Phase separation of +TIP networks regulates microtubule dynamics - PubMed (original) (raw)
Phase separation of +TIP networks regulates microtubule dynamics
Julie Miesch et al. Proc Natl Acad Sci U S A. 2023.
Abstract
Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here, we show that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid networks. When CLIP-170 and EB3 act together, the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells, these liquid networks can concentrate tubulin. In vitro, in the presence of microtubules, phase separation of EB3/CLIP-170 can enrich tubulin all along the microtubule. In this condition, microtubule growth speed increases up to twofold and the frequency of depolymerization events are strongly reduced compared to conditions in which there is no phase separation. Our data show that phase separation of EB3/CLIP-170 adds an additional layer of regulation to the control of microtubule growth dynamics.
Keywords: CLIP-170; EB3; liquid–liquid phase separation; microtubule; tubulin condensation.
Conflict of interest statement
The authors declare no competing interest.
Figures
Fig. 1.
CLIP-170 condenses into droplets in cells and in vitro. (A) Representative confocal images of RPE-1 cells transfected with GFP-CLIP-170 at three different overexpression levels (1: low, 2: high, and 3: very high overexpression). Zoom-in shows +TIP networks with adjusted contrast for visualization; for images with the same contrast, see (
SI Appendix, Fig. S1_A_
). For each cell, the whole cell fluorescence intensity (WCFI) and peak of +TIP network fluorescence intensity (TFI) are indicated; the blue outlining of the image corresponds to colored data points in (B). (Scale bar: 10 µm.) (B) Analysis of A showing the correlation between peak +TIP network fluorescence intensity and WCFI in RPE-1 cells expressing GFP-CLIP-170. The dashed line shows exponential plateau curve fit. Each dot represents 5 analyzed +TIP networks from one cell, data from two independent experiments with a total of 42 cells. (C) Percentage of +TIP networks with trailing foci in fixed cells expressing the indicated CLIP constructs or stained with antibodies to analysis endogenous CLIP-170 or EB3. Mean with SD from three independent experiments. Statistics: one-way ANOVA test. (D) Representative time-lapse TIRF images (Top) and kymograph (Bottom) of +TIP network from GFP-CLIP-170 expressing RPE-1 cell. Cyan and white arrowheads denote trailing foci formation and dissolving, respectively, in both time-lapse images and kymograph. (Scale bar: 2 µm.) (E) Phase diagram of GFP-FL-CLIP in vitro at increasing KCl and protein concentration. The blue shaded dot denotes where phase separation occurred, results of three independent experiments. The red dotted square represents the physiological cell concentration of CLIP-170 where we observe phase separation. (F, Top) representative confocal images of purified GFP-FL-CLIP at indicated concentrations. (Scale bar: 20 µm.) Bottom: quantification of the coverslip surface coverage (Left) and droplet size (Right) for GFP-FL-CLIP condensates at 100, 200, or 400 nM in the absence of PEG. Statistics: two-tailed Student’s t test. Mean with SD from three independent experiments with a total of 27 fields of view per condition. (G) Time-lapse TIRF images of purified GFP-FL-CLIP (1 µM) undergoing fusion. Representative of three experimental replicates. (Scale bar: 10 µm.) (H) Representative TIRF images and recovery curve of purified GFP-FL-CLIP (2 µM) droplets after photobleaching (dashed box). The blue curve shows mean with SD of three individual experiments with a total of 47 condensates. The gray curve shows the FRAP curve for GFP-CLIP droplets in cells. (Scale bar: 5 µm.)
Fig. 2.
The C-terminal region strongly enhances LLPS of CLIP-170. (A) Secondary structure of CLIP-170 (1 to 1,438), H2 (1 to 481), and H1 (1 to 350) drawn to scale, based on (20, 21, 50). (B) Representative confocal images of purified GFP-H1, GFP-H2, GFP-H2-tail, and GFP-FL-CLIP each at 200 nM in the absence (Top) or presence (Bottom) of 2% PEG. (Scale bar: 20 µm.) (C) Condensate surface coverage of the three constructs at indicated PEG concentrations. Mean with SD from three independent experiments with a total of 27 fields of view per condition. Statistics: two-tailed Student’s t test. (D) Droplet size (area) of GFP-H2 (200 nM) and GFP-FL-CLIP (200 nM). Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (E) Size distribution of GFP-FL-CLIP (200 nM) and GFP-H2 (200 nM) droplets in the absence of PEG. The graph shows average size distribution from three independent experiments with a total of 27 fields of view.
Fig. 3.
EB3 undergoes LLPS and cocondenses with CLIP-170 in vitro. (A) Representative DIC images of purified EB3 (1 µM) and GFP-FL-CLIP (400 nM) (Scale bar: 20 µm.) (B, Right) droplet size, Left: condensate surface coverage of EB3 (1 µM) and GFP-FL-CLIP (400 nM). Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (C) Representative TIRF images and recovery curve of purified EB3 (10 µM) + mCherry-EB3 (100 nM) droplets after photobleaching (dashed box). The curve shows mean with SD of three independent experiments with a total of 32 condensates. (Scale bar: 5 µm.) (D) Size distribution of GFP-FL-CLIP (200 nM), EB3 (1 µM), and the EB3/FL-CLIP droplets in the absence of PEG. Mean size distribution from three independent experiments with a total of 27 fields of view. (E) Representative confocal images of EB3/GFP-FL-CLIP droplets (Top) or EB3-ΔY/GFP-FL-CLIP droplets (Bottom) at denoted concentrations with the corresponding line scan. (Scale bar: 4 µm.)
Fig. 4.
Synergistic condensation of CLIP-170 and EB3. (A) Representative SDS-PAGE analysis from the droplet-pelleting assay showing protein fractions in supernatant dilute phase (S) or pellet dense phase (P) under each condition: GFP-FL-CLIP (1 µM); EB3 (10 µM); EB3/GFP-FL-CLIP (10 µM + 1 µM). (B) Quantification of SDS-PAGE analysis showing the fold-change of protein in the pellet fraction at the three conditions. Mean with SD from three independent experiments. Statistics: one-way ANOVA. (C) Representative fluorescence confocal images of purified GFP-FL-CLIP in the absence (Left) or presence (Right) of EB3 and in the absence (Top) or presence (Bottom) of 2% PEG. (Scale bar: 20 µm.) (D) Condensate surface coverage of purified GFP-FL-CLIP (200 nM) in the absence (Left) or presence (Right) of EB3 (1 µM) at indicated PEG concentrations. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (E) Quantification of droplet surface coverage of EB3 (1 µM) and GFP-FL-CLIP (200 nM) alone compared to surface coverage of EB3/GFP-FL-CLIP (1 µM/200 nM) droplet formation when undergoing synergistic LLPS in the absence of PEG. (F) Representative fluorescence confocal images of purified GFP-H1 (Left) or GFP-H2 (Right) in the presence of EB3 and in the absence (Top) or presence (Bottom) of 2% PEG. (Scale bar: 20 µm.) (G) Quantification of condensate surface coverage of indicated GFP-H1 (200 nM) or GFP-H2 (200 nM) in the presence of EB3 (1 µM) and in the presence of the indicated PEG concentrations. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test.
Fig. 5.
CLIP-170 and EB3 droplets concentrate tubulin. (A) Cartoon schematic of domain interactions between EB3, CLIP-170, and tubulin based primarily on (–54). Interaction sites between EB3 and CLIP-170, EB3 and tubulin, and CLIP-170 and tubulin are shown with green, orange, and blue arrows, respectively. For simplification, monomers of EB3 and CLIP-170 are shown. (B) Representative confocal images with the same contrast settings, of Atto565–tubulin (400 nM) in the presence of purified EB3 (1 µM), GFP-H1 (200 nM) + EB3 (1 µM), GFP-H2 (200 nM) + EB3 (1 µM), GFP-FL-CLIP (200 nM) + EB3 (1 µM), and GFP-FL-CLIP (200 nM and 400 nM) alone. (Scale bar: 20 µm.) Note that the treated plastic surface used for this experiment did not allow for a tubulin shell around the FL-CLIP droplet as shown in (E) for details see Materials and Methods. (C) Quantification of the droplet size from (B) under denoted conditions. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (D) Quantification of the integrated density of tubulin fluorescence under denoted conditions with zoom in for the first three conditions. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (E) Representative images of EB3/mCherry-EB3/tubulin-647 droplets (8 µM/2 µM/4 µM), GFP-FL-CLIP/tubulin-647 droplets (200 nM/400 nM), and EB3/mCherry-EB3/GFP-FL-CLIP/tubulin-647 droplets (900 nM/100 nM/200 nM/400 nM), with the corresponding line scans. (Scale bar: 5 µm.)
Fig. 6.
LLPS of +TIPs regulates microtubule dynamics through local tubulin enrichment. (A) Representative microtubule kymographs of denoted +TIP networks in higher salt buffer (60 mM KCl and 85 mM K-acetate, see Materials and Methods). Note that tip-tracking efficiency (GFP channel) is weaker at 5 µM tubulin than at higher tubulin concentrations (
SI Appendix, Fig. S8_B_
). (B) Microtubule growth rate (Top) and catastrophe frequency (Bottom) in high salt buffer. Tubulin (5 µM), EB3 (800 nM), H1 (50 nM), H2 (50 nM), and FL-CLIP (50 nM). Mean with SD of minimum of three independent experiments with the following number of analyzed microtubules: control—48; EB3—31; EB3/H1—28; EB3/H2—28; EB3/FL-CLIP—60. Statistics: One-way ANOVA Fisher’s LSD test. (C) Representative time-lapse TIRF images of GFP-FL-CLIP (50 nM) with tubulin (5 µM) in the presence of unlabeled EB3 (800 nM) and 60 mM KCl. Time denoted in minutes: seconds. (Scale bar: 20 µm.) The zoom-ins (white dashed box) show representative droplet formation along microtubules. (D) Representative microtubule kymographs in the presence of GFP-H2 or GFP-FL-CLIP (50 nM), EB3 (800 nM), tubulin (5 µM), and 60 mM KCl. Arrowheads denote areas of robust tubulin/FL-CLIP condensation on growing microtubule shaft. (E) Representative TIRF images of Atto565–tubulin (5 µM) microtubules growing in the absence (Left, control) and in the presence of EB3/FL-CLIP (800 nM/50 nM) (Right). Right images show tubulin enrichment along microtubule over time. (Scale bar: 2 µm.) (F) Corresponding line scan of E with the gray line scan representing the 5 µM tubulin control condition and the magenta line scans of 5 µM tubulin in the presence of EB3/FL-CLIP (800 nM/50 nM). (G) Tubulin fluorescence intensity in tip-proximal regions in 5 µM tubulin control condition and in the presence of EB3/FL-CLIP. Quantification of perpendicular line scans in tip-proximal regions. Mean with SD from two independent experiments for each condition and a total of 20 microtubules; (scale bar: 2 µm.) (H) Quantification of microtubule growth rate (Left) and catastrophe frequency (Right) in the presence of EB3/H2-networks (800 nM/50 nM) and EB3/FL-CLIP droplets (800 nM/50 nM) in experiments from D (5 µM tubulin and 60 mM KCl). Mean with SD of three independent experiments with the following number of analyzed microtubules: EB3/H2—29; EB3/FL-CLIP—59. Statistics: paired t test.
References
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- Gudimchuk N. B., McIntosh J. R., Regulation of microtubule dynamics, mechanics and function through the growing tip. Nat. Rev. Mol. Cell Biol. 22, 777–795 (2021). - PubMed
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