Paracingulin recruits CAMSAP3 to tight junctions and regulates microtubule and polarized epithelial cell organization.

Paracingulin (CGNL1) is recruited to tight junctions (TJs) by ZO-1 and to adherens junctions (AJs) by PLEKHA7. PLEKHA7 has been reported to bind to the microtubule minus-end-binding protein CAMSAP3, to tether microtubules to the AJs. Here, we show that knockout (KO) of CGNL1, but not of PLEKHA7, results in the loss of junctional CAMSAP3 and its redistribution into a cytoplasmic pool both in cultured epithelial cells in vitro and mouse intestinal epithelium in vivo. In agreement, GST pulldown analyses show that CGNL1, but not PLEKHA7, interacts strongly with CAMSAP3, and the interaction is mediated by their respective coiled-coil regions. Ultrastructure expansion microscopy shows that CAMSAP3-capped microtubules are tethered to junctions by the ZO-1-associated pool of CGNL1. The KO of CGNL1 results in disorganized cytoplasmic microtubules and irregular nuclei alignment in mouse intestinal epithelial cells, altered cyst morphogenesis in cultured kidney epithelial cells, and disrupted planar apical microtubules in mammary epithelial cells. Together, these results uncover new functions of CGNL1 in recruiting CAMSAP3 to junctions and regulating microtubule cytoskeleton organization and epithelial cell architecture.

Exploring the evolution and function of Canoe's intrinsically disordered region in linking cell-cell junctions to the cytoskeleton during embryonic morphogenesis.

One central question for cell and developmental biologists is defining how epithelial cells can change shape and move during embryonic development without tearing tissues apart. This requires robust yet dynamic connections of cells to one another, via the cell-cell adherens junction, and of junctions to the actin and myosin cytoskeleton, which generates force. The last decade revealed that these connections involve a multivalent network of proteins, rather than a simple linear pathway. We focus on Drosophila Canoe, homolog of mammalian Afadin, as a model for defining the underlying mechanisms. Canoe and Afadin are complex, multidomain proteins that share multiple domains with defined and undefined binding partners. Both also share a long carboxy-terminal intrinsically disordered region (IDR), whose function is less well defined. IDRs are found in many proteins assembled into large multiprotein complexes. We have combined bioinformatic analysis and the use of a series of canoe mutants with early stop codons to explore the evolution and function of the IDR. Our bioinformatic analysis reveals that the IDRs of Canoe and Afadin differ dramatically in sequence and sequence properties. When we looked over shorter evolutionary time scales, we identified multiple conserved motifs. Some of these are predicted by AlphaFold to be alpha-helical, and two correspond to known protein interaction sites for alpha-catenin and F-actin. We next identified the lesions in a series of eighteen canoe mutants, which have early stop codons across the entire protein coding sequence. Analysis of their phenotypes are consistent with the idea that the IDR, including the conserved motifs in the IDR, are critical for protein function. These data provide the foundation for further analysis of IDR function.

Age-associated decline in RAB-10 efficacy impairs intestinal barrier integrity.

The age-related decline in the ability of the intestinal barrier to maintain selective permeability can lead to various physiological disturbances. Adherens junctions play a vital role in regulating intestinal permeability, and their proper assembly is contingent upon endocytic recycling. However, how aging affects the recycling efficiency and, consequently, the integrity of adherens junctions remains unclear. Here we show that RAB-10/Rab10 functionality is reduced during senescence, leading to impaired adherens junctions in the Caenorhabditis elegans intestine. Mechanistic analysis reveals that SDPN-1/PACSINs is upregulated in aging animals, suppressing RAB-10 activation by competing with DENN-4/GEF. Consistently, SDPN-1 knockdown alleviates age-related abnormalities in adherens junction integrity and intestinal barrier permeability. Of note, the inhibitory effect of SDPN-1 on RAB-10 requires KGB-1/JUN kinase, which presumably enhances the potency of SDPN-1 by altering its oligomerization state. Together, by examining age-associated changes in endocytic recycling, our study sheds light on how aging can impact intestinal barrier permeability.

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

Relationship between DNA methylation changes and skeletal muscle mass.

BACKGROUND: Sarcopenia is a disease diagnosed in the elderly. In patients with sarcopenia, the muscle mass decreases every year. The occurrence of sarcopenia is greatly affected by extrinsic factors such as eating habits, exercise, and lifestyle. The present study aimed to determine the relationship between muscle mass traits and genes affected by epigenetic factors with three different adjustment methods using Korean Genome and Epidemiology Study (KOGES) data. RESULTS: We conducted a demographic study and DNA methylation profiling by three studies according to the muscle mass index (MMI) adjustment methods: appendicular skeletal muscle mass divided by body weight (MMI1); appendicular skeletal muscle mass divided by square of height (MMI2); appendicular skeletal muscle mass divided by BMI (MMI3). We analyzed differentially methylated regions (DMRs) for each group. We then restricted our subjects to be top 30% (T30) and bottom 30% (B30) based on each MMI adjustment method. Additionally, we performed enrichment analysis using PathfindR to evaluate the relationship between identified DMRs and sarcopenia. A total of 895 subjects were included in the demographic study. The values of BMI, waist, and hip showed a significant difference in all three groups. Among 446 participants, 44 subjects whose DNA methylation profiles were investigated were included for DNA methylation analysis. The results of enrichment analysis showed differences between groups. In the women group through MMI1 method, only the glutamatergic synapse pathway showed a significant result. In the men group through MMI2 method, the adherens junction pathway was the most significant. Women group through MMI2 method showed similar results, having an enriched Rap1 signaling pathway. In men group through MMI3 method, the Fc epsilon RI signaling pathway was the most enriched. Particularly, the notch signaling pathway was significantly enriched in women group through MMI3 method. CONCLUSION: This study presents results about which factor should be concerned first in muscle mass index (MMI) adjustment. The present study suggested that GAB2 and JPH3 in MMI1 method, HLA-DQB1 and TBCD in MMI2 method, GAB2, NDUFB4 and ISPD in MMI3 method are potential genes that can have an impact on muscle mass. It could enable future epigenetic studies of genes based on annotation results. The present study is a nationwide study in Korea with the largest size up to date that compares adjustment indices for MMI in epigenetic research.

Calpain Regulation and Dysregulation-Its Effects on the Intercalated Disk.

The intercalated disk is a cardiac specific structure composed of three main protein complexes-adherens junctions, desmosomes, and gap junctions-that work in concert to provide mechanical stability and electrical synchronization to the heart. Each substructure is regulated through a variety of mechanisms including proteolysis. Calpain proteases, a class of cysteine proteases dependent on calcium for activation, have recently emerged as important regulators of individual intercalated disk components. In this review, we will examine how calcium homeostasis regulates normal calpain function. We will also explore how calpains modulate gap junctions, desmosomes, and adherens junctions activity by targeting specific proteins, and describe the molecular mechanisms of how calpain dysregulation leads to structural and signaling defects within the heart. We will then examine how changes in calpain activity affects cardiomyocytes, and how such changes underlie various heart diseases.

Neurogenesis redirects ß-catenin from adherens junctions to the nucleus to promote axonal growth.

Here, we show that, in the developing spinal cord, after the early Wnt-mediated Tcf transcription activation that confers dorsal identity to neural stem cells, neurogenesis redirects ß-catenin from the adherens junctions to the nucleus to stimulate Tcf-dependent transcription in a Wnt-independent manner. This new ß-catenin activity regulates genes implicated in several aspects of contralateral axon growth, including axon guidance and adhesion. Using live imaging of ex-vivo chick neural tube, we showed that the nuclear accumulation of ß-catenin and the rise in Tcf-dependent transcription both initiate before the dismantling of the adherens junctions and remain during the axon elongation process. Notably, we demonstrated that ß-catenin activity in post-mitotic cells depends on TCF7L2 and is central to spinal commissural axon growth. Together, our results reveal Wnt-independent Tcf/ß-catenin regulation of genes that control the growth and guidance of commissural axons in chick spinal cord.

TiO2 nanoparticles affect spermatogenesis and adhesion junctions via the ROS-mediated mTOR signalling pathway in Eriocheir sinensis testes.

Recent findings found that TiO2 nanoparticles (TiO2-NPs) have male reproductive toxicity. However, few reports have studied the toxicity of TiO2-NPs in crustaceans. In this study, we first chose the freshwater crustacean Eriocheir sinensis (E. sinensis) to explore the male toxicity of TiO2-NP exposure and the underlying mechanisms. Three nm and 25 nm TiO2-NPs at a dose of 30 mg/kg bw induced apoptosis and damaged the integrity of the haemolymph-testis-barrier (HTB, a structure similar to the blood-testis-barrier) and the structure of the seminiferous tubule. The 3-nm TiO2-NPs caused more severe spermatogenesis dysfunction than the 25-nm TiO2-NPs. We initially confirmed that TiO2-NP exposure affected the expression patterns of adherens junctions (α-catenin and ß-catenin) and induced tubulin disorganization in the testis of E. sinensis. TiO2-NP exposure caused reactive oxygen species (ROS) generation and an imbalance of mTORC1-mTORC2 (mTORC1/rps6/Akt levels were increased, while mTORC2 activity was not changed). After using the ROS scavenger NAC to inhibit ROS generation, both the mTORC1-mTORC2 imbalance and alterations in AJs were rescued. More importantly, the mTORC1 inhibitor rapamycin abolished mTORC1/rps6/Akt hyperactivation and partially restored the alterations in AJs and tubulin. Collectively, the mTORC1-mTORC2 imbalance induced by TiO2-NPs was involved in the mechanism of AJ and HTB disruption, resulting in spermatogenesis in E. sinensis.

Rap1 regulates lumen continuity via Afadin in renal epithelia.

The continuity of a lumen within an epithelial tubule is critical for its function. We previously found that the F-actin binding protein Afadin is required for timely lumen formation and continuity in renal tubules formed from the nephrogenic mesenchyme in mice. Afadin is a known effector and interactor of the small GTPase Rap1, and in the current study, we examine the role of Rap1 in nephron tubulogenesis. Here, we demonstrate that Rap1 is required for nascent lumen formation and continuity in cultured 3D epithelial spheroids and in vivo in murine renal epithelial tubules derived from the nephrogenic mesenchyme, where its absence ultimately leads to severe morphogenetic defects in the tubules. By contrast, Rap1 is not required for lumen continuity or morphogenesis in renal tubules derived from the ureteric epithelium, which differ in that they form by extension from a pre-existing tubule. We further demonstrate that Rap1 is required for correct localization of Afadin to adherens junctions both in vitro and in vivo. Together, these results suggest a model in which Rap1 localizes Afadin to junctional complexes, which in turn regulates nascent lumen formation and positioning to ensure continuous tubulogenesis.

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

Neuropilin-1 interacts with VE-cadherin and TGFBR2 to stabilize adherens junctions and prevent activation of endothelium under flow.

Linear and disturbed flow differentially regulate gene expression, with disturbed flow priming endothelial cells (ECs) for a proinflammatory, atheroprone expression profile and phenotype. Here, we investigated the role of the transmembrane protein neuropilin-1 (NRP1) in ECs exposed to flow using cultured ECs, mice with an endothelium-specific knockout of NRP1, and a mouse model of atherosclerosis. We demonstrated that NRP1 was a constituent of adherens junctions that interacted with VE-cadherin and promoted its association with p120 catenin, stabilizing adherens junctions and inducing cytoskeletal remodeling in alignment with the direction of flow. We also showed that NRP1 interacted with transforming growth factor-ß (TGF-ß) receptor II (TGFBR2) and reduced the plasma membrane localization of TGFBR2 and TGF-ß signaling. NRP1 knockdown increased the abundance of proinflammatory cytokines and adhesion molecules, resulting in increased leukocyte rolling and atherosclerotic plaque size. These findings describe a role for NRP1 in promoting endothelial function and reveal a mechanism by which NRP1 reduction in ECs may contribute to vascular disease by modulating adherens junction signaling and promoting TGF-ß signaling and inflammation.

Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo.

Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

Polychaetoid/ZO-1 strengthens cell junctions under tension while localizing differently than core adherens junction proteins.

During embryonic development, dramatic cell shape changes and movements reshape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by mechanosensitive multiprotein complexes assembled via multivalent connections. Here we combine genetic, cell biological, and modeling approaches to define the mechanism of action and functions of an important player, Drosophila polychaetoid, homologue of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways, perhaps in distinct subcomplexes, but combine to produce robust connections.

Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

Peripodial adherens junctions regulate Ajuba-Yorkie signaling to preserve fly eye morphology.

The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. We report here that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components ß-Catenin (ß-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in ß-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of ßPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of ß-Cat. We thus propose that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology.

Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma.

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organised in a variety of ways, ranging from thin discontinuous strands to thick 'pushing' collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by extracellular matrix in three-dimensional assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits - no cell-cell junctions and high proteolysis - exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in squamous cell carcinomas.

Underlying mechanisms that ensure actomyosin-mediated directional remodeling of cell-cell contacts for multicellular movement: Tricellular junctions and negative feedback as new aspects underlying actomyosin-mediated directional epithelial morphogenesis: Tricellular junctions and negative feedback as new aspects underlying actomyosin-mediated directional epithelial morphogenesis.

Actomyosin (actin-myosin II complex)-mediated contractile forces are central to the generation of multifaceted uni- and multi-cellular material properties and dynamics such as cell division, migration, and tissue morphogenesis. In the present article, we summarize our recent researches addressing molecular mechanisms that ensure actomyosin-mediated directional cell-cell junction remodeling, either shortening or extension, driving cell rearrangement for epithelial morphogenesis. Genetic perturbation clarified two points concerning cell-cell junction remodeling: an inhibitory mechanism against negative feedback in which actomyosin contractile forces, which are well known to induce cell-cell junction shortening, can concomitantly alter actin dynamics, oppositely leading to perturbation of the shortening; and tricellular junctions as a point that organizes extension of new cell-cell junctions after shortening. These findings highlight the notion that cells develop underpinning mechanisms to transform the multi-tasking property of actomyosin contractile forces into specific and proper cellular dynamics in space and time.

Organization of cortical microtubules in differentiated cells.

The microtubule cytoskeleton plays a critical role in a variety of cellular activities, and its structures and functions have been extensively studied. However, little is known about cell differentiation-related microtubule remodeling, its regulatory mechanisms, and its physiological functions. Recent studies have shown that microtubule-binding proteins as well as cell junctions, such as desmosomes and adherens junctions, are involved in the remodeling of microtubules in response to cell differentiation. In addition, the microtubule-organizing activity and structural integrity of centrosomes undergo dramatic changes during cell differentiation to promote microtubule remodeling. Here we summarize recent advances revealing the dynamic changes in microtubule organization and functions during cell differentiation. We also highlight the molecular mechanisms underlying microtubule modeling in differentiated cells, focusing on the key roles played by microtubule-binding proteins, cell junctions, and centrosomes.

Remodeling of E-cadherin subcellular localization during cell dissemination.

Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. Here we report that E-cad is necessary for the invasiveness of RasV12-transformed intestinal epithelial cells in Drosophila. E-cad/ß-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of RasV12-transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of RasV12-transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, our observations indicate that E-cad plays a role in the invasiveness of RasV12-transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus our study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.