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, 12 (45), 9142-9150

Biophysical Characterization of Organelle-Based RNA/protein Liquid Phases Using Microfluidics

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Biophysical Characterization of Organelle-Based RNA/protein Liquid Phases Using Microfluidics

Nicole Taylor et al. Soft Matter.

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.

Figures

figure 1
figure 1
bottom-up reconstitution approach to study membrane-less organelles. (a) p granules (yellow) within the c. elegans embryo, (adapted from elbaum-garfinkle et al). nucleoli (green) within the c. elegans hermaphrodite gonad with cell membranes in red, (adapted from weber et al). whi3 assemblies (white circle) in the hypha of the multinucleate fungus ashbya gossypii, (adapted from lee et al). (b) in vitro droplets of fluorescently labeled laf-1 (125 mm nacl, ~3.5 µm laf-1), gar-1δn (150 mm nacl, ~10 µm gar-1δn), and whi3 proteins (150 mm kcl, 9 µm whi3, 50 nm bni1).
figure 2
figure 2
microfluidic-assisted coalescence of protein droplets (e.g., laf-1) into a single protein stream used for microrheology. (a) protein droplets (green) stick to pdms posts and coalesce into a protein-rich stream. right panel shows time-lapse of coalescence; scale bar = 20 µm. (b) brightfield and fluorescent images of a large protein-rich phase in the box microfluidic device. (inset) zoomed in fluorescent image of red tracer beads embedded in the protein-rich phase. (c) msd versus lag time for laf-1 at high salt (250 mm nacl, dark blue dashed line), whi3 at physiological salt in the presence of 53 nm bni1 mrna (150 mm kcl, pink dash-dotted line), laf-1 at low salt (125 mm nacl, cyan solid line), gar-1δn at low salt (150 mm nacl, blue dotted line), and the noise floor (black dashed line). black solid line has a slope of 1; the noise floor is ~ 2 × 10−5 µm2.
figure 3
figure 3
setup of protein stream for flow analysis. (a) protein solution at the phase boundary (e.g., x in inset) and peg-passivated probe particles flow along the main channel from left to right, and protein droplets (e.g., o in inset) and peg-passivated or -cooh probe particles are supplied via the orthogonal inlet channel; arrows indicate the direction of flow. area indicated by red dashed boxes is shown in panels b and c. (b) the position and velocity of the protein stream is manipulated using the main and orthogonal channels. (c) the protein-rich stream fills the entire height of the microfluidic device with the protein-lean phase filling the remaining width of the channel. yellow dashed and solid lines outline the channel walls.
figure 4
figure 4
red peg-passivated probe particles are used to quantify velocity profiles in the protein-rich (green) and protein-lean (black) phase. (a) velocity profiles are quantified far from the t-junction (i.e., red dashed box) where the protein-rich/protein-lean interface is mostly flat. (b) to quantify velocity profiles in the protein-lean phase, the length of probe particle streaks and corresponding exposure times are used. velocity profiles in the protein-rich phase are determined by microparticle tracking velocimetry. the red-dashed line denotes the protein-rich/protein-lean interface and particle streaks and single particles are seen in the protein-lean and protein-rich phases, respectively. (c) measured velocity profiles in the protein-lean (filled black circles) and protein-rich (filled blue circles) phase. (d) zoomed in view of protein-rich (filled blue circles) phase. lines are drawn using Eqs. (1) and (2) with β = −0.15 pa/m and μ1= 26.8 pa.s.
figure 5
figure 5
(a) model symbols and geometry. measured velocity profiles in the (b) protein-lean phase and (c) protein-rich phase at high (black asterisks) and low (blue diamonds) shear rates. cyan and magenta lines are drawn using Eqs. (1) and (2) with β = −0.12 pa/m and μ1 = 65 pa.s and β = −0.06 pa/m and μ1 = 63 pa.s, respectively. (d) protein-rich phase viscosity, μ1, for each protein investigated measured using microrheology (yellow bars) and two-phase flow analysis (blue bars). typical error bars are shown and represent standard deviation of at least three replicates.

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