Mechanical Frustration of Phase Separation in the Cell Nucleus by Chromatin

Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets’ coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Figure 1

Droplet coarsening is suppressed in the cell nucleus. (a) Schematic of light-activated intracellular phase separation. Upon blue-light illumination, up to 24 intrinsically disordered protein regions (IDRs) bind to each 24-mer core. These subsequently phase separate due to multivalent IDR interactions. (b) Optogenetically generated fluorescent protein droplets (green) in the nucleus of a U2OS cell. Large droplets (labeled with numbers) are created by patterned local activation. Subsequently, many much smaller droplets are created by global activation. (The large droplets generated this way initially have higher IDR-to-core ratios than the small droplets due to a diffusive capture mechanism [13], and droplets change their sizes as this ratio equilibrates; we therefore focus on the time evolution of droplet sizes after this effect subsides.) (c) Time evolution of areas of large droplets in (b), starting at 60 min after global activation. (Full time-course trajectories are shown in Fig. S1 [14].)

Figure 2

Coarse-grained MD simulation of phase separation of droplet-forming particles in a cross-linked chromatin network. (a) Schematic: particles (red), chromatin (gray, beads and backbone), and cross-links (gray, dashed lines). (b) Time course of droplets (red particles) embedded in a chromatin network (gray) at cross-link density $7\mu \mathrm{M}$. Chromatin beads are shown at reduced size for better visualization. (c)–(e) Corresponding examples of time evolution of number of particles in individual droplets (colors), where (c) is for the simulation in (b). (f) Mean radius of droplets $⟨R⟩$, (g) mean radius cubed $⟨R{⟩}^3$, and (h) number of droplets as functions of time $t$ for simulations without chromatin (dotted), with chromatin un-cross-linked (dash dotted), and cross-linked at densities $7\mu \mathrm{M}$ (dashed) and $14\mu \mathrm{M}$ (solid).

Figure 3

Mechanical interactions with chromatin quantitatively control droplet number, size, and placement. (a) A shell of stretched chromatin backbones and cross-links (blue) surrounding a droplet (red) in case (iii). Highlighted bonds are stretched by more than 8% of their mean length prior to droplet formation. (b) Pressure $P$ on an inserted sphere of radius $R$ in cross-linked chromatin (red) and un-cross-linked chromatin (blue). Solid and dashed curves are fits to Eq. (6). (c) Comparison of pressure by chromatin on a sphere (radius $R=0.3\mu \mathrm{m}$) at the actual location of a single large droplet (red vertical line) to that on 95 spheres (histogram) randomly inserted in the same chromatin network, for three examples of case (iii). (d) Schematic of droplet number, size, and placement control by the variable local stiffness of cross-linked chromatin. Droplet sizes are determined by the position-dependent chemical potential ${\mu }_{\mathrm{den}}(R_i)$, which in turn depends on the local cross-link density. No droplet forms at location (0) as stretching the chromatin there is too energetically costly.