fluorescent membrane tension probe er flipper tr  (Spirochrome)

Journal: PLOS Pathogens

Article Title: Nucleus softens during herpesvirus infection

doi: 10.1371/journal.ppat.1013873

Figure Lengend Snippet: (A) Nuclear lamina labeled by lamin A chromobody, skeletonization of the lamina, and discretized lamina points showing regions of positive (green) and negative curvature (red) in infected Vero cells at 8 hpi. (B) The fraction of negative curvature points for noninfected cells and cells imaged 8 and 12 hpi (n = 19, 22, and 17, respectively). (C) A zoom-in of the nuclear lamina, showing its motion and maximum intensity projection of the skeleton during 24.8 s. Scale bars, 5 µm. (D) Nuclear lamina mobility, measured as the area covered by the skeleton during one minute for noninfected cells and cells imaged 8 and 12 hpi (n = 19, 22, and 17, respectively). (E) Nuclear envelope (NE) tension analysis by fluorescence lifetime imaging microscopy (FLIM) of noninfected and HSV-1 17 + wild-type virus-infected Vero cells at 12 hpi using fluorescence lifetime-reporting membrane tension probe ER Flipper-TR. A fast FLIM image presented in a pseudocolor scale representing average photon arrival times in each pixel, ranging from 2.0 to 2.6 ns (calibration bar). Representative cells with average lifetime distribution, segmented NE (red) around the Hoechst signal (grey), and average lifetime at the NE region are shown. Scale bar, 10 µm. (F) The longer lifetime component of the ER Flipper probe, responsive to the membrane tension, in noninfected and infected cells at 8 and 12 hpi (n = 51, 58, and 59, respectively). The error bars show the standard deviation. Statistical significance was determined using Tukey’s test, and the significance values are denoted as * (p < 0.05) or **** (p < 0.0001). Nonsignificant differences (p ≥ 0.05) are not labeled.

Article Snippet: Nuclear membrane tension was analyzed by FLIM using fluorescent membrane tension probe ER Flipper-TR (Spirochrome) in noninfected and HSV-1 17 + wt infected live Vero cells at 8 and 12 hpi.

Techniques: Labeling, Infection, Fluorescence, Imaging, Microscopy, Virus, Membrane, Standard Deviation

Journal: iScience

Article Title: Calcium waves and nuclear tension changes coordinate mechanical stress dissipation in locally folded epithelia

doi: 10.1016/j.isci.2025.114477

Figure Lengend Snippet: Level of nuclear envelope tension is essential to trigger nuclear recovery upon epithelial folding (A) Changes in nuclear area upon folding (violet) of confluent monolayers (CM) and highly confluent monolayers (HCM). Data are represented as mean ± SEM, N = 3 independent experiments. (B) Quantification of connexin43 phosphorylation state (Cx43/pCx43) based on the Western blot experiments. Each point represents a separate replicate for CM and HCM; Data are represented as mean ± SEM; n.s. - no statistically significant, Mann-Whitney. (C) Quantification of nuclear area change between CM and HCM (∗∗ p = 0,0079, Mann-Whitney, n > 20 nuclei per condition, N = 2 replicates) and corresponding fluorescent images of CM and HCM stained with anti-Lamin A/C antibody; scale bars: 10 μm. (D) Quantification of τ2 from Flipper-TR (plasma membrane) and ER Flipper-TR (nuclear membrane) stained cells using FLIM; n > 100 cells/condition pooled across three independent experiments; ∗∗∗∗ p < 0.0001, Mann-Whitney; Data are represented as mean ± SEM. (E) Fluorescent images with anti-Lamin A/C staining of control and cells with LBR-RFP overexpression; Scale bars 5 μm. (F) Western blot of control and cells overexpressing LBR-RFP with anti-RFP antibody. (G) Quantification of LBR-RFP overexpression based on the Western blot from F. Each point represents an independent experiment. ∗∗∗∗ p < 0.0001 Mann-Whitney test was used. Data are represented as mean ± SEM. (H) Quantification of τ2 from ER Flipper-TR (nuclear membrane) of control cells and LBR-RFP cells. N = 3 Independent experiments (with n = 140 measurements per condition); data are pooled together. ∗∗∗∗ p < 0.0001, t test was used; Data are represented as mean ± SEM. (I) Normalized intensity profile of calcium wave (Fluo4AM) as a function of distance at t = T max = 18 s for control and LBR-RFP cells, and the corresponding images; data are represented as mean ± SEM; Scale bars 50 μm. (J) Nuclear area changes of LBR-RFP cells upon folding in the function of time; Data are represented as mean ± SEM. N = 3 Independent experiments. Statistical analysis of the change of area at the t stretch and t stretch+250 s can be found in Supplementary Figures. (K) Images of a single nucleus of LBR-RFP cells marked with Hoechst at three time points: before the stretch (t = 6 s), stretched (t = 15 s), and stretched +300 s. The color masks of the nucleus correspond to different time points and come from the image analysis processing. Scale bar 10 μm.

Article Snippet: Flipper-TR fluorescent tension probe (Spirochrome) was used according to the provided protocol in order to quantify plasma membrane tension.

Techniques: Phospho-proteomics, Western Blot, MANN-WHITNEY, Staining, Clinical Proteomics, Membrane, Control, Over Expression

Journal: Nature Communications

Article Title: Spatiotemporal coupling of caveolae mechanosensing and RhoA-GEFs regulates cell polarity and directional migration

doi: 10.1038/s41467-025-67090-z

Figure Lengend Snippet: a Representative Flipper-TR intensity images of a WT-Hs578t cell under isotonic (Iso) condition. White dashed boxes indicate front and rear regions of interest (ROIs). Color bar: Flipper-TR intensity (photons per pixel). Scale bar, 20 µm. b Fluorescence decay curves from front (green) and rear (red) ROIs of a representative cell. Experimental data (dots) were fitted with a two-exponential decay function (blue line) to calculate Flipper-TR lifetime (ns) and adjusted R² values. Insets show ROI locations. c Flipper-TR lifetimes at the front (green) and rear (red) of individual cells. Gray lines connect paired measurements from the same cell. b , c Data are presented as mean values +/- SEM of n = 44 cells from three independent experiments. Statistical analysis: One-tailed, Mann–Whitney test; ****P < 0.0001, **P < 0.001. d Per-cell differences in lifetime (Δ = Front - Rear); each blue circle represents one cell. e Representative Flipper-TR intensity images of a WT-Hs578t cell under isotonic (Iso, left) and hypotonic (Hypo, right) conditions. ROIs at the cell front and rear indicated by dashed boxes. Color bar as in panel a. Scale bar, 10 µm. f Flipper-TR lifetimes at the front and rear under Iso and Hypo conditions. ( g ) Per-cell differences in lifetime (Δ = Front - Rear) under Iso (dark blue circles) and Hypo (light blue circles). Gray lines link paired values; circles show per-cell differences. f , g Data are presented as mean values +/- SEM., n = 19 cells from three independent experiments. One-tailed, Mann–Whitney test; ****P < 0.0001. h Representative time-lapse images of WT-Hs578t cells co-expressing opto-PI3K (iSH2), SiR-actin, and Cav1-RFP. Insets show optogenetically activated ROIs. Timepoints span from -2 min (pre-activation) to +16 min (post-activation). i Quantification of normalized signal intensity and cell area in the activated ROI over time for actin (orange), Cav1 (gray), and cell area (blue). Data are presented as mean values +/- SEM of n = 17 cells from three independent experiments.

Article Snippet: Cells were incubated with the fluorescent membrane tension probe Flipper-TR (Spirochrome, cat. no. #CY-SC020) at a final concentration of 500 nM for 15 minutes at 37 °C in a 5% CO2 atmosphere prior to imaging.

Techniques: Fluorescence, One-tailed Test, MANN-WHITNEY, Expressing, Activation Assay