Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles

Abstract

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including 'high concentration' binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼3-8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

Figure 1:. Measured binodals for the RGG domain and LAF-1.

The latter is measured in the absence or presence of RNA using ultrafast-scanning FCS approach. a, A schematic illustration of the microscope with an acoustically modulated beam that is controlled by a tunable acoustic gradient index of refraction (TAG) lens. The system focus can be axially scanned along the optical-axis at a frequency of 70 kHz. b, Schematic showing a typical binodal with increasing protein concentration along the abscissa and increasing salt concentration along the ordinate. Our measurements show that the salt concentration decreases the strengths of two-body interactions for RGG domain/LAF-1 systems. c, The measured binodals of the RGG domain as well as LAF-1 in the presence and absence of RNA. Error bars represent the standard deviation (N=10).

Figure 2:. RNA and salt influence intermolecular interactions of LAF-1 and RGG.

a, Schematic illustration of mutual-diffusion. b, Mutual diffusion coefficient of LAF-1 strongly depends on the protein concentration. The dashed lines show the linear fits obtained using the equation shown for D in the text. The slope, which is proportional to B₂, decreases with increasing salt concentration. c, The second virial coefficients, B₂, approach the ideal solution limit of zero with increasing salt concentration. The B₂ values are most negative across the entire salt range for the RGG domain implying stronger effective pairwise interactions for the RGG domain when compared to LAF-1 in the absence or presence of RNA. Error bars represent the standard deviation (N=10).

Figure 3:. Summary of computational and theoretical analysis.

a, Results from atomistic simulations of the RGG domain. The plot shows a two-dimensional probability density map of chain dimensions in terms of radius of gyration, R_g and asphericities. RGG samples a broad range of conformations leading to large fluctuations and low values for the overlap volume fraction (Supplementary Fig. 7) b, Comparison of the binodals derived from numerical implementation of Muthukumar’s theory (open symbols) with experimental data (solid symbols). While the data help to identify the critical χ-region, the precise critical point cannot be reliably located because it is characterized by fluctuations that occur on all length scales. Therefore, the analysis was restricted to reproducing the low and high concentration arms of the binodals away from the critical point. The dashed lines are drawn to guide the eye. C,χ -dependent values of ξ are shown for each of the constructs and are calculated as described in Supplementary Information. The horizontal gray stripe corresponds to values of ξ obtained at 125 mM NaCl. The inset shows inferred values of construct-specific three-body interaction coefficients, w and the color-coding of the bars follows the format used in panels (b) and (c). Error bars represent the standard deviation (N=10).

Figure 4:. Nano-scale rheology of RGG and LAF-1 condensed droplets.

a, Increasing the concentration of NaCl decreases the viscosity within LAF-1 droplets. Adding short RNA (poly-rA30 and poly-rA15) also decreases the viscosity within LAF-1 droplets. However, adding long RNA (poly-rA3k) increases viscosity of LAF-1 droplets. b, Viscosity within droplets is proportional to the product of B₂c_D. Upon addition of the short RNA (poly-rA) the droplet viscosity decreases and follows the same universal curve with LAF-1 and RGG. Error bars represent the standard deviation (N=20).

Figure 5:. Low-density semidilute liquid droplets.

a, Apparent viscosities extracted from measurements of diffusion coefficients of dyes, mCherry, dextran, and polystyrene beads within LAF-1 droplets at 125 mM NaCl. The gray bar corresponds to ξ in Fig. 3c b, Permeability of different in vitro droplets to fluorescent dextran (red). 10 kDa dextran permeates droplets, while 70 kDa and 155 kDa dextran molecules are excluded from the droplets. The inset figure shows the bright-field image of NPM1 droplets. c, Permeability of in vivo LAF-1::GFP labeled P granules in C. elegans to fluorescent dextran. Perinuclear P granules in ~16-cell embryos are indicated with arrows. (Scale bar, 10 μm). d, Partition coefficients were calculated from fluorescent intensities inside/outside droplets. The dashed lines are drawn to guide the eye. The gray bar corresponds to ξ in Fig. 5a and 3c. e, Schematic illustrating the void-rich nature of LAF-1 droplets, and their probe size-dependent permeability. The RGG domain in LAF-1 is depicted in blue and envelopes defined by the R_g of LAF-1 are shown in black-dash circles. Error bars represent the standard deviation (N=10).