Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells

Abstract

The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

Figure 1. The nuclear envelope deforms around nanopillars, creating a non-invasive platform for studying in situ nuclear deformation

a, Illustrations of techniques for studying nuclear mechanics. Anticlockwise from top left: micropipette aspiration, atomic force microscopy and nanopillar arrays. b, SEM image of nanopillar array with 75 nm radius, 2 μm pitch and 1.4 μm height. Scale bar, 1 μm. c, Overlay of differential interference contrast (DIC) and fluorescence images of 3T3 cells cultured on a nanopillar array. The red colour shows actin staining, and nanopillars are visible as dark spots in the bright-field image. The grey square in the DIC image is an alignment marker used to locate the nanopillar arrays. Scale bar, 10 μm. d, TEM image showing the nucleus deformed around a nanopillar array with the cytosol visible between the nuclear envelope and the cell membrane. Scale bar, 2 μm. e, Fluorescence (left and middle, different time points) and DIC (right) images of a live 3T3 cell transfected with GFP-Sun2. Nuclear deformation is evident as bright fluorescent spots around the nanopillars and changes as the cell migrates. See Supplementary Movies 1 and 2 for complete time lapse of nuclear deformation during cell migration. Scale bars, 5 μm.

Figure 2. Quantitative analysis of nanopillar-induced nuclear deformation

a, Immunostaining of lamin A reveals nuclear deformation in confocal microscopy. Four optical z slices are shown, beginning from the basal surface of the cell. b, Reconstructed side view of the confocal scan from a. The pinches above each nanopillar in a row are visible (corresponding to the green dotted line in c). c, Corresponding DIC image showing the locations of the nanopillars. d, The z position of the nuclear envelope can be localized precisely by fitting two Gaussian functions to the confocal z scan of the fluorescence signal at each pixel. Top panel: Confocal z scan of the fluorescence signal for a pixel on top of a nanopillar (location indicated by orange boxes in a and c). Bottom panel: Confocal z scan of the fluorescence signal for a pixel between nanopillars (location indicated by purple boxes in a and c). The fitted z positions of the basal nuclear envelope are illustrated by vertical green lines and the difference between the two z positions of the two pixels is evident. e, Nanopillar-induced nuclear deformation is clear in a reconstructed surface of the basal nuclear envelope. The value at each pixel is the height of the nuclear envelope, calculated as the centre of a Gaussian fit to the confocal z scan at that pixel. The nanopillars in this example have the following dimensions: 300 nm radius, 3 μm pitch and 2 μm height. Scale bars, 3 μm.

Figure 3. Nanopillar-induced nuclear deformation shows a strong dependence on nuclear stiffness

a, Reconstructed surfaces of the nuclear envelope reveal differences in the deformability of an untransfected 3T3 cell (top), a GFP-lamin A-transfected 3T3 cell (middle) and a GFP-progerin-transfected 3T3 cell (bottom). The locations of the nanopillars were identified by DIC imaging (left column) and the nuclear envelope was visualized by fixing the cells and immunostaining with anti-lamin A (untransfected cells, green) or anti-GFP (transfected cells, red) antibodies (middle column). The surface of the nuclear envelope (right column, false-coloured in z position) is visibly deformed at nanopillars in the untransfected cell but less so in GFP-progerin-transfected and GFP-lamin A-transfected cells. b, Average nuclear deformation, consistently showing less deformation for lamin A-transfected cells than for untransfected 3T3 cells for all nanopillar geometries tested. Nanopillars with four different radii are shown, but with the same pitch of 3 μm and height of 1.4 μm. c, When normalized to untransfected 3T3 cells on the same geometry, 3T3 cells transfected with lamin A and progerin cells show much less deformation than untransfected cells. Interestingly, progerin-transfected cells show more deformation than lamin A-transfected cells. d, GFP-progerin-transfected 3T3 cell shows characteristic folds in the nuclear lamina (green). These folds align with nanopillar locations (false-coloured white) at the basal nuclear envelope. e, Representative images of hippocampal neurons show significant nuclear deformation on nanopillars. Left: DIC image showing pillar locations. Middle: Immunostaining image with anti-nuclear pore complex illustrating nuclear envelope. Right: Reconstructed z surface of nuclear envelope (false coloured in z) showing that the nuclear envelope is deformed at nanopillar locations. f, Average nuclear deformation in different cell types, normalized to 3T3 cells on the same geometry. HL1 and 3T3 cells are equally deformable, while MCF7 cells show more deformation. Neurons show significantly more deformation than any of the other cell types. All measurements were performed in triplicate. The numbers of nanopillars and nuclei included in each sample are provided in Supplementary Table 1. Comparisons were analysed with two-tailed, unequal variance student t-tests, corrected for multiple comparisons where necessary (c). **P < 0.01, ***P < 0.001. NS, no significant difference. Error bars indicate standard error of the mean. Scale bars, 5 μm.

Figure 4. Nanopillar-induced nuclear deformation depends on the integrity of cytoskeletal components

a, Latrunculin B treatment results in decreased nuclear deformation, whereas acrylamide treatment shows increased nuclear deformation compared with untreated cells. Colchicine did not alter the extent of nuclear deformation. Left column: Fluorescence images showing cell morphology without treatment and under treatment with different cytoskeletal inhibitors: latrunculin B for actin filaments, colchicine for microtubules, and acrylamide for intermediate filaments. Actin was stained with phalloidin-Alexa568 (red) and the nuclear envelope was immunostained for lamin A (green). Scale bars, 10 μm. Right column: Surface plots showing average deformed nuclear surfaces under the different inhibitor treatments. b,c, The change in the depth of nuclear deformation showed the same response to inhibitor treatments on nanopillars with radii of 220 nm (b) and 300 nm (c). All measurements were performed in triplicate. The numbers of nanopillars and nuclei included in each sample are provided in Supplementary Table 1. One-way analysis of variance (ANOVA) was performed on the results in b and c, comparing each group to the untreated cells. NS, no significant difference. ***P < 0.001. Error bars indicate standard error of the mean.

Figure 5. Nanopillar geometry determines the depth and shape of nuclear deformation

a, At constant pitch, larger radii induce shallower, broader deformation. Reconstructed surfaces show the average deformation on nanopillar arrays with constant height (1.4 μm) and pitch (2 μm) but different radii: 75 nm (left) and 350 nm (right). b, The depth of deformation decreases with increasing nanopillar radius. The large plot shows the average depth of nuclear deformation versus nanopillar radius. The two small plots to the left show the profile through the centre of the indentation on 75 nm (top) and 350 nm (bottom) nanopillars, with the depth indicated. Height (1.4 μm) and pitch (2 μm) were held constant for all data points. c, The angle of deformation increases with nanopillar radius. The large plot shows the average angle of nuclear deformation versus nanopillar radius. The two small plots show the nuclear profile on 100 nm (top) and 300 nm (bottom) nanopillars, with the angle measurement indicated. Height (1.4 μm) and pitch (3 μm) were held constant for all data points. d, With the same radius, nanopillar arrays with larger pitch induce deeper, narrower deformation. Reconstructed surface showing average deformation on nanopillar arrays with constant height (1.4 μm) and radius (300 nm) but different pitch: 2 μm (left) and 6 μm (right). e, The depth of deformation increases with increasing pitch. The large plot shows average depth of nuclear deformation versus nanopillar pitch. The two small plots to the right show the nuclear profile for 2 μm (top) and 6 μm (bottom) pitch. f, The angle of deformation decreases with increasing pitch. The large plot shows the average angle of nuclear deformation versus nanopillar pitch. The two small plots show the nuclear profile for 2 μm (top) and 6 μm (bottom) pitch. Height (1.4 μm) and radius (300 nm) were held constant for all data points in e and f. The numbers of nanopillars and nuclei included in each sample are provided in Supplementary Table 1. Error bars indicate standard error of the mean.

Figure 6. Finite-element analysis suggests that nanopillar-induced deformation is a cumulative result of both the actin cap above the nucleus and actin accumulated around the nanopillar

a, Illustration of the mechanical model, in which the force pulling the nucleus toward the nanopillars can either be uniformly applied to the apical surface of the nucleus, locally applied around the nanopillar, or a combination of the two. b, Local actin accumulation around nanopillars. Left: Bright-field image showing nanopillar locations. Right: Fluorescence image of actin staining showing bright rings around nanopillars with 300 nm radii. Scale bars, 5 μm. c, Nuclear deformation surfaces display the differential effects of applied pressure profiles. From left to right: experimental surface; simulated surface under combined uniform and localized pressure; simulated surface under uniform pressure only; simulated surface under localized pressure only. d, Of the simulated pressure profiles, the combined uniform and localized pressure most closely reproduces the experimental dependence of nuclear deformation depth on nanopillar pitch. e, Experimental measurements of the deformation angle, shown in blue, match the results from combined pressure more closely than those from either localized or uniform pressure alone.