Actin polymerization counteracts prewetting of N-WASP on supported lipid bilayers

Abstract

Cortical condensates, transient punctate-like structures rich in actin and the actin nucleation pathway member Neural Wiskott-Aldrich syndrome protein (N-WASP), form during activation of the actin cortex in the Caenorhabditis elegans oocyte. Their emergence and spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, the mechanisms that drive the onset of cortical condensate formation near membranes remain unexplored. Here, using a reconstituted phase separation assay of cortical condensate proteins, we demonstrate that the key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via a prewetting transition. Actin partitions into the condensates, where it polymerizes and counteracts the N-WASP prewetting transition. Taken together, the dynamics of condensate-assisted cortex formation appear to be controlled by a balance between surface-assisted condensate formation and polymer-driven condensate dissolution. This opens perspectives for understanding how the formation of complex intracellular structures is affected and controlled by phase separation.

Keywords: cortical condensates; in vitro actin cortices; prewetting.

Fig. 1.

N-WASP undergoes phase separation without adapter proteins in vitro. (A) Alpha fold predicts large disordered regions for C. elegans N-WASP (WSP-1) (sequence-dependent color code). Sodium dodecyl sulfate gel shows recombinantly expressed and purified MBP-His₆-WSP-1 (for activity controls and results of human N-WASP see SI Appendix). (B) Confocal time-lapse of His₆-WSP-1 (5 $\mathrm{\mu }$M, 10% 488-tagged, MBP-tag cleaved right before experiment; see SI Appendix) forming droplets in bulk upon lowering KCl concentration to 150 mM. Kymograph indicates growth of droplets and fusion (white arrows). (C) Fluorescence recovery after photobleaching (FRAP) images of condensate formed from 5 $\mathrm{\mu }$M His₆-WSP-1 between 5 to 60 min. Diffusion coefficients D inside the condensates were determined for n $=$ 14 condensates. Data are the mean $\pm$ SD. (D) Phase separation assays of His₆-WSP-1 reveal a critical concentration for phase separation of 500 nM in bulk (Upper row; see SI Appendix, Fig. S1E) and 50 nM on a supported lipid bilayer (SLB) with 1% Ni-NTA lipids (Lower row). (All scale bars: 10 $\mathrm{\mu }$m.)

Fig. 2.

N-WASP prewets on supported lipid bilayers. (A) Protein interactions with membranes depend on their bulk concentration. Three protein phases exist: a dissolved phase in the bulk, an adsorbed phase on the SLB, and a condensed phase on the SLB. At low concentrations, proteins adsorb as a thin layer (I, Left). Crossing the critical concentration for prewetting (c_pw) condensed areas form (II, Middle). Above the saturation concentration (c_sat) for liquid–liquid phase separation (III, LLPS) droplets form spontaneously in bulk (Right). (B) Phase diagram depicting these three regions of a binary fluid in the presence of a surface. Prewetting line discriminates between areas of adsorption and surface condensation (prewetting). (C) Confocal images of human His₆-N-WASP (10% AF488 labeled, 100 nM Upper row, 250 nM Middle, and 500 nM Lower) condensates on SLBs after $t$ $=$ 10 min. (Scale bar: 10 $\mathrm{\mu }$m.) Kymographs show adsorption and condensation phase over 10 min (Movie S5). Histogram of the logarithm of pixel intensities (Right) shows enrichment of the condensed phase over time. Two distinct time points marked with light gray and dark gray arrows are chosen respectively for each time series. (D) Condensate number density growth rate (Upper) and condensed phase area (Lower, solid lines) and intensity (Lower, dashed lines) fractions quantified as a function of time from (C). Red filled circles show the time when maximum condensation rate is reached. (E) Left: Confocal images at the time point (red filled circles) identified in (D). Middle: Probability density kymographs of the logarithm of pixel intensities for time-lapse images from (C). Gray lines (single Gaussian) and blue lines (sum-of-two-Gaussians) represent overlays of probability density fits from either method. The time point of high- and low-intensity-branch splitting (red open circles) is determined by comparing fitting residues between the two methods. Right: Histograms and fits (gray dashed lines) for the 3 different N-WASP bulk concentrations each at 2 distinct time points (arrows). In the case of a sum-of-two-Gaussians fit, blue dashed lines show respectively the fitted lower and higher Gaussian peaks. (F) Upper: The time of abrupt switch inversely scales with initial bulk concentrations. Error bars represent the range extracted from 10% variation of the threshold used for determining $t_{\mathit{pw}}$. For segmented images, the threshold is the growth rate of condensate number density; For splits of probability density branches, the threshold is the residue difference between single- and sum-of-two Gaussian fits (SI Appendix). Lower: The curated (SI Appendix) mean pixel intensity at the time of abrupt switch is comparable across different initial bulk concentrations.

Fig. 3.

Actin controls equilibrium size of N-WASP condensates in vitro. (A) Left: Maximum intensity projections of confocal z-stacks of His₆-WSP-1 (5 $\mathrm{\mu }$M, 10% AF488-labeled) condensates formed over 1 h in bulk (Upper row; see Movie S3) and on a SLB (Lower row; see Movie S4) in actin polymerization buffer alone (Left images), and in the presence of actin (10% AF647 labeled, magenta) and Arp2/3. Right: Quantification of N-WASP droplet radii from max projections. (B) Left: Maximum intensity projections of confocal z-stacks of His₆-WSP-1 (5 $\mathrm{\mu }$M, 10% AF488-labeled) condensates formed over 1 h in bulk alone, or in the presence of actin (3 $\mathrm{\mu }$M, 10% AF647 labeled, magenta), actin and Arp2/3 (100 nM), and in the presence of latrunculin A (50 $\mathrm{\mu }$M). For conditions without ATP (Right images), the nucleotide on actin was exchanged with AMP-PNP. Arrows point to example N-WASP bulk condensates. Right: Quantification of bulk condensate radii from maximum intensity projections. Note that lanes 1 and 4 include the data shown in panel (A), Top row. (All scale bars: 10 $\mathrm{\mu }$M.)

Fig. 4.

Actin polymerization counteracts prewetting. (A) Confocal images of binary mixture of His₆-WSP-1 and His₆-N-WASP (500 nM, 10% AF488 labeled, green) alone, and (B) in presence of actin (1 $\mathrm{\mu }$M, 10% AF647 labeled, magenta) and Arp2/3 (10 nM) on SLBs after 30 min incubation. Kymographs along dotted line from 0 to 10 min (Movies S6 and S7 and SI Appendix, Fig. S15). (C) Left: Probability density kymographs of logarithm of N-WASP/WSP-1 pixel intensities for time-lapse images from (A). Gray lines (single Gaussian) and blue lines (sum-of-two-Gaussians) represent overlays from either Gaussian-fitting method, determined for each time point by comparing the fitting residues between the two methods. Right: Histograms at 3 distinct time points (marked with triangle, square, and pentagon) with either a single Gaussian or a sum-of-two-Gaussians fit (gray dashed lines). Blue dashed lines show respectively the fitted lower and higher Gaussian peaks in the case of a sum-of-two-Gaussians fits. (D) Same as in (C), Probability density kymographs of logarithm of N-WASP/WSP-1 pixel intensities for time-lapse images from the data with actin and Arp2/3 (B). (E) Confocal images of actin network (1 $\mathrm{\mu }$M, 10% AF647 labeled, magenta) formed for 30 min on SLB with Arp2/3 (10 nM) and 100 nM His₆-VCA or (F) full-length His₆-N-WASP (green). (Scale bars: 10 $\mathrm{\mu }$M.)