Abstract
Microtubules, as integral part of the eukaryotic cytoskeleton, exert numerous essential functions in cells. A mechanism to control these diverse functions are the posttranslational modifications of tubulin. Despite being known for decades, relatively little insight into the cellular functions of these modifications has been gained so far. The discovery of tubulin-modifying enzymes and a growing number of available knockout mice now allow working with primary cells from those mouse models to address biological functions and molecular mechanisms behind those modifications. However, a number of those mouse models show either lethality or sterility, making it difficult to impossible to obtain a sufficient number of animals for a systematic study with primary cells. Moreover, many of those modifications are controlled by several redundant enzymes, and it is often necessary to knock out several enzymes in parallel to obtain a significant change in a given tubulin modification. Here we describe a method to generate primary cells with combinatorial knockout genotypes using conditional knockout mice. The conditional alleles are converted into knockout in the cultured primary cells by transduction with a lentivirus encoding cre-recombinase. This approach has allowed us to knock out the two main brain deglutamylases in mouse primary neurons, which leads to strongly increased polyglutamylation in these cells. Our method can be applied to measure different cellular processes, such as axonal transport, for which it can be combined with the expression of different fluorescent reporters to label intracellular proteins. Using a panel of conditional knockout mice, our method can further be applied to study the functions of a variety of tubulin modifications that require simultaneous knockout of multiple genes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mandell JW, Banker GA (1995) The microtubule cytoskeleton and the development of neuronal polarity. Neurobiol Aging 16(3):229–237; discussion 238
Brady ST, Morfini GA (2017) Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases. Neurobiol Dis 105:273–282. https://doi.org/10.1016/j.nbd.2017.04.010
Janke C (2014) The tubulin code: Molecular components, readout mechanisms, and functions. J Cell Biol 206(4):461–472. https://doi.org/10.1083/jcb.201406055
Janke C, Bulinski JC (2011) Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat Rev Mol Cell Biol 12(12):773–786. https://doi.org/10.1038/nrm3227
Magiera MM, Singh P, Janke C (2018) SnapShot: functions of tubulin posttranslational modifications. Cell 173(6):1552–1552 e1551. https://doi.org/10.1016/j.cell.2018.05.032
Gadadhar S, Bodakuntla S, Natarajan K, Janke C (2017) The tubulin code at a glance. J Cell Sci 130(8):1347–1353. https://doi.org/10.1242/jcs.199471
Magiera MM, Bodakuntla S, Ziak J, Lacomme S, Marques Sousa P, Leboucher S, Hausrat TJ, Bosc C, Andrieux A, Kneussel M, Landry M, Calas A, Balastik M, Janke C (2018) Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport. EMBO J 37(23):e100440. https://doi.org/10.15252/embj.2018100440
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(41):30707–30716
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. https://doi.org/10.1038/ncomms2962
Rogowski K, van Dijk J, Magiera MM, Bosc C, Deloulme J-C, Bosson A, Peris L, Gold ND, Lacroix B, Bosch Grau M, Bec N, Larroque C, Desagher S, Holzer M, Andrieux A, Moutin M-J, Janke C (2010) A family of protein-deglutamylating enzymes associated with neurodegeneration. Cell 143(4):564–578. https://doi.org/10.1016/j.cell.2010.10.014
Erck C, Peris L, Andrieux A, Meissirel C, Gruber AD, Vernet M, Schweitzer A, Saoudi Y, Pointu H, Bosc C, Salin PA, Job D, Wehland J (2005) A vital role of tubulin-tyrosine-ligase for neuronal organization. Proc Natl Acad Sci U S A 102(22):7853–7858
Rocha C, Papon L, Cacheux W, Marques Sousa P, Lascano V, Tort O, Giordano T, Vacher S, Lemmers B, Mariani P, Meseure D, Medema JP, Bièche I, Hahne M, Janke C (2014) Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon. EMBO J 33(19):2247–2260. https://doi.org/10.15252/embj.201488466
Bosch Grau M, Masson C, Gadadhar S, Rocha C, Tort O, Marques Sousa P, Vacher S, Bieche I, Janke C (2017) Alterations in the balance of tubulin glycylation and glutamylation in photoreceptors leads to retinal degeneration. J Cell Sci 130:938–949. https://doi.org/10.1242/jcs.199091
Gadadhar S, Dadi H, Bodakuntla S, Schnitzler A, Bieche I, Rusconi F, Janke C (2017) Tubulin glycylation controls primary cilia length. J Cell Biol 216(9):2701–2713. https://doi.org/10.1083/jcb.201612050
Giordano T, Gadadhar S, Bodakuntla S, Straub J, Leboucher S, Martinez G, Chemlali W, Bosc C, Andrieux A, Bieche I, Arnoult C, Geimer S, Janke C (2019) Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility. J Cell Sci 132(3). https://doi.org/10.1242/jcs.226951
Silva CG, Peyre E, Adhikari MH, Tielens S, Tanco S, Van Damme P, Magno L, Krusy N, Agirman G, Magiera MM, Kessaris N, Malgrange B, Andrieux A, Janke C, Nguyen L (2018) Cell-intrinsic control of interneuron migration drives cortical morphogenesis. Cell 172(5):1063–1078. https://doi.org/10.1016/j.cell.2018.01.031
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(1):206–219. https://doi.org/10.1083/jcb.201709028
Marcos S, Moreau J, Backer S, Job D, Andrieux A, Bloch-Gallego E (2009) Tubulin tyrosination is required for the proper organization and pathfinding of the growth cone. PLoS One 4(4):e5405
Akella JS, Wloga D, Kim J, Starostina NG, Lyons-Abbott S, Morrissette NS, Dougan ST, Kipreos ET, Gaertig J (2010) MEC-17 is an alpha-tubulin acetyltransferase. Nature 467(7312):218–222. https://doi.org/10.1038/nature09324
Shida T, Cueva JG, Xu Z, Goodman MB, Nachury MV (2010) The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc Natl Acad Sci U S A 107(50):21517–21522. https://doi.org/10.1073/pnas.1013728107
Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang X-F, Yao T-P (2002) HDAC6 is a microtubule-associated deacetylase. Nature 417(6887):455–458
Janke C, Rogowski K, Wloga D, Regnard C, Kajava AV, Strub J-M, Temurak N, van Dijk J, Boucher D, van Dorsselaer A, Suryavanshi S, Gaertig J, Eddé B (2005) Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308(5729):1758–1762. https://doi.org/10.1126/science.1113010
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(3):437–448. https://doi.org/10.1016/j.molcel.2007.04.012
Tort O, Tanco S, Rocha C, Bieche I, Seixas C, Bosc C, Andrieux A, Moutin M-J, Xavier Aviles F, Lorenzo J, Janke C (2014) The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. Mol Biol Cell 25(19):3017–3027. https://doi.org/10.1091/mbc.E14-06-1072
Jeong J-Y, Yim H-S, Ryu J-Y, Lee HS, Lee J-H, Seen D-S, Kang SG (2012) One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies. Appl Environ Microbiol 78(15):5440–5443. https://doi.org/10.1128/AEM.00844-12
Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, Kim M-K, Shin BA, Choi S-Y (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6(4):e18556. https://doi.org/10.1371/journal.pone.0018556PONE-D-11-01024
Magiera MM, Janke C (2013) Investigating tubulin posttranslational modifications with specific antibodies. In: Correia JJ, Wilson L (eds) Methods cell biol, Microtubules, in vitro, vol 115. Academic Press, Burlington, pp 247–267. https://doi.org/10.1016/B978-0-12-407757-7.00016-5
Acknowledgments
This work was supported by the ANR-10-IDEX-0001-02, the LabEx CelTisPhyBio ANR-11-LBX-0038. CJ 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. MMM is supported by the EMBO short-term fellowship ASTF 148-2015 and by the Fondation Vaincre Alzheimer grant FR-16055p, and SB by the FRM grant FDT201805005465. We thank C. Alberti, E. Belloir, F. Bertrand, V. Dangles-Marie, I. Grandjean, C. Caspersen, H. Hermange, A. Thadal, G. Buhagiar, C. Serieyssol, S. Gadadhar, and M. Sittewelle (Institut Curie) for technical assistance. We are grateful to M.-N. Soler, C. Lovo, and L. Besse from the PICT-IBiSA@Orsay Imaging Facility of the Institut Curie supported by the ANR through the “Investment for the future” program (France-BioImaging, ANR-10-INSB-04), and to N. Manel (Institut Curie, Paris) for material and advice for the lentivirus production. We would like to thank F. Del Bene, V. Marthiens (Institut Curie), and C. González-Billault (University of Chile, Santiago, Chile) for instructive discussions and advice.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Bodakuntla, S., Janke, C., Magiera, M.M. (2020). Knocking Out Multiple Genes in Cultured Primary Neurons to Study Tubulin Posttranslational Modifications. In: Maiato, H. (eds) Cytoskeleton Dynamics. Methods in Molecular Biology, vol 2101. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0219-5_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0219-5_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0218-8
Online ISBN: 978-1-0716-0219-5
eBook Packages: Springer Protocols