Actomyosin and vimentin cytoskeletal networks regulate nuclear shape, mechanics and chromatin organization

Abstract

The regulation of nuclear state by the cytoskeleton is an important part of cellular function. Actomyosin stress fibres, microtubules and intermediate filaments have distinct and complementary roles in integrating the nucleus into its environment and influencing its mechanical state. However, the interconnectedness of cytoskeletal networks makes it difficult to dissect their individual effects on the nucleus. We use simple image analysis approaches to characterize nuclear state, estimating nuclear volume, Poisson's ratio, apparent elastic modulus and chromatin condensation. By combining them with cytoskeletal quantification, we assess how cytoskeletal organization regulates nuclear state. We report for a number of cell types that nuclei display auxetic properties. Furthermore, stress fibres and intermediate filaments modulate the mechanical properties of the nucleus and also chromatin condensation. Conversely, nuclear volume and its gross morphology are regulated by intracellular outward pulling forces exerted by myosin. The modulation exerted by the cytoskeleton onto the nucleus results in changes that are of similar magnitude to those observed when the nucleus is altered intrinsically, inducing chromatin decondensation or cell differentiation. Our approach allows pinpointing the contribution of distinct cytoskeletal proteins to nuclear mechanical state in physio- and pathological conditions, furthering our understanding of a key aspect of cellular behaviour.

Figure 1

Epifluorescence–based quantification of cytoskeletal organization, nuclear shape and chromatin condensation. All panels depict the same example cell/nucleus. (A) Overlay of fluorescence images for TRITC (phalloidin) and DAPI channels obtained on an epifluorescence microscope using a 20× objective. Quantification of F-actin fibre fluorescence intensity (B) and fiber orientation (C) obtained from the raw images. Fluorescence intensity of the nucleus before (D) and after (E) band-pass filtering. Fluorescent speckles resulting from areas of high chromatin condensation are clearly visible in the zoomed-in image shown as an inset. (F) Averaged fluorescence intensity profile as a function of radial distance I(r). Black squares correspond to fluorescence intensities recorded, and the imaged nucleus is taller than the depth of focus of the objective lens. Red line corresponds to the ellipse obtained when fitting the fluorescence intensity profile of the outermost pixels. Left axis shows the fluorescence intensity values from the analysed image, while right axis shows the height profile estimated using the calibration factor. (G) x-z reconstruction of the nucleus as obtained from a confocal image stack. Overlaid is the estimated gross nuclear morphology as obtained from the fit shown in (F). Scale bar is 50 µm in panels A–E and 5 µm in panel G. In panels B–E a false colour scale has been used to improve visualization. Inset in (C) exemplifies the computed orientation of the nucleus (θ_nuc) and the average orientation of the fibres (θ_fib).

Figure 2

Cell spread area modulates nuclear state and mechanics. Plot shows results for nuclear height (a), volume (b), Poisson’s ratio (c), apparent elastic modulus (d) and chromatin condensation (e). Values for >50 cells were pooled to compute each individual data point. Data is presented as geometric mean, error bars indicate interquartile range (Q1–Q3). For the single case of Poisson’s ratio, data is presented as mean ± SD. For height data, the red line is the fit to the rational function defined as h = $a+\frac{b}{c+CSA}$. Dotted lines indicate the values for average height of isolated nuclei and minimum nuclear height, as estimated using the fit.

Figure 3

Cytoskeletal organization modulates nuclear state and mechanics. Subplots are arranged according to cytoskeletal protein assessed (columns) and nuclear property measured (rows). Values for >15 cells were pooled to compute each individual data point. Data is presented as geometric mean, error bars indicate interquartile range (Q1–Q3). For the single case of Poisson’s ratio, data is presented as mean ± SD.

Figure 4

Cell spread area modulates cytoskeletal organization. Plot shows cytoskeletal organization in filamentous form for actin (black), myosin (red), tubulin (green) and vimentin (blue). Values for >15 cells were pooled to compute each individual data point. Data is presented as geometric mean, error bars indicate interquartile range (Q1–Q3).

Figure 5

The alignment of cytoskeletal fibres affects nuclear shape and orientation. Top row shows the relationship between fibre anisotropy and nuclear aspect ratio in the x-y plane and bottom row shows the difference between the orientation of the nucleus (θ_nuc) and that of the fibres (θ_fib). Panels depict these relationships for actin (black), myosin (red), tubulin (green) and vimentin (blue). In the top row, values for >15 cells were pooled to compute each individual data point and data is presented as geometric mean, error bars indicate interquartile range (Q1–Q3). Panel e shows the slopes of the linear fits obtained for panels a–d while panel j shows the circular mean and circular standard deviation obtained from the distributions presented in f–i.

Figure 6

Chromatin decondensation and cellular differentiation affect nuclear state. Plot shows population average values for nuclear volume (a), Poisson’s ratio (b), apparent elastic modulus (c) and chromatin condensation (d) for increasing dosages of TSA or two well-established hMSC differentiation treatments (adipogenic and osteogenic differentiation). Dotted lines correspond to maximum and minimum values reachable via CSK-based modulation of nuclear state as obtained in Fig. 2, they are included to aid comparison.

Figure 7

Cell spread area modulates nuclear state and mechanics in a variety of adherent cell types. Plot shows results for nuclear volume (a), Poisson’s ratio (b), apparent elastic modulus (c) and chromatin condensation (d) for the following cell types: hMSC (light blue diamonds), COS-7 (red circles), NIH 3T3 (green triangles), HaCaT (dark blue triangles), HUVEC (yellow triangles), PSC (black squares) and GE 11 (pink triangles). Values for >10 cells were pooled to compute each individual data point. Data is presented as geometric mean but error bars have been omitted for visual clarity. For the large majority of data points, the coefficient of variance computed as (Q1–Q3)/(2·Q2) was found to be smaller than 20%. For the single case of Poisson’s ratio, data is presented as mean.