The aggregation of the amyloid β (Aβ) peptide is one of the molecular hallmarks of Alzheimer’s disease (AD). Although Aβ deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aβ is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aβ (Aβ42). Intracellularly, Aβ42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aβ42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aβ.

Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of alpha-synuclein (alphaS) protein aggregates in the nervous system. While alphaS binding to membranes in its monomeric state is correlated to its physiological role, alphaS oligomerisation 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 alphaS (OalphaS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OalphaS-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 OalphaS-membrane interactions. We find that OalphaS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OalphaS and monomeric alphaS (MalphaS), we further demonstrate that OalphaS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OalphaS compete with and displace bound MalphaS from the membrane surface, suggesting that disruption to the functional binding of MalphaS to membranes may provide an additional toxicity mechanism in PD. These findings present a unique binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.

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Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

The aggregation of the amyloid β peptide (Aβ) is one of the major molecular hallmarks of Alzheimer′s disease. Although Aβ deposits have been mostly observed extracellularly, various studies have reported the presence of also intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of Alzheimer′s disease, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein (APP), from which Aβ is derived by proteolytic cleavage. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of the 42-residue form of Aβ (Aβ42). By using small unilamellar vesicles modelling cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the endoplasmic reticulum (ER), and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation, and suggest the presence of an evolutionary optimization of lipid composition to prevent the intracellular aggregation of Aβ.