scholarly journals RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering

Development ◽  
2021 ◽  
Author(s):  
Eunice H. Y. Chan ◽  
Yanxiang Zhou ◽  
Birgit L. Aerne ◽  
Maxine V. Holder ◽  
Anne Weston ◽  
...  

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. RASSF8 loss elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, whose depletion phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

1997 ◽  
Vol 110 (17) ◽  
pp. 2065-2077 ◽  
Author(s):  
M.G. Lampugnani ◽  
M. Corada ◽  
P. Andriopoulou ◽  
S. Esser ◽  
W. Risau ◽  
...  

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.


2004 ◽  
Vol 286 (5) ◽  
pp. C1159-C1169 ◽  
Author(s):  
Ruei-Jiun Hung ◽  
Ia-Wen J. Hsu ◽  
Jennifer L. Dreiling ◽  
Mon-Juan Lee ◽  
Cicely A. Williams ◽  
...  

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.


Development ◽  
1993 ◽  
Vol 119 (Supplement) ◽  
pp. 163-176 ◽  
Author(s):  
Mark Peifer ◽  
Sandra Orsulic ◽  
Li-Mei Pai ◽  
Joseph Loureiro

Cells must cooperate and communicate to form a multicellular animal. Information about the molecules required for these processes have come from a variety of sources; the convergence between the studies of particular molecules by vertebrate cell biologists and the genes identified by scientists investigating development in Drosophila has been especially fruitful. We are interested in the connection between cadherin proteins that regulate cell-cell adhesion and the wingless/wnt-1 cell-cell signaling molecules controlling pattern formation during development. The Drosophila segment polarity gene armadillo, homolog of the vertebrate adherens junction protein-catenin, is required for both cell adhesion and wg signaling. We review what is known about wingless signaling in Drosophila, and discuss the role of cell-cell junctions in both cell adhesion and cell communication. We then describe the results of our preliminary structure-function analysis of Armadillo protein in both cell adhesion and wingless signaling. Finally, we discuss evidence supporting a direct role for Armadillo and adherens junction in transduction of wingless signal.


EvoDevo ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jennyfer M. Mitchell ◽  
Scott A. Nichols

Abstract The integrity and organization of animal tissues depend upon specialized protein complexes that mediate adhesion between cells with each other (cadherin-based adherens junctions), and with the extracellular matrix (integrin-based focal adhesions). Reconstructing how and when these cell junctions evolved is central to understanding early tissue evolution in animals. We examined focal adhesion protein homologs in tissues of the freshwater sponge, Ephydatia muelleri (phylum Porifera; class Demospongiae). Our principal findings are that (1) sponge focal adhesion homologs (integrin, talin, focal adhesion kinase, etc.) co-precipitate as a complex, separate from adherens junction proteins; (2) that actin-based structures resembling focal adhesions form at the cell–substrate interface, and their abundance is dynamically regulated in response to fluid shear; (3) focal adhesion proteins localize to both cell–cell and cell–extracellular matrix adhesions, and; (4) the adherens junction protein β-catenin is co-distributed with focal adhesion proteins at cell–cell junctions everywhere except the choanoderm, and at novel junctions between cells with spicules, and between cells with environmental bacteria. These results clarify the diversity, distribution and molecular composition of cell junctions in tissues of E. muelleri, but raise new questions about their functional properties and ancestry.


2012 ◽  
Vol 196 (1) ◽  
pp. 115-130 ◽  
Author(s):  
Vivian W. Tang ◽  
William M. Brieher

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that cadherin-enriched junctions can polymerize new actin filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with this hypothesis, inhibition of Arp2/3-dependent nucleation abolished actin assembly at cell–cell junctions. Reconstitution biochemistry using the in vitro actin assembly assay identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as an essential factor. α-Actinin-4 specifically localized to sites of actin incorporation on purified membranes and at apical junctions in Madin–Darby canine kidney cells. Knockdown of α-actinin-4 decreased total junctional actin and inhibited actin assembly at the apical junction. Furthermore, a point mutation of α-actinin-4 (K255E) associated with FSGS failed to support actin assembly and acted as a dominant negative to disrupt actin dynamics at junctional complexes. These findings demonstrate that α-actinin-4 plays an important role in coupling actin nucleation to assembly at cadherin-based cell–cell adhesive contacts.


2005 ◽  
Vol 16 (9) ◽  
pp. 4084-4095 ◽  
Author(s):  
Alisa Vespa ◽  
Sudhir J.A. D'Souza ◽  
Lina Dagnino

Integrin-linked kinase (ILK) is a multidomain protein involved in cell motility and cell-extracellular matrix interactions. ILK is found in integrin-containing focal adhesions in undifferentiated primary epidermal keratinocytes. Induction of keratinocyte differentiation by treatment with Ca2+ triggers formation of cell–cell junctions, loss of focal adhesions, and ILK distribution to cell borders. We now show that Ca2+ treatment of keratinocytes induces rapid (≤1 h) translocation to the cell membrane of the adherens junction (AJ) proteins E-cadherin and β-catenin. This is followed by slower (>6 h) localization of tight junction (TJ) proteins. The kinetics of ILK movement toward the cell periphery mimics that of AJ components, suggesting that ILK plays a role in the early formation of cell–cell contacts. Whereas the N terminus in ILK mediates localization to cell borders, expression of an ILK deletion mutant incapable of localizing to the cell membrane (ILK 191-452) interferes with translocation of E-cadherin/β-catenin to cell borders, precluding Ca2+-induced AJ formation. Cells expressing ILK 191-452 also fail to form TJ and sealed cell–cell borders and do not form epithelial sheets. Thus, we have uncovered a novel role for ILK in epithelial cell–cell adhesion, independent of its well-established role in integrin-mediated adhesion and migration.


2019 ◽  
Vol 30 (16) ◽  
pp. 1938-1960 ◽  
Author(s):  
Lathiena A. Manning ◽  
Kia Z. Perez-Vale ◽  
Kristina N. Schaefer ◽  
Mycah T. Sewell ◽  
Mark Peifer

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell–cell junctions to allow dynamic remodeling while resisting forces generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional–cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction–cytoskeletal linkage during gastrulation. Canoe’s mammalian homologue Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We take these insights back to the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found that Canoe helps cells maintain junctional–cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation, and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe, many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homologue Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest that Canoe and Polychaetoid stabilize Bazooka/Par3 at cell–cell junctions, helping maintain balanced apical contractility and tissue integrity.


2013 ◽  
Vol 203 (5) ◽  
pp. 815-833 ◽  
Author(s):  
Vivian W. Tang ◽  
William M. Brieher

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity.


2001 ◽  
Vol 114 (5) ◽  
pp. 941-952 ◽  
Author(s):  
G.K. Ojakian ◽  
D.R. Ratcliffe ◽  
R. Schwimmer

The extracellular matrix plays an important role in regulation of epithelial development and organization. To determine more precisely the function of extracellular matrix in this process, the initial steps in collagen-mediated formation of epithelial tubules were studied using a model cell culture system. Previous studies have demonstrated that incubation of Madin-Darby canine kidney (MDCK) epithelial cells with a collagen gel overlay induces (beta)1 integrin-regulated epithelial remodeling accompanied by extensive cell rearrangements and formation of epithelial tubules. During epithelial remodeling there was extensive disruption of the epithelial junctional complex. Progressive opening of tight junctions was observed over 8 hours using transepithelial resistance measurements and immunofluorescence microscopy demonstrated that tight and adherens junction proteins were dispersed throughout the apical and basolateral membranes. Junction complex disruption allowed the formation of apical cell extensions and subsequent migration of selected cell sheets from the epithelial monolayer. Confocal microscopy demonstrated the presence of adherens junction (E-cadherin, (alpha)-catenin, (beta)-catenin, plakoglobin) and desmosomal (desmoplakin-1/2, plakoglobin) proteins on, and within, cell extensions demonstrating that cell junctions had undergone considerable disassembly. However, groups of cell extensions appeared to be associated by E-cadherin/catenin-mediated interactions. Association of E-cadherin/catenin complexes with the epithelial cytoskeleton was analyzed by differential detergent extraction. SDS-PAGE and immunoblot analysis demonstrated that adherens junction proteins were primarily cytoskeleton-associated in control cells. During integrin-regulated remodeling, there was a progressive reduction in the interaction of adherens junction proteins with the cytoskeleton suggesting that they play an important role in the maintenance of epithelial integrity. Since loss of transepithelial electrical resistance and disruption of junctional complexes were inhibited by an antifunctional integrin antibody, we propose that activation of integrin signaling pathways regulate junctional complex stability, cell-cell interactions and cell migration. These observations provide evidence that integrin-regulated MDCK epithelial tubule formation can serve as a model system for studying rearrangements of epithelial sheets which occur during development.


Sign in / Sign up

Export Citation Format

Share Document