Tubulin posttranslational modifications have been predicted to control cytoskeletal functions by coordinating the molecular interactions between microtubules and their associating proteins. A prominent tubulin modification in neurons is polyglutamylation, the deregulation of which causes neurodegeneration. Yet, the underlying molecular mechanisms have remained elusive. Here, using in-vitro reconstitution, we determine how polyglutamylation generated by the two predominant neuronal polyglutamylases, TTLL1 and TTLL7, specifically modulates the activities of three major microtubule interactors: the microtubule-associated protein Tau, the microtubule-severing enzyme katanin and the molecular motor kinesin-1. We demonstrate that the unique modification patterns generated by TTLL1 and TTLL7 differentially impact those three effector proteins, thus allowing for their selective regulation. Given that our experiments were performed with brain tubulin from mouse models in which physiological levels and patterns of polyglutamylation were altered by the genetic knockout of the main modifying enzymes, our quantitative measurements provide direct mechanistic insight into how polyglutamylation could selectively control microtubule interactions in neurons.

Institut Curie Université PSL CNRS UMR3348 Orsay France

Institute of Biotechnology Czech Academy of Sciences BIOCEV Prague West Czech Republic

Université Paris Saclay CNRS UMR3348 Orsay France

Zobrazit více v PubMed

Alexander JE, Hunt DF, Lee MK, Shabanowitz J, Michel H, Berlin SC, MacDonald TL, Sundberg RJ, Rebhun LI, Frankfurter A (1991) Characterization of posttranslational modifications in neuron‐specific class III beta‐tubulin by mass spectrometry. Proc Natl Acad Sci USA 88: 4685–4689 PubMed PMC

Audebert S, Desbruyeres E, Gruszczynski C, Koulakoff A, Gros F, Denoulet P, Eddé B (1993) Reversible polyglutamylation of alpha‐ and beta‐tubulin and microtubule dynamics in mouse brain neurons. Mol Biol Cell 4: 615–626 PubMed PMC

Bodakuntla S, Jijumon AS, Janke C, Magiera MM (2020a) Purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization‐Depolymerization cycles. J Vis Exp 10.3791/61826 PubMed DOI

Bodakuntla S, Schnitzler A, Villablanca C, Gonzalez‐Billault C, Bieche I, Janke C, Magiera MM (2020b) Tubulin polyglutamylation is a general traffic‐control mechanism in hippocampal neurons. J Cell Sci 133: jcs241802 PubMed

Bodakuntla S, Yuan X, Genova M, Gadadhar S, Leboucher S, Birling M‐C, Klein D, Martini R, Janke C, Magiera MM (2021) Distinct roles of alpha‐ and beta‐tubulin polyglutamylation in controlling axonal transport and in neurodegeneration. EMBO J 40: e108498 PubMed PMC

Branham K, Matsui H, Biswas P, Guru AA, Hicks M, Suk JJ, Li H, Jakubosky D, Long T, Telenti A et al (2016) Establishing the involvement of the novel gene AGBL5 in retinitis pigmentosa by whole genome sequencing. Physiol Genomics 48: 922–927 PubMed PMC

Braun M, Lansky Z, Fink G, Ruhnow F, Diez S, Janson ME (2011) Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart. Nat Cell Biol 13: 1259–1264 PubMed

Castoldi M, Popov AV (2003) Purification of brain tubulin through two cycles of polymerization‐depolymerization in a high‐molarity buffer. Protein Expr Purif 32: 83–88 PubMed

Chang C‐W, Shao E, Mucke L (2021) Tau: enabler of diverse brain disorders and target of rapidly evolving therapeutic strategies. Science 371: eabb8255 PubMed PMC

Coy DL, Hancock WO, Wagenbach M, Howard J (1999) Kinesin's tail domain is an inhibitory regulator of the motor domain. Nat Cell Biol 1: 288–292 PubMed

Eddé B, Rossier J, Le Caer JP, Desbruyeres E, Gros F, Denoulet P (1990) Posttranslational glutamylation of alpha‐tubulin. Science 247: 83–85 PubMed

Eshun‐Wilson L, Zhang R, Portran D, Nachury MV, Toso DB, Lohr T, Vendruscolo M, Bonomi M, Fraser JS, Nogales E (2019) Effects of alpha‐tubulin acetylation on microtubule structure and stability. Proc Natl Acad Sci USA 116: 10366–10371 PubMed PMC

Gadadhar S, Alvarez Viar G, Hansen JN, Gong A, Kostarev A, Ialy‐Radio C, Leboucher S, Whitfield M, Ziyyat A, Toure A et al (2021) Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility. Science 371: eabd4914 PubMed PMC

Gigant B, Wang W, Dreier B, Jiang Q, Pecqueur L, Pluckthun A, Wang C, Knossow M (2013) Structure of a kinesin‐tubulin complex and implications for kinesin motility. Nat Struct Mol Biol 20: 1001–1007 PubMed

Gilmore‐Hall S, Kuo J, Ward JM, Zahra R, Morrison RS, Perkins G, La Spada AR (2019) CCP1 promotes mitochondrial fusion and motility to prevent Purkinje cell neuron loss in pcd mice. J Cell Biol 218: 206–219 PubMed PMC

Giordano T, Gadadhar S, Bodakuntla S, Straub J, Leboucher S, Martinez G, Chemlali W, Bosc C, Andrieux A, Bieche I et al (2019) Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility. J Cell Sci 132: jcs226951 PubMed

Goedert M, Crowther RA, Garner CC (1991) Molecular characterization of microtubule‐associated proteins tau and MAP2. Trends Neurosci 14: 193–199 PubMed

Goedert M, Eisenberg DS, Crowther RA (2017) Propagation of tau aggregates and neurodegeneration. Annu Rev Neurosci 40: 189–210 PubMed

Henrichs V, Grycova L, Barinka C, Nahacka Z, Neuzil J, Diez S, Rohlena J, Braun M, Lansky Z (2020) Mitochondria‐adaptor TRAK1 promotes kinesin‐1 driven transport in crowded environments. Nat Commun 11: 3123 PubMed PMC

Hoenger A, Thormahlen M, Diaz‐Avalos R, Doerhoefer M, Goldie KN, Müller J, Mandelkow E (2000) A new look at the microtubule binding patterns of dimeric kinesins. J Mol Biol 297: 1087–1103 PubMed

Ikegami K, Mukai M, Tsuchida J‐i, Heier RL, Macgregor GR, Setou M (2006) TTLL7 is a mammalian beta‐tubulin polyglutamylase required for growth of MAP2‐positive neurites. J Biol Chem 281: 30707–30716 PubMed PMC

Janke C (2014) The tubulin code: molecular components, readout mechanisms, and functions. J Cell Biol 206: 461–472 PubMed PMC

Janke C, Magiera MM (2020) The tubulin code and its role in controlling microtubule properties and functions. Nat Rev Mol Cell Biol 21: 307–326 PubMed

Janke C, Rogowski K, Wloga D, Regnard C, Kajava AV, Strub J‐M, Temurak N, van Dijk J, Boucher D, van Dorsselaer A et al (2005) Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308: 1758–1762 PubMed

Jiang K, Rezabkova L, Hua S, Liu Q, Capitani G, Altelaar AFM, Heck AJR, Kammerer RA, Steinmetz MO, Akhmanova A (2017) Microtubule minus‐end regulation at spindle poles by an ASPM‐katanin complex. Nat Cell Biol 19: 480–492 PubMed PMC

Kalebic N, Sorrentino S, Perlas E, Bolasco G, Martinez C, Heppenstall PA (2013) alphaTAT1 is the major alpha‐tubulin acetyltransferase in mice. Nat Commun 4: 1962 PubMed

Kastner S, Thiemann I‐J, Dekomien G, Petrasch‐Parwez E, Schreiber S, Akkad DA, Gerding WM, Hoffjan S, Gunes S, Gunes S et al (2015) Exome sequencing reveals AGBL5 as novel candidate gene and Additional variants for retinitis Pigmentosa in five Turkish families. Invest Ophthalmol Vis Sci 56: 8045–8053 PubMed

Kaul N, Soppina V, Verhey KJ (2014) Effects of alpha‐tubulin K40 acetylation and Detyrosination on Kinesin‐1 motility in a purified system. Biophys J 106: 2636–2643 PubMed PMC

Kellogg EH, Hejab NMA, Poepsel S, Downing KH, DiMaio F, Nogales E (2018) Near‐atomic model of microtubule‐tau interactions. Science 360: 1242–1246 PubMed PMC

Kuo Y‐W, Howard J (2021) Cutting, amplifying, and aligning microtubules with severing enzymes. Trends Cell Biol 31: 50–61 PubMed PMC

Lacroix B, Janke C (2011) Generation of differentially polyglutamylated microtubules. Methods Mol Biol 777: 57–69 PubMed

Lacroix B, van Dijk J, Gold ND, Guizetti J, Aldrian‐Herrada G, Rogowski K, Gerlich DW, Janke C (2010) Tubulin polyglutamylation stimulates spastin‐mediated microtubule severing. J Cell Biol 189: 945–954 PubMed PMC

Lallemand Y, Luria V, Haffner‐Krausz R, Lonai P (1998) Maternally expressed PGK‐Cre transgene as a tool for early and uniform activation of the Cre site‐specific recombinase. Transgenic Res 7: 105–112 PubMed

Lee JE, Silhavy JL, Zaki MS, Schroth J, Bielas SL, Marsh SE, Olvera J, Brancati F, Iannicelli M, Ikegami K et al (2012) CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium. Nat Genet 44: 193–199 PubMed PMC

Lemaitre RP, Bogdanova A, Borgonovo B, Woodruff JB, Drechsel DN (2019) FlexiBAC: a versatile, open‐source baculovirus vector system for protein expression, secretion, and proteolytic processing. BMC Biotechnol 19: 20 PubMed PMC

Lessard DV, Zinder OJ, Hotta T, Verhey KJ, Ohi R, Berger CL (2019) Polyglutamylation of tubulin's C‐terminal tail controls pausing and motility of kinesin‐3 family member KIF1A. J Biol Chem 294: 6353–6363 PubMed PMC

L'Hernault SW, Rosenbaum JL (1985) Chlamydomonas alpha‐tubulin is posttranslationally modified by acetylation on the epsilon‐amino group of a lysine. Biochemistry 24: 473–478 PubMed

Magiera MM, Janke C (2013) Investigating tubulin posttranslational modifications with specific antibodies. In Methods cell biol, Correia JJ, Wilson L (eds), pp 247–267. Burlington: Academic Press; PubMed

Magiera MM, Bodakuntla S, Ziak J, Lacomme S, Marques Sousa P, Leboucher S, Hausrat TJ, Bosc C, Andrieux A, Kneussel M et al (2018) Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J 37: e100440 PubMed PMC

Mahamdeh M, Simmert S, Luchniak A, Schaffer E, Howard J (2018) Label‐free high‐speed wide‐field imaging of single microtubules using interference reflection microscopy. J Microsc 272: 60–66 PubMed PMC

McNally FJ, Roll‐Mecak A (2018) Microtubule‐severing enzymes: from cellular functions to molecular mechanism. J Cell Biol 217: 4057–4069 PubMed PMC

McNally FJ, Vale RD (1993) Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75: 419–429 PubMed

Nitzsche B, Bormuth V, Brauer C, Howard J, Ionov L, Kerssemakers J, Korten T, Leduc C, Ruhnow F, Diez S (2010) Studying kinesin motors by optical 3D‐nanometry in gliding motility assays. Methods Cell Biol 95: 247–271 PubMed

Nurse P (2002) Cyclin dependent kinases and cell cycle control (nobel lecture). Chembiochem 3: 596–603 PubMed

Paturle‐Lafanechere L, Manier M, Trigault N, Pirollet F, Mazarguil H, Job D (1994) Accumulation of delta 2‐tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies. J Cell Sci 107: 1529–1543 PubMed

Piperno G, Fuller MT (1985) Monoclonal antibodies specific for an acetylated form of alpha‐tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol 101: 2085–2094 PubMed PMC

Portran D, Schaedel L, Xu Z, Thery M, Nachury MV (2017) Tubulin acetylation protects long‐lived microtubules against mechanical ageing. Nat Cell Biol 19: 391–398 PubMed PMC

Reed NA, Cai D, Blasius TL, Jih GT, Meyhofer E, Gaertig J, Verhey KJ (2006) Microtubule acetylation promotes Kinesin‐1 binding and transport. Curr Biol 16: 2166–2172 PubMed

Rezabkova L, Jiang K, Capitani G, Prota AE, Akhmanova A, Steinmetz MO, Kammerer RA (2017) Structural basis of katanin p60:p80 complex formation. Sci Rep 7: 14893 PubMed PMC

Roach MC, Boucher VL, Walss C, Ravdin PM, Luduena RF (1998) Preparation of a monoclonal antibody specific for the class I isotype of beta‐tubulin: the beta isotypes of tubulin differ in their cellular distributions within human tissues. Cell Motil Cytoskeleton 39: 273–285 PubMed

Rocha C, Papon L, Cacheux W, Marques Sousa P, Lascano V, Tort O, Giordano T, Vacher S, Lemmers B, Mariani P et al (2014) Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon. EMBO J 33: 2247–2260 PubMed PMC

Rogowski K, van Dijk J, Magiera MM, Bosc C, Deloulme J‐C, Bosson A, Peris L, Gold ND, Lacroix B, Bosch Grau M et al (2010) A family of protein‐deglutamylating enzymes associated with neurodegeneration. Cell 143: 564–578 PubMed

Roll‐Mecak A (2020) The tubulin code in microtubule dynamics and information encoding. Dev Cell 54: 7–20 PubMed PMC

Rüdiger M, Plessman U, Kloppel KD, Wehland J, Weber K (1992) Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435. FEBS Lett 308: 101–105 PubMed

Ruhnow F, Zwicker D, Diez S (2011) Tracking single particles and elongated filaments with nanometer precision. Biophys J 100: 2820–2828 PubMed PMC

Ruhnow F, Klobeta L, Diez S (2017) Challenges in estimating the motility parameters of single Processive motor proteins. Biophys J 113: 2433–2443 PubMed PMC

Santos SDM, Ferrell JE (2008) Systems biology: on the cell cycle and its switches. Nature 454: 288–289 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 (2012) Fiji: an open‐source platform for biological‐image analysis. Nat Methods 9: 676–682 PubMed PMC

Shashi V, Magiera MM, Klein D, Zaki M, Schoch K, Rudnik‐Schoneborn S, Norman A, Lopes Abath Neto O, Dusl M, Yuan X et al (2018) Loss of tubulin deglutamylase CCP1 causes infantile‐onset neurodegeneration. EMBO J 37: e100540 PubMed PMC

Siahaan V, Krattenmacher J, Hyman AA, Diez S, Hernandez‐Vega A, Lansky Z, Braun M (2019) Kinetically distinct phases of tau on microtubules regulate kinesin motors and severing enzymes. Nat Cell Biol 21: 1086–1092 PubMed

Siahaan V, Tan R, Humhalova T, Libusova L, Lacey SE, Tan T, Dacy M, Ori‐McKenney KM, McKenney RJ, Braun M et al (2022) Microtubule lattice spacing governs cohesive envelope formation of tau family proteins. Nat Chem Biol 18: 1224–1235 PubMed PMC

Sirajuddin M, Rice LM, Vale RD (2014) Regulation of microtubule motors by tubulin isotypes and post‐translational modifications. Nat Cell Biol 16: 335–344 PubMed PMC

Skiniotis G, Cochran JC, Muller J, Mandelkow E, Gilbert SP, Hoenger A (2004) Modulation of kinesin binding by the C‐termini of tubulin. EMBO J 23: 989–999 PubMed PMC

Skotheim JM, Di Talia S, Siggia ED, Cross FR (2008) Positive feedback of G1 cyclins ensures coherent cell cycle entry. Nature 454: 291–296 PubMed PMC

Souphron J, Bodakuntla S, Jijumon AS, Lakisic G, Gautreau AM, Janke C, Magiera MM (2019) Purification of tubulin with controlled post‐translational modifications by polymerization–depolymerization cycles. Nat Protoc 14: 1634–1660 PubMed

Sudo H, Baas PW (2010) Acetylation of microtubules influences their sensitivity to severing by katanin in neurons and fibroblasts. J Neurosci 30: 7215–7226 PubMed PMC

Szczesna E, Zehr EA, Cummings SW, Szyk A, Mahalingan KK, Li Y, Roll‐Mecak A (2022) Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing. Dev Cell 57: 2497–2513 PubMed PMC

Tan R, Lam AJ, Tan T, Han J, Nowakowski DW, Vershinin M, Simo S, Ori‐McKenney KM, McKenney RJ (2019) Microtubules gate tau condensation to spatially regulate microtubule functions. Nat Cell Biol 21: 1078–1085 PubMed PMC

Uchimura S, Oguchi Y, Katsuki M, Usui T, Osada H, Nikawa J‐i, Ishiwata SI, Muto E (2006) Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin. EMBO J 25: 5932–5941 PubMed PMC

Valenstein ML, Roll‐Mecak A (2016) Graded control of microtubule severing by tubulin Glutamylation. Cell 164: 911–921 PubMed PMC

Vallee RB (1986) 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 PubMed

van Dijk J, Rogowski K, Miro J, Lacroix B, Eddé B, Janke C (2007) A targeted multienzyme mechanism for selective microtubule polyglutamylation. Mol Cell 26: 437–448 PubMed

Verhey KJ, Gaertig J (2007) The tubulin code. Cell Cycle 6: 2152–2160 PubMed

Verhey KJ, Lizotte DL, Abramson T, Barenboim L, Schnapp BJ, Rapoport TA (1998) Light chain‐dependent regulation of Kinesin's interaction with microtubules. J Cell Biol 143: 1053–1066 PubMed PMC

Vogel P, Hansen G, Fontenot G, Read R (2010) Tubulin tyrosine ligase‐like 1 deficiency results in chronic rhinosinusitis and abnormal development of spermatid flagella in mice. Vet Pathol 47: 703–712 PubMed

Walter WJ, Beranek V, Fischermeier E, Diez S (2012) Tubulin acetylation alone does not affect kinesin‐1 velocity and run length in vitro. PLoS ONE 7: e42218 PubMed PMC

Wang Y, Mandelkow E (2016) Tau in physiology and pathology. Nat Rev Neurosci 17: 22–35 PubMed

Zehr EA, Szyk A, Szczesna E, Roll‐Mecak A (2020) Katanin grips the beta‐tubulin tail through an electropositive double spiral to sever microtubules. Dev Cell 52: e116 PubMed PMC

Polyglutamylation of microtubules drives neuronal remodeling

MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement