Skip to main content
. 2023 Aug 21;120(35):e2301457120. doi: 10.1073/pnas.2301457120

Fig. 6.

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. S8B). (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.