scholarly journals Formin-Dependent Adhesions are Required for Invasion by Epithelial Tissues

2017 ◽  
Author(s):  
Tim B. Fessenden ◽  
Yvonne Beckham ◽  
Mathew Perez-Neut ◽  
Aparajita H. Chourasia ◽  
Kay F. Macleod ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
Mdck Cells ◽  
Collagen Matrix ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
Mouse Tumor ◽  
Collective Invasion ◽  
Shape Changes ◽  
Tumor Organoids

AbstractDeveloping tissues change shape, and tumors initiate spreading, through collective cell motility. Conserved mechanisms by which tissues initiate motility into their surroundings are not known. We investigated cytoskeletal regulators during collective invasion by mouse tumor organoids and epithelial MDCK acini undergoing branching morphogenesis. Inhibition of formins, but not Arp2/3, prevented the formation of migrating cell fronts in both cell types. MDCK cells depleted of the formin protein Dia1 formed polarized acini and could execute planar cell motility, either within the acinus or in 2D scattering assays. However, Dia1 was required to form protrusions into the collagen matrix. Live imaging of actin, myosin, and collagen in control acini revealed adhesions that deformed individual collagen fibrils, while Dia1-depleted acini exhibited unstable adhesions with minimal collagen deformation. This work identifies Dia1-mediated adhesions as essential regulators of tissue shape changes, through their role in focal adhesion maturation.

scholarly journals Dia1-dependent adhesions are required by epithelial tissues to initiate invasion

2018 ◽  
Vol 217 (4) ◽  
pp. 1485-1502 ◽  
Author(s):  
Tim B. Fessenden ◽  
Yvonne Beckham ◽  
Mathew Perez-Neut ◽  
Guillermina Ramirez-San Juan ◽  
Aparajita H. Chourasia ◽  
...  
Keyword(s):  
Cell Motility ◽  
Mdck Cells ◽  
Focal Adhesions ◽  
Collagen Matrix ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
Mouse Tumor ◽  
Shape Changes ◽  
Darby Canine Kidney

Developing tissues change shape and tumors initiate spreading through collective cell motility. Conserved mechanisms by which tissues initiate motility into their surroundings are not known. We investigated cytoskeletal regulators during collective invasion by mouse tumor organoids and epithelial Madin–Darby canine kidney (MDCK) acini undergoing branching morphogenesis in collagen. Use of the broad-spectrum formin inhibitor SMIFH2 prevented the formation of migrating cell fronts in both cell types. Focusing on the role of the formin Dia1 in branching morphogenesis, we found that its depletion in MDCK cells does not alter planar cell motility either within the acinus or in two-dimensional scattering assays. However, Dia1 was required to stabilize protrusions extending into the collagen matrix. Live imaging of actin, myosin, and collagen in control acini revealed adhesions that deformed individual collagen fibrils and generated large traction forces, whereas Dia1-depleted acini exhibited unstable adhesions with minimal collagen deformation and lower force generation. This work identifies Dia1 as an essential regulator of tissue shape changes through its role in stabilizing focal adhesions.

scholarly journals Optogenetic model reveals cell shape regulation through FAK and Fascin

2021 ◽  
Author(s):  
Jean A. Castillo-Badillo ◽  
N. Gautam
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cell Shape ◽  
Spherical Shape ◽  
Cell Types ◽  
Experimental Models ◽  
Molecular Processes ◽  
Different Cell Types ◽  
Shape Changes ◽  
In Cells

Cell shape regulation is important but the mechanisms that govern shape are not fully understood, in part due to limited experimental models where cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we use an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retains its shape and a non-nucleated side which was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types.

scholarly journals Filamin A–β1 Integrin Complex Tunes Epithelial Cell Response to Matrix Tension

2009 ◽  
Vol 20 (14) ◽  
pp. 3224-3238 ◽  
Author(s):  
Scott Gehler ◽  
Massimiliano Baldassarre ◽  
Yatish Lad ◽  
Jennifer L. Leight ◽  
Michele A. Wozniak ◽  
...  
Keyword(s):  
Collagen Matrix ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
High Density ◽  
Actin Binding ◽  
Β1 Integrin ◽  
Filamin A ◽  
Collagen Gels ◽  
Collagen Remodeling ◽  
The Matrix

The physical properties of the extracellular matrix (ECM) regulate the behavior of several cell types; yet, mechanisms by which cells recognize and respond to changes in these properties are not clear. For example, breast epithelial cells undergo ductal morphogenesis only when cultured in a compliant collagen matrix, but not when the tension of the matrix is increased by loading collagen gels or by increasing collagen density. We report that the actin-binding protein filamin A (FLNa) is necessary for cells to contract collagen gels, and pull on collagen fibrils, which leads to collagen remodeling and morphogenesis in compliant, low-density gels. In stiffer, high-density gels, cells are not able to contract and remodel the matrix, and morphogenesis does not occur. However, increased FLNa-β1 integrin interactions rescue gel contraction and remodeling in high-density gels, resulting in branching morphogenesis. These results suggest morphogenesis can be “tuned” by the balance between cell-generated contractility and opposing matrix stiffness. Our findings support a role for FLNa-β1 integrin as a mechanosensitive complex that bidirectionally senses the tension of the matrix and, in turn, regulates cellular contractility and response to this matrix tension.

Mutant cadherin affects epithelial morphogenesis and invasion, but not transformation

2001 ◽  
Vol 114 (6) ◽  
pp. 1237-1246 ◽  
Author(s):  
M.L. Troxell ◽  
D.J. Loftus ◽  
W.J. Nelson ◽  
J.A. Marrs
Keyword(s):  
Tumor Progression ◽  
Mdck Cells ◽  
Collagen Gel ◽  
Collagen Matrix ◽  
Soft Agar ◽  
Epithelial Morphogenesis ◽  
Adhesion System ◽  
Severe Impairment ◽  
Hepatocyte Growth ◽  
E Cadherin

MDCK cells were engineered to reversibly express mutant E-cadherin protein with a large extracellular deletion. Mutant cadherin overexpression reduced the expression of endogenous E- and K-cadherins in MDCK cells to negligible levels, resulting in decreased cell adhesion. Despite severe impairment of the cadherin adhesion system, cells overexpressing mutant E-cadherin formed fluid-filled cysts in collagen gel cultures and responded to hepatocyte growth factor/scatter factor (HGF/SF) that induced cellular extension formation with a frequency similar to that of control cysts. However, cells were shed from cyst walls into the lumen and into the collagen matrix prior to and during HGF/SF induced tubule extension. Despite the propensity for cell dissociation, MDCK cells lacking cadherin adhesion molecules were not capable of anchorage-independent growth in soft agar and cell proliferation rate was not affected. Thus, cadherin loss does not induce transformation, despite inducing an invasive phenotype, a later stage of tumor progression. These experiments are especially relevant to tumor progression in cells with altered E-cadherin expression, particularly tumor samples with identified E-cadherin extracellular domain genomic mutations.

Modulation of alpha-actinin levels affects cell motility and confers tumorigenicity on 3T3 cells

1994 ◽  
Vol 107 (7) ◽  
pp. 1773-1782 ◽  
Author(s):  
U. Gluck ◽  
A. Ben-Ze'ev
Keyword(s):  
Cell Motility ◽  
Colloidal Gold ◽  
Actin Filaments ◽  
Cell Types ◽  
Cell Behavior ◽  
Regulatory Pathway ◽  
3T3 Cells ◽  
Quiescent Cells ◽  
Beta 1 Integrin ◽  
Cdna Construct

alpha-Actinin is an abundant actin crosslinking protein, also localized at adherens type junctions. In adhesion plaques, alpha-actinin can link the actin filaments to integrin via vinculin and talin, or directly by binding to the cytoplasmic domain of beta 1-integrin. The expression of alpha-actinin is rapidly elevated in growth-activated quiescent cells, and is reduced in SV40-transformed 3T3 cells and various differentiating cell types (reviewed by Gluck, U., Kwiatkowski, D. J. and Ben-Ze'ev, A. Proc. Nat. Acad. Sci. USA 90, 383–387, 1993). To study the effect of changes in alpha-actinin levels on cell behavior, alpha-actinin expression was elevated in 3T3 cells by transfection with a full-length human nonmuscle alpha-actinin cDNA. To suppress alpha-actinin levels, 3T3 cells were transfected with an antisense alpha-actinin cDNA construct. Cells overexpressing alpha-actinin by 40–60% displayed a significant reduction in cell motility, as demonstrated by their slower locomotion into an artificial wound, and by forming shorter phagokinetic tracks on colloidal gold-coated substrata. 3T3 cells in which the expression of alpha-actinin was reduced to 25–60% of control levels, after antisense alpha-actinin transfection, had an increased cell motility. Moreover, such alpha-actinin-deficient 3T3 cells formed tumors upon injection into nude mice. The results demonstrate that modulations in alpha-actinin expression can affect, in a major way, the motile and tumorigenic properties of cells, and support the view that decreased alpha-actinin expression could be a common regulatory pathway to malignant transformation of 3T3 cells.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

2000 ◽  
Vol 113 (23) ◽  
pp. 4193-4202 ◽  
Author(s):  
N.R. Meerson ◽  
V. Bello ◽  
J.L. Delaunay ◽  
T.A. Slimane ◽  
D. Delautier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Intracellular Traffic ◽  
Transmembrane Glycoprotein ◽  
Direct Demonstration ◽  
Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway

1998 ◽  
Vol 111 (5) ◽  
pp. 615-624 ◽  
Author(s):  
H. Xie ◽  
M.A. Pallero ◽  
K. Gupta ◽  
P. Chang ◽  
M.F. Ware ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Motility ◽  
Map Kinase ◽  
Signaling Pathway ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Focal Adhesions ◽  
Short Term ◽  
Egfr Kinase

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Deficiency in intercellular communication in two established renal epithelial cell lines (LLC-PK1 and MDCK)

1984 ◽  
Vol 66 (1) ◽  
pp. 81-93
Author(s):  
F.V. Sepulveda ◽  
J.D. Pearson
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Mdck Cells ◽  
Cell Communication ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Renal Epithelial Cell ◽  
Aortic Endothelial Cells ◽  
Epithelial Cell Lines ◽  
Autoradiographic Analysis

We have studied the cell-to-cell passage of uridine nucleotides in two renal epithelial cell lines (LLC-PK1 and MDCK) and in porcine aortic endothelial cells (PAE). All three cell types incorporated tritiated uridine. After a 3 h incubation the radioactivity was predominantly in the form of acid-soluble compounds, mainly UTP. Prelabelled LLC-PK1 or MDCK cells were unable to transfer radioactivity to added adjacent, non-labelled cells, whereas PAE cells readily formed communicating intercellular junctions, as judged by autoradiographic analysis after a 3 h co-culture period. Cell-to-cell communication in either of the renal cell lines was not promoted by treatment with dibutyryl cyclic AMP and methylisobutylxanthine. Radioactivity incorporated into the acid-insoluble pool was not available for intercellular transfer, as assessed in experiments in which cells were prelabelled 24 h before co-culture.

TMOD-29. STANDARDIZED GENERATION OF TUMOR-ORGANOIDS AS NOVEL DRUG SCREENING MODEL IN MENINGIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi221-vi222
Author(s):  
Gerhard Jungwirth ◽  
Tao Yu ◽  
Cao Junguo ◽  
Catharina Lotsch ◽  
Andreas Unterberg ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cancer Biology ◽  
Drug Testing ◽  
Large Scale ◽  
Ex Vivo ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Drug Responses ◽  
Novel Drug ◽  
Tumor Organoids

Abstract Tumor-organoids (TOs) are novel, complex three-dimensional ex vivo tissue cultures that under optimal conditions accurately reflect genotype and phenotype of the original tissue with preserved cellular heterogeneity and morphology. They may serve as a new and exciting model for studying cancer biology and directing personalized therapies. The aim of our study was to establish TOs from meningioma (MGM) and to test their usability for large-scale drug screenings. We were capable of forming several hundred TO equal in size by controlled reaggregation of freshly prepared single cell suspension of MGM tissue samples. In total, standardized TOs from 60 patients were formed, including eight grade II and three grade III MGMs. TOs reaggregated within 3 days resulting in a reducted diameter by 50%. Thereafter, TO size remained stable throughout a 14 days observation period. TOs consisted of largely viable cells, whereas dead cells were predominantly found outside of the organoid. H&E stainings confirmed the successful establishment of dense tissue-like structures. Next, we assessed the suitability and reliability of TOs for a robust large-scale drug testing by employing nine highly potent compounds, derived from a drug screening performed on several MGM cell lines. First, we tested if drug responses depend on TO size. Interestingly, drug responses to these drugs remained identical independent of their sizes. Based on a sufficient representation of low abundance cell types such as T-cells and macrophages an overall number of 25.000 cells/TO was selected for further experiments revealing FDA-approved HDAC inhibitors as highly effective drugs in most of the TOs with a mean z-AUC score of -1.33. Taken together, we developed a protocol to generate standardized TO from MGM containing low abundant cell types of the tumor microenvironment in a representative manner. Robust and reliable drug responses suggest patient-derived TOs as a novel drug testing model in meningioma research.

scholarly journals Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses

2000 ◽  
Vol 149 (7) ◽  
pp. 1419-1432 ◽  
Author(s):  
Ute Schaeper ◽  
Niels H. Gehring ◽  
Klaus P. Fuchs ◽  
Martin Sachs ◽  
Bettina Kempkes ◽  
...  
Keyword(s):  
Binding Site ◽  
Mdck Cells ◽  
Mapk Pathway ◽  
Biological Response ◽  
Adaptor Protein ◽  
Branching Morphogenesis ◽  
Binding Motif ◽  
Yeast Two Hybrid ◽  
Interaction Sites ◽  
Two Hybrid

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met–specific branching morphogenesis. It associates directly with c-Met via the c-Met–binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met–binding site is localized to a 13–amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met–binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1–specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

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