions may still contain functional septae that establish the transepithelial barrier function. Consistent with this view is the observation that Bark localises to recycling endosomes within intracellular compartments. Thus, Bark may shuttle from sites of SJ breakdown to sites of SJ assembly via the recycling endosomal pathway. At the site of SJ assembly Bark might mediate dual functions in cell adhesion and providing anchor points for the SJ core complexes (Hildebrandt, 2015).

    Coordination of septate junctions assembly and completion of cytokinesis in proliferative epithelial tissues

    How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia (Daniel, 2018).

    How permeability barrier function is maintained when epithelial cells divide is largely unknown. This study has investigated how the bicellular septate junctions (BSJs) and tricellular septate junctions (TSJs) are remodeled throughout completion of cytokinesis in Drosophila epithelia. Following cytokinetic ring constriction, the midbody assembles, matures within SJs, and is displaced basally in two phases. In a first slow phase, the neighboring cells remain connected to the dividing cells by means of SJ-containing membrane protrusions pointing to the maturing midbody. Fluorescence recovery after photobleaching (FRAP) experiments revealed that SJs within the membrane protrusions correspond to the old SJs that were present prior to cytokinesis. In contrast, new SJs are assembled below the adherens junctions and spread basally to build a new belt of SJs in a manner analogous to a conveyor belt. Loss of function of a core BSJ component, the Na+/K+-ATPase pump Nervana 2 subunit, revealed that the apical-to-basal spread of BSJs drives the basal displacement of the midbody. In contrast, loss of the TSJ protein Bark beetle indicated that remodeling of TSJs is rate limiting and slowed down midbody migration. In the second phase, once the belt of SJs is assembled, the basal displacement of the midbody is accelerated and ultimately leads to abscission. This last step is temporally uncoupled from the remodeling of SJs. It is proposed that cytokinesis in epithelia involves the coordinated polarized assembly and remodeling of SJs both in the dividing cell and its neighbors to ensure the maintenance of permeability barrier integrity in proliferative epithelia (Daniel, 2018).

    Epithelial growth requires the formation of cell-cell junctions and physical separation of daughters upon cell division. This study has characterized a multicellular mechanism that coordinates completion of cytokinesis and de novo formation of SJ in both cuboidal and columnar epithelia: (1) following actomyosin ring constriction, the midbody forms just basal to the AJ within the SJ and matures into an intercellular bridge that is displaced basally. (2) Neighboring interphase cells maintain SJ contacts, the finger-like protrusions connecting to the maturing midbody of the dividing cells. At this position, the ménage à quatre formed contains pre-existing SJs and is stable. (3) Novel BSJs are assembled below the AJs and above the finger-like protrusions and spread toward the basal side, thereby driving the basal displacement of the finger-like protrusion plus midbody. Once the new BSJ is complete, the midbody is positioned outside the SJ belt and basal displacement accelerated. Overall, it is proposed that this multicellular process is to ensure the maintenance of the permeability barrier throughout cytokinesis (Daniel, 2018).

    The data show that, as in embryos, newly synthesized SJ components assemble into stable protein complexes exhibiting a low rate of diffusion, endocytosis, and recycling. These stable complexes assemble below the AJs and then propagate toward the basal pole, in a manner analogous to a conveyor belt, both in interphase and during cytokinesis (Daniel, 2018).

    Loss of the core SJ component Nrv2 prevents both assembly of stable BSJ complexes and basal displacement of the midbody. Thus, it is proposed that the apico-basal flux of newly assembled BSJs is the propelling force for the basal displacement of the finger-like protrusions connected to the midbody. The conveyor belt model first predicts that newly synthesized and/or slowly recycling SJ components are delivered apically in the vicinity of AJs. The model also predicts that SJ disassembly occurs at the basal rim of SJ belt. Future work will determine what regulates the polarized traffic of SJ components and the disassembly of SJs to control both the positioning and thickness of the SJ belt (Daniel, 2018).

    This study sheds light on the remodeling of TSJs at cytokinesis. Bark and Gli are initially detected as puncta next to the midbody, prior to the pearl necklace distribution along the finger-like protrusions. These dotted structures could represent new TSJs, as, at this location, a new three-way contact is formed between the two daughter cells and a neighbor. However, FRAP analyses suggest that these punctae do not contain exchangeable components and are not moving laterally. Moreover, the appearing of Gli and Bark punctae in mitotic cells is faster (20 min post-anaphase) than the de novo assembly of TSJs in interphase cells (~80% recovery in 90 min). Analyses of the punctae by TEM will be necessary to test whether they are bona fide TSJs or components in the process of assembly. Alternatively, the old TSJs between dividing cells and neighbors prior to mitosis could undergo a change in distribution from a uniform distribution into clusters, giving rise to the pearl necklace. Redistribution could represent an intermediate step in TSJ disassembly and may explain the fast kinetics of assembly of pearl necklace structure. Future work using, for example, photoconvertible probes would help address this issue (Daniel, 2018).

    Finally, loss of Bark resulted in acceleration of the midbody basal displacement. The de novo assembly of TSJs in the pearl necklace structure could be the rate-limiting step that imposes the slower speed of basal displacement. Alternatively, if pearl necklace represents the remodeling of prior TSJ connections with neighboring cells, TSJ disassembly may be the rate-limiting step. Regardless, the data argue that the TSJ components present in the pearl necklace slow down the midbody basal displacement driven by the BSJ-mediated conveyor belt (Daniel, 2018).

    This study reveals differences in midbody maturation and organization of the intercellular bridge between isolated and epithelial cells. In epithelial cells, the densely packed microtubule array that normally sits in the center of the intercellular bridge and the Flemming body were not observed. Instead, an intercellular bridge was found with a uniform density of microtubules that progressively disappear. The electron-dense material detected along the plasma membrane of the intercellular bridge could act as a rigid scaffold preventing membrane constriction. The SJ core components plus MyoII and Pnut at the interface made between the intercellular bridge and the finger-like protrusions might be part of the electron-dense material and therefore contribute to membrane rigidity. Alternatively, this scaffold could prevent tension release within the intercellular bridge, therefore preventing premature abscission (Daniel, 2018).

    It was also found that abscission is asymmetric, with the bridge remnant internalized and then degraded in one of the daughter cells in a mode similar to the internalization and autophagy described for isolated cells. However, it cannot be excluded that abscission could also occur on both sides of the bridge, leading to its release followed by its recapture by endocytosis in some epithelial cells (Daniel, 2018).

    Finally, whereas epithelial cells seem to be set for abscission about 40 min following the onset of anaphase, cytoplasmic isolation had not occurred 5 hr later. Photoconversion data furthermore suggest that cytoplasmic isolation takes place about one hour prior to one of the sisters entering mitosis, possibly at the mid to late G2 phase as reported for germline stem cells (Daniel, 2018).

    Despite the opposite apico-basal positioning of permeability barrier relative to the mechanical barrier, a number of similarities are encountered in vertebrates' and invertebrates' epithelial cells. First, mechanical barrier transmission during epithelial cytokinesis is a multicellular process. Second, the permeability barrier is also maintained throughout epithelial cytokinesis. Third, the recruitment of tricellular junction components in close vicinity to the midbody supports the idea that polarized delivery, membrane composition, and topology at midbody are engineered for tricellular junction remodeling. Fourth, epithelial cytokinesis is polarized along the apico-basal axis, leading to the embedding of midbody en route to abscission within the permeability barrier both in vertebrates and in Drosophila. Based on these findings, it is anticipated that the coordination between permeability barrier transmission and completion of cytokinesis in Drosophila is conserved in vertebrates for maintenance of tissue integrity in proliferative epithelia (Daniel, 2018).

    The Drosophila blood-brain barrier adapts to cell growth by unfolding of pre-existing septate junctions

    The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. This study used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. Septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish (Babatz, 2018).

    This study has dissected the mechanisms underlying growth of occluding septate junctions in non-dividing Drosophila cells. In primary and secondary epithelia, septate junction strands are mostly generated during embryonic stages. Septate junction protein complexes show a remarkable stability and persist over several days. In primary epithelia, newly synthesized linear and unbranched septate junction strands are added basally to the apically localized adherens junctions. In contrast, in secondary epithelial cells of the Drosophila blood-brain barrier, such newly synthesized septate junction strands are added on both sides of the pre-existing septate junctions. This process depends on the G-protein coupled receptor Moody and F-actin formation to integrate preformed vesicular septate junctions into existing strands. In addition, it was shown that moody mutants activate compensatory mechanisms to cope with the loss of junctional integrity. moody mutant cells activate expression of septate junction proteins and form an excess of membrane cell-cell interdigitations, which establish sufficient barrier function (Babatz, 2018).

    Septate junctions are assembled by an astonishing number of proteins in an almost crystalline appearance with considerable stability. This study has demonstrated that septate junctions are indeed stable for several days and that most of the septate junction proteins are generated during early larval development. This is also confirmed by analyzing the developmental expression profile of all septate junction proteins. Moreover, FRAP data are consistent with the notion that septate junctions are arranged in unbranched, long lines connected to tricellular junctions (TCJs). Nevertheless, it is important to note that septate junction strands appear to mature during larval development, and, for example, the loosely associated protein Fas3 is constantly added to the growing septate junctions. Finally, septate junctions of the blood-brain barrier are more stable compared to those found in primary epithelia and use a different mechanism of new strand integration, suggesting alternative characteristics of septate junctions in different tissues (Babatz, 2018).

    When epithelial cells grow or divide, the occluding septate junctions have to be adjusted to the changing cell geometry. During growth of the lateral cell membrane, this could imply an opening of existing strands and the subsequent insertion of new septate junction proteins. However, in freeze fracture studies of reorganizing tissues of Rhodnius or hydra, no evidence for such processes was detected. Consequently, it was postulated that existing septa can be redistributed in the plane of the membrane in cell contact areas, meaning septate junction strands stretch across the entire domain upon changes in cell geometry. This study used high-resolution imaging to show that indeed septate junctions are formed in a wavy manner during embryonic stages and unfold during the larval growth period (Babatz, 2018).

    The corrugated organization of septate junctions appears to be a more general feature. Salivary gland cells have a conical shape, and the perimeter of the cell increases about two times from apical to basal. Similar to the growing subperineurial glial cells, septate junctions are formed as folded structures at the apical side and stretch out as they move to more basal positions. Likewise, during Drosophila oocyte development, follicle cells initially form corrugated adherens junctions that become straight when cells expand. A similar unfolding of tig (Babatz, 2018).

    The unfolding of the bicellular septate junction strands requires force and possibly anchoring of strands to a fixed point. Such a fixed point could be a tricellular junction, which also allows force generation during cytokinesis. Classical freeze fracture studies as well as FRAP data suggest that bicellular occluding junctions are connected to tricellular junctions. This connection might be mediated by the tricellular junction proteins Bark beetle (Anakonda) and Gliotactin, which can also interact with bicellular septate junction proteins (Babatz, 2018).

    During epithelial development, tissue convergence and extension correlate with adherens junction dynamics due to local actomyosin-based contractions. However, the initial corrugated-appearing septate junction strands suggest that the initial mechanical forces do not act on the establishment of septate junctions. Only when the cell grows and thus expands its lateral plasma membrane, the pre-established junctions are straightened. When subperineurial glial growth is suppressed, the initial corrugated shape of the septate junctions is not changed. When additional growth of the subperineurial glia is triggered by, for example, excessive neuroblast proliferation as seen in l(2)gl mutant animals, only rare ruptures of septate junction strands were noticed. In contrast, additional septate junction strands appear to be added to compensate for the increased cell growth. This suggests that the growing subperineurial glia senses barrier integrity, and any leakage results in the addition of new septate junction strands. This plasticity appears to depend on gene activity, since upon block of polyploidization in subperineurial glial cells, septate junctions are interrupted. The ability of subperineurial glial cells to react to changing barrier requirements can also be seen in moody mutants, which show an increase of NrxIV expression (Babatz, 2018).

    The G-protein coupled receptor Moody is initially found along the entire plasma membrane, but after formation of septate junctions, its localization is restricted to the cell membrane facing the cortex glia with some enrichment at the cell-cell contact sites. Septate junctions form in the absence of Moody, but their arrangement in long, corrugated, and possibly anchored strands fails. Recently, special actin-rich structures have been observed along the lateral borders of the subperineurial glia that are regulated by Moody signaling. Gene dose experiments presented in this study indicate that Moody affects septate junction formation by the local activation of F-actin formation possibly to provide the physical force to integrate septate junction building blocks preformed in vesicular structures (Babatz, 2018).

    As the brain grows in size, septate junctions cannot expand in moody mutants. In consequence, the blood-brain barrier established by the subperineurial glial cells becomes leaky, which triggers the expression of additional septate junction proteins, which, however, cannot be properly integrated into the existing strands. Remarkably, moody mutant subperineurial glia also form interdigitating membrane folds, which partially restore barrier integrity. A similar excess of cell interdigitation is induced upon reduction of NrxIV expression, specifically in the subperineurial glia. Interestingly, this type of barrier is also found in several other invertebrates and primitive vertebrates. Thus, moody mutants disclose the principles of an evolutionary ancient form of the blood-brain barrier. The efficient blood-brain barrier based on long, uninterrupted septate junction strands induced by Moody then allowed the evolution of more compact nervous systems, as found in Drosophila (Babatz, 2018).

    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).

    Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila

    This study used the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere (Green, 2018).

    The adult indirect flight muscles of Drosophila have proved to be an excellent system to identify functions for integrin-associated proteins (IAPs) that are not essential for viability. The mechanical linkage between the last Z-line of each myofibril and the plasma membrane is a well ordered and multi-layered structure, ideal for elucidating the mechanisms by which actin can be organized into different structures at subcellular resolution. In the layer closest to the membrane, the integrin signaling layer, an important counterbalancing is found between IAPs, with FAK inhibiting the activation of integrin by tensin, and RSU1 inhibiting excess PINCH activity. It was discovered that the muscle actin regulatory layer (MARL) has a different composition to the fibroblast ARL, containing a mechanotransduction cascade of vinculin and filamin, which, together with WASH and the Arp2/3 complex, builds an actin-rich zone linking the adhesion machinery at the membrane to the first Z-line (Green, 2018).

    The modified terminal Z-lines [MTZ - composed of 4 zones: (1) an integrin signalling layer at the membrane; and then zones containing different actin structures-(2) a force transduction layer (FTL); (3) a muscle actin regulatory layer (MARL); and (4) the first Z-line followed by the first sarcomere] revealed both positive and inhibitory actions of FAK, with the latter consistent with the role of FAK in adhesion disassembly. Both loss of FAK and activated integrin supressed the phenotypes caused by loss of RSU1 or vinculin, but only activated integrin alleviated the defects caused by the absence of tensin, suggesting that FAK inhibition requires tensin activity, and in turn, tensin elevates integrin activity. This fits with the recent discovery that tensin contributes to the inside-out activation of integrins via talin (Georgiadou, 2017). FAK and tensin thus form a balanced cassette that is thought to respond to upstream signals to regulate integrin activity. Further work is needed to discover how tensin increases integrin activity, how this is inhibited by FAK, and what signals control this regulatory cassette. One model would have tensin activating integrin by direct binding to the β subunit cytoplasmic tail, and FAK inhibition by phosphorylation of tensin, but an alternative is that they have antagonistic roles in integrin recycling (Green, 2018).

    RSU1 is part of the complex containing ILK, PINCH and Parvin (IPP complex), and binds the 5th LIM domain of PINCH. Loss of RSU1 causes milder phenotypes than loss of ILK, PINCH or parvin, and these phenotypes have previously been interpreted as a partial loss of IPP activity. The current findings indicate that the phenotypes observed in the absence of RSU1 are due to too much PINCH activity, and therefore the role of RSU1 is to keep PINCH activity in check. This suggests that PINCH is perhaps the key player of the IPP complex, and is recruited to adhesions by integrin via ILK, and kept in check by integrin and RSU1. The importance of regulating active PINCH levels is consistent with the dosage sensitivity of PINCH: reducing PINCH partially rescues the dorsal closure defect in embryos lacking the MAPK Misshapen, and elevating PINCH rescues hypercontraction caused by loss of Myosin II phosphatase. Reducing the interaction of PINCH with ILK had unexpectedly no phenotype, but in combination with the loss of RSU1 becomes lethal; the lethality can now be interpreted as being caused by too much PINCH activity, rather than too little. Excess 'free' PINCH results in elongated membrane interdigitations and elevated paxillin levels. This suggests that PINCH has an important role at the cell cortex, consistent with cortical proteins in the PINCH interactome. Too much parvin activity also causes lethality, which is suppressed by elevating ILK levels. Thus, it is increasingly clear that the functions of IPP components need to be tightly controlled. This study gained some insight into how RSU1 inhibits PINCH activity by demonstrating that ΔLIM4, 5 PINCH still caused longer interdigitations. This rules out RSU1 blocking the binding of another protein from binding LIM5, and suggests instead that RSU1 bound to LIM5 must be inhibiting the activity of LIM1-3 (Green, 2018).

    Vinculin has a dual function in the MTZ: its head domain promotes force transduction layer (FTL; containing actin, the C-terminus of talin and vinculin) stability via binding talin, and its tail promotes muscle actin regulatory layer (MARL) formation. This analysis of the vinculin mutant by electron microscopy showed a phenotype within the electron dense layer close to the membrane that is presumed to corresponds to the integrin signalling layer. It suggests that vinculin may mediate interactions between IAPs that aid in keeping this as an even layer. The fact that the disruption to this layer is only evident on the muscle side of the interaction raises the question of how similar the integrin junctions are on the two sides of this cell-cell interaction via an intervening ECM. Many other sites of integrin-mediated adhesion in Drosophila involve integrins on both sides of the interaction and by electron microscopy the electron dense material looks similar on the two sides, and it would be expected that both sides need to resist the same forces. Even with structured illumination microscopy the two sides of the membrane cannot be resolved, but the results show that the C-terminus of talin and vinculin are not pulled away from the membrane in the adult tendon cells. This suggests either that vinculin has a different role in the tendon cell, with a different configuration, as was observed for talin in the pupal wing, or it is absent (Green, 2018).

    The vinculin tail function in MARL formation does not require that vinculin is bound to talin, but it is suspected that in the wild type it is talin-binding that converts vinculin into an open conformation, permitting the tail to trigger MARL formation with filamin, as outlined in a working model (see Model of IAP function in the IFM MTZ). A key function of vinculin tail in the MARL is to aid the mechanical opening of the filamin mechanosensitive region. This study presents evidence suggesting this is achieved by the vinculin tail anchoring the C-terminus to actin, but further work is required to determine if there is direct binding between the two proteins. Similarly, the results indicate that the Arp2/3 nucleation promoting factor WASH is part of the same pathway as filamin and acts downstream of it, but the connection between the two has yet to be resolved. This new function for WASH is distinct from its best characterized role regulating actin on intracellular vesicles during endosomal sorting and recycling, but WASH also has additional roles in the nucleus and the oocyte cortex, showing that it is a versatile protein (Green, 2018).

    Given the myofibril defects seen with loss of RSU1, tensin, vinculin and filamin it might be expected that mutations in genes encoding these IAPs might be implicated in muscle disease. Indeed, mutations in integrin α7, talin and ILK are associated with muscular myopathies in humans and mice. Mutations in the genes encoding RSU1, tensin and vinculin have not been linked to muscle myopathies, but mutations in filamin are linked to myofibrillar myopathies. However, given the subtlety of these defects in Drosophila, one might predict that mutations in genes encoding these IAPs are associated with subtle defects in humans such as reduced sporting performance or susceptibility to muscle injury. The authors were unaware of any mutations in genes encoding these IAPs being related to athletic performance or injury susceptibility, but these IAPs would be good candidates for further study in this area (Green, 2018).

    One way that these IAPs may contribute to athletic performance is by building a muscle shock absorber, the MARL, which protects the myofibrils from contraction-induced damage. The concept of muscle shock absorbers is well established since tendons perform this function. The presence of filamin, Arp3, vinculin and α-actinin in the MARL suggests that the MARL contains branched and bundled actin filaments. Branched actin networks have been shown to be viscoelastic and actin crosslinkers such as filamin have been shown to reduce viscosity and increase elasticity of actin networks. Further study into the functional nature of the MARL should increase understanding of athletic performance and injury susceptibility (Green, 2018).

    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).

    Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

    In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).

    In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).

    This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).

    An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).

    Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).

    To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbtP1 null cells (evaluated at ~70% for Rap1IR and 45% for mbtP1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbtP1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbtP1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).

    The force-sensitive protein Ajuba regulates cell adhesion during epithelial morphogenesis

    The reorganization of cells in response to mechanical forces converts simple epithelial sheets into complex tissues of various shapes and dimensions. Epithelial integrity is maintained throughout tissue remodeling, but the mechanisms that regulate dynamic changes in cell adhesion under tension are not well understood. In Drosophila melanogaster, planar polarized actomyosin forces direct spatially organized cell rearrangements that elongate the body axis. This study shows that the LIM-domain protein Ajuba is recruited to adherens junctions in a tension-dependent fashion during axis elongation. Ajuba localizes to sites of myosin accumulation at adherens junctions within seconds, and the force-sensitive localization of Ajuba requires its N-terminal domain and two of its three LIM domains. Ajuba stabilizes adherens junctions in regions of high tension during axis elongation, and that Ajuba activity is required to maintain cell adhesion during cell rearrangement and epithelial closure. These results demonstrate that Ajuba plays an essential role in regulating cell adhesion in response to mechanical forces generated by epithelial morphogenesis (Razzell, 2018).

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

    Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. This study demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, it was observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, this study presents a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact (Yang, 2018).

    Adherens junction length during tissue contraction is controlled by the mechanosensitive activity of actomyosin and junctional recycling

    During epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. This study shows that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. This study reveals that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis (Sumi, 2018).

    This study has shown that adherens junction length is actively controlled during epithelial contraction, leading to a constant junctional straightness upon cell contraction. It is proposed that two different cellular mechanisms are at the origin of this length control. First, a modulation of actomyosin localization from the medial areas toward junctions allows for integrity maintenance by increasing actomyosin contraction along the junctions upon junctional stretch. Possibly, E-cadherin dilution triggered by junctional stretching is responsible for the myosin recruitment, a possibility supported by the anti-correlation between junctional myosin and E-cadherin. Second, the junction removal rate depends on junctional straightness, maintaining a preferred junctional straightness. Presumably, the endocytosis machinery is at the origin of this maintenance. As junctional tension is dependent on junctional straightness, maintaining junctional straightness could also allow it to maintain junctional tension, which remains approximately constant during DC (Sumi, 2018).

    These mechanisms rely on a complex interplay between junctional E-cadherin, actomyosin, and tension. Indeed, this study finds that (1) higher junctional E-cadherin levels are associated with lower myosin levels and (2) decreased junctional straightness is associated with lower junctional tension. These observations suggest that E-cadherin is an essential component for the control of adherens junction length and for actomyosin localization in the cell. In cell culture experiments, E-cadherin has already been determined to be a key player in cell-cell mechanical interplay and shown to interact closely with the actomyosin cortex. In the case of the AS tissue, an enrichment of actomyosin was observed at junctions when E-cadherin levels are reduced. A similar interaction has been previously identified in the context of cytokinesis. Ghd results indicate a general interplay between E-cadherin and myosin that could play a role in several morphogenetic rearrangements. Such an interplay is likely to involve actin-associated proteins that could respond to changes in mechanical states of the adherens junction. Consistent with this idea, previous studies identified dynamic enrichment of the formin Diaphanous or of vinculin specifically at adherens junctions (Sumi, 2018).

    Interestingly, this study observed that when junctions are stretched and E-cadherin density decreased, the contractile pulses of the AS tissue are arrested, possibly due to the observed myosin recruitment to junctions. Such pulses of contraction have been identified in several tissues during Drosophila, chicken, and mouse embryo development. They have been associated with global tissue contraction and epithelial remodeling. This study has shown that the pulsatile activity can be tuned by junctional stretch and E-cadherin levels. The results therefore uncover a direct coupling between cell-cell adhesion and the contractile activity of the cell (Sumi, 2018).

    Finally, the rate of junction removal appears to depend on junctional straightness. How is junction straightness sensed to dictate junction removal? Laser ablation experiments indicate that junctions with lower straightness also have lower mechanical tension. In single cells, it is known that the endocytic rate is modulated by the tension of the cell membrane. In a developmental context, endocytosis could also be tension sensitive, resulting in the control of junctional straightness through junctional tension. Alternatively, E-cadherin density could play a role in the control of the junction turnover rate. Indeed, an increase of E-cadherin density associated to a decrease in junction straightness could trigger an enhanced endocytic rate, therefore controlling junction straightness and tension. The mechanisms regulating junction length and integrity identified here involve the activity of two essential cellular machineries. They are therefore likely to act during many morphogenetic events that involve tissue contraction, such as neural tube closure, optic cup morphogenesis, or wound healing (Sumi, 2018).

    Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis

    Coordinated rearrangements of cytoskeletal structures are the principal source of forces that govern cell and tissue morphogenesis. However, unlike for actin-based mechanical forces, knowledge about the contribution of forces originating from other cytoskeletal components remains scarce. This study has establish microtubules as central components of cell mechanics during tissue morphogenesis. Individual cells were found to be mechanically autonomous during early Drosophila wing epithelium development. Each cell contains a polarized apical non-centrosomal microtubule cytoskeleton that bears compressive forces, whereby acute elimination of microtubule-based forces leads to cell shortening. It was further established that the Fat planar cell polarity (Ft-PCP) signalling pathway couples microtubules at adherens junctions (AJs) and patterns microtubule-based forces across a tissue via polarized transcellular stability, thus revealing a molecular mechanism bridging single cell and tissue mechanics. Together, these results provide a physical basis to explain how global patterning of microtubules controls cell mechanics to coordinate collective cell behaviour during tissue remodelling. These results also offer alternative paradigms towards the interplay of contractile and protrusive cytoskeletal forces at the single cell and tissue levels (Singh, 2018).

    During development individual cells assemble into complex tissues and organs with specialized forms and functions. Tissue morphogenesis is driven by mechanical forces that are generated by the cytoskeleton within cells and transmitted in a coordinated manner through adhesion molecules across neighbouring cells. The best-studied cytoskeletal component is actin, which, together with other proteins, forms protrusive and contractile arrays, a fundamental constituent of rearrangements on the single cell and tissue levels. Recent work has suggested that microtubules, similar to actin, can also generate forces in cells. However, understanding of the contribution of microtubules to cell mechanics, cell shape changes and force coordination during morphogenesis remains poor. This is mainly due to the fact that many current models describing the mechanical state of tissues during shape changes focus on actomyosin dynamics and/or rely on continuum mechanics. These studies, which are based on coarse-grain observations of cell movements or cell shape changes, reveal only part of the physical mechanisms that drive morphogenesis and do not directly investigate the physicomechanical context of tissue remodelling. To understand the relationship between cell mechanics, force patterning and molecular structure, this study investigated the mechanical properties of microtubules at high spatiotemporal resolution using wing development in Drosophila melanogaster as a paradigm (Singh, 2018).

    During pupal wing development, non-centrosomal microtubules form an apical array of parallel microtubule bundles that are globally aligned along the proximal-distal (P-D) axis. Patterning of the microtubule cytoskeleton depends on the Ft-PCP signalling pathway and occurs during the early phase of wing reshaping (that is, between 14 and 18 h after puparium formation, or APF). This patterning is associated with extensive changes in cell shape, cell divisions and cell-cell rearrangements. In the Drosophila wing, the Ft-PCP pathway further orients cell elongation and cell divisions along the P-D axis to induce wing tissue elongation. Intriguingly, rescue of the Hippo pathway in Ft-PCP mutant animals, in which microtubule alignment is impaired, aberrant development results in perturbed cell elongation and an abnormal rounded wing shape, suggesting that there is an interdependence between these events. Therefore this study explored the possibility that microtubule-based cell mechanics control cell and tissue shape during early wing development between 16 and 18 h APF (Singh, 2018).

    Tissue remodelling is driven by intrinsic and extrinsic mechanisms, and it has previously been shown that extrinsic mechanical forces act during the late phase of wing reshaping (starting 18 h APF). These forces are generated by hinge contraction of the wing that is attached to the cuticle on the distal side. This study evaluated the mechanical autonomy of individual cells before hinge contraction at an earlier developmental stage (that is, between 16 and 18 h APF). This was done by isolating a single cell (or a small patch of cells) using a single-pulse multipoint procedure to cut AJs, thus mechanically uncoupling individual cells from their surrounding. Strikingly, the shape of individual isolated cells did not change significantly upon laser ablation at 17-18 h APF, when cells in the wing are already elongated. The same result was obtained when patches of cells were isolated. Additional analyses of the Feret's diameter before and after ablation showed a small isotropic decrease in cell size, providing evidence that at this early stage, individual cells are not influenced by the neighbouring cells or by tissue-scale forces in a polarized fashion. Consistently, analysis of animals expressing a mutant form of dumpy protein, an extracellular matrix protein associated with tissue remodelling at later developmental stages, showed no substantial differences in wing shape compared to wild-type wings at 18 h APF. Together, these experiments argue that unlike later stages, cell autonomous forces are the major drivers of initial cell shape changes between 16 and 18 h APF (Singh, 2018).

    To identify the molecular mechanism underlying cell autonomous shape formation, the distribution and dynamics of two cytoskeletal force-generators were investigated: microtubules and non-muscle myosin II (MyoII) as a component of the actomyosin cytoskeleton. MyoII was detected at the apical cell cortex at the level of AJs. A subsequent analysis of the signal distribution within single cells revealed a planar polarized distribution of MyoII along the P-D axis, which correlated with increased tension along the same junctions. As MyoII provides contractile forces, this should result in P-D junctional shortening upon laser ablation. However, this is inconsistent with the current ablation experiments, suggesting that there is an opposing force present. Interestingly, staining of microtubules showed planar polarized apical microtubules along the P-D axis at the level of AJs. Microtubules are the stiffest cytoskeletal filaments, with a persistence length on the order of millimetres. Microtubules are therefore well suited to balance the tension generated by actomyosin contraction. Consistently, the distribution of microtubules and MyoII in mechanically isolated cells remain polarized. In addition, microtubule and MyoII polarity was preserved in dumpy mutant wings at 18 h APF, indicating that they are polarized in a cell autonomous fashion. The possible role of the atypical myosin Dachs, a downstream component of the Fat signalling pathway, was also analyzed. Dachs mutant wings showed no change in cell elongation or microtubule polarity, which is consistent with recent work reporting that recombinant Dachs does not have ATPase activity and can therefore not function as a molecular motor. Together, these observations argue that planar polarized microtubules may balance actomyosin tensional forces that pull on P-D junctions and stabilize cell shape (Singh, 2018).

    To validate this hypothesis, and to elucidate the dynamic and functional role of microtubules in cell mechanics, their properties were investigated during wing development. Live cell imaging of EOS-α-tubulin (EOS-Tub) showed that microtubules were not static but engaged in complex and dynamic rearrangements. An analysis of microtubule straightness showed that in wing cells, virtually all microtubules along the P-D axis were bent, consistently undergoing short wavelength buckling (~3 μm) near the cell cortex. It was further observed that growing microtubules remain straight and only start to buckle after they reach the cell cortex, exhibiting local short wavelength buckling near these sites. This result indicates that microtubule polymerization can generate considerable compressive forces to induce microtubule buckling (Singh, 2018).

    Next, whether buckling of microtubules in Drosophila wing epithelium is indeed a result of forces acting on microtubules was also investigated, as suggested by the current experiments and in vitro studies, or whether the cellular environment yields more flexible microtubules. This is important, as buckling of flexible microtubules would rule out a role in balancing actomyosin contractility. To probe the forces of single microtubule filaments in vivo, individual microtubules were cut by laser ablation and the subsequent relaxation was monitored using live imaging. Previously curved microtubules rapidly straighten out, thus verifying that microtubules are indeed loaded with compressive forces. Finally, it was also observed that local ablation of microtubules triggers a rapid translocation of the adjacent junction. This finding supports the idea that non-centrosomal microtubules continuously generate pushing forces via polymerization that may then be stored as compressive forces in a polarized fashion to balance contractile forces generated by junctional actomyosin (Singh, 2018).

    How are microtubules polarized along the P-D axis? While the molecular mechanism has remained elusive, previous work has established that the Ft-PCP signalling pathway aligns the apical microtubule network along the P-D axis by regulating association sites of microtubules with AJs. Considering the observed stability of aligned microtubules, whether directional differences in microtubule dynamics could serve as a mechanism for the planar polarization of microtubules was tested. Monitoring of EB1 tagged with green fluorescent protein (EB1-GFP) revealed two populations of microtubule-plus ends: fast growing microtubules with a growth velocity of 24.43 ± 0.43 7mi;m min-1 (mean ± s.e.m.), and slow growing microtubules with a velocity of 17.06 ± 0.26 7mi;m min-1. A further analysis showed that the microtubule growth rates depended on relative localization within cells as well as the growth angle relative to the P-D axis. Microtubule growth rates in the cell interior were higher compared to the cell cortex. Similarly, microtubules along the P-D axis grew faster than microtubules growing perpendicular to the P-D axis along the A-P axis, establishing a spatial gradient in microtubule growth velocity. The lower growth rate along the A-P axis close to the cell periphery suggests that there is more frequent pausing and switching between polymerization and depolymerization of microtubules, thus indicating a decreased stability of A-P oriented microtubules (Singh, 2018).

    It was reasoned that over time, such differences in dynamics and stability may result in predominantly P-D aligned microtubules. To test this hypothesis, the cortical residence time was analyzed of microtubules as a function of their angle with respect to the P-D axis. Intriguingly, it found that microtubules that interact with the P-D cell cortex have a longer lifetime than microtubules interacting with the A-P cortex. Upon closer inspection, dynamic cycles of short-lived interactions of microtubules with A-P junctions were noted followed by depolymerization. Importantly, A-P oriented microtubules do not show buckling behaviour, which is in contrast to P-D oriented microtubules, but rather undergo catastrophe soon after interaction with A-P oriented cell junctions. This result suggests that microtubule-plus ends are less stable at these sites and thus cannot sustain long-lasting interactions with the cell cortex, which are required to generate compressive forces. Building on these observations, in silico probing was performed to see whether the angular difference in lifetime may indeed be sufficient for microtubule polarization. Assuming a random orientation for de novo formed microtubules, the lifetime of each microtubule was defined as a function of the angle with a maximal lifetime along the P-D axis. Upon expiration, individual microtubules were re-introduced into the system at random angles, therefore keeping the total number of microtubules constant. Consistent with the in vivo observations, the simulation reached a steady-state at which a constant fraction of microtubules polarized along the P-D axis. Taken together, these observations point to a mechanism whereby microtubule stability regulates the planar alignment of the microtubule cytoskeleton along the P-D axis, which in turn directs cell mechanics along this axis. These data place directional microtubule stability upstream of proposed mechanisms of how cell shape influences microtubule alignment. Furthermore, these results are consistent with previous findings that microtubule association with P-D oriented AJs during the initial stage of wing development depends on Ft-PCP signalling (Singh, 2018).

    Having established that planar polarized microtubule-based forces shape single cells, their mechanical coupling and integration into tissue-level mechanics were investigated. In a first round of experiments, transcellular coupling of microtubules were investigated on the ultrastructural level using transmission electron microscopy (TEM). In agreement with previous work, AJs were juxtaposed in neighbouring cells associated with microtubule filaments that span across cells in wild-type wings, forming supracellular cables analogous to myosin cables. Notably, no such association was observed in ftl(2)fdd1 / ftl(2)fd dGC13 (ft d) and ftl2 fd/ftGRV;ActP-Gal4/UAS-FtΔECDΔN-1 (N1) mutant wings, in which microtubules are randomly oriented in wing cells, therefore providing structural support for the Ft-PCP-dependent stabilization of microtubule-based forces at P-D oriented AJs. Consistently, ft mutant clones showed a fragmented microtubule cytoskeleton, arguing that there is Ft-PCP-dependent stabilization of microtubules via coupling at AJs (Singh, 2018).

    To further validate the role of polarized transcellular microtubule stability in tissue mechanics and organization, tissue shape changes were observed upon acute perturbation of microtubule-based forces. To control microtubule dynamics in a precise spatial and temporal manner, he recently developed photostatin (PST1)35, a photo-switchable analogue of combretastatin A-4 (CA4)36 was used. The drug was applied to directly test the requirement of microtubules for cell shape maintenance. Notably, it was found that the exposed wing area contracted along the P-D axis upon microtubule inhibition. Quantitative cell shape analysis showed a small but significant reduction in the elongation index (EI) in selective regions where the drug was activated, arguing that polarized tissue stabilization is via microtubule-based forces. Finally, overexpression of the microtubule-severing protein Spastin increased cell shape heterogeneity. These results are consistent with the hypothesis that an intact polarized microtubule cytoskeleton is not only required for the maintenance of anisotropic cell shape but also critically involved in shaping the whole tissue during morphogenesis via polarized transcellular force stability (Singh, 2018).

    Understanding the role of microtubules during animal development has so far been limited, especially because of a shortage of methods suitable to demonstrate causality in vivo. Taking advantage of complementary genetic, chemical, numerical and microscopy approaches, these experiments unveil polarized microtubule-based compressive forces as an alternative principle for stabilizing and maintaining cell and tissue shape during morphogenesis. Alignment of microtubules along the P-D axis was found to be based on increased longevity and polymerization of microtubules interacting with P-D oriented AJs compared to non-polarized microtubules. The result of this microtubule patterning along the P-D axis is an asymmetric distribution of protruding forces, which are stored in a polarized fashion via compressive loads on microtubules. Considering that actomyosin and microtubules are both planar polarized, it is plausible to envision that the observed compressive load on microtubules plays an active role in balancing actin-based contractile forces, resulting in the cell mechanical autonomy observed in the laser ablation experiments. Intriguingly, it was recently shown that acetylation of microtubules increases their mechanical resistance and that microtubules undergo self-repair upon damage. These important features support the role of the microtubule cytoskeleton as a site of long-term compressive force storage. Finally, evidence is provided that planar polarized microtubules are coupled at AJs across individual cells, bridging forces on the tissue level via polarized transcellular stability. Although the molecular identity remains elusive, the data suggests an involvement of AJ-associated proteins organized by the Ft-PCP pathway in this process (Singh, 2018).

    Collectively, this work provides evidence that PCP-based planar patterning of the microtubule cytoskeleton not only results in polarized cell-autonomous forces but also coordinates global force patterning during tissue morphogenesis. The proposed mechanism establishes the Ft-PCP pathway at the onset of cell and wing elongation, before shape changes, due to extrinsic mechanical forces. Consistently, in a Ft-PCP mutant, in which initial elongation fails, consecutive remodelling by extrinsic tensile forces cannot rescue these length defects, therefore leading to shorter and rounder adult wings. Considering that the Ft-PCP signalling pathway controls a variety of dynamic cell population in vertebrates, the microtubule-based mechanism described in this study is likely to be physiologically relevant beyond wing development (Singh, 2018).

    Recruitment of Jub by alpha-catenin promotes Yki activity and Drosophila wing growth

    The Hippo signaling network controls organ growth through YAP family transcription factors, including the Drosophila Yorkie protein. YAP activity is responsive to both biochemical and biomechanical cues, with one key input being tension within the F-actin cytoskeleton. Several potential mechanisms for biomechanical regulation of YAP proteins have been described, including tension-dependent recruitment of Ajuba family proteins, which inhibit kinases that inactivate YAP proteins, to adherens junctions (AJ). This study investigated the mechanism by which the Drosophila Ajuba family protein, Ajuba LIM protein (Jub) is recruited to adherens junctions, and the contribution of this recruitment to the regulation of Yorkie. Alpha-catenin is identifed as the mechanotransducer responsible for tension-dependent recruitment of Jub by identifying a region of α-catenin that associates with Jub, and by identifying a region, which when deleted, allows constitutive, tension-independent recruitment of Jub. Increased Jub recruitment to alpha-catenin is associated with increased Yorkie activity and wing growth, even in the absence of increased cytoskeletal tension. These observations establish alpha-catenin as a multi-functional mechanotransducer and confirm Jub recruitment to alpha-catenin as a key contributor to biomechanical regulation of Hippo signaling (Alegot, 2019).

    To evaluate the role of α-catenin in the recruitment of Jub to AJ, Jub localization was examined in wing discs isolated from animals expressing truncated forms of α-catenin. This was achieved by using RNAi to knock-down expression of endogenous α-catenin and expressing RNAi-insensitive Venus- or V5-tagged α-catenin transgenes under UAS-Gal4 control. Transgenes were expressed in posterior cells using an en-Gal4 driver, so that anterior cells could serve as a control for Jub localization. Full-length α-catenin expressed under UAS-Gal4 control rescued both the lethality associated with knockdown of α-catenin, and Jub localization. α-catenin has similarity to Vinculin in N-terminal, middle, and C-terminal regions termed VH1, VH2, and VH3. An initial series of constructs deleted regions of α-catenin centered around these Vinculin homology regions, as well as separate deletions of either the N-terminal or C- terminal half of VH2. Constructs lacking either the N- or C-terminus of α-catenin (ΔVH1 or ΔVH3) could not rescue the lethality associated with α-catenin knockdown. Thus, to enable visualization of the consequences of these deletions on Jub, conditional knockdown and expression of α-catenin was achieved using a temperature sensitive-allele of Gal80 (Gal80ts) that represses Gal4-driven expression at 18°C but not at 29°C. After a 30 hour shift to higher temperature, Jub was mostly lost from apical cell junctions. However, E-cadherin was also mostly lost, indicating that these regions of α-catenin are required to maintain AJ. This is consistent with the roles of F-actin and α-catenin in stabilizing AJ, as the VH1 region is required for association with β-catenin and thus localization to AJ, and the VH3 region is required for association with F-actin. The ΔVH1 and ΔVH3 constructs also did not themselves localize to junctions. The loss of Jub from cell junctions under these conditions is consistent with studies indicating its association with AJ, but does not provide specific information about its interactions with α-catenin (Alegot, 2019).

    Conversely, deletions within the central region of α-catenin increased Jub recruitment to AJ. Deletion of the entire VH2 region increased Jub recruitment but was also associated with abnormal cell morphology and E-cadherin localization. However, two smaller deletions comprising the N- and C-terminal halves of the VH2 region (ΔVH2-N and ΔVH2-C) rescued knock-down of α-catenin, resulting in cells that appear to have normal localization of E-cadherin and α-catenin. The ΔVH2-C deletion also appears to have normal Jub localization. In contrast, the ΔVH2-N deletion is associated with increased Jub localization at AJ. Moreover, in cells with the ΔVH2-N deletion, Jub is distributed relatively uniformly around cell junctions, whereas in cells with wild-type α-catenin, Jub localization to junctions is often punctate. The ΔVH2-N deletion was not only associated with increased Jub recruitment when expressed in place of endogenous α-catenin; it could also promote Jub recruitment even when expressed in the presence of endogenous α-catenin. Thus, expression of this isoform dominantly increases Jub recruitment to AJ (Alegot, 2019).

    To further investigate the influence of α-catenin on Jub, smaller deletion constructs were created. To aid in the design of these additional constructs, Phyre2 was used to predict the structure of Drosophila α-catenin, based on its sequence similarity to mammalian α-catenin proteins with experimentally-determined structures. Mammalian α-catenin consists largely of a series of α-helical bundles: two N-terminal 4-helix bundles (N1 and N2) that share one long α-helix; three central 4-helix bundles (M1, M2, and M3), and a C-terminal 5-helix bundle. These structural features are also predicted for Drosophila α-catenin (Alegot, 2019).

    The ΔVH2-N deletion that increases Jub recruitment to AJ deletes both the M1 and M2 helical bundles. Thus, separate deletions were created of either the M1 or M2 bundles. Deletion of the M2 bundle (ΔM2) slightly increased Jub localization to AJ, but a much stronger increase in Jub binding was detected when the M1 bundle was deleted (ΔM1a or ΔM1b). Recruitment of Jub to AJ is normally promoted by cytoskeletal tension. To examine the possibility that M1 deletions increase Jub recruitment by increasing tension, levels were examined of junctional myosin (using a GFP-tagged myosin light chain) that correlate with junctional tension, but no difference was observed. F-actin levels were similarly unaffected. Staining was carried out specifically for the phosphorylated (activated) form of myosin regulatory light chain (pMLC) in discs expressing Jub:GFP. This revealed similar levels of pMLC in control cells and cells expressing α-catenin ΔM1b, whereas expression of rok RNAi or an activated form of Rok visibly decreased or increased, respectively, junctional levels of both pMLC and Jub. Since Jub associates with α-catenin, the observation that deletion of the M1 bundle increases Jub localization to AJ without increasing tension suggests that deletion of this region alters the structure of α-catenin in a way that makes a Jub-binding region more accessible. The M1 bundle might thus act as an 'inhibitory' region that prevents Jub binding, with this inhibition normally alleviated in wild-type α-catenin by a conformational change induced by high tension. Alternatively, deletion of the M1 bundle might destabilize a non-Jub binding conformation of α-catenin and thereby indirectly increase accessibility of a Jub-binding region (Alegot, 2019).

    While both Jub and Vinculin exhibit tension-dependent association with α-catenin, the results suggest that their interactions are distinct. A region initially identified as inhibitory for Vinculin binding, corresponds to the M3 helical bundle and the linker between VH2 and VH3. Moreover, the M1 helical bundle includes the Vinculin binding interface. To directly compare the influence of α-catenin deletions on Jub and Vinculin binding, advantage was taken of a Drosophila genomic GFP-tagged Vinculin (Vinc:GFP). Both of the M1 deletions substantially reduced, but did not eliminate, Vinculin localization to AJ, which fits with observations that Vinculin interacts with sequences in the M1 bundle, but implies that additional mechanisms also contribute to Vinculin localization. Deletion of the M2 bundle increased Vinculin localization to AJ, consistent with observations that M2 participates in interactions that stabilize a conformation of α-catenin that is 'closed' with respect to Vinculin binding (Alegot, 2019).

    If M1 deletions mimic the influence of cytoskeletal tension on the ability of α-catenin to bind to Jub, the increased recruitment of Jub in M1 deletion isoforms could occur even in the absence of tension. To test this, Jub localization was examined in flies expressing α-catenin constructs and with cytoskeletal tension decreased by RNAi of Rho-associated protein kinase (Rok), a promoter of myosin activity. In the presence of full-length α-catenin, rok RNAi decreases Jub recruitment to junctions. Conversely, in the presence of deletions that include the M1 bundle, Jub recruitment to junctions remains elevated. Quantitation of Jub at junctions, normalized to E-cadherin, revealed a decrease in Jub recruitment when tension is lowered even in cells expressing M1 deletions, but Jub recruitment nonetheless remains above that in control cells. Thus, these deletions recruit Jub even under low-tension conditions, which implicates α-catenin as the key mechanotransducer responsible for tension-dependent recruitment of Jub (Alegot, 2019).

    Cytoskeletal tension promotes Yki activity, and Yki activity is suppressed by knockdown of Jub. However, other mechanisms by which tension might increase Yki activity have also been suggested, such as an influence on spectrins. The observation that deletions of the M1 bundle lead to increased Jub at AJ without increasing myosin identifies a condition under which it was possible to determine whether recruitment of Jub to junctions is sufficient to increase Yki activity, and thus distinguish the contribution of Jub from other potential influences of cytoskeletal tension (Alegot, 2019).

    Yki activity was evaluated by examining ex-lacZ expression, which is a reporter for the Yki target gene expanded (ex). Expression of α-catenin with deletion of the M1 bundle increased ex-lacZ, whereas expression of full length α-catenin did not. Increased Yki activity is associated with increased accumulation of Yki in the nucleus, and nuclear levels of Yki were slightly increased in wing disc cells expressing M1 region deletions. To further examine the influence of the M1 deletion, adult wing size was examined, as wing growth is promoted by Yki activity. Expression of M1 deletion isoforms throughout the developing wing under nub-Gal4 control increased wing size as compared to wings expressing wild-type α-catenin. The increased Yki activity and wing growth in M1 deletion mutants are Jub-dependent, because they are reversed by RNAi of jub. As mutation or knock-down of Vinculin has only minor phenotypic consequences in Drosophila, the effects of M1 deletion on Yki activity and wing growth cannot be attributed to loss of Vinculin. Altogether, these observations establish that increased Jub recruitment to junctions can be sufficient to elevate Yki activity. This strongly supports the conclusion that Jub recruitment to AJ is a key component of the biomechanical response linking cytoskeletal tension to Yki activity (Alegot, 2019).

    However, analysis of the larger ΔVH2N deletion suggests that the role of α-catenin in promoting Yki activity is more complex. Although expression of ΔVH2N α-catenin increased Jub recruitment at AJ, it did not detectably increase ex-lacZ expression or wing size. Similarly, even though deletion of the M2 helical bundle modestly increased Jub recruitment to AJ, it did not detectably increase ex-lacZ expression or wing size. Thus, it is inferred that additional features of α-catenin may contribute to Yki regulation (Alegot, 2019).

    Jub requires α-catenin to localize to junctions. To map regions of α-catenin that mediate association with Jub, co-immunoprecipitation experiments were performed in Drosophila S2 cells expressing transfected constructs. Initially, co- immunoprecipitation of Jub with α-catenin full length, VH1, VH2, and VH3 region constructs was compared. Significant association of Jub and a VH1 region construct was detected. Conversely, association of Jub with full length, VH2, or VH3 constructs was weaker, and close to the non- specific background. The association with the VH1 region of α-catenin was not as strong as binding of Jub to Warts, which was included as a positive control. Association of Jub with the VH1 region is consistent with reports that the mammalian Ajuba protein can associate with an N- terminal fragment of α-catenin, corresponding roughly to VH1. The stronger association of Jub with a VH1 fragment as compared to full length α-catenin is consistent with normal binding depending upon junctional tension, as S2 cells are not epithelial, so it would be expected that the transfected α-catenin would be in a low-tension conformation. Moreover, co-immunoprecipitation experiments comparing M1 bundle deletion constructs to full length α-catenin revealed that M1 deletions significantly increased association of Jub with α-catenin (Alegot, 2019).

    To further refine the Jub-binding region, the predicted Drosophila α-catenin structure was used to design constructs corresponding to the N1 or N2 helical bundles. No significant co-immunoprecipitation with the N1 bundle was detected, whereas co- immunoprecipitation with N2 was comparable to that for the VH1 region construct. Thus, it is inferred that the primary site of association of Jub with α-catenin is within N2. While the simplest model would be that Jub directly binds to this region of α-catenin, it remains possible that their association is mediated through other proteins. The N2 bundle is near the M1 bundle, but they do not directly contact each other in the predicted structure. Thus, the effect of M1 deletion on Jub binding may be an indirect consequence of a conformational change in α-catenin when this region is deleted, or reduced stability of a more closed conformation, rather than the M1 bundle directly obscuring a binding site within N2. Attempts were also mad to examine the consequences of loss of Jub recruitment to junctions by expressing α-catenin with N2 deleted in vivo. However, this protein failed to rescue the viability of wing disc cells expressing α-catenin RNAi, so this could not be examined. Moreover, when expressed in wild-type cells, the ΔN2 construct failed to localize to adherens junctions (Alegot, 2019).

    These results establish key features of α-catenin mechanotransduction and tension- dependent regulation of Yki activity. They implicate α-catenin as a multi-functional mechanotransducer that associates with both Vinculin and Jub upon a tension-induced conformational change in α-catenin, but through distinct sites. The complete structure of the open conformation of α-catenin has not been determined, but it is inferred that it makes Jub- associating regions accessible. M1 deletions also make the Jub-associating region more accessible, but it's not clear whether they do so in a manner similar to, or distinct from, the effect of tension. The observation that M1 deletions increase Yki activity also supports the crucial role of Jub recruitment to AJ in promoting Yki activity. This does not exclude the possibility that of other consequences of cytoskeletal tension contribute to Yki regulation, but clearly implicates recruitment of Jub as a key factor (Alegot, 2019).


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    Zygotically transcribed genes

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