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Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours

Abstract

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule–MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid–liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

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Fig. 1: A pipeline to analyse microtubule–MAP assemblies in cell lysates.
Fig. 2: Systematic characterization of 45 MAP candidates.
Fig. 3: Unique activities of MAPs on growing microtubules.
Fig. 4: The effect of MAP–GFP concentrations on microtubule behaviour in lysates.
Fig. 5: The effect of a disease-related mutation of EML1 on microtubule binding.
Fig. 6: Structural analyses of microtubule–MAP assemblies in lysates using cryo-electron microscopy.
Fig. 7: Characterization of droplet formation by CLIP170.
Fig. 8: Generation of microtubules from CLIP170–tubulin co-condensates.

Data availability

The cryo-EM map for EML1–GFP bound to a microtubule has been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code EMD-32033. Gene sequences of newly identified MAPs (DCX_L, MAP11; Supplementary Table 1) have been deposited at GenBank under the accession codes OK539808 (DCX_L) and OK539809 (MAP11). Deposited data are listed in Supplementary Table 5. All commercial resources used here are detailed in Supplementary Table 3; primers for molecular cloning are listed in Supplementary Table 1; antibodies are listed in Supplementary Table 4. All data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Free software was used for most of the analyses and details of paid software are included in Supplementary Table 3. Home-made macros used in the analyses (Fig. 7d,g) have been deposited at Zenodo (https://doi.org/10.5281/zenodo.5648066).

References

  1. Hirokawa, N., Noda, Y., Tanaka, Y. & Niwa, S. Kinesin superfamily motor proteins and intracellular transport. Nat. Rev. Mol. Cell Biol. 10, 682–696 (2009).

    CAS  PubMed  Google Scholar 

  2. Veigel, C. & Schmidt, C. F. Moving into the cell: single-molecule studies of molecular motors in complex environments. Nat. Rev. Mol. Cell Biol. 12, 163–176 (2011).

    CAS  PubMed  Google Scholar 

  3. Roberts, A. J., Kon, T., Knight, P. J., Sutoh, K. & Burgess, S. A. Functions and mechanics of dynein motor proteins. Nat. Rev. Mol. Cell Biol. 14, 713–726 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. McNally, F. J. & Roll-Mecak, A. Microtubule-severing enzymes: from cellular functions to molecular mechanism. J. Cell Biol. 217, 4057–4069 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Akhmanova, A. & Steinmetz, M. O. Control of microtubule organization and dynamics: two ends in the limelight. Nat. Rev. Mol. Cell Biol. 16, 711–726 (2015).

    CAS  PubMed  Google Scholar 

  6. Olmsted, J. B. Microtubule-associated proteins. Annu. Rev. Cell Biol. 2, 421–457 (1986).

    CAS  PubMed  Google Scholar 

  7. Mandelkow, E. & Mandelkow, E.-M. Microtubules and microtubule-associated proteins. Curr. Opin. Cell Biol. 7, 72–81 (1995).

    CAS  PubMed  Google Scholar 

  8. Bodakuntla, S., Jijumon, A. S., Villablanca, C., Gonzalez-Billault, C. & Janke, C. Microtubule-associated proteins: structuring the cytoskeleton. Trends Cell Biol. 29, 804–819 (2019).

    CAS  PubMed  Google Scholar 

  9. Dogterom, M. & Surrey, T. Microtubule organization in vitro. Curr. Opin. Cell Biol. 25, 23–29 (2013).

    CAS  PubMed  Google Scholar 

  10. Monroy, B. Y. et al. A combinatorial MAP code dictates polarized microtubule transport. Dev. Cell 53, 60–72.e64 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hooikaas, P. J. et al. MAP7 family proteins regulate kinesin-1 recruitment and activation. J. Cell Biol. 218, 1298–1318 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Alfaro-Aco, R. & Petry, S. Building the microtubule cytoskeleton piece by piece. J. Biol. Chem. 290, 17154–17162 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Field, C. M., Pelletier, J. F. & Mitchison, T. J. Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis. Methods Cell. Biol. 137, 395–435 (2017).

    CAS  PubMed  Google Scholar 

  14. Bergman, Z. J., Wong, J., Drubin, D. G. & Barnes, G. Microtubule dynamics regulation reconstituted in budding yeast lysates. J. Cell Sci. 132, jcs.219386 (2018).

    Google Scholar 

  15. Soppina, V. et al. Dimerization of mammalian kinesin-3 motors results in superprocessive motion. Proc. Natl Acad. Sci. USA 111, 5562–5567 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sun, F., Zhu, C., Dixit, R. & Cavalli, V. Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility. EMBO J. 30, 3416–3429 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Cai, D., Verhey, K. J. & Meyhofer, E. Tracking single kinesin molecules in the cytoplasm of mammalian cells. Biophys. J. 92, 4137–4144 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ayloo, S. et al. Dynactin functions as both a dynamic tether and brake during dynein-driven motility. Nat. Commun. 5, 4807 (2014).

    CAS  PubMed  Google Scholar 

  19. Schimert, K. I., Budaitis, B. G., Reinemann, D. N., Lang, M. J. & Verhey, K. J. Intracellular cargo transport by single-headed kinesin motors. Proc. Natl Acad. Sci. USA 116, 6152–6161 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Budaitis, B. G. et al. Neck linker docking is critical for kinesin-1 force generation in cells but at a cost to motor speed and processivity. eLife 8, e44146 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Blasius, T. L. et al. Sequences in the stalk domain regulate auto-inhibition and ciliary tip localization of the immotile kinesin-4 KIF7. J. Cell Sci. 134, jcs258464 (2021).

    CAS  PubMed  Google Scholar 

  22. Pierre, P., Pepperkok, R. & Kreis, T. E. Molecular characterization of two functional domains of CLIP-170 in vivo. J. Cell Sci. 107, 1909–1920 (1994).

    CAS  PubMed  Google Scholar 

  23. Jeong, J.-Y. et al. One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl. Environ. Microbiol. 78, 5440–5443 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Cleveland, D. W. Autoregulated control of tubulin synthesis in animal cells. Curr. Opin. Cell Biol. 1, 10–14 (1989).

    CAS  PubMed  Google Scholar 

  25. Lin, Z. et al. TTC5 mediates autoregulation of tubulin via mRNA degradation. Science 367, 100–104 (2020).

    CAS  PubMed  Google Scholar 

  26. Hiller, G. & Weber, K. Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues. Cell 14, 795–804 (1978).

    CAS  PubMed  Google Scholar 

  27. Roll-Mecak, A. & Vale, R. D. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 451, 363–367 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Akhmanova, A. & Steinmetz, M. O. Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat. Rev. Mol. Cell Biol. 9, 309–322 (2008).

    CAS  PubMed  Google Scholar 

  29. Wieczorek, M., Bechstedt, S., Chaaban, S. & Brouhard, G. J. Microtubule-associated proteins control the kinetics of microtubule nucleation. Nat. Cell Biol. 17, 907–916 (2015).

    CAS  PubMed  Google Scholar 

  30. Brandt, R. & Lee, G. Functional organization of microtubule-associated protein tau. Identification of regions which affect microtubule growth, nucleation, and bundle formation in vitro. J. Biol. Chem. 268, 3414–3419 (1993).

    CAS  PubMed  Google Scholar 

  31. Spector, I., Shochet, N. R., Kashman, Y. & Groweiss, A. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 493–495 (1983).

    CAS  PubMed  Google Scholar 

  32. Coue, M., Brenner, S. L., Spector, I. & Korn, E. D. Inhibition of actin polymerization by latrunculin A. FEBS Lett. 213, 316–318 (1987).

    CAS  PubMed  Google Scholar 

  33. Eichenmüller, B., Everley, P., Palange, J., Lepley, D. & Suprenant, K. A. The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus. J. Biol. Chem. 277, 1301–1309 (2002).

    PubMed  Google Scholar 

  34. Bulinski, J. C. & Bossler, A. Purification and characterization of ensconsin, a novel microtubule stabilizing protein. J. Cell Sci. 107, 2839–2849 (1994).

    CAS  PubMed  Google Scholar 

  35. Metzger, T. et al. MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function. Nature 484, 120–124 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Yadav, S., Verma, P. J. & Panda, D. C-terminal region of MAP7 domain containing protein 3 (MAP7D3) promotes microtubule polymerization by binding at the C-terminal tail of tubulin. PLoS ONE 9, e99539 (2014).

    PubMed  PubMed Central  Google Scholar 

  37. Sung, H.-H. et al. Drosophila ensconsin promotes productive recruitment of kinesin-1 to microtubules. Dev. Cell 15, 866–876 (2008).

    CAS  PubMed  Google Scholar 

  38. Nédélec, F. J., Surrey, T., Maggs, A. C. & Leibler, S. Self-organization of microtubules and motors. Nature 389, 305–308 (1997).

    PubMed  Google Scholar 

  39. Backer, C. B., Gutzman, J. H., Pearson, C. G. & Cheeseman, I. M. CSAP localizes to polyglutamylated microtubules and promotes proper cilia function and zebrafish development. Mol. Biol. Cell 23, 2122–2130 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Bompard, G. et al. CSAP acts as a regulator of TTLL-mediated microtubule glutamylation. Cell Rep. 25, 2866–2877.e2865 (2018).

    CAS  PubMed  Google Scholar 

  41. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    CAS  PubMed  Google Scholar 

  42. Cuveillier, C. et al. MAP6 is an intraluminal protein that induces neuronal microtubules to coil. Sci. Adv. 6, eaaz4344 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Leung, C. L., Sun, D., Zheng, M., Knowles, D. R. & Liem, R. K. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J. Cell Biol. 147, 1275–1286 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, Y. et al. An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell 86, 655–665 (1996).

    CAS  PubMed  Google Scholar 

  45. Sun, D., Leung, C. L. & Liem, R. K. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins. J. Cell Sci. 114, 161–172 (2001).

    CAS  PubMed  Google Scholar 

  46. Matus, A. Microtubule-associated proteins: their potential role in determining neuronal morphology. Annu Rev Neurosci 11, 29–44 (1988).

    CAS  PubMed  Google Scholar 

  47. Kindler, S., Schulz, B., Goedert, M. & Garner, C. C. Molecular structure of microtubule-associated protein 2b and 2c from rat brain. J. Biol. Chem. 265, 19679–19684 (1990).

    CAS  PubMed  Google Scholar 

  48. Ludin, B., Ashbridge, K., Funfschilling, U. & Matus, A. Functional analysis of the MAP2 repeat domain. J. Cell Sci. 109, 91–99 (1996).

    CAS  PubMed  Google Scholar 

  49. Murphy, D. B. & Borisy, G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc. Natl Acad. Sci. USA 72, 2696–2700 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Bowne-Anderson, H., Hibbel, A. & Howard, J. Regulation of microtubule growth and catastrophe: unifying theory and experiment. Trends Cell Biol. 25, 769–779 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Roger, B., Al-Bassam, J., Dehmelt, L., Milligan, R. A. & Halpain, S. MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Curr. Biol. 14, 363–371 (2004).

    CAS  PubMed  Google Scholar 

  52. Sandoval, I. V. & Vandekerckhove, J. S. A comparative study of the in vitro polymerization of tubulin in the presence of the microtubule-associated proteins MAP2 and tau. J. Biol. Chem. 256, 8795–8800 (1981).

    CAS  PubMed  Google Scholar 

  53. Monroy, B. Y. et al. Competition between microtubule-associated proteins directs motor transport. Nat. Commun. 9, 1487 (2018).

    PubMed  PubMed Central  Google Scholar 

  54. Siahaan, V. et al. Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes. Nat. Cell Biol. 21, 1086–1092 (2019).

    CAS  PubMed  Google Scholar 

  55. Kielar, M. et al. Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human. Nat. Neurosci. 17, 923–933 (2014).

    CAS  PubMed  Google Scholar 

  56. Uzquiano, A. et al. Mutations in the heterotopia gene Eml1/EML1 severely disrupt the formation of primary cilia. Cell Rep. 28, 1596–1611.e1510 (2019).

    CAS  PubMed  Google Scholar 

  57. Kellogg, E. H. et al. Near-atomic model of microtubule–tau interactions. Science 360, 1242–1246 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Adib, R. et al. Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression. Sci. Signal. 12, aaw2939 (2019).

    Google Scholar 

  59. Manka, S. W. & Moores, C. A. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem. 62, 737–751 (2018).

    PubMed  PubMed Central  Google Scholar 

  60. Zhang, R., Roostalu, J., Surrey, T. & Nogales, E. Structural insight into TPX2-stimulated microtubule assembly. eLife 6, e30959 (2017).

    PubMed  PubMed Central  Google Scholar 

  61. Su, C.-C. et al. A ‘build and retrieve’ methodology to simultaneously solve cryo-EM structures of membrane proteins. Nat. Methods 18, 69–75 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Perez, F., Diamantopoulos, G. S., Stalder, R. & Kreis, T. E. CLIP-170 highlights growing microtubule ends in vivo. Cell 96, 517–527 (1999).

    CAS  PubMed  Google Scholar 

  63. Bieling, P. et al. Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105 (2007).

    CAS  PubMed  Google Scholar 

  64. Bieling, P. et al. CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Pierre, P., Scheel, J., Rickard, J. E. & Kreis, T. E. CLIP-170 links endocytic vesicles to microtubules. Cell 70, 887–900 (1992).

    CAS  PubMed  Google Scholar 

  66. Wu, Y.-F.O. et al. Overexpression of the microtubule-binding protein CLIP-170 induces a +TIP network superstructure consistent with a biomolecular condensate. PLoS ONE 16, e0260401 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Tan, R. et al. Microtubules gate tau condensation to spatially regulate microtubule functions. Nat. Cell Biol. 21, 1078–1085 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. King, M. R. & Petry, S. Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation. Nat. Commun. 11, 270 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Steigemann, P. et al. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484 (2009).

    PubMed  Google Scholar 

  71. Roux, K. J., Kim, D. I., Burke, B. & May, D. G. BioID: a screen for protein–protein interactions. Curr. Protoc. Protein Sci. 91, 19.23.11–19.23.15 (2018).

    Google Scholar 

  72. Ben-Sasson, A. J. et al. Design of biologically active binary protein 2D materials. Nature 589, 468–473 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Dogterom, M. & Koenderink, G. H. Actin–microtubule crosstalk in cell biology. Nat. Rev. Mol. Cell Biol. 20, 38–54 (2019).

    CAS  PubMed  Google Scholar 

  74. Gentili, M. et al. Transmission of innate immune signaling by packaging of cGAMP in viral particles. Science 349, 1232–1236 (2015).

    CAS  PubMed  Google Scholar 

  75. Alexopoulou, A. N., Couchman, J. R. & Whiteford, J. R. The CMV early enhancer/chicken β actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors. BMC Cell Biol. 9, 2 (2008).

    PubMed  PubMed Central  Google Scholar 

  76. Magiera, M. M. & Janke, C. in Methods Cell Biol, Vol. 115, Edn 2013/08/27 (eds Correia, J. J. & Wilson, L.) 247–267 (Academic Press, 2013).

  77. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Smith, P. K. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85 (1985).

    CAS  PubMed  Google Scholar 

  79. Brady, P. N. & Macnaughtan, M. A. Evaluation of colorimetric assays for analyzing reductively methylated proteins: biases and mechanistic insights. Anal. Biochem. 491, 43–51 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Bodakuntla, S., Jijumon, A. S., Janke, C. & Magiera, M. M. Purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization–depolymerization cycles. J. Vis. Exp. https://doi.org/10.3791/61826 (2020).

  81. Gell, C. et al. Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods Cell. Biol. 95, 221–245 (2010).

    CAS  PubMed  Google Scholar 

  82. Li, W. et al. Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin. J. Cell Biol. 199, 849–862 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Castoldi, M. & Popov, A. V. Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88 (2003).

    CAS  PubMed  Google Scholar 

  84. Vallee, R. B. Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments. Methods Enzymol. 134, 89–104 (1986).

    CAS  PubMed  Google Scholar 

  85. Nakata, T. & Hirokawa, N. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport. J. Cell Biol. 131, 1039–1053 (1995).

    CAS  PubMed  Google Scholar 

  86. Lacroix, B. et al. Tubulin polyglutamylation stimulates spastin-mediated microtubule severing. J. Cell Biol. 189, 945–954 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Dutta, P. et al. Presence of actin binding motif in VgrG-1 toxin of Vibrio cholerae reveals the molecular mechanism of actin cross-linking. Int. J. Biol. Macromol. 133, 775–785 (2019).

    CAS  PubMed  Google Scholar 

  88. Souphron, J. et al. Purification of tubulin with controlled post-translational modifications by polymerization–depolymerization cycles. Nat. Protoc. 14, 1634–1660 (2019).

    CAS  PubMed  Google Scholar 

  89. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Scheres, S. H. W. A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406–418 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. He, S. & Scheres, S. H. W. Helical reconstruction in RELION. J. Struct. Biol. 198, 163–176 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Cook, A. D., Manka, S. W., Wang, S., Moores, C. A. & Atherton, J. A microtubule RELION-based pipeline for cryo-EM image processing. J. Struct. Biol. 209, 107402 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  Google Scholar 

  96. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).

    CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the ANR-10-IDEX-0001-02, the LabEx Cell’n’Scale ANR-11-LBX-0038 and the Institut de convergence Q-life ANR-17-CONV-0005. C.J. is supported by the Institut Curie, the French National Research Agency (ANR) awards ANR-12-BSV2-0007 and ANR-17-CE13-0021, the Institut National du Cancer (INCA) grant 2014-PL BIO-11-ICR-1, and the Fondation pour la Recherche Medicale (FRM) grant DEQ20170336756. A.S.J. was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 675737, and the FRM grant FDT201904008210. S.B. was supported by the FRM grant FDT201805005465. M.S. acknowledges funding support from DBT/Wellcome Trust India Alliance Intermediate Fellowship (IA/I/14/2/501533), EMBO Young Investigator Programme award, CEFIPRA (5703-1), Department of Science and Technology, SERB-EMR grant (CRG/2019/003246) and DBT-BIRAC (BT/PR40389/COT/142/6/2020). The authors acknowledge the National Cryo-EM Facility at Bangalore Life Science Cluster, Bangalore, India and the funding by B-life grant from Department of Biotechnology (DBT/PR12422/MED/31/287/2014). M.M.M. is supported by the Fondation Vaincre Alzheimer grant FR-16055p. We thank the team of T. Surrey for technical advice and training; L. Kainka, F. Lautenschläger and G. M. Montalvo Bereau for experimental support; S. Citi, F. Francis, D. Gerlich, N. Manel, and C. Nahmias for providing essential reagents; C. Messaoudi, M.-N. Soler and C. Lovo from the Multimodal Imaging Center (MIC; CNRS UMR2016 / Inserm US43) for support with imaging and image analyses; K. R. Vinothkumar for help with EM data collection and deposition of the maps; and T. Müller-Reichert and P. Tran for insightful discussions and advice.

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Authors and Affiliations

Authors

Contributions

A.S.J. established the lysate-based pipeline and performed all experiments related to it. Lysate experiments shown in Extended Data Fig. 10 and Supplementary Video 19 were performed by M.G. S.B., F.M. and V.H. performed molecular cloning and sequence analyses with the help of J.A.S. and M.G. S.B. performed all cell biology experiments. S.B. and V.S. performed live-cell imaging experiments. M.G. purified proteins and established quantitative approaches with A.S.J. M.B. performed electron microscopy studies and analyses. A.S.J. and L.B. analysed and quantified imaging data with the help of S.B. A.S.J., S.B. and C.J. analysed data and prepared figures and videos with the help of M.B. and L.B. M.S. supervised electron microscopy studies and analyses. C.J. supervised the study with the help of M.M.M. A.S.J., S.B., M.M.M., M.S. and C.J. acquired funding. A.S.J., S.B. and C.J. wrote the manuscript with the help of M.G., M.B., M.M.M. and M.S.

Corresponding author

Correspondence to Carsten Janke.

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The authors declare no competing interests.

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Nature Cell Biology thanks Eva Nogales and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Schematic representation of the SLIC cloning approach (complement to Fig. 1).

a, Schematic representation (SnapGene® map) of the principal cloning vector used in this study, pTRIP, which is designed for the generation of lentivirus74, but can also be used to transiently transfect cells as done in our study. Zoom images show two possible insertion points of coding sequence for either amino- or carboxy-terminal tagging with GFP. Primer overhang sequences shown here were completed with the forward and reverse sequences from the open reading frame of the coding sequence (see Supplementary Table 1). Note that the P2A site after the GFP sequence is discarded when MAPs are cloned with an amino-terminal GFP tag. A similar vector with mCherry instead of GFP was used for experiments in which two MAPs are visualised concomitantly. b, Schematic representation of the SLIC cloning procedure23. Step 1: amplification of open reading frames of proteins of interest; digestion of plasmid vector with appropriate enzymes for amino- or carboxy-terminal tagging (a). Step 2: treatment of plasmid and PCR product with T4 polymerase without dNTPs. Under these conditions, the 3’ exonuclease activity of the enzyme generates single-strand overhangs on both, PCR product and plasmid. Step 3: transformation into competent E. coli cells, which will fuse PCR product and plasmid using endogenous DNA repair mechanism23. Step 4: plasmid DNA from single bacteria colonies is extracted and verified by enzymatic digestion and subsequent sequencing. We identified alternative splice isoforms for: DCLK1, MAP2, MAP4, SNPH, Tau, and so-far non-described isoforms for DCX (called here DCX_L) and MAP11. Accession numbers, ORF size, position of the fluorescent tag, and PCR primers are listed in Supplementary Table 1, and are also schematically represented in Fig. 2c.

Extended Data Fig. 2 Systematic characterisation of the intracellular behaviour of 45 MAP candidates (complement to Fig. 2a,c).

Representation of all 45 GFP-tagged MAPs, or presumed MAPs (Supplementary Table 1), expressed in U-2 OS cells. MAPs with strong expression levels were fixed after 24 h for immunostaining, while weakly expressed MAPs were fixed after 48 h. Cells were stained with anti-α-tubulin antibody 12G10. Separate images for tubulin and the GFP are shown in Supplementary Fig. 1. All proteins analysed here were classified into three distinct categories (Fig. 2a,c). Details of the experimental repeats and number of cells are provided as source data and in the Statistics & Reproducibility section. Numerical data are available in source data.

Source data

Extended Data Fig. 3 Characterisation of the behaviour of selected GFP-MAPs with growing microtubules in cell lysates (complement to Fig. 2b,c; see also Supplementary Fig. 2; Supplementary Videos 2a,3d,7b,c).

Lysates of HEK 293 cells expressing GFP-tagged MAP candidates: a, control, b, CSAP, c, MACF1(C1023), d, MACF2(C1079). Frames from videos observed by TIRF microscopy to visualise the dynamic decoration of growing microtubules by GFP-MAPs. Microtubule (MT) seeds are shown in green and GFP-MAPs in white (left panels). At the end of the time-lapse imaging (12 min), microtubules and actin are visualised with YL1/2 antibody and SiR-actin, respectively, allowing to determine whether a MAP associates with microtubules alone, or co-aligns microtubules and actin fibres in the lysate (right panels). All experiments were performed under equivalent conditions. To determine whether actin polymerisation played a role in the observed microtubule-MAP interactions, all experiments were performed using the same lysates and equivalent conditions in the presence of 11 µM of Latrunculin A to prevent actin polymerisation31,32. Details of the experimental repeats are provided as source data and in the Statistics & Reproducibility section. Numerical data are available in source data.

Source data

Extended Data Fig. 4 Characterisation of the behaviour of selected GFP-MAPs with growing microtubules in cell lysates (complement to Fig. 2c; see also Supplementary Fig. 2, Supplementary Video. 5).

Lysates of HEK 293 cells expressing GFP-tagged MAP candidates: a, EML1, b, EML2, c, EML3, d, EML4. Frames from videos observed by TIRF microscopy to visualise the dynamic decoration of growing microtubules by GFP-MAPs. Microtubule (MT) seeds are shown in green and GFP-MAPs in white (left panels). At the end of the time-lapse imaging (12 min), microtubules and actin are visualised with YL1/2 antibody and SiR-actin, respectively, allowing to determine whether a MAP associates with microtubules alone, or co-aligns microtubules and actin fibres in the lysate (right panels). All experiments were performed under equivalent conditions. To determine whether actin polymerisation played a role in the observed microtubule-MAP interactions, all experiments were performed using the same lysates and equivalent conditions in the presence of 11 µM of Latrunculin A to prevent actin polymerisation31,32. Details of the experimental repeats are provided as source data and in the Statistics & Reproducibility section. Numerical data are available in source data.

Source data

Extended Data Fig. 5 Characterisation of the behaviour of selected GFP-MAPs with growing microtubules in cell lysates (complement to Fig. 2c; see also Supplementary Fig. 2; Supplementary Video 9).

Lysates of HEK 293 cells expressing GFP-tagged MAP candidates: a, MAP7, b, MAP7D1, c, MAP7D2, d, MAP7D3. Frames from videos observed by TIRF microscopy to visualise the dynamic decoration of growing microtubules by GFP-MAPs. Microtubule (MT) seeds are shown in green and GFP-MAPs in white (left panels). At the end of the time-lapse imaging (12 min), microtubules and actin are visualised with YL1/2 antibody and SiR-actin, respectively, allowing to determine whether a MAP associates with microtubules alone, or co-aligns microtubules and actin fibres in the lysate (right panels). All experiments were performed under equivalent conditions. To determine whether actin polymerisation played a role in the observed microtubule-MAP interactions, all experiments were performed using the same lysates and equivalent conditions in the presence of 11 µM of Latrunculin A to prevent actin polymerisation31,32. Details of the experimental repeats are provided as source data and in the Statistics & Reproducibility section. Numerical data are available in source data.

Source data

Extended Data Fig. 6 Characterisation of the behaviour of selected GFP-MAPs with growing microtubules in cell lysates (complement to Fig. 2c; see also Supplementary Fig. 2; Supplementary Video 7d,8a,12c,d).

Lysates of HEK 293 cells expressing GFP-tagged MAP candidates: a, MAP2C, b, MAP2D, c, Tau(0N-3R), d, Tau(0N-4R). Frames from videos observed by TIRF microscopy to visualise the dynamic decoration of growing microtubules by GFP-MAPs. Microtubule (MT) seeds are shown in green and GFP-MAPs in white (left panels). At the end of the time-lapse imaging (12 min), microtubules and actin are visualised with YL1/2 antibody and SiR-actin, respectively, allowing to determine whether a MAP associates with microtubules alone, or co-aligns microtubules and actin fibres in the lysate (right panels). All experiments were performed under equivalent conditions. To determine whether actin polymerisation played a role in the observed microtubule-MAP interactions, all experiments were performed using the same lysates and equivalent conditions in the presence of 11 µM of Latrunculin A to prevent actin polymerisation31,32. Details of the experimental repeats are provided as source data and in the Statistics & Reproducibility section. Numerical data are available in source data.

Source data

Extended Data Fig. 7 Quantification of unique activities of MAPs on growing microtubules (complement to Fig. 3).

a, Inverted grey-scale TIRF images of CSAP-GFP-decorated microtubules, showing that CSAP induces microtubule helices / coils, as well as straight microtubules. Red dashes in the images show where the measurements of the width of the microtubule coils were taken. The diagram (also shown in Fig. 3c) shows a plot of all 41 measurements, with a mean coil width of 872 nm (± s.d.: 125 nm). The quantification was performed from n = 4 independent TIRF assays using 3 independent cell lysate preparations. Data are presented as scatter plots with mean ± s.d.. b, Inverted grey-scale TIRF images of MACF1(C1023)-GFP-decorated microtubules, showing the induction of microtubule hooks. Two red arrow heads show where individual measurements of the diameters of the microtubule hooks were taken. The plot (also shown in Fig. 3d) shows all 136 measurements, with a mean hook diameter of 1.025 µm (± s.d.: 0.15 µm). The quantification was performed from n = 3 independent TIRF assays. Data are presented as scatter plots with mean ± s.d.. c, Inverted grey-scale TIRF images of two zoomed time series of GFP-MAP2C. In the first time series, GFP-MAP2C reveals the formation of transitional hooks that were fainter than the microtubules, suggesting that they could consist of some protofilaments decorated by GFP-MAP2C. These filaments appeared to initiate the formation of a GFP-MAP2C cluster. The cluster remains attached to the microtubule (MT), which subsequently continues growing. In the second series, a hook-like structure at the growing microtubule (MT) end is converted into a GFP-MAP2C cluster, which however arrests microtubule growth. Note that the fluorescence intensity of both GFP-MAP2C clusters increases continuously, suggesting an accumulation of GFP-MAP2C. The time-lapse shown here is a representative experiment from n = 7 independent TIRF assays using 4 independent set of cell lysates. Numerical data are available in source data.

Source data

Extended Data Fig. 8 The impact of a disease-related mutation of EML1 on microtubule binding (complement to Fig. 5; Supplementary Video 16b).

Time-lapse images of microtubules polymerising in a 1:1 mixture of lysates from HEK 293 cells expressing wild-type EML1-mCherry and EML1(T243A)-GFP, respectively. Note that the behaviour of wild-type and mutated EML1 is similar to the observations in (Fig. 5b) despite the inversion of the fluorescence tags. N = 3 independent TIRF assays from one set of cell lysate preparations. Numerical data are available in source data.

Source data

Extended Data Fig. 9 Characterisation of concentration-dependence of CLIP170 droplet formation (complement to Fig. 7).

a, Formation of GFP-CLIP170 droplets in lysates of HEK 293 cells at three different concentrations. Images shown are 10 min after the start of the experiment. Red lines indicate microtubules on which CLIP170 droplets were quantified (b; Fig. 7g,h). White rectangles indicate image sections shown in Fig. 7f. N = 3 independent TIRF assays from 2 sets of independent cell lysate preparation. b, Line scans of GFP-CLIP170 intensity profiles. Numbers indicate the identity of the scanned microtubule in (a). Note that the intensity profiles were only used to determine local maxima, from which distances between maxima were measured and plotted in Fig. 7h. The overall fluorescence intensity of the profiles was thus not considered for calculating droplet spacing. Numerical data are available in source data.

Source data

Extended Data Fig. 10 Characterisation of CLIP170 droplet behaviour in the presence of 1,6-hexanediol (complement to Fig. 7; Supplementary Video 19).

a, Formation of GFP-CLIP170 droplets (white) in lysates with polymerising microtubules. GMPCPP seeds are shown in green. N = 2 independent TIRF assays from 2 sets of independent cell lysate preparation. b, Development of GFP-CLIP170 droplets formed in (a) after the addition of 5% and 10% 1,6-hexanediol (w/v). Note that droplets do not dissolve at any of the tested conditions. BRB80 buffer was used as a control. N = 2 independent TIRF assays from 2 sets of independent cell lysate preparation. Numerical data are available in source data.

Source data

Supplementary information

Supplementary Information

The supplementary information pdf file contains legends, Supplementary Figs. 1, 2, 3, and references for all supplementary material including tables (which are provided as Excel files).

Reporting Summary

Peer Review Information

Supplementary Tables 1–5

Supplementary Video 1

Spastin and EB3 behaviour in cell lysates (corresponding to Fig. 1b,c). a, Spastin–GFP from cell lysates severs taxol-stabilized microtubules (green). See still images in Fig. 1b. b, EB3–GFP (white) from cell lysates tracks growing microtubule ends. GMPCPP seeds are shown in green. Note the enrichment of EB3–GFP at a growing microtubule tip. Still images are shown in Fig. 1c.

Supplementary Video 2

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 3a and Supplementary Fig. 2a). Untransfected control lysates (a), GFP-tagged ATIP3 (b), CCDC66 (c) and CFAP (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 3a for a, Supplementary Fig. 2a), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 3a and Supplementary Fig. 2a.

Supplementary Video 3

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 3b, Supplementary Fig. 2b). GFP-tagged Cingulin (a), CKAP2 (b), CKAP2L (c) and CSAP (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 3b for d, Supplementary Fig. 2b), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 3b and Supplementary Fig. 2b.

Supplementary Video 4

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2b,c, Supplementary Fig. 2c). GFP-tagged DCLK1_4 (a), DCLK1_5 (b), DCX (c) and DCX_L (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Fig. 2b for c, Supplementary Fig. 2c), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Fig. 2b and Supplementary Fig. 2c.

Supplementary Video 5

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 4, Supplementary Fig. 2d). GFP-tagged EML1 (a), EML2 (b), EML3 (c) and EML4 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 4, Supplementary Fig. 2d), while in the lower row, actin polymerisation in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 4 and Supplementary Fig. 2d.

Supplementary Video 6

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2b,c, Supplementary Fig. 2e). GFP-tagged GLFND1 (a), GLFND2 (b), HICE1 (c) and JPL1 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Fig. 2b for d, Supplementary Fig. 2e), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Fig. 2b and Supplementary Fig. 2e.

Supplementary Video 7

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2b,c, Extended Data Figs. 3c,d,6a, Supplementary Fig. 2f). GFP-tagged JPL2 (a), MACF1(C1023) (b), MACF2(C1079) (c) and MAP2C (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Fig. 2b for b, Extended Data Fig. 3c,d for b,c, Extended Data Fig. 6a for d, Supplementary Fig. 2f), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Fig. 2b, Extended Data Figs. 3c,d,6a and Supplementary Fig. 2f.

Supplementary Video 8

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 6b, Supplementary Fig. 2g). GFP-tagged MAP2D (a), MAP4_X16 (b), MAP4_X20 (c) and MAP6 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 6b for a, Supplementary Fig. 2g), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 6b and Supplementary Fig. 2g.

Supplementary Video 9

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 5, Supplementary Fig. 2h). GFP-tagged MAP7 (a), MAP7D1 (b), MAP7D2 (c) and MAP7D3 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 5, Supplementary Fig. 2h), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 5 and Supplementary Fig. 2h.

Supplementary Video 10

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Supplementary Fig. 2i). GFP-tagged MAP8 (a), MAP9 (b), MAP10 (c) and MAP11 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown Supplementary Fig. 2i), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Supplementary Fig. 2i.

Supplementary Video 11

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Supplementary Fig. 2j). GFP-tagged Parkin (a), PRC1 (b), SAXO1 (c) and SPEF1 (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown Supplementary Fig. 2j), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Supplementary Fig. 2j.

Supplementary Video 12

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Extended Data Fig. 6c,d, Supplementary Fig. 2k). GFP-tagged SNPH_A (a), SNPH_C (b), Tau(0N-3R) (c) and Tau(0N-4R) (d) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown in Extended Data Fig. 6c,d for c,d, Supplementary Fig. 2k), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Extended Data Fig. 6c,d and Supplementary Fig. 2k.

Supplementary Video 13

Characterization of MAP candidates with growing microtubules in cell lysates (corresponding to Fig. 2c, Supplementary Fig. 2l). GFP-tagged TRAK1 (a) and TRAK2 (b) in lysates with growing microtubules. GFP-tagged MAPs are shown in white, GMPCPP seeds are in green. In the upper row, actin can polymerize (visualized by actin staining shown Supplementary Fig. 2l), while in the lower row, actin polymerization in the lysates was prevented by the addition of 11 µM of latrunculin A. Still images of all videos are shown in Supplementary Fig. 2l.

Supplementary Video 14

Unique activities of MAPs on growing microtubules (corresponding to Fig. 3a). Representative videos of selected MAPs inducing unique microtubule behaviour in lysates. MAPs are false-colour-coded. MAP7 (cyan) generated microtubule asters. CSAP (blue) induced formation of microtubule coils. MACF1(C1023) (green) formed hooks at growing microtubule ends. MAP2C (red) induced the formation of temporary hooks along microtubules and at growing microtubule ends, which further accumulated MAP2C to form droplet-like clusters. Still images of all videos are shown in Fig. 3a.

Supplementary Video 15

The impact of MAP–GFP concentration on microtubule behaviour in lysates (corresponding to Fig. 4b). The impact of MACF1(C1023)–GFP concentrations on microtubule hook formation was determined in lysates with four defined concentrations of this MAP. TIRF videos of MACF1(C1023)–GFP (GFP shown in inverted greyscale) show that hook-formation propensity decreases with decreasing MACF1(C1023)–GFP concentrations. Still images of all videos are shown in Fig. 4b.

Supplementary Video 16

The impact of a disease-related mutation of EML1 on microtubule binding (corresponding to Fig. 5, Extended Data Fig. 8). a, Microtubules polymerizing in a 1:1 mixture of lysates from HEK 293 cells expressing wild-type EML1 and EML1(T243A), respectively. microtubule seeds are visualized in red together with EML1(T243A)–mCherry, and wild-type EML1–GFP in green. b, Microtubules polymerising in a 1:1 mixture of lysates from HEK 293 cells expressing wild-type EML1–mCherry and EML1(T243A)–GFP, respectively. Note that the behaviour of wild-type and mutated EML1 is similar to the observations in a despite the inversion of the fluorescence tags. Still images of all videos are shown in Fig. 5b and Extended Data Fig. 8.

Supplementary Video 17

Helical reconstructions of EML1–GFP and EML4(N207) bound to microtubules (corresponding to Fig. 6c). Animated views of a symmetrized helical reconstruction (resolution 3.7 Å, low-pass filtered to 15 Å) of 14-protofilament microtubules (grey) with extra densities corresponding to EML1–GFP (green). Comparison with a similar helical reconstruction (EMDB 0331; resolution 3.6 Å, low-pass filtered to 15 Å) obtained for 13-protofilament microtubules (polymerized from brain tubulin) with purified EML4(N207) (red; Adib et al., 2019). Note that both MAPs bind along the microtubule protofilament ridge. Still images of all videos are shown in Fig. 6c.

Supplementary Video 18

Droplet formation by CLIP170 (corresponding to Fig. 7,8). a, A video showing the accumulation of overexpressed GFP–CLIP170 in a U-2 OS cell. The cell contours are visualized by bright-field microscopy. Note that initially small droplets are formed, which are later fusing. Note also the constant deformation of the GFP patches. Still images of this video are shown in Fig. 7a. b, Time-lapse of a TIRF assay with lysate of HEK 293 cells expressing GFP–CLIP170 (white). GFP–CLIP170 is forming regularly spaced droplets along the microtubules. The evolution of these droplets along one microtubule (orange line) is plotted as profiles of relative GFP intensity. Still images of these videos are shown in Fig. 7c. c, Microtubules grown in lysates of HeLa Kyoto cells stably expressing mCherry-α-tubulin (green), and transfected with GFP–CLIP170 (white). This video was recorded after GFP–CLIP170 droplets had formed and the new cell lysate with only mCherry-α-tubulin was added. Still images of this video are shown in Fig. 8c. d, At the end of the time-lapse imaging (c), microtubules were stained with the antibody YL1/2 for better visualization. Note that microtubules have grown almost exclusively from GFP–CLIP170 droplets (image is also shown in Fig. 8d).

Supplementary Video 19

Characterization of CLIP170 droplet behaviour in the presence of 1,6-hexanediol (complement to Extended Data Fig. 10). a, Formation of GFP–CLIP170 droplets (white) in lysates with polymerising microtubules. GMPCPP seeds are shown in green. b, Development of GFP–CLIP170 droplets formed in a after the addition of 5% and 10% 1,6-hexanediol (w/v). Note that droplets do not dissolve at any of the tested conditions. BRB80 buffer was used as a control.

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Original Coomassie brilliant blue stained gel used for Fig. 4a with visible molecular weight markers and full-size immunoblot as in Fig. 4a. An additional immunoblot with visible light shows molecular weight markers (red boxes show the sections of the gels or blots shown in the main figure).

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Jijumon, A.S., Bodakuntla, S., Genova, M. et al. Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours. Nat Cell Biol 24, 253–267 (2022). https://doi.org/10.1038/s41556-021-00825-4

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