Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Abstract

Nuclei are often under external stress, be it during migration through tight constrictions or compressive pressure by the actin cap, and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area, respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

FIGURE 1:

Combined atomic force microscopy and side-view light sheet microscopy (AFM-LS) extracts dynamics of nuclear morphology and applied force under whole-cell compression. (A) Cartoon schematic of our AFM-LS system. A full description is provided in our previous work. (B) A subset of fluorescence images collected by our AFM-LS during indentation of a live, SKOV3 cell (scale bar = 5 µm). The cell is stably expressing snap-tagged KRas-tail (magenta) and Halo-tagged H2B (green) labeled with Janelia Fluor 503 and 549, respectively. Custom workflow (see Materials and Methods) allows for extraction of nuclear perimeter (NP) and nuclear cross-sectinoal area (NCSA). (C) Force vs. indentation data for the previously displayed compression experiment. Left inset provides a scale for the noise in the force data. Right inset shows a scanning electron microscope image of a bead glued to the end of an AFM cantilever (see Materials and Methods). Beads are nominally 6 µm in diameter; this bead was measured to be 5.4 µm in diameter (scale bar = 2 µm). (D) Nuclear morhpology as a percentage vs. indentation for the previously displayed compression experiment. Orange and blue represent NP and NCSA, respectively. Δδ is defined to be the difference in indentation at which NP and NCSA reach 1% change. (E) Δδ for n = 17 separate compression experiments. The red bar represents the standard error in the mean.

FIGURE 2:

Correlating nuclear morphology and applied force informs an emperical model for strain-stiffening response. (A) Force as recorded by the AFM vs. change in NCSA plotted on a log-log scale. Two distinct power-law regimes are observed. (B) Force as recorded by the AFM minus the force response in regime 1 plotted against change in NP on a log-log scale, showing a single power-law relationship in regime 2. (C) α, the exponent for , and β, the exponent for , as determined for n = 16 cells. Red bars represent mean and SEM. (D) The strain in NP at the transition point between regime 1 and regime 2 as determined for n = 16 cells. Red bars represent mean and SEM. (E) An emperical model for nuclear deformation as shown over force vs. indentation. We display our full emperical model (magenta), the individual contributions required to deform the nuclear volume and surface area (blue and orange, respectively), and the AFM data over the full indentaiton. (F) Resistance to nuclear volume change, , and resistance to nuclear surface area change, , as determined for n = 17 cells. Red bars represent mean and SEM.

FIGURE 3:

Chromatin decompaction and lamin A/C depletion weaken resistance to volume and surface area changes, respectively, while behaving similarly to the empirical model. (A) Resistance to nuclear volume change, , is decreased by TSA but unchanged by LA/C KD. n = 17, 14, and 13 for WT, TSA, and LA/C KD, respectively. (B) Resistance to nuclear surface area change, , is unchanged by TSA but decreased by LA/C KD. n = 17, 14, and 13 for WT, TSA, and LA/C KD, respectively. (C) α, the exponent for , is unchanged by TSA and LA/C KD. n = 16, 14, and 13 for WT, TSA, and LA/C KD, respectively. (D) β, the exponent for , is unchanged by TSA and LA/C KD. n = 16, 14, and 11 for WT, TSA, and LA/C KD, respectively. (E) The difference in indentation at the onset of change in NP and NCSA is unchanged by TSA and LA/C KD. n = 17, 14, and 13 for WT, TSA, and LA/C KD, respectively. (F) The strain in NP at the transition point between regime 1 and regime 2 is unchanged by TSA and LA/C KD. n = 16, 14, and 11 for WT, TSA, and LA/C KD, respectively. Red bars represent mean and SEM. NS, not significant. *, p < 0.05.

FIGURE 4:

Dynamic nuclear curvature analysis during AFM indentation. (A) Nuclear curvature vs. position along perimeter for an undeformed nucleus. The inset displays the mask of the nucleus and the discretization of the perimeter. (B) Nuclear curvature vs. position along the perimeter for a deformed nucleus. The inset displays the mask of the nucleus and the discretization of the perimeter. Note the additional peak centered around 10 µm representing the site of indentation. (C) A Gaussian fit to the peak at the site of indentation extracts maximum curvature under the AFM bead (red dashed box in B). (D) Maximum curvature plotted during the entire indentation. A linear fit is performed for the region of changing curvature. (E) Maximum curvature as a function of indentation plotted for n = 14 WT cells and n = 14 TSA-treated cells. ** represnts p < 0.01 for a t test comparing the mean slope of maximum curvature vs. indentation. (F) Maximum curvature as a function of indentation plotted for n = 14 WT cells and n = 13 LA/C KD cells. Black dashed line represents curvature of the AFM bead. NS represents no significance for a t test comparing the mean slope of maximum curvature vs. indentation. (G) Representative images of SKOV3 nuclei (H2B) under maximum compression with various treatments. Scale bar = 5 µm.

FIGURE 5:

Finite element analysis (FEA) model of AFM indentation. (A) Selected frames from a FEA simulation of a nucleus under compression. The nucleus has an elastic modulus, E, and a separate stretch modulus, γ. (B) Force vs. indentation shown for varied E and γ. (C) Resistance to nuclear volume change, , plotted against variations in both E and γ. A significant correlation (p < 0.001) is seen between and E, but no significant correlation is seen between and E. (D). Resistance to nuclear surface area change, E_SA, plotted against variations in both E and γ. A significant correlation (p < 0.05) between and γ is seen, but no significant correlation is seen between and E.