Cell cultures and enucleation
Two cell lines were used in the study, REF52 rat fibroblasts and HT1080 human fibrosarcoma cells. The cells were cultured in the DMEM medium supplemented with 1 × GlutaMax, 15 mM HEPES, 10% FBS and 1% antibiotic/antimycotic solution (all by Thermo Fisher, USA) in a humidified 5% CO2 atmosphere at 37 °C.
Enucleation was performed by the previously described approach [23,24,25] with some modifications (Fig. 1). Small pieces of culture plastic (plastic slides) with an approximate size of 8 × 20 mm were cut from standard cell culture Petri dishes (Corning, USA). The plastic slides were sterilized by incubation in 95% ethanol and exposed to a germicidal UV lamp (Microcide, Electronic Medicine, Russia) for 10 min, then coated with a 10 µg/mL fibronectin (Sigma, USA) solution for 20 min. Cells growing as monolayers on plastic slides were inserted in 1.5 mL Eppendorf tubes filled with 2 µM of Cytochalasin D (CytD) (Sigma, USA) in the cell medium. After 15 min of pre-incubation at 37 °C, the cells were centrifuged at 13,400 rpm (12,000 g) in a MiniSpin microcentrifuge (Eppendorf, Germany) for 9 to 25 min (longer duration needed for REF52 cells). After centrifugation, the cells on the plastic slides were washed with PBS, trypsinized, and reseeded on fibronectin-coated glass-bottom cell culture dishes (WillCo Wells B.V., Amsterdam, Netherlands). After 3 to 5 h, the cells (including cytoplasts) were fully spread and recovered from the effects of CytD. The medium was exchanged for the fresh one before the AFM experiment. The pellet containing nucleoplasts was resuspended in HBSS (Hanks balanced salt solution, Thermo Fischer, USA) and placed on poly-L-lysine-coated glass-bottom dishes. After 15 min of incubation, unattached nucleoplasts were removed by washing with HBSS.
The nuclei were also isolated using a detergent-based approach. Briefly, the cells were transferred to suspension by trypsinization, centrifuged and resuspended in a 0.1% Triton X-100 detergent (Thermo Fischer, USA) solution in HBSS, mildly pipetted and centrifuged for 10 s at 12,000 g. The detergent treatment was performed twice, on ice with ice-cold solutions to minimize the nuclei’s damage and degradation due to cellular proteases. Then the pellet was resuspended in HBSS and a small amount of the solution was transferred to poly-L-lysine-coated glass-bottom dishes for AFM experiments. After 15 min of incubation, the unattached nuclei were removed by washing with HBSS.
Atomic force microscopy
All AFM measurements were performed using a Bioscope Resolve AFM (Bruker, Santa Barbara, USA) mounted on an Axio Observer inverted fluorescent microscope (Carl Zeiss, Germany). The microscope was equipped with a heated stage, and the sample temperature was kept constant at 37 °C during the experiments with cells. PeakForce QNM-Live Cell probes (PFQNM-LC-A-CAL, Bruker AFM Probes, Camarillo, CA, USA), short paddle-shaped cantilevers with a pre-calibrated spring constant (values were in the range of 0.06–0.08 N/m) were used. The cantilever deflection sensitivity (nm/V) was calibrated from the thermal spectrum directly in the dish with a sample using the pre-calibrated value of the spring constant [28]. The nanomechanical and topography maps were acquired in the fast force volume (FFV) mode with a map size from 40 × 40 to 100 × 100 µm and from 32 × 32 to 128 × 128 point-measurements. The force curves (F-Z curves) had a vertical ramp distance of 3 μm, a vertical piezo speed of 183 μm/s, and the trigger force of 0.5–1 nN. The topography and local height were calculated from the force curves by the contact point position versus contact position over the substrate, the global tilt correction was performed if needed.
The numerical processing of the F-Z curves was done using MATLAB scripts (The MathWorks, Natick, MA) developed in the previous works [29, 30] with the utilization of the Ting’s model [31]:
$$F\left( {t,\delta \left( t \right)} \right) = \frac{4\sqrt R }{{3\left( {1 - \nu^{2} } \right)}}\mathop \smallint \limits_{0}^{{t_{1} \left( t \right)}} f_{BEC} (\delta )E\left( {t - \xi } \right)\frac{{\partial \delta^{\frac{3}{2}} }}{\partial \xi }d\xi ;$$
(1)
$$t_{1} \left( t \right) = \left\{ {\begin{array}{*{20}c} {t_{1} \left( t \right) = t, 0 \le t \le t_{m} } \\ {\mathop \smallint \limits_{{t_{1} \left( t \right)}}^{t} E\left( {t - \xi } \right)\frac{\partial \delta }{{\partial \xi }}d\xi = 0, t > t_{m} } \\ \end{array} } \right.;$$
(2)
where F is the force acting on the cantilever tip; \(\delta\) is the indentation depth; \(t\) is the time initiated at the contact; \(t_{m}\) is the duration of the approach phase; \(t_{1}\) is the auxiliary function determined by Eq. 2; \(\xi\) is the dummy time variable required for the integration; \(\nu\) is the Poisson’s ratio of the sample (assumed to be time-independent and equal to 0.5); \(R\) is the radius of the indenter; \(f_{BEC} (\delta )\) is the bottom-effect correction factor [32]; and \(E(t)\) is the Young’s relaxation modulus for the selected rheology model. Here we used the power-law rheology (PLR) model (also known as a springpot in parallel with a dashpot) [33, 34]:
$$E\left( t \right) = E_{1} t^{ - \alpha } + \eta \delta_{D} (t),$$
(3)
where \(E_{1}\) is the relaxation modulus at t = 1 s (scale factor of the relaxation modulus); \(\alpha\) is the power-law exponent; \(\eta\) is the Newtonian viscous term (with Pa*s units); and \(\delta_{D} (t)\) is the Dirac delta function. A larger \(\alpha\) value means a larger amount of relaxation; materials exhibit a solid-like behavior at \(\alpha = 0\), and a fluid-like behavior at \(\alpha = 1\). The PLR model described by Eq. [3] was successfully used for the description of cell mechanics in several previous studies [33, 35,36,37]. The Young’s modulus with the assumptions of the Hertz’s theory, YM (“apparent” elastic modulus), was also calculated from the approach part of the force curves [38].
We used the top 50% of each cell data set over a cell to define the nuclear part, and the lower areas were discarded in the analysis, since their local properties were highly affected by the high F-actin concentration at the periphery. The same part of the dataset was used for the nucleoplasts and isolated nuclei as well, to exclude the peripheral regions where the data can potentially be affected by the local tilt of the hemispherical sample. From the datasets, we used the mean geometric values of YM and \(E_{1}\), and mean arithmetic values of \(\alpha\) and \(\eta\) for the further comparison between the samples [36]. In the text, the data are presented as mean ± SD. All the statistical analyses were performed using the MATLAB software (MathWorks, USA). A non-parametric Mann–Whitney U test was used to determine the statistically significant differences between the groups. The change in the parameters was calculated as that relative to the median value. The percentiles in the box-and-whisker plots are 10%, 25%, 50%, 75%, and 90%, the dots correspond to each value of the set.
Fluorescent and confocal microscopy
The nuclei in the prepared AFM samples were stained with Hoechst 33342 dye (2 µg/mL) to detect enucleated cells or to distinguish the nucleoplasts and isolated nuclei from the cell debris. Phase-contrast and fluorescent images were recorded with 10x/0.3 or 20x/0.4 objectives using the ZEN software (Carl Zeiss, Germany) of the Axio Observer inverted fluorescent microscope.
For the confocal microscopy studies, the samples were fixed in a 4% formaldehyde solution in PBS for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin for 10 min and stained with Alexa Fluor 488 phalloidin (Life Technologies, USA). The samples were washed with PBS and mounted with the ProLong Gold antifade reagent (Invitrogen, USA). Fluorescent images (Z-stacks) were acquired using an LSM 880 confocal laser scanning microscope equipped with an AiryScan module and a GaAsP detector (Carl Zeiss, Germany) with a Plan-Apochromat 63x/1.4 N.A. oil immersion objective.