The mitotic chromosome periphery modulates chromosome mechanics

Abstract

In dividing cells, chromosomes are coated in a sheath of proteins and RNA called the mitotic chromosome periphery. This sheath is thought to confer biophysical properties to chromosomes, critical for successful cell division. However, the details of chromosome mechanics, and specifically, if and how the chromosome periphery contributes to them, remain poorly understood. In this study, we present a comprehensive characterisation of single-chromosome mechanics using optical tweezers and an improved broadband microrheology analysis. We extend this analysis to direct measurements of the chromosome periphery by manipulating levels of Ki-67, its chief organiser, and apply a rheological model to isolate its contribution to chromosome mechanics. We report that the chromosome periphery governs dynamic self-reorganisation of chromosomes and acts as a structural constraint, providing force-damping properties. This work provides significant insight into chromosome mechanics and will inform our understanding of the mitotic chromosome periphery's role in cell division.

Fig. 1. Chromosome mechanics are rate-dependent.

ai Examples of Bright-field and Fluorescence HILO images of Ki-67 over-expression (OE), wild-type (WT) and knock-down (KD) chromosomes in dumbbell configuration. Scale bar = 2 μm. aii Schematic of MCP load with Ki-67 expression. b Schematic of the force-extension experiments where one optical trap is displaced while the other is kept stationary, to apply stretching forces at a known speed. Fluorescence intensities of chromosomes were analysed to quantify MCP load. Individual examples of force-extension experiments at either 0.02 μm/s (bi) or 0.2 μm/s rate (bii) in WT chromosomes to illustrate the difference in response, showing linear and non-linear behaviour respectively. c Occurrence of linear and non-linear mechanical response with different force-loading rates in WT chromosomes. d Stretch modulus 'S' was acquired from chromosomes showing linear behaviour at force-extension of 0.02 μm/s (WT n = 20 chromosomes, KD n = 37 chromosomes and OE n = 24 chromosomes). e Stiffening exponent γ from chromosomes showing non-linear behaviour at force-extension of 0.2 μm/s (WT n = 36 chromosomes, KD n = 29 chromosomes and OE n = 43 chromosomes) compared to γ values for the worm-like chain (WLC) and hierarchical worm-like chain (HWLC) models. Comparisons to WT (Kruskal–Wallis test), p = 0.001. d, e Box plots; Centre: Median, Box bounds: 25th to 75th percentile, Whiskers: minimum and maximum data points (excluding outliers). Data are provided in a Source Data file.

Fig. 2. Broadband microrheology of chromosomes.

a Schematic of microrheology experimental procedure. Dashed lines represent data at a force-loading rate of 0.2 μm/s and solid lines for 100 μm/s. Data in purple were analysed to provide broadband mechanical response. b Schematic representation of the tweezer and chromosome positions from (a) c Opposing forces experienced at bead handles (one shown) in the non-equilibrium state. d Zoomed-in sub-region of the analysed force at one bead and chromosome extension data. e Complex stiffness ${\kappa }^{*}\left(\omega \right)$ with frequency (bottom axis in black) and lag time $\tau$ (top axis in green) from broadband microrheology (BM) of WT chromosomes at 100 μm/s (median and 95% CI; n = 14 chromosomes), highlighting regions of viscous reorganisation and gel-like behaviour. Data in blue are the viscous modulus ${\kappa }^{″}\left(\omega \right)$ and in red are the elastic modulus ${\kappa }^{\prime }\left(\omega \right)$. f $\kappa *\left(\omega \right)$ at 0.2 μm/s force-loading rate (median and 95% CI; n = 15 chromosomes) of WT chromosomes. e and f are both overlaid with oscillatory microrheology (OM) data from Meijering et al. (2022)^,. Schematics shown are not to scale. Data are provided in a Source Data file.

Fig. 3. Chromosomes show distinct relaxation dynamics in the presence and absence of the MCP.

a Complex stiffness ${\kappa }^{*}\left(\omega \right)$ of KD chromosomes (median and 95% CI; n = 16 chromosomes) and b OE chromosomes (median and 95% CI; n = 15 chromosomes) compared to WT chromosomes in Fig. 2e. Data in blue are the viscous modulus ${\kappa }^{″}\left(\omega \right)$ and in red are the elastic modulus ${\kappa }^{\prime }\left(\omega \right)$. c $\tan \delta$ values for the three conditions (mean ± 95% CI). Blue line represents $\tan \delta$ value of 1 and values higher than this indicate viscosity is greater than elasticity. Inverted triangles highlight peak reorganisation times for WT and both KD and OE chromosomes. Dashed vertical lines with coloured regions show mean ± 95% CI of time at relaxation minimum. d Maximum value of $\tan \delta$ for individual chromosomes. Comparisons to WT (Kruskal–Wallis test): KD p = 0.001, OE p = 0.008. e $\tan \delta$ value at 10 ms for individual chromosomes. Comparisons to WT (Kruskal–Wallis test): KD p = 0.0002, OE p = 0.002. f $\tan \delta$ minimum after relaxation highlighted by the vertical dashed lines in c, comparisons to WT (Kruskal–Wallis test): KD p = 0.0004, OE p = 0.618. c–f WT n = 14 chromosomes, KD n = 16 chromosomes, OE n = 14 chromosomes. Box plots: Centre: Median, Box bounds: 25th to 75th percentile, Whiskers: minimum and maximum data points (excluding outliers). Significance values: *p < 0.05, **p < 0.001, ***p < 0.0001. Data are provided in a Source Data file.

Fig. 4. Chromosome mechanical behaviour explained using the Burgers model.

a Burgers model fits to average WT (n = 14 chromosomes), KD (n = 16 chromosomes) and OE data (n = 15 chromosomes). b Spring-dashpot schematic of the Kelvin representation of Burgers Model. c, d Comparisons of parameters extracted by fitting experimental data to the Burgers model individually ${\kappa }_1$ comparisons to WT (Kruskal–Wallis test): KD p = 0.57, OE p = 0.99; ${\eta }_1$ comparisons to WT (Kruskal–Wallis test): KD p = 0.23, OE p = 0.94; ${\kappa }_2$ comparisons to WT (Kruskal–Wallis test): KD p = 0.001, OE p = 0.015; ${\eta }_2$ comparisons to WT (Kruskal–Wallis test): KD p < 0.0001, OE p < 0.0001. e Ratios of fit parameters; ${\eta }_1/{\kappa }_1$ comparisons to WT (Kruskal–Wallis test): KD p = 0.001, OE p = 0.77; ${\eta }_2/{\kappa }_1$ comparisons to WT (Kruskal–Wallis test): KD p < 0.0001, OE p < 0.0001; relative $\kappa$ comparisons to WT (Kruskal–Wallis test): KD p = 0.0001, OE p = 0.022. For c–e, WT n = 14 chromosomes, KD n = 16 chromosomes, OE n = 15 chromosomes. Box plots: Centre: Median, Box bounds: 25th to 75th percentile, Whiskers: minimum and maximum data points (excluding outliers). Significance values: *p < 0.05, **p < 0.001, ***p < 0.0001. f Schematic of a chromosome to show gel-like properties are associated with the whole chromosome, while the MCP shows liquid-like dynamics. Data are provided in a Source Data file.