Biology subjects

Makasewicz, K.

Publications and source records attributed to Makasewicz, K..

4 recordsLinked to original sources

bioRxiv2026

Single-particle light scattering reveals the dynamic heterogeneity of biomolecular condensates

Biomolecular condensates can be heterogeneous in composition, morphology, and material properties, but resolving this heterogeneity at the level of individual submicrometer condensates remains challenging. Here, we combine off-axis holographic imaging with quantitative light-scattering analysis and particle tracking to simultaneously measure the size, optical properties, interfacial structure, and hydrodynamic mobility of hundreds of individual condensates per minute. Applied to condensates formed by the N-terminal domain of Ddx4 (Ddx4N1), the method reveals two populations with distinct scattering signatures: compact particles with well-defined interfaces and structurally heterogeneous, non-compact assemblies. Their relative abundance varies systematically with ionic strength. Synthetic polymer controls show that pronounced structural heterogeneity is not a generic consequence of phase separation and suggest a role for interaction heterogeneity in promoting competing mesoscale structures. Mass-size scaling and Brownian aggregation simulations further show that aggregation with incomplete fusion of smaller protein-rich units provides a physical model for the non-compact population. Simultaneous optical and diffusion measurements further show that the two populations have distinct hydrodynamic properties. These results demonstrate that chemically homogeneous condensate-forming systems can populate distinct mesoscale structures that are obscured by ensemble-averaged measurements.

biophysics↗

bioRxiv2025

Competition between protein-RNA clustering and phase separation drives re-entrant phase behavior of hnRNPA1

Phase separation of RNA-binding proteins plays crucial roles in the cell and is modulated by RNA, including promotion and suppression at low and high RNA concentrations, respectively. In complex coacervates, suppression of phase separation is rationalized by charge inversion when increasing the concentration of one component. Here, we show that suppression of biomolecular condensates of the RNA-binding protein hnRNPA1 at high RNA concentration is driven by a different mechanism, namely the competition with formation of nano-sized protein-RNA clusters in the dilute phase. We show that the competition is modulated not only by RNA concentration, but also by the type of RNA, with specific RNA being more effective in promoting cluster formation than unspecific RNA. We further show that protein-RNA clusters convert into amyloid fibrils over a longer time-scale compared to condensates, therefore providing higher kinetic stability. The competition between clustering and phase separation reported in this study provides a unifying framework to understand the distinct assemblies of hnRNPA1 in the nucleus and the cytoplasm, where the protein is exposed to different types and concentrations of RNA.

biophysics↗

bioRxiv2025

On the transient interactions of α-synuclein in different dimensions

0.-Synuclein (Syn) is a neuronal protein predominantly found in the brain, whose native function seems to be associated with vesicle trafficking. While intrinsically disordered in solution, the first ca. 100 residues adopt an amphipathic -helical structure when the protein adsorbs onto membranes. Additionally, the aggregation of Syn into highly ordered {beta}-sheet rich amyloid fibrils is associated with Parkinso[n]s disease. The different regions of Syn and the interactions between them have been reported to play a key role in the behaviour of the protein in solution, its membrane binding, and its aggregation into fibrils. This study employs photo-induced cross-linking of unmodified proteins (PICUP) to capture and identify the transient contacts of Syn in different conformational states: free in solution, adsorbed to membranes, and aggregated into fibrils. By using tyrosine-to-phenylalanine mutations to block the reactivity of specific amino acid residues, we establish key cross-links in each state. In solution, we identify internal contacts between the N and C termini of monomers, as well as inter-monomer contacts between C termini in oligomers. When Syn is adsorbed to membranes, the internal cross-linking is blocked, while cross-linking between C-terminal regions persists. In fibrils, cross-linking is significantly reduced, primarily occurring between C-terminal residues of adjacent monomers. This work highlights the utility of PICUP for reporting on the transient contacts that occur on the pathways of self- and co-assembly of Syn.

biochemistry↗

bioRxiv2024

Tipping point in α-synuclein-membrane interactions: stable protein-covered vesicles or amyloid aggregation

-synuclein is a neuronal protein implicated in neurotransmitter release. Its function is thought to critically depend on the dynamic equilibrium between free and membrane-bound protein. -synuclein amyloid formation implicated in Parkinsons Disease was also shown to be modulated by lipid membranes. However, it remains elusive whether -synuclein-related pathology is due to loss-of-function or gain-of-toxic-function. To help address this question, we studied the coupling of the equilibrium between free and membrane-bound -synuclein and membrane-induced amyloid formation - phenomena that are usually treated separately. We present a description of the system on a wide range of length scales and timescales for lipid-to-protein ratio conditions where amyloid formation is either accelerated or inhibited by lipid membranes. We find a clear difference between the dynamics and heterogeneity of the protein-covered membrane interface in the two sets of conditions. In aggregation-accelerating conditions, the membrane interface is dynamic and heterogeneous with rapid exchange between free and membrane-bound protein, and disordered protein segments of varying lengths exposed to solution. All these characteristics of the membrane interface are likely to decrease the free energy barrier for amyloid formation. Conversely, the membrane interface is homogeneous and less dynamic in conditions where amyloid formation is inhibited. Importantly, any factors affecting the equilibrium between free and membrane-bound -synuclein may trigger a change from non-aggregating to aggregating conditions. Altogether, our results highlight a strong coupling of the dynamic equilibrium between the free and membrane-bound -synuclein and membrane-modulated amyloid formation and thus of the physiological function of -synuclein and its aberrant aggregation.

biochemistry↗