E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape - PubMed (original) (raw)
E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape
Kevin C Hart et al. Proc Natl Acad Sci U S A. 2017.
Abstract
Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.
Keywords: cell division orientation; cell–cell adhesion; mechanotransduction; mitotic spindle.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Epithelial cell divisions align with a low level of uniaxial stretch. (A) Design of uniaxial cell stretching device, in which a stretchable silicone membrane is bonded to the bottom of microfabricated PDMS channels. The central channel containing cells is surrounded with pneumatic channels in which vacuum pressure is controlled to induce outward deflection of the channel walls surrounding the cell chamber and stretching of the underlying membrane. (B) Substrate strain measured by displacement of microsphere beads in the normal (εyy), transverse (εxx), and shear (εxy) directions, with the mean percentage of strain and SD from three independent experiments. (C) Representative example of 12% axial cell strain based on the displacement of nuclei in confluent MDCK monolayers, showing the distribution of nuclei before (magenta) and after (green) application of stretch. (D) Visualization of the mitotic spindle in MDCK cells expressing GFP–α-tubulin with and without application of 12% uniaxial stretch (3 h of stretch in this representative image). (E) Circular frequency histograms (Rose diagrams) of the mitotic spindle angle relative to the direction of stretch, in MDCK GFP–α-tubulin cells with (n = 279) or without (n = 153) application of at least 1 h of uniaxial stretch, from three independent experiments. Orientation of cell divisions in the direction of stretch is observed within 1 h of stretch (Fig. S1_C_). (Scale bars, 10 µm.)
Fig. S1.
A low level of uniaxial stretch orients cell divisions, whereas only having a minimal effect on cell shape. (A) Unbinned data of the mitotic spindle angle relative to the direction of 12% stretch, from three independent experiments (shown as Rose diagrams of binned data in Fig. 1_E_). Gray bars show the mean spindle angle with SD from the independent experiments. ****P < 0.0001 using a Mann–Whitney _U_ test. (_B_) Bar graph of the distribution of the division orientation relative to the direction of 12% stretch, showing the mean with SD from three independent experiments (shown as Rose diagrams in Fig. 1_E_). **_P_ < 0.007 using an unpaired Student _t_ test. (_C_) Circular frequency histograms (Rose diagrams) of the mitotic spindle angle relative to the direction of stretch, in MDCK GFP–α-tubulin cells with application of 1 h of uniaxial stretch (_n_ = 108 cells), from three independent experiments. (_D_) Bar graph of the distribution of the cell long axis relative to the direction of 12% stretch, showing the mean with SD from six independent experiments (shown as binned Rose diagrams in Fig. 2_B_). **_P_ < 0.007 using an unpaired Student _t_ test. (_E_) Circular frequency histograms (Rose diagrams) of the distribution of the long axis angle relative to the direction of stretch in MDCK E-cadherin:DsRed cells after 4 h of uniaxial stretching, from three independent experiments with at least 500 cells per experiment (for data of long axis orientation of unstretched cells and cells stretched for 2 min, see Fig. 2_B_). (_F_ and _G_) Aspect ratio of the long and short axes of MDCK E-cadherin:DsRed cells before and after 12% uniaxial stretch (_F_) and distribution of the aspect ratio relative to the angle of applied stretch (_G_). Data are pooled from seven independent experiments; _n_ = 122 unstretched and 160 stretched cells (of which 96 and 120 had an aspect ratio of >1.4, respectively). n.s., not significant, using a Mann–Whitney U test. (H) Angle of the cell long axis (in cells with aspect ratio >1.4) that have their mitotic spindle aligned with uniaxial stretch (within 15°) or in the same direction before application of stretch, showing that cell divisions that are aligned with uniaxial stretch have no strong bias in long axis orientation. Gray bars indicate the mean long axis angle with SD from data from seven independent experiments (15 unstretched and 35 stretched cells). ****P < 0.009.
Fig. 2.
Alignment of cell divisions with low-level uniaxial stretch does not correspond to interphase cell shape. (A) Representative image of the analysis of the angle of the cell long axis, indicated with yellow arrows, in MDCK cells expressing E-cadherin:DsRed (shown in white) before and after the application of 12% uniaxial stretch. (B) Circular frequency histograms (Rose diagrams) of the distribution of the long axis angle relative to the direction of stretch in MDCK E-cadherin:DsRed cells before and after uniaxial stretching, from six independent experiments with at least 500 cells per experiment. Images were taken upon 2 min of applied stretch [similar results were obtained with 4 h of applied stretch (Fig. S1_E_)]. (C) Example of the measurement of the angle of the cell long axis before entering mitosis, and of the angle of the mitotic spindle in late metaphase, in MDCK cells expressing GFP–α-tubulin in which the plasma membrane is visualized with Cellmask. (D) Error in alignment of mitotic spindle orientation with the interphase long axis in cells with a well-defined long axis (aspect ratio >1.4) with and without the application of at least 1 h of 12% uniaxial stretch. Gray bars show the mean alignment error with SD from seven independent experiments; n = 96 unstretched and 120 stretched cells. (E) Error in alignment of mitotic spindle orientation with the interphase long axis (aspect ratio >1.4) plotted against the error in alignment with the stretch axis in 12% uniaxially stretched monolayers. The gray area indicates divisions that align equally well with the interphase long axis and stretch axis (within 20°). The population of cells that align divisions better with the stretch axis than interphase long axis is indicated in green dots. Data are pooled from seven independent experiments. (Scale bars, 10 µm.) ****P < 0.0001. w.r.t., with respect to.
Fig. 3.
Uniaxial stretch-oriented division requires E-cadherin adhesion. (A) Localization of GFP-myosin IIA in MDCK cells before and after application of 1 h of 12% uniaxial stretch, showing the cortical accumulation of GFP-myosin IIA following uniaxial stretch. (B) Quantification of the number of cells with cortical GFP-myosin IIA before and upon application of (at least 1 h of) 12% uniaxial stretch, showing mean with SD of three independent experiments (n = 4,176 unstretched and 3,159 stretched cells). (C) Quantification of the distribution of the angle of cortices that contained cortical myosin IIA enrichment upon (at least 1 h of) 12% uniaxial stretch, with mean and SD from three independent experiments (n = 278 cells). (D) Schematic representation of the T151 E-cadherin mutant with a truncated extracellular domain. (E) Circular frequency histograms (Rose diagrams) of the distribution of the long axis angle relative to the direction of stretch, in MDCK cells expressing T151 E-cadherin, before and after application of 12% stretch, from five independent experiments with at least 500 cells per experiment. (F) Circular frequency histograms (Rose diagrams) of the mitotic spindle angle relative to the direction of stretch, with and without application of 12% stretch in parental MDCK (n = 148 cells) and MDCK T151 (n = 285) cells from three independent experiments. (Scale bars, 10 µm.) *P < 0.05.
Fig. S2.
E-cadherin adhesion and LGN are required for uniaxial stretch-induced division orientation. (A) The mean with SD of the fraction of cells of which the long axis is oriented within 0–15° of applied strain, from data shown in Figs. 3_E_ and 5_E_. n.s., not significant using an unpaired Student t test. (B) Bar graph of the distribution of the division orientation relative to the direction of 12% stretch, in parental, T151, and LGN shRNA-expressing MDCK cells, showing the mean with SD from three independent experiments (shown as Rose diagrams in Figs. 3_F_ and 5_D_). **P < 0.002 using an unpaired Student t test. (C) Unbinned data of the mitotic spindle angle relative to the direction of 12% stretch, in parental, T151, and LGN shRNA-expressing MDCK cells, from three independent experiments (shown as Rose diagrams of binned data in Figs. 3_F_ and 5_D_). Gray bars show the mean spindle angle with SD from the independent experiments. *P < 0.015; ***P < 0.0001, using a Mann–Whitney U test.
Fig. 4.
Recruitment of LGN to the cell cortex is regulated by tension. (A) Immunostaining of endogenous LGN and E-cadherin in low-density and high-density MDCK cell monolayers. The quantification shows the ratio of LGN at cell–cell contacts (marked with E-cadherin) versus cytosol in monolayers at different cell densities. Pearson coefficient = −0.893. (B) Immunostaining of endogenous LGN and E-cadherin in high-density monolayers MDCK cells upon 10% biaxial stretch. The quantification shows the mean ratio ± SD of LGN at the cell–cell contacts versus cytosol from three independent experiments, with stretch applied for at least 1 h. (C) Localization of endogenous LGN and E-cadherin in low-density MDCK monolayers incubated for 5 min with 25 µM ML-7 or vehicle control (DMSO). The quantification shows the mean ratio ± SD of LGN at cell–cell contacts versus cytosol from three independent experiments. (Scale bars, 10 µm.) **P < 0.005.
Fig. S3.
Density-dependent cortical localization of myosin IIA. Representative images of the distribution of GFP-myosin IIA in MDCK cells grown at confluency showing the cortical enrichment of GFP-myosin IIA in low-density monolayers, which is reduced in high-density monolayers. (Scale bars, 10 µm.)
Fig. S4.
Tension-dependent recruitment of LGN to E-cadherin adhesions. (A) Immunostaining of endogenous LGN at cell–cell contacts in MDCK cells expressing T151 E-cadherin that were cultured at low density, high density, or at high density with 10% biaxial stretch. The quantification shows the mean ratio ± SD of LGN at cell–cell contacts versus cytosol. Similar to wild-type MDCK cells (Fig. 4_A_), LGN localized to cell–cell contacts in low cell density MDCK T151 cells, but LGN levels at cell–cell contacts were decreased at high cell density. This is explained by the fact that the actomyosin cytoskeleton exerts force on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is involved in cell–cell contacts (31), and thus regardless of whether it forms homotypic interactions with E-cadherin on neighboring cells (which are absent in MDCK T151 cells). Stretch only increases tension on E-cadherin involved in cell–cell contacts (31), and biaxial stretch did not result in the enrichment of LGN at cell–cell contacts in MDCK cells expressing T151 E-cadherin. This finding indicates that LGN recruitment to cell–cell contacts upon stretch requires the transmission of force through the extracellular domain of E-cadherin. (Scale bars, 10 μm.) (B) Schematic overview of E-cadherin:Fc–coated sidewalls in which a vertical silicone sidewall is coated with E-cadherin:Fc (E-cad:Fc). (C and D) Localization of E-cadherin:DsRed and endogenous LGN at the sidewall–cell junction in MDCK cells, incubated for 5 min with 25 µM ML-7 or vehicle control (C), with the mean ratio ± SD of LGN at the cell–sidewall E-cadherin junction versus cytosol from three independent experiments (16 control cells and 13 ML-7–treated cells) (D). Arrowheads indicate enrichment of E-cadherin or LGN at the sidewall–cell junction. (Scale bars, 10 µm.) **P < 0.006, n.s., not significant.
Fig. 5.
Uniaxial stretch orients cell divisions through LGN. (A) Immunostaining of endogenous LGN in MDCK cells upon application (at least 1 h) of 12% uniaxial stretch, showing the polarized distribution of LGN, which becomes mostly enriched at cell–cell contacts perpendicular to the stretch axis and less at cell–cell contacts parallel to the stretch axis. (B) Quantification of the intensity of LGN at the cell–cell contact relative to the underlying cytosol, with respect to the angle of the cell–cell contact relative to the stretch axis (0° is perpendicular to the stretch axis), with mean and SD from three independent experiments (n = 76 cells). *P < 0.007; **P < 0.0001, using a Mann–Whitney U test. (C) Immunostaining of endogenous LGN and E-cadherin in MDCK cells upon application of (at least 1 h of) 12% uniaxial stretch, showing the polarization of LGN upon uniaxial stretch with enrichment at the cell cortex perpendicular to the stretch axis (arrowheads) and less at the cortex parallel to the stretch axis (white asterisks), whereas E-cadherin remains uniformly localized at the plasma membrane. (D) Circular frequency histograms (Rose diagrams) of the mitotic spindle angle relative to the direction (at least 1 h) of 12% stretch in MDCK cells expressing LGN shRNA (n = 162) from three independent experiments. The distribution of the mitotic spindle angle in stretched control cells (as shown in Fig. 3_F_) is indicated in dotted lines. (E) Circular frequency histograms (Rose diagrams) of the distribution of the long axis angle relative to the direction of stretch in MDCK cells expressing LGN shRNA, before and after application of (at least 1 h of) 12% stretch, from three independent experiments with at least 500 cells per experiment. (Scale bars, 10 µm.)
Fig. S5.
LGN and NuMA localization in uniaxially stretched MDCK cells. (A) Immunostaining of endogenous LGN in MDCK cells upon application of different periods of 12% uniaxial stretch (shown as representative duplicates for each time point). (B) Loss of cortical staining with the LGN antibody upon shRNA-mediated depletion of LGN, compared with control (scr) shRNA-infected MDCK cells. (C) Immunostaining of endogenous LGN and E-cadherin in MDCK cells in metaphase upon application of 12% uniaxial stretch. (D) Immunostaining of endogenous NuMA and E-cadherin in MDCK cells upon application of 12% uniaxial stretch. (Scale bars, 10 µm.)
Fig. S6.
DLG1, Afadin, and phospho-ERM in uniaxially stretched cells. Immunostaining of endogenous E-cadherin and DLG1 (A), Afadin (B), or phospho-Ezrin (T567)/phosho-Radixin (T564)/phospho-Moesin (T558) (pERM) (C) in MDCK cells with and without 1 h application of 12% uniaxial stretch, showing the lack of polarization of DLG1, Afadin, and pERM upon uniaxial stretch. Similar results were obtained with longer duration of uniaxial stretch. Note that pERM stains the apical brush border and not cell–cell contacts. (Scale bars, 10 µm.)
References
- Pease JC, Tirnauer JS. Mitotic spindle misorientation in cancer: Out of alignment and into the fire. J Cell Sci. 2011;124:1007–1016. -PubMed
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