Biophysical characterization of organelle-based RNA/protein liquid phases using microfluidics

Abstract

Living cells contain numerous membrane-less RNA/protein (RNP) bodies that assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function within living cells, and also through the link to protein aggregation pathologies. However, techniques such as droplet coalescence analysis or standard microrheology do not always enable robust property measurements of model RNA/protein droplets in vitro. Here, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using passive microrheology and/or active two-phase flow analysis. We use this technique to study various phase separating proteins from structures including P granules, nucleoli, and Whi3 droplets. In each case, droplets exhibit simple liquid behavior, with shear rate-independent viscosities, over observed timescales. Interestingly, we find that a reported order of magnitude difference between the timescale of Whi3 and LAF-1 droplet coalescence is driven by large differences in surface tension rather than viscosity, with implications for droplet assembly and function. The ability to simultaneously perform active and passive microrheological measurements enables studying the impact of ATP-dependent biological activity on RNP droplets, which is a key area for future research.

Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates

“…There are many considerations for microrheology including the material of and size of the bead, the passivation of the bead surface, the need for a stable microscope set-up to minimize drift, and a means of adding beads to droplets and ensuring that analyzed beads are in the central regions of droplets so as to avoid boundary-effect behavior. A microfluidics-based approach has been implemented that aids in incorporation of beads and avoiding boundary effects by forming only two distinct liquid phases under flow (by fusing all droplets), and this helps avoid some artifacts commonly encountered and promotes incorporation of tracer beads into the dense phase (Taylor et al, 2016). Surveying the behavior of a variety of sizes of beads can help determine whether, when, and where the material may show elastic properties.…”

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics

“…These and related RNA-binding proteins are components of membrane-less organelles found in the nucleus (e.g., nuclear speckles and nucleoli) and cytoplasm (e.g., processing bodies and stress granules, SGs) of neurons and other cell types (Brangwynne et al, 2011; Collier et al, 1988; Huang and Spector, 1992; Sheth and Parker, 2003; Taylor et al, 2016b). It has recently emerged that biophysical properties encoded in prion-like, low complexity sequence domains (LCDs) of RNA-binding proteins promote the assembly of membrane-less organelles through the process of liquid-liquid phase separation (LLPS) (Kato et al, 2012; Lin et al, 2015; Molliex et al, 2015; Patel et al, 2015).…”

Clinical and neuropathological features of ALS/FTD with TIA1 mutations

“…Similar to several other ALS/FTD related proteins (e.g. transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma (FUS) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1)), TIA1 is an RNA binding protein that contains a C-terminal, prion-like, low complexity domain (LCD) which promotes its self-assembly and the formation of membrane-less organelles through the process of liquid-liquid phase separation (LLPS) [ 16 , 22 , 31 ]. Specifically, TIA1 plays a central role in the formation of stress granules (SG) that form in response to environmental stress to temporarily store and protect mRNA [ 1 , 9 , 14 , 25 ].…”