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Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.

Sorce B, Escobedo C, Toyoda Y, Stewart MP, Cattin CJ, Newton R, Banerjee I, Stettler A, Roska B, Eaton S, Hyman AA, Hierlemann A, Müller DJ - Nat Commun (2015)

Bottom Line: Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis.The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape.The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Mattenstrasse 26, Basel 4058, Switzerland.

ABSTRACT
Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

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Mitosis in polarized MDCK cells.(a) (i) MDCK cells expressing actin-mCherry (red) and eGFP-labelled histones (H2B-eGFP, green) and (ii) MDCK cells expressing E-cadherin-eGFP (green) and nuclei stained with Hoechst 33342 (grey). Cells were plated on permeable filter supports and grown for 3 days before data acquisition (see Supplementary Fig. 1 for MDCK cells grown for 14 days). The eGFP-labelled histones allowed us to determine the mitotic phases of MDCK cells (see Methods). (i) Confocal microscopy images of polarized MDCK cells expressing actin-mCherry and H2B-eGFP showing mitotic cells rounded up in metaphase (yellow arrow). (ii) Confocal microscopy image of MDCK cells expressing E-cadherin-eGFP that show extensive contacts between metaphase (yellow arrow) and interphase cells. (b) Schematic drawing of cell division in the epithelial layer. Scale bars, 10 μm.
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f1: Mitosis in polarized MDCK cells.(a) (i) MDCK cells expressing actin-mCherry (red) and eGFP-labelled histones (H2B-eGFP, green) and (ii) MDCK cells expressing E-cadherin-eGFP (green) and nuclei stained with Hoechst 33342 (grey). Cells were plated on permeable filter supports and grown for 3 days before data acquisition (see Supplementary Fig. 1 for MDCK cells grown for 14 days). The eGFP-labelled histones allowed us to determine the mitotic phases of MDCK cells (see Methods). (i) Confocal microscopy images of polarized MDCK cells expressing actin-mCherry and H2B-eGFP showing mitotic cells rounded up in metaphase (yellow arrow). (ii) Confocal microscopy image of MDCK cells expressing E-cadherin-eGFP that show extensive contacts between metaphase (yellow arrow) and interphase cells. (b) Schematic drawing of cell division in the epithelial layer. Scale bars, 10 μm.

Mentions: We wanted to characterize morphological changes of mitotic cells in the epithelium. As an established epithelial in vitro model, we chose MDCK cells. When cultivated on membrane supports, MDCK cells differentiate in a cuboidal epithelial layer28 and retain properties characteristic of kidney epithelial cells, such as tight junctions and distinct basal and apical membrane domains2029. Using confocal microscopy we monitored mitotic MDCK cells in the epithelial layer (Fig. 1). We followed the mitotic state by expressing enhanced green fluorescent protein (eGFP)-labelled histones (H2B-eGFP), the actomyosin cortex by expressing actin-mCherry and cell junctions by expressing eGFP-labelled E-cadherin. Confocal microscopy images show epithelial mitotic cells rounding and increasing in height by ≈5 μm while retaining tight junctions. Mitotic rounding was observed for MDCK cells plated on permeable filter supports and grown for 3 days (Fig. 1a) and 14 days (Supplementary Fig. 1). This observation is in accord with previous reports that mitotic epithelial cells lose their cuboidal architecture and round towards the apical surface of the epithelia2028. Furthermore, the confocal images reveal that mitotic cells mechanically deform adjacent cells and orient their metaphase plate perpendicular to the substrate while rounding. Having observed that mitotic MDCK cells round in the epithelial layer, increase in height and deform adjacent cells (Fig. 1b), we sought to answer the following questions regarding these processes: what is the driving force behind mitotic MDCK cells rounding against the confinement of the epithelium? By which mechanisms do mitotic MDCK cells increase in height in the epithelium? What role does mechanical confinement play in epithelial cells conducting mitosis?


Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement.

Sorce B, Escobedo C, Toyoda Y, Stewart MP, Cattin CJ, Newton R, Banerjee I, Stettler A, Roska B, Eaton S, Hyman AA, Hierlemann A, Müller DJ - Nat Commun (2015)

Mitosis in polarized MDCK cells.(a) (i) MDCK cells expressing actin-mCherry (red) and eGFP-labelled histones (H2B-eGFP, green) and (ii) MDCK cells expressing E-cadherin-eGFP (green) and nuclei stained with Hoechst 33342 (grey). Cells were plated on permeable filter supports and grown for 3 days before data acquisition (see Supplementary Fig. 1 for MDCK cells grown for 14 days). The eGFP-labelled histones allowed us to determine the mitotic phases of MDCK cells (see Methods). (i) Confocal microscopy images of polarized MDCK cells expressing actin-mCherry and H2B-eGFP showing mitotic cells rounded up in metaphase (yellow arrow). (ii) Confocal microscopy image of MDCK cells expressing E-cadherin-eGFP that show extensive contacts between metaphase (yellow arrow) and interphase cells. (b) Schematic drawing of cell division in the epithelial layer. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Mitosis in polarized MDCK cells.(a) (i) MDCK cells expressing actin-mCherry (red) and eGFP-labelled histones (H2B-eGFP, green) and (ii) MDCK cells expressing E-cadherin-eGFP (green) and nuclei stained with Hoechst 33342 (grey). Cells were plated on permeable filter supports and grown for 3 days before data acquisition (see Supplementary Fig. 1 for MDCK cells grown for 14 days). The eGFP-labelled histones allowed us to determine the mitotic phases of MDCK cells (see Methods). (i) Confocal microscopy images of polarized MDCK cells expressing actin-mCherry and H2B-eGFP showing mitotic cells rounded up in metaphase (yellow arrow). (ii) Confocal microscopy image of MDCK cells expressing E-cadherin-eGFP that show extensive contacts between metaphase (yellow arrow) and interphase cells. (b) Schematic drawing of cell division in the epithelial layer. Scale bars, 10 μm.
Mentions: We wanted to characterize morphological changes of mitotic cells in the epithelium. As an established epithelial in vitro model, we chose MDCK cells. When cultivated on membrane supports, MDCK cells differentiate in a cuboidal epithelial layer28 and retain properties characteristic of kidney epithelial cells, such as tight junctions and distinct basal and apical membrane domains2029. Using confocal microscopy we monitored mitotic MDCK cells in the epithelial layer (Fig. 1). We followed the mitotic state by expressing enhanced green fluorescent protein (eGFP)-labelled histones (H2B-eGFP), the actomyosin cortex by expressing actin-mCherry and cell junctions by expressing eGFP-labelled E-cadherin. Confocal microscopy images show epithelial mitotic cells rounding and increasing in height by ≈5 μm while retaining tight junctions. Mitotic rounding was observed for MDCK cells plated on permeable filter supports and grown for 3 days (Fig. 1a) and 14 days (Supplementary Fig. 1). This observation is in accord with previous reports that mitotic epithelial cells lose their cuboidal architecture and round towards the apical surface of the epithelia2028. Furthermore, the confocal images reveal that mitotic cells mechanically deform adjacent cells and orient their metaphase plate perpendicular to the substrate while rounding. Having observed that mitotic MDCK cells round in the epithelial layer, increase in height and deform adjacent cells (Fig. 1b), we sought to answer the following questions regarding these processes: what is the driving force behind mitotic MDCK cells rounding against the confinement of the epithelium? By which mechanisms do mitotic MDCK cells increase in height in the epithelium? What role does mechanical confinement play in epithelial cells conducting mitosis?

Bottom Line: Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis.The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape.The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Mattenstrasse 26, Basel 4058, Switzerland.

ABSTRACT
Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells.

Show MeSH
Related in: MedlinePlus