Non-centrosomal microtubule bundles play important roles in cellular organization and function. Although many diverse proteins are known that can bundle microtubules, biochemical mechanisms by which cells could locally control the nucleation and formation of microtubule bundles are understudied. Here, we demonstrate that the concentration of tubulin into a condensed, liquid-like compartment composed of the unstructured neuronal protein tau is sufficient to nucleate microtubule bundles. We show that, under conditions of macro-molecular crowding, tau forms liquid-like drops. Tubulin partitions into these drops, efficiently increasing tubulin concentration and driving the nucleation of microtubules. These growing microtubules form bundles, which deform the drops while remaining enclosed by diffusible tau molecules exhibiting a liquid-like behavior. Our data suggest that condensed compartments of microtubule bundling proteins could promote the local formation of microtubule bundles in neurons by acting as non-centrosomal microtubule nucleation centers and that liquid-like tau encapsulation could provide both stability and plasticity to long axonal microtubule bundles.

B CUBE Center for Molecular Bioengineering Technische Universität Dresden Dresden 01307 Germany; Institute of Biotechnology CAS BIOCEV Vestec 25250 Czech Republic

Department Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA

Max Planck Institute of Molecular Cell Biology and Genetics Dresden 01307 Germany

Max Planck Institute of Molecular Cell Biology and Genetics Dresden 01307 Germany; B CUBE Center for Molecular Bioengineering Technische Universität Dresden Dresden 01307 Germany

Max Planck Institute of Molecular Cell Biology and Genetics Dresden 01307 Germany; BIOTEC Biotechnology Center of the Technische Universität Dresden Dresden 01307 Germany

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Arendt T, Stieler JT, Holzer M. Tau and tauopathies. Brain Res. Bull. 2016;126:238–292. PubMed

Aronov S, Aranda G, Behar L, Ginzburg I. Axonal tau mRNA localization coincides with tau protein in living neuronal cells and depends on axonal targeting signal. J. Neurosci. 2001;21:6577–6587. PubMed PMC

Baas PW, Karabay A, Qiang L. Microtubules cut and run. Trends Cell Biol. 2005;15:518–524. PubMed

Banani SF, Lee HO, Hyman AA, Rosen MK. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 2017;18:285–298. PubMed PMC

Banjade S, Rosen MK. Phase transitions of multivalent proteins can promote clustering of membrane receptors. eLife. 2014;3:e04123. PubMed PMC

Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F, Hyman AA. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 2009;324:1729–1732. PubMed

Chen J, Kanai Y, Cowan NJ, Hirokawa N. Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons. Nature. 1992;360:674–677. PubMed

Chen W-S, Chen Y-J, Huang Y-A, Hsieh B-Y, Chiu H-C, Kao P-Y, Chao C-Y, Hwang E. Ran-dependent TPX2 activation promotes acentrosomal microtubule nucleation in neurons. Sci. Rep. 2017;7:42297. PubMed PMC

Chung PJ, Song C, Deek J, Miller HP, Li Y, Choi MC, Wilson L, Feinstein SC, Safinya CR. Tau mediates microtubule bundle architectures mimicking fascicles of microtubules found in the axon initial segment. Nat. Commun. 2016;7:12278. PubMed PMC

Dickson JR, Kruse C, Montagna DR, Finsen B, Wolfe MS. Alternative polyadenylation and miR-34 family members regulate tau expression. J. Neurochem. 2013;127:739–749. PubMed PMC

Drechsel DN, Hyman AA, Cobb MH, Kirschner MW. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol. Biol. Cell. 1992;3:1141–1154. PubMed PMC

Elbaum-Garfinkle S, Kim Y, Szczepaniak K, Chen CC-H, Eckmann CR, Myong S, Brangwynne CP. The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics. Proc. Natl. Acad. Sci. USA. 2015;112:7189–7194. PubMed PMC

Gell C, Friel CT, Borgonovo B, Drechsel DN, Hyman AA, Howard J. Purification of tubulin from porcine brain. Methods Mol. Biol. 2011;777:15–28. PubMed

Goedert M, Jakes R, Spillantini MG, Hasegawa M, Smith MJ, Crowther RA. Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature. 1996;383:550–553. PubMed

Guharoy M, Szabo B, Contreras Martos S, Kosol S, Tompa P. Intrinsic structural disorder in cytoskeletal proteins. Cytoskeleton. 2013;70:550–571. PubMed

Herrmann H, Aebi U. Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. Annu. Rev. Biochem. 2004;73:749–789. PubMed

Herzmann S, Krumkamp R, Rode S, Kintrup C, Rumpf S. PAR-1 promotes microtubule breakdown during dendrite pruning in Drosophila. EMBO. J. 2017;36:1981–1991. PubMed PMC

Hinrichs MH, Jalal A, Brenner B, Mandelkow E, Kumar S, Scholz T. Tau protein diffuses along the microtubule lattice. J. Biol. Chem. 2012;287:38559–38568. PubMed PMC

Igaev M, Janning D, Sündermann F, Niewidok B, Brandt R, Junge W. A refined reaction-diffusion model of tau-microtubule dynamics and its application in FDAP analysis. Biophys. J. 2014;107:2567–2578. PubMed PMC

Janning D, Igaev M, Sündermann F, Brühmann J, Beutel O, Heinisch JJ, Bakota L, Piehler J, Junge W, Brandt R. Single-molecule tracking of tau reveals fast kiss-and-hop interaction with microtubules in living neurons. Mol. Biol. Cell. 2014;25:3541–3551. PubMed PMC

Jiang H, Wang S, Huang Y, He X, Cui H, Zhu X, Zheng Y. Phase transition of spindle-associated protein regulate spindle apparatus assembly. Cell. 2015;163:108–122. PubMed PMC

Kadavath H, Hofele RV, Biernat J, Kumar S, Tepper K, Urlaub H, Mandelkow E, Zweckstetter M. Tau stabilizes microtubules by binding at the interface between tubulin heterodimers. Proc. Natl. Acad. Sci. USA. 2015;112:7501–7506. PubMed PMC

Kornreich M, Avinery R, Malka-Gibor E, Laser-Azogui A, Beck R. Order and disorder in intermediate filament proteins. FEBS Lett. 2015;589(19 Pt A):2464–2476. PubMed

Li X-H, Rhoades E. Heterogeneous tau-tubulin complexes accelerate microtubule polymerization. Biophys. J. 2017;112:2567–2574. PubMed PMC

Li P, Banjade S, Cheng HC, Kim S, Chen B, Guo L, Llaguno M, Hollingsworth JV, King DS, Banani SF, et al. Phase transitions in the assembly of multivalent signalling proteins. Nature. 2012;483:336–340. PubMed PMC

Li X-H, Culver JA, Rhoades E. Tau binds to multiple tubulin dimers with helical structure. J. Am. Chem. Soc. 2015;137:9218–9221. PubMed PMC

Ma Q-L, Zuo X, Yang F, Ubeda OJ, Gant DJ, Alaverdyan M, Kiosea NC, Nazari S, Chen PP, Nothias F, et al. Loss of MAP function leads to hippocampal synapse loss and deficits in the Morris water maze with aging. J. Neurosci. 2014;34:7124–7136. PubMed PMC

Malmqvist T, Anthony K, Gallo J-M. Tau mRNA is present in axonal RNA granules and is associated with elongation factor 1A. Brain Res. 2014;1584:22–27. PubMed

Matamoros AJ, Baas PW. Microtubules in health and degenerative disease of the nervous system. Brain Res. Bull. 2016;126:217–225. PubMed PMC

Matus A. Microtubule-associated proteins: their potential role in determining neuronal morphology. Annu. Rev. Neurosci. 1988;11:29–44. PubMed

Melo AM, Coraor J, Alpha-Cobb G, Elbaum-Garfinkle S, Nath A, Rhoades E. A functional role for intrinsic disorder in the tau-tubulin complex. Proc. Natl. Acad. Sci. USA. 2016;113:14336–14341. PubMed PMC

Mukhopadhyay R, Kumar S, Hoh JH. Molecular mechanisms for organizing the neuronal cytoskeleton. BioEssays. 2004;26:1017–1025. PubMed

Mukrasch MD, Bibow S, Korukottu J, Jeganathan S, Biernat J, Griesinger C, Mandelkow E, Zweckstetter M. Structural polymorphism of 441-residue tau at single residue resolution. PLoS Biol. 2009;7:e34. PubMed PMC

Pak CW, Kosno M, Holehouse AS, Padrick SB, Mittal A, Ali R, Yunus AA, Liu DR, Pappu RV, Rosen MK. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell. 2016;63:72–85. PubMed PMC

Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, Stoynov S, Mahamid J, Saha S, Franzmann TM, et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 2015;162:1066–1077. PubMed

Rosenberg KJ, Ross JL, Feinstein HE, Feinstein SC, Israelachvili J. Complementary dimerization of microtubule-associated tau protein: implications for microtubule bundling and tau-mediated pathogenesis. Proc. Natl. Acad. Sci. USA. 2008;105:7445–7450. PubMed PMC

Saha S, Weber CA, Nousch M, Adame-Arana O, Hoege C, Hein MY, Osborne-Nishimura E, Mahamid J, Jahnel M, Jawerth L, et al. Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism. Cell. 2016;166:1572–1584. PubMed PMC

Sanchez AD, Feldman JL. Microtubule-organizing centers: from the centrosome to non-centrosomal sites. Curr. Opin. Cell Biol. 2017;44:93–101. PubMed PMC

Sánchez-Huertas C, Freixo F, Viais R, Lacasa C, Soriano E, Lüders J. Non-centrosomal nucleation mediated by augmin organizes micro-tubules in post-mitotic neurons and controls axonal microtubule polarity. Nat. Commun. 2016;7:12187. PubMed PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. PubMed PMC

Su X, Ditlev JA, Hui E, Xing W, Banjade S, Okrut J, King DS, Taunton J, Rosen MK, Vale RD. Phase separation of signaling molecules promotes T cell receptor signal transduction. Science. 2016;352:595–599. PubMed PMC

Takei Y, Teng J, Harada A, Hirokawa N. Defects in axonal elongation and neuronal migration in mice with disrupted tau and map1b genes. J. Cell Biol. 2000;150:989–1000. PubMed PMC

Tanaka EM, Kirschner MW. Microtubule behavior in the growth cones of living neurons during axon elongation. J. Cell Biol. 1991;115:345–363. PubMed PMC

Tortosa E, Kapitein LC, Hoogenraad CC. Microtubule organization and microtubule-associated proteins (MAPs) In: Emoto K, Wong R, Huang E, Hoogenraad C, editors. Dendrites. Springer; 2016. pp. 31–75.

Voter WA, Erickson HP. The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J. Biol. Chem. 1984;259:10430–10438. PubMed

Wieczorek M, Chaaban S, Brouhard GJ. Macromolecular crowding pushes catalyzed microtubule growth to near the theoretical limit. Cell. Mol. Bioeng. 2013;6:383–392.

Wieczorek M, Bechstedt S, Chaaban S, Brouhard GJ. Microtubule- associated proteins control the kinetics of microtubule nucleation. Nat. Cell Biol. 2015;17:907–916. PubMed

Woodruff JB, Ferreira Gomes B, Widlund PO, Mahamid J, Honigmann A, Hyman AA. The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin. Cell. 2017;169:1066–1077. PubMed

Zempel H, Mandelkow E. Lost after translation: missorting of Tau protein and consequences for Alzheimer disease. Trends Neurosci. 2014;37:721–732. PubMed

Zhang X, Lin Y, Eschmann NA, Zhou H, Rauch JN, Hernandez I, Guzman E, Kosik KS, Han S. RNA stores tau reversibly in complex coacervates. PLoS Biol. 2017;15:e2002183. PubMed PMC

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins

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