Phase transition of spindle-associated protein regulate spindle apparatus assembly

Abstract

Spindle assembly required during mitosis depends on microtubule polymerization. We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. BuGZ coacervation and its binding to microtubules and tubulin are required to promote assembly of spindle and spindle matrix in Xenopus egg extract and in mammalian cells. Since several previously identified spindle-associated components also contain low-complexity regions, we propose that coacervating proteins may be a hallmark of proteins that comprise a spindle matrix that functions to promote assembly of spindles by concentrating its building blocks.

Figure 1. BuGZ promotes spindle assembly independent of kinetochores

A. Western blotting of xBuGZ depletion (left) and add-back (right) in extracts. xBuGZ depletion (dep) efficiency and xBuGZ addition are shown in titrations. B–E. Representative images (B) show that xBuGZ depletion reduced astral MT length, bipolar spindle formation and length, which were all rescued by His-xBuGZ. ~50 (C, D) or ~500 (E) structures were measured in each experiment and condition. White dashed lines in B indicate Aurora A spindle length and longest astral MTs measured. F–H. xBuGZ depletion caused multiple sperm spindle defects (F), which was rescued by His-xBuGZ (G, H). ~500 (G) and ~50 (H) structures were analyzed in each experiment and condition. I. hBuGZ depletion reduced astral MT re-growth in mitotic HeLa cells, and was rescued by mBuGZ. Cold-treated cells were examined at 3 or 9 min after returning to 37°C. MTs, centromeres, and chromosomes were stained by tubulin antibody, CREST serum, and DAPI, respectively. J. Western blotting analyses of HeLa and U2OS cells treated by hBuGZ siRNA and transfected with indicated plasmids. K–L. HeLa or U2OS images (K) show that hBuGZ depletion by 72 h of RNAi diminished MT intensity in spindles, and was rescued fully by mBuGZ, partially by mBuGZAA, but not by mBuGZΔN. Cells were blocked with 10 μM MG132 for 1 h before immunostaining. ~30 cells were measured for each experiment and condition (L). Error bars, standard error of the means (SEM). Student’s t-test: *p<0.05, **p<0.01, ***p<0.001, three independent experiments. Scale bars, 5 μm.

Figure 2. Effects of BuGZ and temperature on spindle matrix

A–C. Both BuGZ and temperature influenced spindle matrix assembly. Spindle matrices were prepared with or without xBuGZ after nocodazole (Noc) treatment at RT or on ice and assayed by Western blotting (A) or immunostaining (B) using indicated markers. Tubulin, negative control. LB3 intensity of ~30 spindle matrices associated with one or two beads was quantified (C). Less spindle matrix was present in the cold than at RT. Green Aurora A beads appear larger than 2.8 μm due to secondary anti-rabbit antibody staining. D. Taxol treated HeLa cells were incubated at RT or on ice. Metaphase cells were visualized by immunostaining with tubulin (green), hBuGZ (red) antibodies, and DAPI (blue). ~100 cells were quantified for each condition. E. Treatment of taxol-stabilized Aurora A spindles on ice for 5 min diminished xBuGZ signal on spindles visualized by fluorescein-labeled MTs (red) and xBuGZ immunostaining (green). ~50 spindles were quantified for each condition. F. Extraction of taxol-stabilized and cold-treated metaphase spindles in HeLa cells diminished hBuGZ signal on spindles compared to RT extraction. Metaphase cells shown were immunostained using tubulin and hBuGZ antibodies and DAPI. ~100 cells were quantified for each condition. Error bars, SEM. Student’s t-test: p*<0.05, **p<0.01, ***p<0.001, three independent experiments. Scale bar, 5 μm.

Figure 3. Temperature- and concentration-dependent phase transition of BuGZ

A. Structural features of xBuGZ. The line at 0.5 (y-axis) is the cutoff for disorder (>0.5) and order (<0.5) predictions. P-FIT, VSL2B, VL3, and VLXT, predictors for disordered dispositions. LC1, LC2, and LC3 indicate low complexity regions determined at three stringencies. ZnF, zinc fingers, predicted structured region. B. YFP-xBuGZ formed spheres in Sf9 cells. YFP served as control. Scale bar, 20 μm. C. Gel filtration chromatography of YFP-xBuGZ. Arrowheads, positions of size markers (in kDa): thyroglobulin, apoferritin, amylase, alcohol dehydrogenase, albumin, and carbonic anhydrase. Fractions 7–14 were analyzed. D. YFP-xBuGZ formed droplets in vitro as visualized by DIC and fluorescence microscopy. Scale bar, 20 μm. E. Temperature-dependent droplet formation by YFP-xBuGZ in XB buffer as visualized by Hoffman modulation contrast microscopy. Temperature ramp, 4 to 20°C at 1°C/min. Scale bar, 20 μm. F–G. Turbidity assay of reversibility (F) and repeatability (G) of phase transition by YFP-xBuGZ. Increase (4°C to 35°C) and decrease (35°C to 4°C) in temperature in F had the same temperature ramp. The temperature ramp in G was 4°C–30°C. Ramp rate, 0.5°C/min. H. Concentration-dependent accumulation of YFP-xBuGZ coacervates at the bottom of cuvettes after turbidity measurements. Scale bar, 1 cm. I–K. Concentration- (color-coded) and temperature-dependent phase transition of YFP-xBuGZ (I) and YFP-xBuGZΔN (J), but not YFP (K), based on the turbidity assay. L–M. xBuGZ5S (L) and xBuGZ13S (M) coacervation at increasingly higher protein concentrations and temperatures. N. The temperature at which the turbidity was half (T_1/2) of the difference between maximum and 4°C absorbance was plotted against log₁₀ protein concentration. YPF-xBuGZ13S did not reach maximum turbidity at 3.125 and 1.56 μM even at 60°C. Error bars, standard deviation from three independent experiments.

Figure 4. Bundling of MTs by BuGZ depends on its MT binding and phase transition

A. YFP-xBuGZ, but not YFP-BuGZΔN, YFP-BuGZ13S, or YFP, induced MT bundling from taxol-stabilized and rhodamine-labeled MT seeds. B–C. Quantifications of length (B) and average intensity (C) of MTs or MT bundles formed in the presence of indicated proteins and concentrations. MT images were randomly captured under a 63× objective. ~100 individual MTs or MT bundles were measured. D. YFP-xBuGZ droplets (green, white arrowheads) along some MT bundles (red). Line scans of tubulin and YFP-BuGZ intensity of the indicated segments of MTs (red arrowheads) are shown. E–F. Purified YFP-xBuGZ, but not YFP-xBuGZ13S, had increased binding to preformed MTs at 37°C compared to 4°C, but YFP-xBuGZΔN fail to bind. Quantification is shown in F. Error bars, SEM. Student’s t-test: *p<0.05, **p<0.01, ***p<0.001, three independent experiments. Scale bars, 10 μm.

Figure 5. BuGZ coacervation promotes MT polymerization by concentrating tubulin

A. Tubulin was concentrated in droplets formed by YFP-xBuGZ, but not by YFP-xBuGZΔN. Scale bar, 20 μm. B. Anti-His-tag antibody pulled down indicated xBuGZ and tubulin at 4°C. C. Illustration of the spin down assay. D. Higher concentrations of YFP-xBuGZ and tubulin are found in droplets (Y-axis) than in initial solution concentrations (X-axis). E–F. Compared to initial solution concentrations (X-axis), YFP-xBuGZΔN (E) coacervation only concentrated itself, but not tubulin, in droplets (Y-axis), whereas YFP-xBuGZ13S (F) did not coacervate or concentrate itself or tubulin (Y-axis). G–H. Representative fields of MTs polymerized. MTs were counted in 20 random microscopic fields using a 63× objective. Scale bar, 10 μm. Error bars, SEM. Student’s t-test: ns, not significant, *p<0.05, **p<0.01, ***p<0.001, three independent experiments.

Figure 6. Tubulin/MT binding and phase transitions of BuGZ promote spindle matrix assembly and function

A. Incorporation of YFP-xBuGZ into preformed His-xBuGZ droplets in vitro required Fs and Ys but not the N-terminus of xBuGZ. YFP intensity was quantified in ~50 droplets. B. Incorporation of 0.1 μM YFP-xBuGZ into isolated spindle matrix required Fs and Ys but not the N-terminus of xBuGZ. ~30 structures were analyzed. C. Incubation of YFP-xBuGZΔN, but not YFP, YFP-xBuGZ, or YFP-xBuGZ13S, with isolated spindle matrix disrupted matrix-mediated MT assembly. ~30 asters were analyzed. D. Spindle matrix assembly required MT-binding of BuGZ. YFP-xBuGZ, but not YFP-xBuGZΔN, rescued spindle matrix assembly upon endogenous xBuGZ depletion, as assayed by Western blotting analyses using spindle matrix markers. E. Spindle matrix assembly required BuGZ coacervation. YFP-xBuGZ, but not YFP-xBuGZ13S, rescued spindle matrix assembly upon endogenous xBuGZ depletion. The addition of GFP-BuGZ-B, but not -BuGZ-B3S, into unperturbed extract disrupted spindle matrix assembly. Error bars, SEM. Student’s t-test: ns, not significant, **p<0.01, ***p<0.001, three independent experiments. The numbers of structures quantified are for each experiment and condition. Scale bars, 10 μm.

Figure 7. Tubulin/MT binding and coacervation of BuGZ promotes spindle assembly

A. Incorporation of 0.1 μM YFP-xBuGZ into isolated spindles required Fs and Ys and MT binding. ~30 spindles were analyzed. Scale bars, 10 μm. B–D. xBuGZ, but not xBuGZΔN or xBuGZ13S, rescued astral MT length (B), percentages of normal spindles (C), and length of spindles (D) induced by Aurora A beads. ~50 (B, D) or ~500 (C) structures were analyzed. E–F. Only wild-type xBuGZ, but not xBuGZΔN or xBuGZ13S, rescued defective morphology (E) or MT intensity (F) of spindles in xBuGZ-depleted extracts. ~500 sperm-associated MT structures (E) or ~50 spindles (F) were analyzed. G–I. Expression of mBuGZ, but not mBuGZ15S, in hBuGZ depleted HeLa cells (G) rescued normal spindle MT intensity (H, I). ~30 spindles were analyzed. Spindles, centromeres, and chromosomes were stained. Scale bars, 5 μm. J–M. Models for BuGZ phase transition in vitro (J–K) or during spindle assembly (L–M). See explanations in the Discussion. Error bars, SEM. Student’s t-test: ns, not significant, *p<0.05, **p<0.01, ***p<0.001 from three independent experiments. The numbers of structures analyzed are for each experiment and condition.