One-Step Generation of Multisomes from Lipid-Stabilized Double Emulsions

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α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

Publication status: PublishedParkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions.

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions.

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches

Direct digital sensing of protein biomarkers in solution.

Funder: Please see main manuscript file.The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches