Initiation of hnRNPA1 Low-Complexity Domain Condensation Monitored by Dynamic Light Scattering

Abstract

Biomolecular condensates (BMCs) exhibit physiological and pathological relevance in biological systems. Both liquid and solid condensates play significant roles in the spatiotemporal regulation and organization of macromolecules and their biological activities. Some pathological solid condensates, such as Lewy Bodies and other fibrillar aggregates, have been hypothesized to originate from liquid condensates. With the prevalence of BMCs having functional and dysfunctional roles, it is imperative to understand the mechanism of biomolecular condensate formation and initiation. Using the low-complexity domain (LCD) of heterogenous ribonuclear protein A1 (hnRNPA1) as our model, we monitored initial assembly events using dynamic light scattering (DLS) while modulating pH and salt conditions to perturb macromolecule and condensate properties. We observed the formation of nanometer-sized BMCs (nano-condensates) distinct from protein monomers and micron-sized condensates. We also observed that conditions that solubilize micron-sized protein condensates do not solubilize nano-condensates, indicating that the balance of forces that stabilize nano-condensates and micron-sized condensates are distinct. These findings provide insight into the forces that drive protein phase separation and potential nucleation structures of macromolecular condensation.

Keywords: biomolecular condensates; hnRNPA1; nano-clusters; nano-condensates; neurodegenerative diseases; protein phase separation.

Figure 1

hnRNPA1^LCD monomer, nano-condensate, and micro-condensate populations. (A) hnRNPA1 domain organization. hnRNPA1 consists of two RNA-binding domains (RBDs) and a C-terminal prion-like low-complexity domain (LCD). Adapted from Tsoi et al. [19]. Representative conditions where major hnRNPA1^LCD populations are primarily monomeric (B), 20 μM hnRNPA1^LCD, water), primarily nano-condensates (C), 20 μM hnRNPA1^LCD, 0 mM NaCl, pH 7, αβγ buffer), or a coexistence of nano-condensates and micro-condensates (D), and 20 μM hnRNPA1^LCD, 200 mM NaCl, pH 7, αβγ buffer) was observed using DLS and optical microscopy. Scale bar = 10 μm.

Figure 2

Micro-condensate and nano-condensate electrostatic modulation. (A) hnRNPA1^LCD monomer, nano-condensate, and micro-condensates observed for 20 μM and 5 μM protein in 0–500 μM NaCl, αβγ buffer, pH 7, (B) pH 6, (C) pH 5, (D) pH 4, and (E) pH 8. Circles refer to 20 μM hnRNPA1^LCD and squares refer to 5 μM hnRNPA1^LCD. * Filled circles refer to the presence of micro-condensates.

Figure 3

Hexanediol solubilized micro-condensates and decreased nano-condensate size. (A) hnRNPA1^LCD monomer and nano-condensate formation for 20 μM protein in 0–500 mM NaCl, pH 4–8, αβγ buffer. (B) hnRNPA1^LCD monomer and nano-condensate formation observed for 20 μM protein in 0–500 mM NaCl, pH 4–8, αβγ buffer, in the presence of 1% 1,6-hexanediol. (C) hnRNPA1^LCD monomer and nano-condensate formation observed for 5 μM protein in 0–500 mM NaCl, pH 4–8, αβγ buffer. (D) hnRNPA1^LCD monomer and nano-condensate formation observed for 5 μM protein in 0–500 mM NaCl, pH 4–8, αβγ buffer, in the presence of 1% 1,6-hexanediol. Grayed-over open circles refer to conditions wherein only monomers were detected.

Figure 4

Model for hnRNPA1^LCD condensation nucleation. Observed nanometer-sized condensates (nano-condensates) were hypothesized as nuclei and initiation sites for biomolecular condensation. These nano-condensates exhibited slower, more linear growth characteristics as a function of monomer protein concentration, as compared to micron-sized condensates (micro-condensates). Phase separation was enhanced by increasing protein and salt concentrations, as well as pH changes approaching optimal protein charge (approximately neutral pH, for hnRNPA1^LCD in the solution conditions used). 1,6-hexanediol partially reversed the phase separation process, transforming micro-condensates to more stable nano-condensates.