Fig. 1: cPla2–INM adsorption correlates with ER–NM disruption during osmotic stress.
From: Endoplasmic reticulum disruption stimulates nuclear membrane mechanotransduction
a, Left: representative confocal midplane slices of U2OS cells expressing the luminal ER marker eGFP–KDEL and zebrafish cPla2–mKate2 at baseline and 5 min after ΔΠ = 270 mOsm osmotic shock. Scale bar, 20 μm. Top middle: a plot of normalized ER surface area versus time after hypo-osmotic shock treatment (∆Π = 270 mOsm). The data represent six independent experiments. The line shows the average and error bars are the s.e.m. Bottom middle: a plot of normalized ER circularity, and cPla2–mKate2–INM adsorption versus time after hypo-osmotic shock (∆Π = 270 mOsm). The line shows the average and error bars are the s.e.m. Note that the bottom middle image shows the result for a subset of the data presented in the top middle image. The data represent three independent experiments. Top right: a scatter plot showing the correlation between cPla2–mKate2–INM adsorption and ER vesiculation (as measured by a decrease of ER surface area) in cells after being exposed to hypo-osmotic (∆Π = 303 mOsm, n = 33) or isosmotic medium (∆Π = 0 mOsm, n = 34) for 5 min. Bottom right: a scatter plot showing the correlation between cPla2–mKate2–INM adsorption and ER fragmentation (as measured by an increase of ER network circularity) in cells after being exposed to hypo-osmotic (∆Π = 303 mOsm, n = 33) or isosmotic solution (∆Π = 0 mOsm, n = 34) for 5 min. The data represent one independent experiment performed on cells from two distinct biological sources (separate frozen vials). b, Left: representative, confocal midplane slices of U2OS cells expressing the ER membrane marker eGFP–SEC61B acquired at the indicated times after ∆Π = 270 mOsm or 303 mOsm hypo-osmotic shock. Right: time-lapse quantification of ER circularity upon osmotic shock. The lines show the average and the shaded areas are the s.e.m. Data represent two independent experiments for 222 mOsm, three independent experiments for 270 mOsm and two independent experiments for 303 mOsm. Scale bar, 20 µm. c, Left: representative lattice SIM2apotome super-resolution maximum intensity projections (MIPs) of U2OS cells expressing the ER membrane marker eGFP–SEC61B (∆Π = 0 mOsm, n = 6). Right: a representative super-resolution MIP of U2OS cells 10 min after hypo-osmotic shock (∆Π = 270 mOsm, n = 4). Scale bars, 5 μm. d, A cartoon scheme of the FLIP experiment. The ER–NE sites that are bleached (red, flash) or measured (green) are indicated. e, Fluorescence dynamics in response to bleaching the indicated sites. Left: FLIP of eGFP–KDEL at baseline (∆Π = 0 mOsm, n = 10) or during osmotic shock (∆Π = 270 mOsm, n = 8). Right: FLIP of membrane, eGFP–SEC61B at baseline (∆Π = 0 mOsm, n = 6) and osmotic shock (∆Π = 270 mOsm, n = 10). The line shows the average and error bars the 95% CI. The data represent three independent experiments. If not otherwise indicated, n denotes the number of analysed cells or nuclei. Note that ER morphology measurements (normalized circularity/area) are always analysed on an entire FOV (that is, no structure segmentation, n represents FOV, 1–8 cells per FOV). For time-lapse movies, values were normalized to baseline at t = 0. For single images captured at the indicated time point, values were normalized to measurements obtained under isosmotic conditions. A, area; S, ratiometric signal; Cir, circularity. I, intensity; Ctr, non-targeted/bleached neighbouring cell control in whole field of view.
