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A new strategy to measure intercellular adhesion forces in mature cell-cell contacts

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ABSTRACT

Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.

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Disassembly of cell-cell contacts upon upregulation of MSX1 in a HUAEC monolayer.(a–c) Immunofluorescence staining on HUAECs transduced with Cherry- (a; control) or MSX1-encoding (b) lentivirus for the adherens junction marker VEC (green) and nuclear marker TO-PRO-3 (blue) and the corresponding quantification in (c). A reduced expression of VEC is observed in MSX1-overexpressing cells. Data in (c) are expressed as mean area (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). (d–f) Staining of Zonula Occludens-1 (ZO1; green) and nuclear marker TO-PRO-3 (blue). MSX1-overexpressing cells (e) show a reduction of ZO1 expression as compared to control cells (d). Panel (f) shows the corresponding quantification in which data are expressed as mean area of positive ZO1 signal (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). Scale bars: 25 μm.
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f3: Disassembly of cell-cell contacts upon upregulation of MSX1 in a HUAEC monolayer.(a–c) Immunofluorescence staining on HUAECs transduced with Cherry- (a; control) or MSX1-encoding (b) lentivirus for the adherens junction marker VEC (green) and nuclear marker TO-PRO-3 (blue) and the corresponding quantification in (c). A reduced expression of VEC is observed in MSX1-overexpressing cells. Data in (c) are expressed as mean area (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). (d–f) Staining of Zonula Occludens-1 (ZO1; green) and nuclear marker TO-PRO-3 (blue). MSX1-overexpressing cells (e) show a reduction of ZO1 expression as compared to control cells (d). Panel (f) shows the corresponding quantification in which data are expressed as mean area of positive ZO1 signal (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). Scale bars: 25 μm.

Mentions: In the early stages of EndMT, cell-cell contacts are disassembled resulting in a more motile, mesenchymal phenotype40. The loss of cell junctions is molecularly characterised by reduced expression of the adherens junction protein VEC18, and the diffusion of zonula occludens, namely ZO126. We observe this reduction upon overexpression of MSX1 by immunostaining for VEC and ZO1 (Fig. 3a,b,d,e). Overexpression of MSX1 resulted in a 50% and 45% reduction of the area taken up by VEC and ZO1 staining at the cell perimeter, respectively, and this reduction was similar in both HUAEC clones (Fig. 3c,f). While reduced expression was most prominent in areas where cells were no longer in contact with neighbouring cells, there was also a reduction in expression in regions where cells were still in contact (Fig. 3b,e). The reduction in ZO1 protein expression was further confirmed by Western blot (Supplementary Fig. S1).


A new strategy to measure intercellular adhesion forces in mature cell-cell contacts
Disassembly of cell-cell contacts upon upregulation of MSX1 in a HUAEC monolayer.(a–c) Immunofluorescence staining on HUAECs transduced with Cherry- (a; control) or MSX1-encoding (b) lentivirus for the adherens junction marker VEC (green) and nuclear marker TO-PRO-3 (blue) and the corresponding quantification in (c). A reduced expression of VEC is observed in MSX1-overexpressing cells. Data in (c) are expressed as mean area (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). (d–f) Staining of Zonula Occludens-1 (ZO1; green) and nuclear marker TO-PRO-3 (blue). MSX1-overexpressing cells (e) show a reduction of ZO1 expression as compared to control cells (d). Panel (f) shows the corresponding quantification in which data are expressed as mean area of positive ZO1 signal (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). Scale bars: 25 μm.
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f3: Disassembly of cell-cell contacts upon upregulation of MSX1 in a HUAEC monolayer.(a–c) Immunofluorescence staining on HUAECs transduced with Cherry- (a; control) or MSX1-encoding (b) lentivirus for the adherens junction marker VEC (green) and nuclear marker TO-PRO-3 (blue) and the corresponding quantification in (c). A reduced expression of VEC is observed in MSX1-overexpressing cells. Data in (c) are expressed as mean area (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). (d–f) Staining of Zonula Occludens-1 (ZO1; green) and nuclear marker TO-PRO-3 (blue). MSX1-overexpressing cells (e) show a reduction of ZO1 expression as compared to control cells (d). Panel (f) shows the corresponding quantification in which data are expressed as mean area of positive ZO1 signal (in μm2) per cell perimeter (in μm) ± s.e.m. of the total number of measured cells (n = 16 for all conditions; *P < 0.05 versus corresponding control by Student’s t-test). Scale bars: 25 μm.
Mentions: In the early stages of EndMT, cell-cell contacts are disassembled resulting in a more motile, mesenchymal phenotype40. The loss of cell junctions is molecularly characterised by reduced expression of the adherens junction protein VEC18, and the diffusion of zonula occludens, namely ZO126. We observe this reduction upon overexpression of MSX1 by immunostaining for VEC and ZO1 (Fig. 3a,b,d,e). Overexpression of MSX1 resulted in a 50% and 45% reduction of the area taken up by VEC and ZO1 staining at the cell perimeter, respectively, and this reduction was similar in both HUAEC clones (Fig. 3c,f). While reduced expression was most prominent in areas where cells were no longer in contact with neighbouring cells, there was also a reduction in expression in regions where cells were still in contact (Fig. 3b,e). The reduction in ZO1 protein expression was further confirmed by Western blot (Supplementary Fig. S1).

View Article: PubMed Central - PubMed

ABSTRACT

Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.

No MeSH data available.


Related in: MedlinePlus