Nucleation of the destruction complex on the centrosome accelerates degradation of β-catenin and regulates Wnt signal transmission

Abstract

Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate β-catenin stability. Overexpressed DC scaffolds undergo liquid-liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in β-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of β-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3β partitioning on the centrosome controls β-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle.

Keywords: LLPS; Wnt; destruction complex; optogenetics; stem cells.

Fig. 1.

Endogenously expressed β-cat puncta are inversely correlated with lrp6-mediated Wnt pathway activation and β-cat accumulation. (A) Schematic of tdmRuby3 CRISPR tag strategy. (B) Representative tdmRuby3-β-cat images of cells treated with Wnt-3a or media vehicle. Arrows indicate β-cat puncta. (Scale bars, 10 μm.). Insets show closeup examples of presence (arrows) or lack of puncta. (C) Fraction of t0 population with visible β-cat puncta, presented as mean ± SEM (n = 12 imaging fields per condition). (D) Representative cells from Wnt-3a condition. Arrows indicate puncta, and asterisks indicate cells lacking puncta. (Scale bars, 10 μm.) (E) Comparison of mean cytoplasmic β-cat fluorescence between Wnt-3a cells with and without visible β-cat puncta. (F) Schematic of Wnt I/O cells containing lentivirally expressed Cry2-LRP6c and CRISPR-tagged tdmRuby3-β-cat. Stimulation of Cry-2-Lrp6c with blue light results in reversible clustering of lrp6c and downstream pathway activation. (G) Representative tdmRuby3-β-cat images of cells stimulated with blue light or left in the dark throughout imaging time course. Insets show closeup examples of presence (arrows) or lack of puncta. (Scale bars, 10 μm.) (H) Fraction of t0 population with visible β-cat puncta, presented as mean ± SEM (n = 12 imaging fields per condition). (I) Representative cells from Wnt-3a condition. Arrows indicate puncta, and asterisks indicate cells lacking puncta. (Scale bars, 10 μm.) (J) Comparison of mean cytoplasmic β-cat fluorescence between Light ON cells with and without visible β-cat puncta. (K and L) Measurements of CRISPR cytoplasmic tdmRuby3-β-cat in live 293Ts; data presented as mean fluorescent intensity fraction of t0 ± SEM (n = 30 cells per condition). (M) Time course montage of single CHIR+ cells containing β-cat puncta undergoing dynamic fission and fusion. Arrows indicate puncta. Images are from consecutive frames of time course, separated by 5-min intervals. (Scale bars, 10 μm.)

Fig. 2.

Canonical DC components reside in liquid droplets nucleated at the centrosome. (A) Representative images of CRISPR-integrated tdmRuby3-CK1α, tdmRuby3-GSK3β, and Axin1-tdmRuby3 cells. (Insets) Close-up views of singular perinuclear puncta. (Scale bar, 10 μm.) (B) Representative cells bearing the indicated DC component fixed and stained for endogenous γ-tubulin. (Scale bar, 10 μm.) (C) Representative timelapse images from live cells bearing dox- and cumate-inducible Axin1 and APC cassettes under induction. Montages depict the same cell increasing its DC scaffold concentration through time. (Scale bar, 10 μm.) (D) Representative cells bearing the indicated DC component fixed and stained for endogenous γ-tubulin. (Scale bar, 10 μm.) (E) FRAP traces of mean puncta:cytoplasm fluorescence ratio for indicated DC components. Data are presented as mean ± SEM normalized to extent of bleaching (n = 39, 20, 33, 17, and 22 for Axin1, APC, CK1α, GSK3β, and β-cat, respectively). Individual FRAP traces were fit to the equation: f(t) = a(1-e^(-bt)) to obtain a and b parameters and half-max recovery time (τ1/2). Mean τ1/2 for each DC component is displayed on each plot.

Fig. 3.

In silico modeling of β-cat processing efficiency from a nucleated liquid droplet. (A) Nucleation interaction topology that describes the pairwise interactions between each component of the simulation. Connected components minimize free energy by mixing, and unconnected components either demix or remain in a noninteracting neutral state. (B) Schema describing the phosphorylation reactions and rates modeled in the simulation. (C) Simulation at steps 0 and 100 comparing a system with and without a centrosome. (D) Quantification of each form of β-cat with and without a centrosome. (E) Nucleation efficiency as a function of both rate parameters k₁ and k₂. (F) Nucleation efficiency in simulations as a function of the interaction parameters between a single client and the cytoplasm.

Fig. 4.

Optogenetic clustering of GSK3β increases centrosomal droplet partitioning and suppresses Wnt pathway activation. (A) Schematic of Opto-GSK3 and possible spatial outcomes of blue light stimulation. (B) Representative images of cells bearing Opto-GSK3 responding to blue light stimulation. Montage depicts the same cells throughout the activation time course. (Scale bar, 10 μm.) (C) Quantification of cells in B. Mean fluorescence fold change from t0 for each compartment ± SEM (n = 20 cells). (D) Representative images of cells bearing Opto-GSK3 + tdmRuby3-β-cat following treatment with Wnt-3a. (Scale bar, 10 μm.) (E) Quantification of cells in D. Mean fluorescence fold change from t0 ± SEM is shown (n = 20 cells per condition). (F) Representative images of cells bearing Opto-GSK3 + TOPFlash-IRFP following treatment with Wnt-3a. (Scale bar, 10 μm.) (G) Quantification of F. Mean fluorescence fold change from t0 ± SEM is shown (n = 24 cells per condition).

Fig. 5.

Optogenetic clustering of GSK3β suppresses Wnt pathway-mediated differentiation of embryonic stem cells. (A) Representative images of H9 embryonic stem cells bearing Opto-GSK3 following 24 h in described conditions, fixed and stained for endogenous Brachyury. (B) Quantification of experiment from A. Mean nuclear fluorescence for cells measured in each condition is presented.