Early Stages of FUS Droplet Formation via Liquid-Liquid Phase Separation.

The phase behavior of complex biomolecular solutions may explain different cellular processes, including the organization of cells by membraneless organelles. The early stages of phase separation are crucial to understanding the underlying mechanism and identifying biomolecules that trigger or drive the transition. Here, we analyze the early events of liquid-liquid phase separation (LLPS) of FUS by multiangle time-resolved static and dynamic light scattering. LLPS was triggered by TEV-catalyzed cleavage of the MBP-tag from FUS-MBP. The light scattering measurements revealed the existence of at least two fractions of FUS-MBP aggregates already prior to the onset of LLPS. The orders of magnitude of the aggregate size in these two fractions are 10 and 100 nm, respectively. LLPS started after an induction period, which depended on the concentration of FUS-MBP. The data from time-dependent light scattering revealed a coalescence of droplets also denoted as a step growth process. A step growth process instead of nucleation and growth via monomer addition suggests that LLPS takes place within the spinodal rather than between the binodal and the spinodal.

Ethylene glycol energetically disfavours oligomerization of pseudoisocyanine dyestuffs at crowded concentrations.

The intriguing role of the intracellular crowded environment in regulating protein aggregation remains elusive. The convolution of several factors such as the protein sequence-dependence, crowder's shape and size and diverse intermolecular interactions makes it complex to identify systematic trends. One of the ways to simplify the problem is to study a synthetic model for self-assembling proteins. In this study, we examine the aggregation behaviour of the cationic pseudoisocyanine chloride (PIC) dyestuff which is known to self-assemble and form fibril-like J-aggregates in aqueous solutions, similar to those formed by amyloid-forming proteins. Prior experimental studies have shown that polyethylene glycol impedes and Ficoll-400 promotes the self-assembly of PIC dyes. To achieve molecular insights, we examine the effect of crowding by ethylene glycol on the solvation thermodynamics of oligomerization of dyes into H-type and J-type oligomers using extensive molecular dynamics simulations. The binding free energy calculations show that the formation of J-oligomers is more favourable than that of H-oligomers in water. The stability of H- and J- tetramers and pentamers decreases in crowded solutions. The formation of oligomers is supported by the favourable change in dye-solvent interaction energy in both pure water and aqueous ethylene glycol solution although it is opposed by the reduced dye-solvent entropy. Ethylene glycol, as a molecular crowder, disfavours the H- as well as J-oligomerization via preferential binding to the dye oligomers. An unfavourable change in dye-crowder and dye-dye interaction energy on dye association makes the H-oligomer formation less favourable in crowded solution than in pure water solution. In the case of J-oligomers, however, the unfavourable change in dye-crowder interaction energy primarily contributes to making total dye-solvent energy unfavourable. The results are supported by isothermal titration calorimetry measurements where the binding of ethylene glycol to PIC molecules is found to be endothermic. The results provide an emerging view that a crowded environment can disfavour self-assembly of PIC dyes by interactions with the oligomeric states. The findings have implications in understanding the role of a crowded environment in shaping the free energy landscapes of proteins.

Sequestration of Proteins in Stress Granules Relies on the In-Cell but Not the In Vitro Folding Stability.

Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

Self-Assembly of Pseudo-Isocyanine Chloride as a Sensor for Macromolecular Crowding In Vitro and In Vivo.

Pseudo-isocyanine chloride (PIC) is a cationic dyestuff that exhibits self-assembly in aqueous solution, promoted either by increasing the PIC concentration or by decreasing the temperature. PIC-aggregates exhibit a characteristic and sharp absorption band as well as a fluorescence band at a wavelength of 573 nm making PIC an interesting candidate to analyze the self-assembly process in various environments. The present work developed PIC-based, synthetic model systems, suitable to investigate how macromolecular crowding influences self-assembly processes. Four synthetic additives were used as potential crowders: Triethylene glycol (TEG), polyethylene glycol (PEG), Ficoll 400 as a highly branched polysaccharide, and sucrose corresponding to the monomeric unit of Ficoll. Combined UV/Vis spectroscopy and time-resolved light scattering revealed a strong impact of crowding based on excluded volume effects only for Ficoll 400. Sucrose had hardly any influence on the self-assembly of PIC and PEG and TEG impeded the PIC self-assembly. Development of such a PIC based model system led over to in-cell experiments. HeLa cells were infiltrated with PIC solutions well below the aggregation threshold in the infiltrating solution. In the cellular environment, PIC was exposed to a significant crowding and immediately started to aggregate. As was demonstrated by fluorescence imaging, the extent of aggregation can be modulated by exposing the cells to salt-induced osmotic stress. The results suggest future use of such a system as a sensor for the analysis of in vitro and in vivo crowding effects on self-assembly processes.

Cell stress and phase separation stabilize the monomeric state of pseudoisocyanine chloride employed as a self-assembly crowding sensor.

Cellular stress and ageing involve an increase in crowding and aggregation of amylogenic proteins. We here investigate if crowding is the intrinsic cause of aggregation and utilise a previously established non-protein aggregation sensor, namely pseudoisocyanine chloride (PIC). PIC shows fibrillization in cells into a highly fluorescent J-aggregated state and is sensitive to crowding. Surprisingly, cell stress conditions stabilise the monomeric rather than the aggregated state of PIC both in the cytoplasm and in stress granules. Regarding the different physiochemical changes of the cytoplasm occurring upon cell stress, involving volume reduction, phase separation and solidification, the intrinsic crowding effect is not the key factor to drive associated self-assembly processes.

A Comparative Study on Cyanine Dyestuffs as Sensor Candidates for Macromolecular Crowding In Vitro and In Vivo.

Pseudo isocyanine chloride (PIC) has been identified in a preceding work as a sensor suited to probe macromolecular crowding both in test tubes with solutions of synthetic crowding agents and in HeLa cells as a representative of living systems. The sensing is based on a delicate response of the self-assembly pattern of PIC towards a variation in macromolecular crowding. Based on a suitable selection of criteria established in the present study, four additional cyanine dyestuffs (TDBC, S071, S2275, and PCYN) were scrutinized for their ability to act as such a sensor, and the results were compared with the corresponding performance of PIC. UV-VIS and fluorescence spectroscopy were applied to investigate the photo-physical properties of the four candidates and, if possible, light scattering was used to characterize the self-assembly of the dyestuffs in solution. Finally, HeLa cells were exposed to solutions of the most promising candidates in order to analyze their ability to infiltrate the cells and to self-assemble therein. None of the dyestuff candidates turned out to be as similarly promising in probing crowding effects in cells as PIC turned out to be. S0271 and S2275 are at least stable enough and meet the photophysical requirements necessary to act as sensors responding to changes in macromolecular crowding.

Spatial Distribution of Intracellular Ion Concentrations in Aggregate-Forming HeLa Cells Analyzed by µ-XRF Imaging.

Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal-ion homeostasis is associated with disease progression. Here we utilize µ-X-ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon-1-mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10-fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate-containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon-1 amyloid formation.