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. 2023 Jun 30;14:3867. doi: 10.1038/s41467-023-39563-6

Fig. 5. Mechanical strain in nucleoplasmic lamin A/C filaments.

Fig. 5

a Representative acceptor (venus) intensity image together with donor (mTFP1) fluorescence lifetime microscopy images of Lamin-SS and Lamin-TM expressing cells. Scale bars, 20 µm. Blowup images show equal distribution of lifetimes throughout the nucleus. b Donor fluorescence lifetime histograms show highly similar lifetimes and thus FRET for nuclear rim and nucleoplasm, indicating similar strain in lamin A/C in the nuclear lamina and in the nuclear interior (n = 26 and n = 26 cells for Lamin-SS and Lamin-TM, respectively, two biological replicates). c Schematic representation of the FRET based lamin A/C - histone H2A strain sensor (Lamin-histone-SS), truncated control sensor (Lamin-histone-TM), and the working mechanism of the force sensing between lamins and chromatin. d Representative confocal Airyscan xy-sections of immunolabeled lamin A/C (top, magenta), histone H2A (magenta, bottom) and the expressed Lamin-histone-SS sensor (cyan) along with corresponding fluorescence line-profiles (n = 5 and n = 4 cells, respectively, two biological replicates). Scale bar 5 µm. e sFRET efficiency images and quantified sFRET efficiency (mean ± SEM) of Lamin-histone-SS and Lamin-histone-TM sensors (n = 10 fields, two biological replicates). Scale bar 20 µm. Unpaired two-tailed Student’s t test ((****) p < 0.0001, t = 5.14, df = 18). f WT and LMNA KO cells transiently transfected with Lamin-histone-SS. Scale bars 10 µm. g Quantified riFRET (mean ± SEM) of Lamin-histone-SS (n = 67 and n = 72 cells, respectively, two biological replicates) and Lamin-histone-TM (n = 67 and n = 78 cells, 2 biological replicates) in WT and LMNA KO cells. Ordinary one-way ANOVA Tukey’s multiple comparisons (for Lamin-histone-SS (***) p = 0.0003 and Lamin-histone-TM (ns) p = 0.4756).