Amyloid Formation in Nanoliter Droplets

Abstract

Processes that monitor the nucleation of amyloids and characterize the formation of amyloid fibrils are vital to medicine and pharmacology. In this study, we observe the nucleation and formation of lysozyme amyloid fibrils using a facile microfluidic system to generate nanoliter droplets that can control the flow rate and movement of monomer-in-oil emulsion droplets in a T-junction microchannel. Using a fluorescence assay, we monitor the nucleation and growth process of amyloids based on the volume of droplets. Using the microfluidic system, we demonstrate that the lag phase, which is vital to amyloid nucleation and growth, is reduced at a lower droplet volume. Furthermore, we report a peculiar phenomenon of high amyloid formation at the edge of a bullet-shaped droplet, which is likely due to the high local monomer concentration. Moreover, we discovered that amyloid fibrils synthesized in the nanoliter droplets are shorter and thicker than fibrils synthesized from a bulk solution via the conventional heating method. Herein, a facile procedure to observe and characterize the nucleation and growth of amyloid fibrils using nanoliter droplets is presented, which is beneficial for investigating new features of amyloid fibril formation as an unconventional synthetic method for amyloid fibrils.

Atomic force microscopy of spherical intermediates on the pathway to fibril formation of influenza A virus nuclear export protein

“…Moreover, at present, there are some considerations that spherical nanoparticles of various sizes and morphologies are “obligate” intermediates on the pathway to amyloid formation (Ke et al, 2020). And, apparently, it is the morphological diversity of these nanospheres that largely determines the polymorphism of fibrils (Cheong et al, 2022; Zielinski et al, 2021).…”

“…However, structured aggregates exhibit a wide variety of morphologically different shapes: rigid rods, flexible (worm‐like) “beads on a string” fibrils, bundles of fibrils twisted around each other, and so forth (Iadanza et al, 2018; Tycko, 2014; Wei et al, 2017). Many studies have revealed the presence of an intermediate step on the pathway of protein molecules to amyloids under certain conditions (in particular, at a high protein concentration), i.e., self‐assembly of monomers into morphologically different spherical particles with dimensions ranging from ~20 nm to ~1 μm (Breydo & Uversky, 2015; Cheong et al, 2022; Harper et al, 1997; Lomakin et al, 1996; Moore et al, 2007; Shin & Brangwynne, 2017; Uversky, 2010). These colloidal nanoparticles (sometimes referred to as micelles (Lomakin et al, 1996; Redwan et al, 2015), amylospheroids (Matsumura et al, 2011), spheroidal or spherical oligomers (Drombosky et al, 2018; Ruggeri et al, 2015), coacervates (Abbas et al, 2021; Jang et al, 2019), droplets (Abbas et al, 2021; Shin & Brangwynne, 2017), condensates (Banani et al, 2017), “acunas” (Fauerbach et al, 2012), etc.)…”

“…These colloidal nanoparticles (sometimes referred to as micelles (Lomakin et al, 1996; Redwan et al, 2015), amylospheroids (Matsumura et al, 2011), spheroidal or spherical oligomers (Drombosky et al, 2018; Ruggeri et al, 2015), coacervates (Abbas et al, 2021; Jang et al, 2019), droplets (Abbas et al, 2021; Shin & Brangwynne, 2017), condensates (Banani et al, 2017), “acunas” (Fauerbach et al, 2012), etc.) are capable to transform into fibrils of various morphology (Babinchak & Surewicz, 2020; Cheong et al, 2022; Ke et al, 2020). This phenomenon has been mostly demonstrated for eukaryotic amyloidogenic proteins, that is, α‐synuclein (Ray et al, 2020), β‐amyloid (A‐beta) (Matsumura et al, 2011; Wang, Eom, & Kwon, 2021a), prions PrP (Dalal et al, 2015), tau (Barracchia et al, 2022; Wegmann et al, 2018), huntingtin exon1 (Drombosky et al, 2018; Peskett et al, 2018), caseins (Bahraminejad et al, 2022; Ecroyd et al, 2008), yeast prions (Vitrenko et al, 2007), some RNA‐binding proteins, like hnRNPA1 (Molliex et al, 2015), FUS (Berkeley et al, 2021; Murakami et al, 2015), TDP‐43 (Babinchak & Surewicz, 2020; Sun et al, 2019), and so forth.…”

“…These colloidal nanoparticles (sometimes referred to as micelles (Lomakin et al, 1996;Redwan et al, 2015), amylospheroids (Matsumura et al, 2011), spheroidal or spherical oligomers (Drombosky et al, 2018;Ruggeri et al, 2015), coacervates (Abbas et al, 2021;Jang et al, 2019), droplets (Abbas et al, 2021;Shin & Brangwynne, 2017), condensates (Banani et al, 2017), "acunas" (Fauerbach et al, 2012), etc.) are capable to transform into fibrils of various morphology (Babinchak & Surewicz, 2020;Cheong et al, 2022;Ke et al, 2020).…”