FluidFM stimulation combined with FLIM readout for measuring subcellular mechanoresponse upon mechanical stimulation of nuclei.

A, B) SEM micrographs of a pyramidal probe (A) and a cylindrical probe (B) before (left) and after FIB-cut (top right). The top view of the cylinder is also shown in panel B. Scale bars represent 1 µm.

C) Lifetime images of HeLa cells stained with molecular tension probe (ER Flipper-TR) before (top), during (middle) and after (bottom) indentation at 100 nN, with pyramidal (left panel) and sharpened cylindrical probes (right panels). Arrows denote the indentation site when either pyramidal or cylindrical probes are used. Dashed lines indicate a representative selection of ROIs, encompassing 1) the ER, 2) nuclear membrane (Nuc-mem), and 3) the area around the indentation site (Indent-site). Scale bar represents 5 µm.

D) Change in lifetime during (top panel, “mech” in C) and after (bottom panel) mechanical stimulus with varied indentation forces at different ROIs: ER, nucleus membrane (Nuc-mem) and incident indentation site (“Indent-site”) (Mean ± SD, N ≥ 2, n ≥ 4, ***p < 0.001, **p < 0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test). For mechanical stimulation, pyramidal (“Pyr”) and cylindrical (“Cyl”) probes with similar stiffness (2 ± 0.5 Nm-1) were utilized.

E) Lateral view of nucleus before (top panels) and during indentation (bottom panels) (indicated by “mech” in panel C) at 100 nN using pyramidal and cylindrical probes. Nuclei are stained with Hoechst dye (blue) and probes are filled with sulforhodamine dye (SuR, red). Scale bar represents 5 µm.

F) Change in perimeter of nucleus measured from confocal images at varied indentation forces, with pyramidal and sharpened cylindrical probes (Mean ± SD, N = 2, n ≥ 3).

FluidFM-FLIM mechanochemical stimulation of HeLa cell nuclei using pyramidal and cylindrical probes.

A) Schematic showing the mechanotransduction at LINC complexes before (top) and after external stimulus: mechanical (middle) or mechanochemical (bottom) stimulus. ΔP represents the fluidic pressure difference between the probe reservoir and the aperture. When this pressure difference is positive, it indicates that the injection mode of FluidFM is being utilized.

B) Concurrent readout of AFM-measured indentation force, ER Flipper-TR lifetime, and fluidic pressure pulse for injection over time. For assured injection, 150 nN was chosen as the set point for indentation using both probe geometries. Parts i, ii and iii on the graph in B and the schematic in A represent: i) before stimulus, ii) during mechanical stimulus when the probe has reached the set point on the cell without fluidic pressure application, and iii) during mechanochemical stimulus when the probe remains in contact with the cell at the set point, and a fluidic pressure pulse is applied for drug (or control) injection.

C) Absolute lifetime values before, during and after injecting HeLa cells with CytoD with pyramidal (“Pyr”) and cylindrical (“Cyl”) probes at indentation site (Mean ± SD, N ≥ 3, n ≥ 11). The orange arrow indicates the injection pulse at the specified time in the mechanochemical triggers.

D) Time-lapse lifetime images of ER Flipper-TR-stained HeLa cells before, upon indentation (“mech”), after indentation plus injection (“mech + inj”) of either DMSO (as control) or 50 µM CytoD, and after retracting pyramidal (left) and cylindrical (right) probes. The numbers represent time (min:s), with time 0 corresponding to the indented state (mechanical stimulus, noted as “mech”). Inserts represent magnified nuclear membrane at locations specified with arrows. Scale bars in time-lapse images and inserts represent 5 µm and 1 µm, respectively.

E) Change in lifetime at different ROIs: ER, nucleus membrane (Nuc-mem) and indentation site (“Indent-site”) when mechanochemically challenged by either CytoD or Control using pyramidal (top row) and cylindrical (bottom row) probes (Mean ± SD, N ≥ 3, n ≥ 11, ***p < 0.001, **p < 0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test). Orange arrows indicate the injection pulse at the specified time in the mechanochemical process.

F) Box plots showing lifetime change upon retracting probes from the injected cells at different ROIs: ER, Nuc-mem and indentation site. Mean values are shown with triangles (N ≥ 3, n ≥ 11, ***p < 0.001, **p < 0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, ns: not significant, t-test).

G) Schematic represents the tension report of ER Flipper-TR dye inside the membrane of the nucleus and the ER before (left panel) and after mechanochemical triggering of the nucleus with DMSO (as control, middle panel) or CytoD (right panel). In contrast to control cells, delivery of CytoD induces more accumulated tension (i.e., FLIM data) at the nuclear membrane and the ER because it disrupts load-bearing actin component of cytoskeleton. ONM: outer nuclear membrane, INM: inner nuclear membrane, PNS: perinuclear space, and σ: tension exerted by indenting the nucleus using FluidFM probes.

ER and nuclear envelope crosstalk as a response to mechanochemical triggering of different intracellular components of HeLa cells.

A) Schematic shows targeting different organelles for mechanochemical stimulation: nucleus (top), the ER (middle) and cell periphery (bottom).

B) Lifetime images of HeLa cells stained with the ER Flipper-TR before, at mechanochemical stimulated state (“mech + inj”) and after stimulation targeting different subcellular compartments: nucleus (top panel), the ER (middle panel) and cell periphery (bottom panel) for indentation and injection of either control (Cont) or CytoD using sharpened cylindrical probes. Arrows indicate where the cylindrical probe penetrates the cell (“Indent-site”). Scale bar represents 5 µm.

C, D) Change in lifetime at the ER and nucleus membrane (Nuc-mem) during (C, noted as “mech + inj” in B) and after (D) stimuli imposing injection of DMSO as control (light) or CytoD (dark) at different subcellular compartments: nucleus (top panel), ER (middle panel) and cell periphery (bottom panel) (Mean ± SD, N = 2, n ≥ 5, **p < 0.01, *p < 0.05, t-test). Orange arrows in C indicate the injection pulse at the specified time in the mechanochemical process. Mean values are shown with triangles in boxplots in D (N = 2, n ≥ 5, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, ns: not significant, t-test).

A-type lamins contribute to elasticity, while B-type lamins may influence viscosity in the nucleus’s viscoelastic behavior under sustained deformation with disrupted actin polymerization.

A, B) Lifetime images (A) and boxplots showing the absolute lifetime values (B) of HeLa cells with varied levels of downregulation of lamin proteins: WT-siCtrl (WT), WT-siLMNB (A-KO), LMNA KO-siLMNB (B-KD) and LMNA KO-siLMNB (A-KO/B-KD). Cells were stained with ER Flipper-TR dye. Scale bar in A represents 5 µm. Mean values are shown with triangles in B (N = 2, n ≥ 8, ***p < 0.001, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, ns: not significant, t-test).

C) Lifetime images of WT, A-KO, B-KD and A-KO/B-KD cells stained with ER Flipper-TR before, upon indentation (“mech”), upon indentation + CytoD injection, “mech + inj”, and after stimulation using sharpened cylindrical probes. Note the extended range for lifetime, compared to A, revealing distinguished cell responses to the stimuli, thus indicating the initial tension state for different cell versions in the same color. Scale bar represents 5 µm.

D) Lifetime change at the ER, nucleus membrane (Nuc-mem) and indentation site during mechanochemical stimuli. All cells were injected with CytoD. In each panel, the tension response of WT cells were compared with either of A-KO, B-KD and A-KO/B-KD cells. Orange arrows indicate the injection pulse at the specified time in the mechanochemical process. Time zero corresponds to the indented state (indicated by “mech” in C) which follows by an injection (indicated by “mech + inj” in C) (Mean ± SD, N = 3, n ≥ 9).

E, top) Viscoelastic response of cells with varied depletion of lamins to the mechanochemical stimuli composed of lifetime change originating from the instantaneous cell response (Elastic) upon indentation (first time points in D) and from the time-dependent response (Creep) due to AFM probe being in contact with cell (at 150 nN) for around 4 min after CytoD injection (last time points in D). Data represent the mean ± SEM (N = 3, n ≥ 9, ***p < 0.001, **p < 0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test). E, bottom) Relaxation behavior of WT, A-KO, B-KD and A-KO/B-KD cells after stimuli reported as lifetime change right after probe retraction compared to last time point during stimuli (last time points in D). Mean values are shown with triangles in boxplots (N = 3, n ≥ 9, reported p-values of 0.05 ≤ p < 0.1, ns: not significant, t-test).

Dynamic role of microtubules and actinomyosin in regulating mechanoresponse of nuclei in cells with and without lamin A/C-depletion to the external stimulus.

A) Lifetime images of WT- and LMNA KO-HeLa cells stained with ER Flipper-TR before, upon indentation (“mech”), upon indentation + injection (“mech + inj”) of various drugs and after stimulation using sharpened cylindrical probes. For chemical stimulation, interruption of different cytoskeleton components including actin filaments, microtubules and both of actin filaments and microtubules was targeted realized by injection of 50 µM CytoD (left column), 50 µM Noco (middle column) and 50µM CytoD + 50µM Noco (right column) dissolved in DMSO. Scale bar represents 5 µm.

B) Lifetime change at ER, nucleus membrane (Nuc-mem) and indentation site during stimuli in WT and LMNA KO cells. Arrows indicate the injection pulse at the specified time in the mechanochemical process. Time zero corresponds to indented state (indicated by “mech” in A) which follows by injection, indicated by “mech + inj” in A (Mean ± SEM, N = 2, n ≥ 6, **p <0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test with Holm correction).

C) Relaxation behavior of WT and LMNA KO cells after mechanochemical stimuli involving various drugs (CytoD, Noco and CytoD-Noco). Relaxation is reported as lifetime change at 5 min after probe retraction compared to the last time point of stimuli (last time points in B). Mean values are shown with triangles in boxplots (N = 2, n ≥ 6, **p <0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test with Holm correction).

Selection of ROIs of 1) the ER, 2) nuclear membrane (Nuc-mem), and 3) a spot around the indentation site (Indent-site) using pyramidal (top) and cylindrical (bottom) probes.

Representative lifetime images of a HeLa cell stained with ER Flipper-TR dye are shown. Scale bar represents 5 µm.

Co-staining HeLa cells with NucBlue (targeting DNA inside the nucleus) and ER Flipper-TR dye.

The significant overlap signals obtained provide confirmation of the precision of the designated ROI as representing the nuclear membrane in this investigation. Scale bars represent 5 µm.

Lifetime values before, during and after injecting HeLa cells with DMSO as control group with pyramidal and cylindrical probes at incident point (Mean ± SD, N ≥ 3, n ≥ 11).

Different sections on the graph represent lifetime measurements before stimulus, during mechanical stimulus when the probe has reached the set point on the cell without fluidic pressure application (“mech”), and during mechano-chemical stimulus when the probe remains in contact with the cell at the set point, and a fluidic pressure pulse is applied for drug (or here control) injection (“mech + inj”), and after retraction of probe and removal of the stimulus.

Western blot analysis verifying the absence of lamin A/C in LMNA KO cells and the successful downregulation of lamins B1 and B2 in both WT and LMNA KO upon treatment with siLMNB.

Three biological replicates were utilized for mechano-chemical triggering assays and FLIM-based tension measurements.

Lifetime images (A) and boxplots showing absolute lifetime values at the ER and nuclear membrane (Nuc-mem) (B) of HeLa cells stained with ER Flipper-TR tension reporter molecule.

The lamin A/C level of cells was downregulated with altering methods: WT, LMNA KO and WT siLMNA. Cells. Scale bar in A represents 5 µm. Mean values are shown with triangles in boxplots in B (N = 2, n ≥ 6, *p < 0.05, ns: not significant, t-test with Holm correction).

Michaelis-Menten fit to lifetime response (top) and slope value of fitted curve (bottom) in HeLa cells with and without A-type and B-type lamins at the ER, nuclear membrane (Nuc-mem) and indentation site (Indent-site) (Mean ± SD, N = 3, n ≥ 9).

Relaxation behavior of cell with different levels of lamin proteins: WT, A-KO, B-KO and A-KO/B-KD at the ER, nuclear membrane (Nuc-mem) and indentation site (Indent-site) after stimulus.

Relaxation is reported as lifetime change 5 min after probe retraction compared to last time point of stimuli. Mean values are shown with triangles in boxplots (N = 3, n ≥ 9, ns: not significant, t-test).

Relaxation behavior of WT and LMNA KO cells during and after mechano-chemical stimuli involving various drugs (CytoD, Noco and CytoD-Noco), respectively, at the ER, nuclear membrane (Nuc-mem) and indentation site (Indent-site).

Viscoelastic response is composed of lifetime change originating from instantaneous cell response (Elastic) upon indentation and from time-dependent response (Creep) due to AFM probe being in contact (at 150 nN) with cell for around 4 min after different drug injection including CytoD, Noco, CytoD-Noco. Data represent the mean ± SEM (N ≥ 2, n ≥ 6, **p <0.01, *p < 0.05, reported p-values of 0.05 ≤ p < 0.1, t-test with Holm correction). Statistical significance shown on the bars indicates differences between WT and LMNA KO cells, emphasizing variations in either the elastic or viscous components under specific drug conditions. The significance levels adjacent to dashed or solid lines signify disparities in the creep or elastic segments of the response, respectively. Relaxation is reported as lifetime change following the retraction of probe compared to last time point of stimuli. Mean values are shown with triangles in boxplots (N ≥ 2, n ≥ 6, no significant differences were captured from t-test with Holm correction).