Cell Surface Mechanics Gate Embryonic Stem Cell Differentiation

Abstract

Cell differentiation typically occurs with concomitant shape transitions to enable specialized functions. To adopt a different shape, cells need to change the mechanical properties of their surface. However, whether cell surface mechanics control the process of differentiation has been relatively unexplored. Here we show that membrane mechanics gate exit from naive pluripotency of mouse embryonic stem cells. By measuring membrane tension during early differentiation, we find that naive stem cells release their plasma membrane from the underlying actin cortex when transitioning to a primed state. By mechanically tethering the plasma membrane to the cortex by enhancing Ezrin activity or expressing a synthetic signaling-inert linker, we demonstrate that preventing this detachment forces stem cells to retain their naive pluripotent identity. We thus identify a decrease in membrane-to-cortex attachment as a new cell-intrinsic mechanism that is essential for stem cells to exit pluripotency.

Keywords: atomic force spectroscopy; exit from pluripotency; mESC; membrane tension; membrane-to-cortex attachment (MCA); naive-to-primed transition.

Graphical abstract

Figure 1

During Exit from Naive Pluripotency, Cells Spread with a Concomitant Reduction in Apparent Membrane Tension and MCA (A) Representative bright-field (differential interference contrast [DIC]) images of Rex1-GFPd2 mESCs during exit from pluripotency in plain N2B27 medium. The bottom panel corresponds to a magnification of the boxed region in the top panel. See also Video S1. Scale bars, 50 μm (top panel), 10 μm (bottom panel). (B) Representative scanning electron microscopy images of naive (2i/LIF) and primed (FGF2/ActA) Rex1-GFPd2 mESCs. Scale bar, 10 μm. (C) Single-cell spreading area quantified from scanning electron microscopy images. n, number of cells analyzed; p value, Welch’s t test. (D) Schematic of static tether pulling using atomic force spectroscopy. (E) Schematic of dynamic tether pulling using atomic force spectroscopy. (F) Mean static tether force of naive (2i/LIF) and primed (FGF2/ActA) Rex1-GFPd2 mESCs. n, number of cells analyzed in 2 independent experiments; p value, Mann-Whitney U test. (G) Force-velocity curve from dynamic tether pulling on Rex1-GFPd2 mESCs in 2i/LIF medium, during exit from pluripotency in N2B27 medium at 48 h, and primed in FGF2/ActA medium. Data points are mean tether force f ± SEM at 2, 5, 10, and 30 μm/s pulling velocity. n, number of cells analyzed in 3 independent experiments. (H) Mean and standard deviation of the MCA parameter $a$ obtained from Monte Carlo-based fitting (see STAR Methods for details); p value, Z test. (I) Normalized GFP geometric mean intensities for Rex1-GFPd2 mESCs in 2i/LIF medium, during exit from pluripotency in N2B27 medium at 48 h, and primed in FGF2/ActA medium. n_Exp, number of independent experiments; error bars, SEM; p values, Welch’s t test. (J) Representative scanning electron microscopy images of naive (2i/LIF) Rex1-GFPd2 mESCs on gelatin or on Laminin 511 (L511). Scale bar, 10 μm. (K) Single-cell spreading area quantified from scanning electron microscopy images. n, number of cells analyzed; p value, Welch’s t test. (L) Force-velocity curve from dynamic tether pulling on naive (2i/LIF) Rex1-GFPd2 mESCs plated on gelatin or on L511. Data points are mean f ± SEM at 2, 5, 10, and 30 μm/s pulling velocity. n, number of cells analyzed in 3 independent experiments. The inset shows mean and standard deviation of the MCA parameter $a$ obtained from Monte Carlo-based fitting (see STAR Methods for details); p value, Z test. (M) Force-velocity curve from dynamic tether pulling on Rex1-GFPd2 mESCs in 2i/LIF medium after plating for 48 h on L511-coated hydrogels of 25-kPa or 0.5-kPa stiffness. Data points are mean f ± SEM at 2, 5, 10, and 30 μm/s pulling velocity. n, number of cells analyzed in 4 independent experiments. The inset shows mean and standard deviation of the MCA parameter$a$obtained from Monte Carlo-based fitting (see STAR Methods for details); p value, Z test.

Figure 2

CAEzrin and the Synthetic iMC-Linker Gate Exit from Naive Pluripotency by Maintaining High MCA (A) Representative bright-field (DIC) and fluorescent images of naive Rex1-GFPd2 ind-CAEz mESCs expressing CAEz-mCherry in 2i/LIF+Dox medium. Scale bar, 10 μm. (B) Force-velocity curve from dynamic tether pulling on Rex1-GFPd2 ind-CAEz mESCs in 2i/LIF medium and during exit from pluripotency in N2B27 ± Dox medium at 48 h. Data points are mean f ± SEM at 2, 5, 10, and 30 μm/s pulling velocity. n, number of cells analyzed in 3 independent experiments. The inset shows mean and standard deviation of the MCA parameter $a$ obtained from Monte Carlo-based fitting (see STAR Methods for details); p value, Z test. (C) Schematic of the iMC-linker. PMBD, plasma membrane-binding domain; ABD, actin-binding domain. (D) Representative bright-field (DIC) and fluorescent images of naive Rex1-GFPd2 ind-iMC mESCs expressing the iMC-linker in 2i/LIF+Dox medium. Scale bar, 10 μm. (E) Force-velocity curve from dynamic tether pulling on Rex1-GFPd2 ind-iMC mESCs in 2i/LIF medium and during exit from pluripotency in N2B27 ± Dox medium at 48 h. Data points are mean f ± SEM at 2, 5, 10, and 30 μm/s pulling velocity. n, number of cells analyzed in 3 independent experiments. The inset shows mean and standard deviation of the MCA parameter $a$ obtained from Monte Carlo-based fitting (see STAR Methods for details); p value, Z test. (F) Normalized geometric mean intensities of Nanog immunofluorescence levels for Rex1-GFPd2 ind-CAEz and ind-iMC mESCs plated for 48 h in N2B27 medium or N2B27+Dox medium. Error bars, SEM; p values, Welch’s t test. (G) Normalized GFP geometric mean intensities for Rex1-GFPd2 ind-CAEz, ind-mCherry, ind-iMC, ind-PMBD, and ind-ABD mESCs in 2i/LIF medium and during exit from pluripotency in N2B27 ± Dox medium at 48 h. Error bars, SEM; p value, Welch’s t test. (H) Comparison of mRNA fold-changes for Rex1-GFPd2 ind-CAEz and ind-iMC mESCs grown in 2i/LIF and N2B27+Dox media (48 h) with plain N2B27 medium (48 h). Naive pluripotency genes (Kalkan et al., 2017) were upregulated in cells grown in 2i/LIF medium and N2B27+Dox medium (top left quadrant; green: significantly enriched genes, log-fold change [LFC] > 1 and false discovery rate [FDR] < 0.05). Data are from 3 independent RNA-seq experiments. (I) RNA-seq-derived enriched pathway maps for Rex1-GFPd2 ind-CAEz and ind-iMC mESCs in N2B27+Dox medium compared with plain N2B27 medium at 48 h. Significantly enriched genes (LFC > 1 and FDR < 0.05) from differential RNA-seq expression analysis were used to identify the enriched pathway maps from the KEGG database (see STAR Methods for details). Shown are the 4 most enriched pathway maps. (J) Top: representative images of the re-plating assay for Rex1-GFPd2 ind-CAEz and ind-iMC mESCs. Scale bar, 500 μm. Bottom: normalized colony number (Dox/Ctrl) for Rex1-GFPd2 ind-CAEz, ind-mCherry, ind-iMC, ind-PMBD, and ind-ABD mESCs re-plated after 48-h exit in N2B27 ± Dox medium. Error bars, SEM; p value, Welch’s t test. (K) RNA-seq-derived mRNA fold changes of general and naive pluripotency markers (Kalkan et al., 2017) and markers for neuroectoderm and mesendoderm formation on day 4 of embryoid body differentiation for Rex-GFPd2 ind-CAEz (top) or ind-iMC (bottom) mESCs (data from 4 independent experiments). Green indicates higher and blue indicates lower expression in Dox-induced cells dissociated from embryoid bodies. N/A, expression below detection limits. All LFCs are significant (p < 0.01) except when noted otherwise (n.s.).