Apical Adhesion Research Articles

Apical adhesion of extracellular bacteria modulates host cell mechanics through a novel actomyosin structure

Apical adhesion of extracellular bacteria modulates host cell mechanics through a novel actomyosin structure

A balance of noncanonical Semaphorin signaling from the cerebrospinal fluid regulates apical cell dynamics during corticogenesis.

During corticogenesis, dynamic regulation of apical adhesion is fundamental to generate correct numbers and cell identities. While radial glial cells (RGCs) maintain basal and apical anchors, basal progenitors and neurons detach and settle at distal positions from the apical border. Whether diffusible signals delivered from the cerebrospinal fluid (CSF) contribute to the regulation of apical adhesion dynamics remains fully unknown. Secreted class 3 Semaphorins (Semas) trigger cell responses via Plexin-Neuropilin (Nrp) membrane receptor complexes. Here, we report that unconventional Sema3-Nrp preformed complexes are delivered by the CSF from sources including the choroid plexus to Plexin-expressing RGCs via their apical endfeet. Through analysis of mutant mouse models and various ex vivo assays mimicking ventricular delivery to RGCs, we found that two different complexes, Sema3B/Nrp2 and Sema3F/Nrp1, exert dual effects on apical endfeet dynamics, nuclei positioning, and RGC progeny. This reveals unexpected balance of CSF-delivered guidance molecules during cortical development.

Transient opening of tricellular vertices controls paracellular transport through the follicle epithelium during Drosophila oogenesis.

Paracellular permeability is regulated to allow solute transport or cell migration across epithelial or endothelial barriers. However, how cell-cell junction dynamics controls paracellular permeability is poorly understood. Here, we describe patency, a developmentally regulated process in Drosophila oogenesis, during which cell vertices in the follicular epithelium open transiently to allow paracellular transport of yolk proteins for uptake by the oocyte. We show that the sequential removal of E-cadherin, N-cadherin, NCAM/Fasciclin 2, and Sidekick from vertices precedes their basal-to-apical opening, while the subsequent assembly of tricellular occluding junctions marks the termination of patency and seals the paracellular barrier. E-cadherin-based adhesion is required to limit paracellular channel size, whereas stabilized adherens junctions, prolonged NCAM/Fasciclin 2 expression, blocked endocytosis, or increased actomyosin contractility prevent patency. Our findings reveal a key role of cell vertices as gateways controlling paracellular transport and demonstrate that dynamic regulation of adhesion and actomyosin contractility at vertices governs epithelial barrier properties.

THE ROLE OF THE ARP2/3 INHIBITORY PROTEIN ARPIN IN THE REGULATION OF ENDOTHELIAL BARRIER FUNCTIONS

The endothelium forms a semi‐permeable barrier in the vasculature that separates blood from underlying tissues. During inflammation, this barrier is altered usually leading to increased permeability and leukocyte recruitment. Such alterations include endothelial actin remodeling regulated by actin‐binding proteins (ABP) and junction architecture modulation. An ABP critical for actin remodeling is the Arp2/3 complex, which induces formation of branched actin networks. The Arp2/3 complex needs to be tightly regulated which is achieved by activation via nuclear promoting factors (NPF) and inhibition via proteins that locally antagonize the activity of NPFs such as PICK1, gadkin and arpin. Arpin contains a COOH‐terminal acidic motif that binds directly to the Arp2/3 complex in competition with NPF to inhibit its activity. Arpin has been shown to localize at the lamellipodial edge, where it colocalizes with the WAVE2 complex and antagonizes WAVE2 to regulate Arp2/3 activity in this specific subcellular location. However, nothing is known about the role of arpin in endothelial barrier regulation.First, we analyzed expression of arpin in different endothelial cell types and found that it is expressed at high levels in Human vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HMEC‐1), brain‐derived Endothelial cells.3 (bEnd3) and primary murine lung endothelial cells (MLEC). To analyze arpin functions in endothelial barrier regulation, we generated arpin‐depleted HUVEC by transfecting them with lentiviruses encoding specific short hairpin RNA (shRNA) against arpin and a scrambled control sequence, and selecting stable clones using puromycin. Western blot analysis revealed arpin downregulation of more than 90%. Expression of the junctional proteins VE‐cadherin, claudin‐1, occludin, ZO‐1 and the apical adhesion molecule ICAM‐1 did not change in arpin‐depleted HUVECs. However, VE‐Cadherin staining revealed significant gaps at cell contacts and redistribution into the cytoplasm. Moreover, we consistently observed more actin filaments in arpin‐depleted HUVEC. As ROCK1 and ZIPK kinases were overexpressed in the absence of arpin, we conclude that these filaments are contractile stress fibers that destabilize junctions by generating pulling forces. In vitro filter‐based permeability assays showed that arpin‐depleted HUVEC are much more permeable compared to control cells transduced with lentivirus expressing scrambled shRNA. These data suggest that arpin regulates the endothelial barrier by two possible mechanisms: First, arpin may inhibit Arp2/3 to control junction protein internalization and vesicle trafficking; and second, arpin may inhibit Arp2/3 to control actin dynamics at junctions. Whether arpin additionally regulates Arp2/3‐independent processes is currently unknown, and will be investigated in the future. Our data reveal arpin as a new critical regulator of endothelial barrier integrity.Support or Funding InformationThis work was supported by a grant of the Mexican Council for Science and Technology (CONACyT, 284292 to MS).

P2.05-05 Availability of Four Dimensional Computed Tomography (4DCT) for Lung Cancer

Four-dimensional computed tomography (4DCT) imaging is a new form of chest CT that can detect continuous movement of the airways and lungs under free breathing conditions. This method is likely to be used increasingly to evaluate pulmonary function or predict intraoperative adhesion or tumor invasion. A total of 31 cases that underwent 4DCT at the Kanagawa Cardiovascular Respiratory Center from October 2017 to February 2019 were reviewed. Dynamic ventilatory scans were performed using a 320-row multi detector CT (Aquilion ONE GENESIS, Canon Medical Systems, Otawara, Tochigi, Japan). The frame rate was 0.25sec/volume and the total estimated radiation exposure was 3.9 mSV. In surgical cases of lung cancer, the 4DCT findings were compared with the intraoperative assessment. We obtained 2 sets (upper lung region and lower region) of 4DCT covering the whole lung. These volume sets were connected and evaluated using imaging processing software. Intrathoracic adhesion and tumor invasion were defined by the differential movement between the tumor and adjacent structures, and laterality of the respiratory motion. There were 16 cases of non-neoplastic diseases, such as non-tuberculous mycobacteriosis, giant bulla and lung sequestration. The 15 neoplastic diseases included 2 solitary fibrous tumors and 13 lung cancers. Among them, 12 cases in the lung cancer group underwent surgical procedures and were able to be evaluated. The purposes of the preoperative 4DCT were as follows: evaluation of tumor invasion (n=8), reoperation cases (n=2), medical history of tuberculous mycobacteriosis (n=2) and suspicion of intrathoracic adhesion on chest X-ray (n=1). Adjacent structures were the descending aorta (n=2), pulmonary artery (n=1), subclavian artery (n=1) and chest wall (n=5). The surgical procedures included 10 lobectomies, 1 wedge resection and 1 open lung biopsy. The 4DCT findings were comparable with the intraoperative findings in eleven cases; intraoperative adhesions were noted at the same locations and there were no signs of direct tumor invasion to vital structures, as indicated by 4DCT. In only one case of suspected invasion of the subclavian artery, was unexpected adhesion to the upper mediastinum observed during surgery. As the motion of the apical region was restricted in all cases, we were not able to evaluate apical adhesion. The intraoperative findings supported those of 4DCT in 11/12cases. 4DCT of the whole lung was an useful and reliable tool for the preoperative assessment and determination of the surgical indication for lung cancers.

Borderline pneumothorax

Extraordinary demanding case requires creative solution within expenditure of intellectual energy supported by mature experience, and discerning intuition. This all with single rule: patients benefit. Previous and subsequent sharing and exchange yield full-blown versatility. Pneumothorax in breastfeeding mother, brachial plexus injury caused by lysis of apical adhesions, support of pulmonary plication by autologous fascia lata in advanced pulmonary emphysema, dorsal chest tube insertion through first intercostal space, stubborn pneumothorax in a musician of brass band, successful conservative treatment by chest tube as an alternative of thoracoplasty, talc slurry solution in elderly man with life threatening pneumothorax from advanced lung cancer decay cavity, and in young woman afflicted by lymphangioleiomyomatosis (LAM) are briefly reported.

Scribble, Erbin, and Lano redundantly regulate epithelial polarity and apical adhesion complex.

The basolateral protein Scribble (Scrib), a member of the LAP protein family, is essential for epithelial apicobasal polarity (ABP) in Drosophila However, a conserved function for this protein in mammals is unclear. Here we show that the crucial role for Scrib in ABP has remained obscure due to the compensatory function of two other LAP proteins, Erbin and Lano. A combined Scrib/Erbin/Lano knockout disorganizes the cell-cell junctions and the cytoskeleton. It also results in mislocalization of several apical (Par6, aPKC, and Pals1) and basolateral (Llgl1 and Llgl2) identity proteins. These defects can be rescued by the conserved "LU" region of these LAP proteins. Structure-function analysis of this region determined that the so-called LAPSDb domain is essential for basolateral targeting of these proteins, while the LAPSDa domain is essential for supporting the membrane basolateral identity and binding to Llgl. In contrast to the key role in Drosophila, mislocalization of Llgl proteins does not appear to be critical in the scrib ABP phenotype.

Apical Cell-Cell Adhesions Reconcile Symmetry and Asymmetry in Zebrafish Neurulation

SummaryThe symmetric tissue and body plans of animals are paradoxically constructed with asymmetric cells. To understand how the yin-yang duality of symmetry and asymmetry are reconciled, we asked whether apical polarity proteins orchestrate the development of the mirror-symmetric zebrafish neural tube by hierarchically modulating apical cell-cell adhesions. We found that apical polarity proteins localize by a pioneer-intermediate-terminal order. Pioneer proteins establish the mirror symmetry of the neural rod by initiating two distinct types of apical adhesions: the parallel apical adhesions (PAAs) cohere cells of parallel orientation and the novel opposing apical adhesions (OAAs) cohere cells of opposing orientation. Subsequently, the intermediate proteins selectively augment the PAAs when the OAAs dissolve by endocytosis. Finally, terminal proteins are required to inflate the neural tube by generating osmotic pressure. Our findings suggest a general mechanism to construct mirror-symmetric tissues: tissue symmetry can be established by organizing asymmetric cells opposingly via adhesions.

Further dissection of pleural reflection and release of additional apical adhesions

Further dissection of pleural reflection and release of additional apical adhesions

根尖癒合を伴う歯根が下歯槽神経血管束を取り囲んだ下顎第三大臼歯の2例

根尖癒合を伴う歯根が下歯槽神経血管束を取り囲んだ下顎第三大臼歯の2例

Cavity morphogenesis: imaging mitotic forces in action

Cavity morphogenesis: imaging mitotic forces in action

Biphasic influence of Miz1 on neural crest development by regulating cell survival and apical adhesion complex formation in the developing neural tube

Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle- and cell adhesion-related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest-selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein.

Ephrin B1 maintains apical adhesion of neural progenitors

Apical neural progenitors are polarized cells for which the apical membrane is the site of cell-cell and cell-extracellular matrix adhesion events that are essential for maintaining the integrity of the developing neuroepithelium. Apical adhesion is important for several aspects of the nervous system development, including morphogenesis and neurogenesis, yet the mechanisms underlying its regulation remain poorly understood. Here, we show that ephrin B1, a cell surface protein that engages in cell signaling upon binding cognate Eph receptors, controls normal morphogenesis of the developing cortex. Efnb1-deficient embryos exhibit morphological alterations of the neuroepithelium that correlate with neural tube closure defects. Using loss-of-function experiments by ex vivo electroporation, we demonstrate that ephrin B1 is required in apical progenitors (APs) to maintain their apical adhesion. Mechanistically, we show that ephrin B1 controls cell-ECM adhesion by promoting apical localization of integrin β1 and we identify ADP-ribosylation factor 6 (Arf6) as an important effector of ephrin B1 reverse signaling in apical adhesion of APs. Our results provide evidence for an important role for ephrin B1 in maintaining the structural integrity of the developing cortex and highlight the importance of tightly controlling apical cell-ECM adhesion for neuroepithelial development.

自然気胸における肺尖部索状癒着のCTによる検出能評価―新しい手術適応の提案

【目的】自然気胸における肺尖部の索状癒着は血気胸を生じる危険性がある．これをCTでどれほど検出できるか検討した．【対象と方法】2005年1月から2012年12月まで，当院で胸腔鏡手術を行った本人申告初発自然気胸154例に対し，MPRも併用し，胸部CTによる索状癒着の検出を試みた．【結果】154例中，索状癒着が指摘された症例は31例で，全例胸腔鏡でも索状癒着を確認した．一方，残る123例は指摘出来なかったが，その内4例に術中に索状癒着を確認した．結局，154例中35例（22.7%）に索状癒着が存在し，その35例中31例（88.6%）はCTで検出可能であった．【結語】肺尖部索状癒着は血気胸の原因となるため，これを認める症例は初発再発に関係なく手術適応としても良いと考える．CTはそのようなリスクを有する症例の約90%を検出でき有用であった．

Cell Adhesion Signaling and Its Impact on Tumorigenesis

Cell adhesion can be divided into cell-cell adhesion mediated by three major structures, tight junctions, adherens junctions, desmosomes, and cell-matrix adhesion, which is for the most part integrin-based. Cell-cell interactions play an important role in the maintenance and integrity of tissues and organs but similar to cell-matrix complexes may also transduce cell signaling. While this is the basics for understanding cell adhesion and its function, the role of cell adhesion proteins in disease, especially tumorigenesis, is far more diverse. A summary is given in the paper presented by Dr. Andl in this issue where she also focuses on recently discovered cell adhesion components and the regulation of cell adhesion molecules by miRNAs. In cancer, cell adhesion molecules are generally thought to be deactivated in order to aid in cancer cell dissemination and dispersal. However, aside from the loss of function, various gain-of-function events occur during induction and progression of cancer that can be mediated by cell adhesion molecules. Many of the molecules involved in these processes, such as the components of desmosomes, were initially identified as targets of autoimmune diseases. This special issue outlines the latest findings in research and review articles. Tight junctions mediate apical cell-cell adhesion and regulate epithelial cell polarity. Members of the claudin family are involved in a number of diseases including cancer; however, little is known regarding the mechanisms behind their pathophysiological function. Drs. Singh, Sharma, and Dhawan give an overview of the current state of knowledge in this issue. To identify cell transformation mechanisms other than previously described, Lynch et al. investigated the effect of MMP-7 mediated cleavage of E-cadherin. They demonstrate that loss of cell-cell adhesion is induced following E-cadherin processing, which results in increased cell migration, loss of polarization, and activation of RhoA. Interactions of cell adhesion molecules with their environment are central to their regulation and to cell migration and invasion. In the tumor context, the array of integrin receptors expressed causes the cells to differentially respond to external signals, resist the effects of cytotoxic drugs, and facilitate growth and invasion through a range of different tissue environments encountered by invasive cancer cells. In particular, the regulation of integrin receptor interaction with the extracellular matrix is thought to be critical for those cancers that invade and metastasize using a mesenchymal mode of migration. Jossen et al. show a novel prostate cancer bone metastatic model to assay drug sensitivity in three-dimensional cultures that mimic the preclinical or clinical setting more accurately. In here, they show that prostate cells revert to more epithelial cells, mesenchymal-epithelial transition (MET) in the presence of bone stroma modeling later stages of metastatic colonization, and are sensitized to radiation treatment when blocking E-cadherin or α-integrin. Mesenchymal migration as a therapeutic target for glioblastoma is discussed by Zhong et al. Another potential therapeutic agent, a Src inhibitor, is described by Yaseem et al. Comparison of SKI-606 and Iressa effects, Src/Abl and EGFR inhibitors, respectively, on cervical cancer cell lines shows inhibition of cell proliferation. Additionally, SKI-606 decreases cell migration and invasion through MET accompanied by the re-expression of E-cadherin and the recruitment of β-catenin to the cell membrane. Hironobu Yamashita et al. demonstrate that lysophosphatidic acid, a phospholipid growth factor, induces the stimulation of lamellipodia formation and enhanced cell migration through the upregulation of the TGFβ1 target gene Laminin-322. The role of focal adhesion kinase (FAK) phosphorylation in the dynamic regulation of integrin-based adhesions and cellular migration is shown in a paper by Hamadi et al. Upon pervanadate-induced phosphorylation, FAK delocalizes from focal adhesions to newly formed membrane ruffles in a src-dependent event. Finally, advances in nanotechnology that allow the study of cellular transformation in engineered cellular environments are introduced by Siti Hawa Ngalmi et al. Such cross-disciplinary approaches have begun to advance our knowledge of how adhesion signaling is organized and how cells relay information regarding the composition and structure of the external environment into biological activity. In particular, these advances are being driven by novel microscopy technologies and the continuing development of new techniques that allow us to visualize the intricate workings of adhesion-dependent signaling processes. This synopsis gives a glimpse at the current leading edge science published in this special issue of the Journal of Oncology. We hope that these papers inspire interest in multidisciplinary approaches as well as in the identification of novel therapeutics. Claudia D. Andl Ala-Eddin Al Moustafa Therese B. Deramaudt Geraldine M. O'Neill

Horner’s syndrome in a patient presenting with a spontaneous pneumothorax

Spontaneous pneumothoraces are a common thoracic problem presenting to an Accident and Emergency (A&E) department. The symptoms and signs are well described and a chest x-ray examination is usually diagnostic....

The role of trophinin, an adhesion molecule unique to human trophoblasts, in progression of colorectal cancer

Trophinin is a unique adhesion molecule expressed by human trophoblastic cells. Its activity and in vivo expression pattern implicate trophinin in the initial attachment of trophectoderm cells to maternal epithelia. Subsequent to apical adhesion, trophoblasts aggressively invade maternal tissue to form the placenta, a process resembling tumor invasion. Here, we report that trophinin is expressed in tumors from 64% of colon cancer patients (n = 50) and high trophinin expression is closely associated with poor prognosis. To determine the link between trophinin expression and malignancy, colon adenocarcinoma SW480 cells were stably transfected with trophinin. An invasion assay showed that trophinin-expressing SW480 cells were more invasive than mock-transfected cells. Microarray analysis comparing SW480 cells transfected with trophinin with mock-transfected cells identified high-mobility group box 1 (HMGB1) as the most significantly elevated transcript. Immunohistochemical analysis of tumors from the colorectal cancer patients confirmed positive correlation of HMGB1 protein expression in the nucleus to trophinin expression in tumor. HMGB1 and its ligand RAGE (the receptor for advanced glycation end product) proteins were coexpressed in 65.6% of trophinin-positive patients (n = 32). These results suggest that trophinin promotes invasion through a mechanism involving HMGB1/RAGE.

Surface mechanics mediate pattern formation in the developing retina.

Pattern formation of biological structures involves organizing different types of cells into a spatial configuration. In this study, we investigate the physical basis of biological patterning of the Drosophila retina in vivo. We demonstrate that E- and N-cadherins mediate apical adhesion between retina epithelial cells. Differential expression of N-cadherin within a sub-group of retinal cells (cone cells) causes them to form an overall shape that minimizes their surface contact with surrounding cells. The cells within this group, in both normal and experimentally manipulated conditions, pack together in the same way as soap bubbles do. The shaping of the cone cell group and packing of its components precisely imitate the physical tendency for surfaces to be minimized. Thus, simple patterned expression of N-cadherin results in a complex spatial pattern of cells owing to cellular surface mechanics.

Association of ARVCF with zonula occludens (ZO)-1 and ZO-2: binding to PDZ-domain proteins and cell-cell adhesion regulate plasma membrane and nuclear localization of ARVCF.

ARVCF, an armadillo-repeat protein of the p120(ctn) family, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.

Human Uterine Epithelial RL95-2 Cells Reorganize Their Cytoplasmic Architecture with Respect to Rho Protein and F-Actin in Response to Trophoblast Binding

Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners. Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids. We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells. Before binding of spheroids, significantly higher fluorescence signals of RhoA [37 ± 6 grey scale values (gsv)] and of F-actin (41 ± 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 ± 3 gsv, F-actin: 28 ± 2 gsv). After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 ± 10 gsv apical vs. 25 ± 3 gsv basal; F-actin: 108 ± 17 gsv apical vs. 57 ± 7 gsv basal). Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published. Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells. These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm. This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration.