Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics.
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ABSTRACT:
SUBMITTER: Chambers KF
PROVIDER: S-EPMC3078910 | biostudies-literature | 2011 Apr
REPOSITORIES: biostudies-literature
Publications
Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics.
Chambers Karen F KF Pearson Joanna F JF Aziz Naveed N O'Toole Peter P Garrod David D Lang Shona H SH
PloS one 20110418 4
<h4>Background</h4>Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism fu ...[more]
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Project description:In order to metastasize, few cells need to detach from the primary tumor, and transition through the circulation to distant sites to establish new solid tumor metastases. The gene expression changes occurring between these transitions are poorly understood, and controversial findings have been reported regarding transitions between epithelial (E) and mesenchymal (M) cell states. To model the metastasis process in vitro, here we examined the dynamics of gene expression changes and respective cell fates that are occurring in cells that are transitioning from adhesion culture to suspension 3D culture (mammospheres) and back to adhesion culture in vitro. 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We cultured cell lines under 3D suspension conditions resembling mammosphere growth conditions that are typically used to enrich for stem-like cells (Dontu et al., 2003), and examined gene expression of cells under mammosphere conditions (_sus, GSE66527), or after replating (_re) in a kinetic examining gene expression from 6, 24, 96, and 240 hrs after cells were allowed to readhere to normal dishes for the heterogeneous but mostly epithelial (HP) cell lines and mesenchymal (M4) cell lines. To validate our findings we also examined gene expression of cells replated after mammosphere culture for about two weeks from three different E (E3, E4, E5) and M clones (M3, M4, M1). All gene expression samples were taken from biological replicates (at least duplicates) of cells grown in different wells or even at different times. Gene expression of replated (_re) cells was compared to the respective cell lines grown under adhesion conditions (_adh), as well as to the respective cell line grown under suspension mammosphere condition (_sus) which have been deposited previously under accession number GSE66527. HP replicates (re): HP_re_AGW120913, HP_re_RK12026, HP_re_AGW1209_5, HP_re_AGW1209_6, HP_re_AGW1209_7, HP_re_AGW1209_8, HP_re_AGW1209_9, HP_re_0607_1, HP_re_0607_2 E3 replicates (re): E3_re_RK1202, E3_re_RK12028; E4 replicates: E4_re_0622, E4_re_0613; E5 replicates: E5_re_0613_1, E5_re_0613_2 M1 replicates (re): M1_re_0607_1, M1_re_0607_2, M2 replicates: M2_re_RK1202, M2_re_RK12029, M2_re_AGW1209_3, M2_re_AGW1209_4, M2_re_AGW1209_5, M3 replicates: M4_re_0607_1, M4_re_0607_2 HP replicates (adh): HP_adh_0629, HP_adh_2210, HP_adh_0607sc, HP_adh_0607c E3 replicates (adh): E3_adh_0629, E3_adh, E3_adh_0826. E4 replicates (adh): E4_adh_0607, E4_adh_0210, E5 replicates (adh): E5_adh_0210, E5_adh_0607 M1 replicates (adh): M1_adh_0210, M1_adh_0607, M4 replicates (adh): M4_adh_0607, M4_adh_0210, M3 replicates (adh): M3_adh, M3_adh_2210, M3_adh_2810 HP replating kinetic (adh): HP_adh_0607c (GSE66527), HP_adh_0607sc (GSE66527), HP (mammospheres, suspension): HP_sus_0603_2 (GSE66527), HP_sus_0603_1 (GSE66527), HP (replated 6 hrs): HP_re_6_0603_1, HP_re_6_0603_2, HP (replated 24hrs): HP_re_24_0607_1, HP_re_24_0607_2, HP (replated 96 hrs): HP_re_96_0607_1, HP_re_96_0607_2, HP (replated 240hrs): HP_re_0607_1, HP_re_0607_2, M4 replating kinetic (adh): M4_adh_0210 (GSE66527), M4_adh_0607 (GSE66527), M4 (mammospheres, suspension): M4_sus_0603_2 (GSE66527), M4_sus_0603_1 (GSE66527), M4 (replated 24 hrs): M4_re_24_0603_1, M4_re_24_0603_2, M4 (replated 96 hrs), M4_re_96_0607_1, M4_re_96_0607_2, M4 replated 240hrs): M4_re_0607_1, M4_re_0607_2
2015-07-10 | E-GEOD-70279 | biostudies-arrayexpress
Project description:Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell-cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.
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We found that mammosphere and replating cultures of epithelial (E) cell lines (HP cells and E clones) led to enrichment for mesenchymal (M) signatures characteristic of the mesenchymal cell lines, indicative of an epithelial to mesenchymal transition (EMT), but retainment of E-specific signatures (Grosse-Wilde et al., 2015). This co-expression of E and M signatures was stably maintained between the mammosphere state in suspension and in replated cells, indicating a stable E/M gene expression state by partial EMT in E cell lines, which has been associated with a stem-like state (Grosse-Wilde et al., 2015; Jolly et al., 2014; Lu et al., 2014; Zhang et al., 2014). Interestingly, mammosphere suspension culture of mesenchymal M clones also resulted in co-expression of E and M signatures generated by partial mesenchymal to epithelial transition (MET). However, in stark contrast to E clones and HP cells, immediately upon replating M cell-derived mammospheres to adhesion lost the intermediate E/M state and converted back to stable expression of M signatures indicative of rapid EMT. This suggests that the intermediate state in M cells is very unstable. Together, these replating data suggest that prolonged detachment of epithelial-derived cells leads to loss of expression of E signatures and EMT. Life-threatening macrometastases typically recapitulate the gene expression of the primary tumor, which requires expression of E signatures, while the mesenchymal micrometastases are frequent but not-proliferative and harmless. Since all HMLER cell lines independent of their initial E or M state converged to an M state upon expansion in suspension, our data suggest that induction of EMT in suspended HMLER cells may be one reason for the observed benign non-metastatic character of HMLER cells when used in mouse xenograft experiments, and for their metastatic inefficiency. Our datasets of the replating kinetic may be useful to identify which pathways are involved in the processes enabling the mixed HP population to maintain a stable E/M expression signature and a more stem-like state, which we have identified as caused by cell-cell cooperation between phenotypically different E and M cell-types. By contrast, M cells can only transiently assume this E/M gene expression state, and not generate a stable phenotypic heterogeneity and therefore absence of cooperation between E and M cell-types. 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Zhang, J., Tian, X.-J., Zhang, H., Teng, Y., Li, R., Bai, F., Elankumaran, S., and Xing, J. (2014). TGF- -induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci. Signal. 7, ra91–ra91.
2015-07-10 | GSE70279 | GEO
Project description:The adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies proteins with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. Here we use ERC to identify proteins with evolutionary histories similar to the Drosophila E-cadherin (DE-cad) ortholog. Core adherens junction components α-catenin and p120-catenin displayed positive ERC correlations with DE-cad, indicating that they evolved under similar selective pressures during evolution between Drosophila species. Further analysis of the DE-cad ERC profile revealed a collection of proteins not previously associated with DE-cad function or cadherin-mediated adhesion. We then analyzed the function of a subset of ERC-identified candidates by RNAi during border cell (BC) migration and identified novel genes that function to regulate DE-cad. Among these, we found that the gene CG42684, which encodes a putative GTPase activating protein (GAP), regulates BC migration and adhesion. We named CG42684 raskol ("to split" in Russian) and show that it regulates DE-cad levels and actin protrusions in BCs. We propose that Raskol functions with DE-cad to restrict Ras/Rho signaling and help guide BC migration. Our results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion.
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Project description:Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, β-catenin, and the filamentous (F)-actin binding protein αE-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.
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Project description:Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.
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