Trowbridge, J. W. | bioRxiv Science

bioRxiv2026

Lipid-Coated Water-in-Oil Droplets as a Passivation-Free Platform for Cost-Effective Fluorescence Spectroscopy

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@199c622org.highwire.dtl.DTLVardef@9f195eorg.highwire.dtl.DTLVardef@13a8969org.highwire.dtl.DTLVardef@138692_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

biochemistry↗

bioRxiv2025

Small heat shock proteins HspB1 and HspB5 differentially alter the condensation and aggregation of the TDP-43 low complexity domain

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that regulates processes of mRNA metabolism, during which it undergoes condensation mediated by its C-terminal low complexity domain (TDP-43LCD). TDP-43 aggregation and condensation are associated with neurodegenerative disease. However, the proteostasis mechanisms that regulate these processes remain elusive. Some evidence has shown that the molecular chaperone small heat shock protein HspB1 binds to and regulates the cytoplasmic phase separation of TDP-43, indicating that other small heat shock proteins may have similar effects. Here, we demonstrate divergent behaviours for HspB1 and its homolog HspB5 on TDP-43LCD condensation and aggregation. In addition to inhibiting TDP-43LCD aggregation, HspB1 partitions into TDP-43LCD condensates and increases the dynamic exchange of TDP-43LCD within condensates and with the surrounding solution. These effects of HspB1 are enhanced by mutations that mimic phosphorylation. HspB5 inhibits TDP-43LCD aggregation more effectively than HspB1 and partitions into TDP-43LCD condensates, where it delays pathological transition of the condensate to a gel/solid. We localise the chaperone effects of HspB1 and HspB5 to the N- and C-terminal regions of the protein, emphasising the role of sequence diversity in these regions in defining small heat shock protein function. These findings demonstrate that HspB1 and HspB5 are regulators of TDP-43 phase separation and aggregation and may be potential therapeutic targets in mitigating toxic TDP-43 aggregation in neurodegenerative disease. StatementThis work describes how two small heat shock proteins (proteins that bind to misfolded proteins) impact the condensation and aggregation of TDP-43, a protein implicated in most cases of amyotrophic lateral sclerosis. In doing so, it highlights their divergent behaviours and therapeutic potential.

biochemistry↗