The septate junction protein Tetraspanin 2A is critical to the structure and function of Malpighian tubules in Drosophila melanogaster

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.

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Topographical variations in the structure of the smooth septate junction

Journal of Cell Science ◽

10.1242/jcs.37.1.373 ◽

1979 ◽

Vol 37 (1) ◽

pp. 373-389

Author(s):

H.B. Skaer ◽

J.B. Harrison ◽

W.M. Lee

Keyword(s):

Musca Domestica ◽

Functional Significance ◽

Freeze Fracture ◽

Rhodnius Prolixus ◽

Malpighian Tubules ◽

Septate Junction ◽

Lanthanum Hydroxide ◽

Septate Junctions ◽

Standard Fixation

Smooth septate junctions in the midgut of Musca domestica and in Malpighian tubules of both Musca and Rhodnius prolixus are described. Details of the structures revealed after standard fixation, fixation in the presence of the stain, lanthanum hydroxide, and after freeze-fracture are discussed in the light of models previously put forward to explain the interrelations of the images obtained by these different methods. The organization of the junction between cells of the midgut varies in the apical-to-basal axis. At the apical border the septa (or ridges in freeze-fracture replicas) are packed tightly and follow an undulating but strictly parallel course. This packing loosens towards the middle of the junction until, at its basal extremity, the septa (ridges in replicas) are widely separated and follow independent meandering courses. That these features are found both in lanthanum-infiltrated specimens and freeze-fracture replicas allows a correlation to be made between the septa and the freeze-fracture ridges. The functional significance of these smooth septate junctions is discussed.

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The septate junction protein Mesh is required for epithelial morphogenesis, ion transport, and paracellular permeability in the Drosophila Malpighian tubule

AJP Cell Physiology ◽

10.1152/ajpcell.00492.2019 ◽

2020 ◽

Vol 318 (3) ◽

pp. C675-C694 ◽

Cited By ~ 2

Author(s):

Sima Jonusaite ◽

Klaus W. Beyenbach ◽

Heiko Meyer ◽

Achim Paululat ◽

Yasushi Izumi ◽

...

Keyword(s):

Ion Transport ◽

Malpighian Tubule ◽

Paracellular Permeability ◽

Malpighian Tubules ◽

Septate Junction ◽

Cell Junctions ◽

Chloride Permeability ◽

Transepithelial Potential ◽

Junction Protein ◽

Urine Production

Septate junctions (SJs) are occluding cell-cell junctions that have roles in paracellular permeability and barrier function in the epithelia of invertebrates. Arthropods have two types of SJs, pleated SJs and smooth SJs (sSJs). In Drosophila melanogaster, sSJs are found in the midgut and Malpighian tubules, but the functions of sSJs and their protein components in the tubule epithelium are unknown. Here we examined the role of the previously identified integral sSJ component, Mesh, in the Malpighian tubule. We genetically manipulated mesh specifically in the principal cells of the tubule at different life stages. Tubules of flies with developmental mesh knockdown revealed defects in epithelial architecture, sSJ molecular and structural organization, and lack of urine production in basal and kinin-stimulated conditions, resulting in edema and early adult lethality. Knockdown of mesh during adulthood did not disrupt tubule epithelial and sSJ integrity but decreased the transepithelial potential, diminished transepithelial fluid and ion transport, and decreased paracellular permeability to 4-kDa dextran. Drosophila kinin decreased transepithelial potential and increased chloride permeability, and it stimulated fluid secretion in both control and adult mesh knockdown tubules but had no effect on 4-kDa dextran flux. Together, these data indicate roles for Mesh in the developmental maturation of the Drosophila Malpighian tubule and in ion and macromolecular transport in the adult tubule.

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Septate junctions regulate gut homeostasis through regulation of stem cell proliferation and enterocyte behavior in Drosophila

10.1101/582148 ◽

2019 ◽

Author(s):

Yasushi Izumi ◽

Kyoko Furuse ◽

Mikio Furuse

Keyword(s):

Cell Proliferation ◽

Stem Cell ◽

Intestinal Barrier ◽

Septate Junction ◽

Intestinal Barrier Function ◽

Barrier Dysfunction ◽

Septate Junctions ◽

Stem Cell Proliferation ◽

Associated Proteins ◽

Intestinal Hypertrophy

AbstractSmooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route of epithelial cells in the Drosophila midgut. We previously identified three sSJ-associated membrane proteins, Ssk, Mesh, and Tsp2A, and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. Here, we investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ-proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAP kinase and Jak-Stat pathways in stem cells. Loss of cytokines Unpaired2 and Unpaired3, which are involved in Jak-Stat pathway activation, suppressed the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut.Summary statementDepletion of smooth septate junction-associated proteins from enterocytes in the Drosophila adult midgut results in intestinal hypertrophy accompanied by accumulation of morphologically aberrant enterocytes and increased stem cell proliferation.

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A novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut

10.1101/2020.11.10.376210 ◽

2020 ◽

Author(s):

Yasushi Izumi ◽

Kyoko Furuse ◽

Mikio Furuse

Keyword(s):

Stem Cell ◽

Membrane Protein ◽

Intestinal Barrier ◽

Septate Junction ◽

Barrier Dysfunction ◽

Septate Junctions ◽

Cell Homeostasis ◽

Intestinal Barrier Dysfunction ◽

Epithelial Tumors ◽

Junction Protein

AbstractSmooth septate junctions (sSJs) regulate the paracellular transport in the intestinal and renal system in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh, and Tsp2A. Here, we report a novel sSJ membrane protein Hoka, which has a single membrane-spanning segment with a short extracellular region having 13-amino acids, and a cytoplasmic region with three repeats of the Tyr-Thr-Pro-Ala motif. The larval midgut in hoka-mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh, and Tsp2A and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors. In hoka-knockdown midguts, aPKC is up-regulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ-proteins Ssk, Mesh, and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut.Summary statementDepletion of hoka, a gene encoding a novel septate junction protein, from the Drosophila midgut results in the disruption of septate junctions, intestinal barrier dysfunction, stem cell overproliferation, and epithelial tumors.

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CircCDYL Acts as a Tumor Suppressor in Wilms’ Tumor by Targeting miR-145-5p

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.668947 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rui Zhou ◽

Wei Jia ◽

Xiaofeng Gao ◽

Fuming Deng ◽

Kai Fu ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cells ◽

Wilms Tumor ◽

Expression Level ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Junction Protein

Circular RNAs (circRNA) have been reported to exert evident functions in many human carcinomas. However, the possible mechanisms concerning the circRNA in various tumors are still elusive. In this research, we analyzed the expression profile and biological functions of circular RNA CDYL (circCDYL, circBase ID: hsa_circ_0008285) in Wilms’ tumor. Here, miRNA and gene expression were examined by real-time PCR in Wilms’ tumor tissues and cell lines. The functions of circCDYL and its potential targets to influence cell proliferation, migration, and invasion in Wilms’ tumor cells were determined by biological functional experiments in vitro and in vivo. We predicted and analyzed potential miRNA targets through online bioinformatic tools. To validate the interactions between circCDYL and its targets, we performed RNA fluorescence in situ hybridization, biotin-coupled miRNA capture assay, and biotin-coupled probe pull-down assay. Tight junction protein l (TJP1) was proved to be the target gene of the predicted miRNA by dual-luciferase reporter assay. The expression level of TJP1 in Wilms’ tumor cells was identified via Western blot. We showed that circCDYL was downregulated in WT tissue compared with adjacent non-tumor tissue. Upregulation of circCDYL could reduce cell proliferation, migration, and invasion. Mechanically, circCDYL, functioning as a miRNA sponge, decreased the expression level of miR-145-5p and TJP1 3′UTR was validated as the target of miR-145-5p, facilitating the circCDYL/miR-145-5p/TJP1 axis. In conclusion, our study suggested circCDYL as a novel biomarker and therapeutic target for WT treatment.

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The septate junction protein Snakeskin is critical for epithelial barrier function and tissue homeostasis in the Malpighian tubules of adult Drosophila

10.1101/2020.12.14.422678 ◽

2020 ◽

Author(s):

Anthony J Dornan ◽

Kenneth A Halberg ◽

Liesa-Kristin Beuter ◽

Shireen-Anne Davies ◽

Julian A.T. Dow

Keyword(s):

Barrier Function ◽

Tissue Homeostasis ◽

Malpighian Tubules ◽

Septate Junction ◽

Epithelial Barrier ◽

Renal Tubules ◽

Epithelial Barrier Function ◽

Adhesion Protein ◽

Junction Protein ◽

Secretory Transport

Transporting epithelia provide a protective physical barrier while directing appropriate transport of ions, solutes and water. In invertebrates, epithelial integrity is dependent on formation, and maintenance, of ′tight′ septate junctions (SJs). We demonstrated that Drosophila Malpighian (renal) tubules undergo an age-dependent decline in secretory transport capacity, which correlates with mislocalisation of SJ proteins and coincident progressive degeneration in cellular morphology and tissue homeostasis. By restrictively impairing, in adult tubules, the cell adhesion protein Snakeskin, which is essential for smooth SJ formation, we observed progressive changes in cellular and tissue morphology that phenocopied these effects, including mislocalisation of junctional proteins with concomitant loss of cell polarity and barrier function. Resulting in significant accelerated decline in tubule secretory capacity and organismal viability. Our investigations highlight the tubule′s essential role in maintenance of organismal health, while providing measurable markers of compromised epithelial barrier and tissue function that manifest in advanced morbidity and death.

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The wingless product is required for cell proliferation in the Malpighian tubule anlage of Drosophila melanogaster

Development ◽

10.1242/dev.116.3.745 ◽

1992 ◽

Vol 116 (3) ◽

pp. 745-754 ◽

Cited By ~ 19

Author(s):

H. Skaer ◽

A. Martinez Arias

Keyword(s):

Cell Proliferation ◽

Drosophila Melanogaster ◽

Cell Division ◽

Malpighian Tubule ◽

Malpighian Tubules ◽

Temperature Sensitive ◽

Over Expression ◽

Normal Expression ◽

Segment Polarity ◽

Sensitive Allele

Cell division in the Malpighian tubules of Drosophila melanogaster depends on the presence of a specialised cell at the tip of each tubule (Skaer, H. le B (1989) Nature 342, 566–569). Here we show that cell division also depends on the normal expression of the segment polarity gene, wingless. The pattern of wingless RNA and protein in developing tubules is consistent with a requirement for wingless for cell division. Analysis of the temporal requirement for wingless using a temperature- sensitive allele confirms that the normal expression of wingless is necessary during cell proliferation in the Malpighian tubules. Over-expression of the gene, induced in a stock containing the wg gene under the control of a heat-shock promoter, results in supernumerary cells in the tubules. We discuss the role of wingless in the cell interactions that govern cell division in the Malpighian tubules.

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Characterization of transepithelial transport of salicylate by the Malpighian tubules of Drosophila melanogaster and the effects of changes in fluid secretion rate

Physiological Entomology ◽

10.1111/j.1365-3032.2007.00563.x ◽

2007 ◽

Vol 32 (2) ◽

pp. 157-166 ◽

Cited By ~ 6

Author(s):

ESAU RUIZ-SANCHEZ ◽

MICHAEL J. O?DONNELL

Keyword(s):

Drosophila Melanogaster ◽

Fluid Secretion ◽

Malpighian Tubules ◽

Secretion Rate ◽

Transepithelial Transport

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Role of 4-O Galloylchlorogenic Acid in Lung Cancer- An Insilico Approach

International Journal of Pharmaceutical and Clinical Research ◽

10.25258/ijpcr.v9i5.8595 ◽

2017 ◽

Vol 9 (5) ◽

Author(s):

Jaynthy C. ◽

N. Premjanu ◽

Abhinav Srivastava

Keyword(s):

Lung Cancer ◽

Cell Proliferation ◽

In Silico ◽

Computational Study ◽

Regulation Mechanism ◽

Down Regulation ◽

Fruit Extract ◽

In Vitro Techniques ◽

Discovery Studio

Cancer is a major disease with millions of patients diagnosed each year with high mortality around the world. Various studies are still going on to study the further mechanisms and pathways of the cancer cell proliferation. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. In cancer development increased core fucosylation by FUT8 play an important role in cell proliferation. Down regulation of FUT8 expression may help cure lung cancer. Therefore the computational study based on the down regulation mechanism of FUT8 was mechanised. Sapota fruit extract, containing 4-Ogalloylchlorogenic acid was used as the inhibitor against FUT-8 as target and docking was performed using in-silico tool, Accelrys Discovery Studio. There were several conformations of the docked result, and conformation 1 showed 80% dock score between the ligand and the target. Further the amino acids of the inhibitor involved in docking were studied using another tool, Ligplot. Thus, in-silico analysis based on drug designing parameters shows that the fruit extract can be studied further using in-vitro techniques to know its pharmacokinetics.

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