ABSTRACT In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate

structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical

properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid–liquid phase separation (LLPS) to constitute the microtubule

plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a

interactions that guide microtubule dynamics. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access

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support SIMILAR CONTENT BEING VIEWED BY OTHERS EVIDENCE FOR A HURP/EB FREE MIXED-NUCLEOTIDE ZONE IN KINETOCHORE-MICROTUBULES Article Open access 10 August 2022 MULTIVALENCY ENSURES

PERSISTENCE OF A +TIP BODY AT SPECIALIZED MICROTUBULE ENDS Article Open access 19 December 2022 CLASP-MEDIATED COMPETITIVE BINDING IN PROTEIN CONDENSATES DIRECTS MICROTUBULE GROWTH Article

Open access 02 August 2024 DATA AVAILABILITY Previously published protein sequence data that were re-analysed here are available from UniProt under accession codes EB1__Homo sapiens_ (ID:

Q15691), EB1__Bovin_ (ID Q3ZBD9) EB1__Mouse_ (ID: Q61166), EB1__Xenopus_ (ID: Q6P848), EB1__Chicken_ (ID: Q5ZLC7), EB1__Danre_ (ID: Q6NUY9), EB1__fly_ (ID: A1Z6P3) and Mal3__yeast_ (ID:

Q10113). All nucleotide sequences used in the design or synthesis were from genome annotation databases under accession codes EB1__Homo sapiens_ (ID: ENSG00000101367), EB1__Bovin_ (ID:

ENSBTAT00000044474.4) EB1__Mouse_ (ID: ENSMUSG00000027479), EB1__Xenopus_ (ID: ENSXETT00000055099.4), EB1__Chicken_ (ID: ENSGALG00000006657), EB1__Danre_ (ID: ENSDART00000004474.9),

EB1__fly_ (ID: FBtr0300285) and Mal3__yeast_ (ID: SPAC18G6.15.1). Previously published structures are available from the Protein Data Bank under accession codes EB1 CH domain (PDB entry 1PA7

(ref. 8)) and the EB1 EBH domain (PDB entry 3GJO26). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding

author on reasonable request. CODE AVAILABILITY The custom code for coarse-grained computational modelling and statistical analysis along with its output dataset are available at

http://zenodo.org with the identifier https://doi.org/10.5281/zenodo.7113746. RPA and shuffling data analysis is deposited at https://github.com/Carolinge/EB1_rpashuffle. Source data are

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Lennard–Jones chains. _J. Chem. Phys._ 129, 144703 (2008). Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to Mingjie Zhang, Peace Cheng, Yeguang Chen

and Yi Luo for critical reading, and to Iain M. Cheeseman, Li Yu and Clifford P. Brangwynne for reagents. This work was supported by MOST-NSFC grants (2017YFA0503600, 32090040, 91854203,

31621002, 21922706, 92153302, 92254302, 2022YFA1303100, 2022YFA0806800 and 91853115 to X.L.; 92059102 and 92253305 to X.S.; 2019YFA0508403 and 31971128 to S.X.; 92253301 to C.X.;

2022YFA1302700 to Z.W.); the Ministry of Education (IRT_17R102, 20113402130010 and YD2070006001) to X.L.; the Strategic Priority Research Program of the Chinese Academy of Sciences

(XDB19040000 to X.L.; XDB37040202 to S.X.); the Fundamental Research Funds for the Central Universities (WK2070000194) to X.L.; Anhui Provincial Natural Science Foundation Grant

(1908085MC64) and China Postdoctoral Science Foundation Grant (2019M662184) to X.S., and NIH grants (CA146133 and DK115812). The funders had no role in study design, data collection and

analysis, decision to publish or preparation of the manuscript. AUTHOR INFORMATION Author notes * These authors contributed equally: Xiaoyu Song, Fengrui Yang, Tongtong Yang, Yong Wang,

Mingrui Ding, Linge Li. * These authors jointly supervised this work: Xing Liu, Xuebiao Yao. AUTHORS AND AFFILIATIONS * MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics,

Hefei National Center for Cross-disciplinary Sciences, University of Science & Technology of China School of Life Sciences, Hefei, China Xiaoyu Song, Fengrui Yang, Tongtong Yang, Mingrui

Ding, Panpan Xu, Shuaiyu Liu, Ming Dai, Changbiao Chi, Shengqi Xiang, Chao Xu, Zhikai Wang, Chuanhai Fu, Jianye Zang, Yunyu Shi, Xing Liu & Xuebiao Yao * Keck Center for Organoids

Plasticity, Morehouse School of Medicine, Atlanta, GA, USA Xiaoyu Song, Fengrui Yang, Mingrui Ding, Zhikai Wang & Xing Liu * Frontier Science Center for Immunology and Metabolism,

Medical Research Institute, Wuhan University, Wuhan, China Yong Wang & Kai Jiang * Anhui Key Laboratory for Chemical Biology & Hefei National Center for Cross-disciplinary Sciences,

Hefei, China Linge Li, Shuaiyu Liu & Zhonghuai Hou * Institute of Biophysics, Beijing, China Dong Li * CAS Center of Excellence in Molecular Cell Sciences, Chinese Academy of Sciences,

Shanghai, China Lin Li * Department of Pathology, University of Alabama, Birmingham, AL, USA Donald L. Hill * Hunan Key Laboratory of Molecular Precision Medicine, Central South University

School of Life Sciences, Changsha, China Kai Yuan * School of Life Sciences, Tsinghua University, Beijing, China Pilong Li Authors * Xiaoyu Song View author publications You can also search

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search for this author inPubMed Google Scholar * Xuebiao Yao View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.Y. and X.L. conceived the

project. X.S., T.Y. and M. Ding designed and performed most of the biochemical experiments. X.S. performed OptoDroplet assay, and F.Y. performed live-cell imaging and IF analyses. X.S. and

D.L. performed GI-SIM and lattice-SIM imaging. Y.W., Z.W. and K.J. performed in vitro TIRF assays. Linge L. and Z.H. performed molecular modelling. P.X. and S.X. performed NMR experiments.

T.Y. and C.X. performed ITC assays. C.C. performed the surface charge analysis and prepared structure-modelling images. S.L. and M. Dai engineered the EB1-GFP knock-in HeLa cell line. X.S.,

Y.W. and F.Y. performed data analyses. P.L. contributed reagents. X.S., X.L. and X.Y. wrote the manuscript. K.Y., C.F., J.Z., Y.S., Lin L. and D.L.H. edited the manuscript. All authors have

commented on and approved the manuscript. CORRESPONDING AUTHORS Correspondence to Xing Liu or Xuebiao Yao. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests.

PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks Carsten Janke, Claire Walczak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA

FIG. 1 EB1 COMETS EXHIBIT LIQUID-LIKE PROPERTY. (A) GI-SIM image of a typical GFP-EB1 comet fusion at the microtubule plus-ends. COS-7 cells were transfected with EB1-GFP and

mCherry-tubulin. The boxed area is magnified in the right panels, which shows time-lapse imaging of EB1 comets over 4 s. White arrows highlight the fusion process of two adjacent comets.

Scale bar, 10 μm in original image and 5 μm in magnified montages. (B) Time-lapse analysis of EB1-GFP in HeLa cells by Lattice-SIM. A representative cell with EB1 comet fusion (arrowheads)

at different z-plane was shown. Scale bar, 10 μm. (C) Schematic representation of engineering design of the endogenous EB1 gene with an mEGFP tag via CRISPR-Cas9 mediated gene editing to

generate EB1-GFP knock-in (KI) in HeLa cells. The mEGFP were inserted before the TAA stop codon of EB1. (D) The EB1-GFP KI HeLa cells were verified by Western blotting using an anti-EB1

antibody. (E) Time-lapse analysis of EB1-GFP KI HeLa cells by Lattice-SIM. The arrows indicate EB1 comets before and after fusion. Scale bar: in original images, 10 μm; in zoom-in images, 5 

μm. (F) Schematic of the OptoEB1 construct, which contains mCherry and Cry2 (blue light-induced oligomerization). (G) Representative snapshots of mCherry-Cry2 (mCh-Cry2) in living COS-7

cells upon blue light activation. Note that Cry2 alone does not exhibit comet-like structure after blue-light activation. Scale bar, 10 μm. Source data EXTENDED DATA FIG. 2 PHASE SEPARATION

PROPERTY OF EB1 IS EVOLUTIONARILY CONSERVED IN EUKARYOTIC CELLS. (A) Sequence alignment of EB1 from yeast to human. The residues with positive charge were highlighted. (B) Schematic

representation of the domain structures of human EB1, fission yeast Mal3 and budding yeast Bim1. (C) _In vitro_ phase separation assay. Representative fluorescence images of Mal3-GFP phase

separation in BRB80 buffer with various concentrations of KCl. Scale bar, 5 µm. (D) Representative SDS-PAGE gel of EB1 solubility in solution. Samples of EB1-GFP protein at various

concentration were centrifuged as described under “Methods”. Equal volumes of supernatant (S) and pellet (P) fractions were resolved by electrophoresis and resolved proteins were visualized

by CBB staining. (E) Determination of a saturation concentration (_c_sat) of EB1-GFP. The absorbance at 340 nm (condensed protein has a distinctly different absorbance profile, leading to a

pronounced uptick in A340 absorbance) was measured. The tendency of soluble and condensed concentration were labeled by red line, and the intersection was measured as _c_sat. Data represent

mean ± s.e.m. from three independent experiments. (F) _In vitro_ phase separation assay. Representative fluorescence and DIC images of EB1-GFP (20 μM) phase separation with or without 5%

1,6-Hexanediol (Hex) in BRB80 buffer with 150 mM KCl. Scale bar, 5 µm. (G) Schematic illustration of an ultra-coarse-grained modeling for CH domain, EBH domain, the IDR region and C-terminal

region. (H) Representative image of EB1 phase separation based on the ultra-coarse-grained modeling. Source data EXTENDED DATA FIG. 3 CHARACTERIZATION OF MULTIVALENT INTERACTIONS AMONG EB1

REGIONS BY NMR. (A) Combined chemical shifts perturbation (CSP) of IDR in the presence of EBH (IDR vs IDR + EBH). The dash lines indicated the level of standard deviation values. Proteins

boxed in red were labeled by 15N. Gray bars indicated overlapping or unassigned peaks, while the asterisks labeled the proline residues which had no signal in NMR assay. (B) Combined

chemical shifts perturbation of IDR + EBH in the presence of CTT (IDR + EBH vs IDR + EBH + CTT. *, proline residues. (C) Peak intensity change of IDR + EBH + CTT (15N labeling) upon CH

domain titration. The error bars were calculated based on every peak’s signal-to-noise ratios as described in Methods. *, proline residues. (D) Representative spectra of indicated regions

were overlaid to show the peak shifts. The spectra of IDR, IDR + EBH, and IDR + EBH + CTT were shown in red, blue, and green, respectively. The peak shifts of residues showed the interaction

between indicated regions. (E) Summary of the interactions among different EB1 regions. Source data EXTENDED DATA FIG. 4 LYSINE/ARGININE RESIDUES ON IDR ARE ESSENTIAL FOR EB1 PHASE

SEPARATION. (A) Characterization of recombinant purified proteins used in _in vitro_ phase separation assay. Recombinant EB1 proteins were subjected to electrophoresis followed by CBB

staining. (B) Diagram of EB1 wild type and IDR chimeras. Their phase separation capacities at 10 μM were also annotated. “−” no phase separation events were observed; “+” phase separation

events were observed. (C) Representative images of EB1 chimeras in COS-7 cells (Upper) and corresponding phase separation test _in vitro_ (Lower). Scale bar, 10 μm. (D) Sequence alignment of

EB1-IDR and Pub1-IDR. The residues with positive charge were highlighted. (E) Summary of _in vitro_ phase separation behavior of EB1 RDI and KR6A mutants. EB1 mutants were purified and

assessed for _in vitro_ phase separation assay. The saturation concentration was measured by turbidity assay at indicated salt conditions. The red circles indicate phase separation (above

saturation concentration), and the blue circles indicate no phase separation (below saturation concentration). (F) EB1 binding with purified +TIPs _in vitro_. Purified GST- or MBP-tagged

+TIPs were used as affinity matrices to absorb His-EB1 wild type or KR6Q mutant as in Fig. 2k. The bound His-EB1 protein was assessed by Western blotting using an anti-His antibody. Source

data EXTENDED DATA FIG. 5 BINDING AFFINITY BETWEEN EB1 AND ITS BINDING PARTNERS WAS NOT CHANGED BY KR6Q MUTATION. ITC binding curves for interaction of EB1 and its binding partners upon KR6Q

mutation. (A) MCAK peptide, aa 85-118; (B) TIP150 peptide, aa 823-856; (C) CLASP1 peptide, aa 713-747; (D) CLASP2 peptide, aa 490-523; (E) p150Glued CAP-GLY domain (aa 1-105). For ITC

assay, the peptides and p150Glued CAP-GLY domain were used at 1 mM in syringe, while the purified EB1 wild type and KR6Q mutant were used at 50 μM in cell. Data are representative from three

independent replicates. All peptide sequences were listed in Supplementary Table 1. EXTENDED DATA FIG. 6 CHARACTERIZATION OF POSITIVE CHARGES AND THEIR POSITIONS IN IDR FOR EB1 PHASE

SEPARATION. (A) Schematic diagram of three classes of EB1 mutants: 1) positive charges in IDR were mutated (KR1Q-KR6Q); 2) the positions of charges in IDR were rearranged in cluster as 1-10 

K/R; 3) a scramble sequence of IDR (scr9). The positive charges and mutations in IDR were annotated. All the sequences of EB1 mutants were listed in Supplementary Table 2. (B)

Ultra-coarse-grained computational modeling to elucidate the phase separation behaviors of the three classes of EB1 mutants as in (A). (C) The random phase approximation (RPA) theory was

applied to estimate the IDR electrostatic effects on EB1 phase separation. (D) The _in vitro_ phase separation assay of EB1 mutants (10 μM) in the presence of 150 mM KCl. Scale bar, 5 µm.

(E) The phase diagram of EB1 mutants generated by _in vitro_ phase separation assay. The saturation concentration was measured by turbidity assay at indicated salt and protein

concentrations. The red circles indicate phase separation (above saturation concentration), and the blue circles indicate no phase separation (below saturation concentration). Data are

representative from three independent replicates. EXTENDED DATA FIG. 7 PHASE SEPARATION DIAGRAM OF EB1 FROM DIFFERENT SPECIES _IN VITRO_. Summary of _in vitro_ phase separation behavior of

wild-type and lysine/arginine mutated EB1 in response to changes in salt concentration. Proteins of both wild-type and mutated EB1 from different species were purified and assessed for _in

vitro_ phase separation propensity as described in Methods. The saturation concentration was measured by turbidity assay at indicated salt and protein concentrations. The red circles

indicate phase separation (above saturation concentration), and the blue circles indicate no phase separation (below saturation concentration). Data are representative from three independent

replicates. EXTENDED DATA FIG. 8 CHARACTERIZATION OF EB1 PHASE SEPARATION IN THE PRESENCE OF MICROTUBULE AND +TIPS _IN VITRO_. (A) Characterization of EB1 phase separation in microtubule

plus-end tracking at different KCl concentrations using _in vitro_ TIRFM assay. The concentration of EB1-GFP was 50 nM in the presence of various concentrations of KCl. Scale bar, 5 μm.

(B-C) Quantitative analyses of fluorescence intensity of EB1 at microtubule plus-ends (B) and microtubule growth rate (C) in (A). For each group, n = 30 microtubules pooled from three

independent experiments. Data represent mean ± s.e.m. Ordinary one-way ANOVA followed by Tukey’s _post hoc_ test was used to determine statistical significance. _****p_ < 0.0001; ns (not

significant) indicates _p_ > 0.05. (D) Electron micrograph of EB1-hnRNPA1 chimeras (2 μM) from _in vitro_ phase separation assay as carried out in Fig. 4a. Scale bar, 500 nm. (E)

Representative images of co-phase separation of FITC-labeled His-EB1 (EB1-FITC) and other +TIPs co-condensates in the presence of 150 mM KCl. Scale bar, 5 μm. (F) Partition coefficients

(_PCs_) for EB1 and other +TIPs from experiment in (E). Data represent mean ± s.e.m. from three independent experiments. Dotted line, _PC_ = 1. (G) Quantitative analyses of droplet areas in

(E). For EB1-FITC, n = 30; EB1-FITC + TIP150, n = 50; EB1-FITC + MCAK, n = 30; EB1-FITC + p150Glued, n = 30; EB1-FITC + CLIP170, n = 30; for +TIPs only, no droplet was observed in ten fields

of view under microscope; n represents the number of EB1 droplets pooled from three independent experiments. Data represent mean ± s.e.m. Ordinary one-way ANOVA followed by Tukey’s post hoc

test was used to determine statistical significance. ****_p_ < 0.0001; ns (not significant) indicates _p_ > 0.05. Source data EXTENDED DATA FIG. 9 PHASE SEPARATION IS A FUNCTION OF

EB1 IN MICROTUBULE PLUS-END TRACKING. (A-B) Representative immunofluorescence images of FLAG-EB1 (wild type and KR6Q) and GFP-tagged +TIPs proteins (MCAK and p150Glued) co-expressing in

endogenous EB1-depleted HeLa cells. DNA was stained with DAPI and microtubule was stained with an anti-tubulin antibody. Scale bar, 10 μm. EXTENDED DATA FIG. 10 DYNAMIC PHASE SEPARATION OF

EB1 GUIDES CHROMOSOME MOVEMENTS. (A) Characterization of expression levels of GFP-tagged EB1 wild type, KR6Q mutant and EB1-hnRNPA1 chimeras in endogenous EB1-depleted HeLa cells were

analyzed by Western blot with an anti-EB1 antibody. Doxycycline (Dox) was added to induce EB1 knockout. (B) Representative mitotic phenotypes in endogenous EB1-depleted HeLa cells expressing

EB1-hnRNPA1 chimeras shown by time-lapse microscopy. Chromosomes were visualized with mCherry-H2B. Arrows, spindle poles; arrowheads, lagging chromosomes. Scale bar, 10 μm. (C)

Quantification of mitotic phenotypes of live HeLa cells in (B). For each group, n = 30 cells pooled from three independent experiments. Data represent mean ± s.e.m. Statistical significance

was assessed by two-way ANOVA. ****_p_ < 0.0001; ***_p_ = 0.001; **_p_ = 0.0041; *_p_ = 0.0107; ns (not significant) indicates _p_ > 0.05. Source data SUPPLEMENTARY INFORMATION

REPORTING SUMMARY SUPPLEMENTARY TABLES Sequence of peptides for ITC assay and sequence of EB1 mutants. SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical

source data. SOURCE DATA FIG. 2 Unprocessed WBs and/or gels. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source

data. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Unprocessed WBs and/or gels. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED

DATA FIG. 2 Unprocessed WBs and/or gels. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed WBs and/or gels. SOURCE DATA EXTENDED DATA

FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 10 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 10 Unprocessed WBs and/or gels. RIGHTS AND PERMISSIONS Springer

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CITE THIS ARTICLE Song, X., Yang, F., Yang, T. _et al._ Phase separation of EB1 guides microtubule plus-end dynamics. _Nat Cell Biol_ 25, 79–91 (2023).

https://doi.org/10.1038/s41556-022-01033-4 Download citation * Received: 15 October 2021 * Accepted: 21 October 2022 * Published: 19 December 2022 * Issue Date: January 2023 * DOI:

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