Neurochemical Research

, Volume 42, Issue 9, pp 2566–2576 | Cite as

Post-Translational Tubulin Modifications in Human Astrocyte Cultures

Original Paper
First Online:
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Revised:
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Abstract

The cytoskeletal protein tubulin plays an integral role in the functional specialization of many cell types. In the central nervous system, post-translational modifications and the expression of specific tubulin isotypes in neurons have been analyzed in greater detail than in their astrocytic counterparts. In this study, we characterized post-translational specifications of tubulin in human astrocytes using the normal human astrocyte (NHA; Lonza) commercial cell line of fetal origin. Immunocytochemical techniques were implemented in conjunction with confocal microscopy to image class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin using fluorescent antibody probes. Fluorescent probe intensity differences and colocalization were quantitatively assessed with the ‘EBImage’ package for the statistical programming language R. Colocalization analysis revealed that, although both acetylated tubulin and polyglutamylated tubulin showed a high degree of correlation with βIII-tubulin, the correlation with acetylated tubulin was stronger. Quantification and statistical analysis of fluorescence intensity demonstrated that the fluorescence probe intensity ratio for acetylated tubulin/βIII-tubulin was greater than the ratio for polyglutamylated tubulin/βIII-tubulin. The open source GEODATA set GSE819950, comprising RNA sequencing data for the NHA cell line, was mined for the expression of enzymes responsible for tubulin modifications. Our analysis uncovered greater expression at the mRNA level for enzymes reported to function in acetylation and deacetylation as compared to enzymes implicated in glutamylation and deglutamylation. Taken together, the results represent a step toward unraveling the tubulin isotypic expression profile and post-translational modification patterns in astrocytes during human brain development.

Keywords

Acetylation Polyglutamylation Astrocyte Immunocytochemistry Tubulin Post-translational modifications 

Abbreviations

β

Beta

BSA

Bovine serum albumin

FPKM

Fragments per kilobase of exon per million reads mapped

MT

Microtubule

NHA

Normal human astrocyte

PBS

Phosphate buffered saline

PBST

Phosphate buffered saline with 0.1% TWEEN® 20

RNA

Ribonucleic acid

RNA-seq

RNA sequencing

RRID

Research resource identifiers

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Notes

Acknowledgements

We would like to thank Dr. Armando Varela-Ramirez of the BBRC-CSIC Facility for assistance with confocal imaging.

Funding

This research was supported by the New Mexico State University Manasse Chair Endowment. Confocal microscopy experiments used instrumentation and software located at the University of Texas El Paso Border Biomedical Research Center Cytometry, Screening and Imaging Core (BBRC-CSIC), a facility that is supported by the National Institute on Minority Health and Health Disparities (NIMHD 2G12MD007592).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest. The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or New Mexico State University.

Supplementary material

11064_2017_2290_MOESM1_ESM.tif (25.8 mb)
Online Resource 1 βIII-Tubulin and acetylated tubulin. Confocal microscopy was used to obtain the fluorescence signal from optical sections; merged signal for each section shown on the right. The fluorescent nucleic acid probe Hoescht 33342 (blue) was used in conjunction with immunocytochemical methods that labelled βIII-tubulin (green) and acetylated tubulin (red) in normal human astrocytes cultured for 5 days. Settings for image capture are described in Table 1. (TIF 26419 KB)
11064_2017_2290_MOESM2_ESM.tif (25.8 mb)
Online Resource 2 βIII-Tubulin and polyglutamylated tubulin. Confocal microscopy was used to obtain the fluorescence signal from optical sections; merged signal for each section shown on the right. The fluorescent nucleic acid probe Hoescht 33342 (blue) was used in conjunction with immunocytochemical methods that labelled βIII-tubulin (green) and polyglutamylated tubulin (red) in normal human astrocytes cultured for 5 days. Settings for image capture are described in Table 1. (TIF 26419 KB)
11064_2017_2290_MOESM3_ESM.tif (2.4 mb)
Online Resource 3 Western blot analyses. Chemiluminescent signal was detected in nitrocellulose membranes containing NHA protein isolated from both donors after probing with antibodies against βIII-tubulin (a; 1:1000), acetylated tubulin (b; 1:1000), and polyglutamylated tubulin (c; 1:250). (TIF 2418 KB)

References

  1. 1.
    Chakraborti S, Natarajan K, Curiel J, Janke C, Liu J (2016) The emerging role of the tubulin code: from the tubulin molecule to neuronal function and disease. Cytoskeleton (Hoboken) 73:521–550CrossRefGoogle Scholar
  2. 2.
    Li L, Yang XJ (2015) Tubulin acetylation: responsible enzymes, biological functions and human diseases. Cell Mol Life Sci 72:4237–4255CrossRefPubMedGoogle Scholar
  3. 3.
    Tischfield MA, Cederquist GY, Gupta ML, Engle EC (2011) Phenotypic spectrum of the tubulin-related disorders and functional implications of disease-causing mutations. Curr Opin Genet Dev 21:286–294CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lee MK, Rebhun LI, Frankfurter, A (1990) Posttranslational modification of class III beta-tubulin. Proc Natl Acad Sci USA 87:7195–7199CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Song Y, Brady ST (2015) Post-translational modifications of tubulin: pathways to functional diversity of microtubules. Trends Cell Biol 25:125–136CrossRefPubMedGoogle Scholar
  6. 6.
    Janke C (2014) The tubulin code: molecular components, readout mechanisms, functions. J Cell Biol 206:461–472CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yu I, Garnham CP, Roll-mecak A (2015) Writing and reading the tubulin code*. J Biol Chem 290:17163–17172CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Janke C, Kneussel M (2010) Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton. Trends Neurosci 33:362–372CrossRefPubMedGoogle Scholar
  9. 9.
    Cambray-Deakin MA, Burgoyne RD (1987) Posttranslational modifications of α-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum. J Cell Biol 104:1569–1574CrossRefPubMedGoogle Scholar
  10. 10.
    Romaniello R, Arrigoni F, Bassi MT, Borgatti R (2015) Mutations in α- and β-tubulin encoding genes: implications in brain malformations. Brain Dev 37:273–280CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang F, Su B, Wang C, Siedlak SL, Mondragon-Rodriguez S, Lee H-G, Wang X, Perry G, Zhu X (2015) Posttranslational modifications of α-tubulin in alzheimer disease. Transl. Neurodegener. 4:9CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Audebert S, Desbruyères 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 4:615–626CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Saragoni L, Hernández P, Maccioni RB (2000) Differential association of tau with subsets of microtubules containing posttranslationally-modified tubulin variants in neuroblastoma cells. Neurochem Res 25:59–70CrossRefPubMedGoogle Scholar
  14. 14.
    Dráberová E, Del Valle L, Gordon J, Marková V, Smejkalová B, Bertrand L, de Chadarévian J-P, Agamanolis DP, Legido A, Khalili K, Dráber P, Katsetos CD (2008) Class III beta-tubulin is constitutively coexpressed with glial fibrillary acidic protein and nestin in midgestational human fetal astrocytes: implications for phenotypic identity. J Neuropathol Exp Neurol 67:341–354CrossRefPubMedGoogle Scholar
  15. 15.
    Knight VB, Serrano EE (2017) Hydrogel scaffolds promote neural gene expression and structural reorganization in human astrocyte cultures. PeerJ 5:e2829CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Noristani HN, Sabourin JC, Boukhaddaoui, H, Chan-Seng E, Gerber YN, Perrin FE (2016) Spinal cord injury induces astroglial conversion towards neuronal lineage. Mol Neurodegener 11:68CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Duan CL, Liu CW, Shen SW, Yu Z, Mo JL, Chen XH, Sun FY (2015) Striatal astrocytes transdifferentiate into functional mature neurons following ischemic brain injury. Glia 63:1660–1670CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Katsetos CD, Del Valle L, Geddes JF, Assimakopoulou M, Legido A, Boyd JC, Balin B, Parikh, NA, Maraziotis T, de Chadarevian JP, Varakis JN, Matsas R, Spano A, Frankfurter A, Herman MM, Khalili K (2001) Aberrant localization of the neuronal class III beta-tubulin in astrocytomas. Arch Pathol Lab Med 125:613–624PubMedGoogle Scholar
  19. 19.
    Katsetos CD, Del Valle L, Geddes JF, Aldape K, Boyd JC, Legido A, Khalili K, Perentes E, Mörk SJ (2002) Localization of the neuronal class III beta-tubulin in oligodendrogliomas: comparison with Ki-67 proliferative index and 1p/19q status. J Neuropathol Exp Neurol 61:307–320CrossRefPubMedGoogle Scholar
  20. 20.
    Casale CH, Previtali G, Barra HS (2003) Involvement of acetylated tubulin in the regulation of Na+,K+-ATPase activity in cultured astrocytes. FEBS Lett 534:115–118CrossRefPubMedGoogle Scholar
  21. 21.
    Yoshiyama Y, Zhang B, Bruce J, Trojanowski JQ, Lee, VM-Y (2003) Reduction of detyrosinated microtubules and golgi fragmentation are linked to tau-induced degeneration in astrocytes. J Neurosci 23:10662–10671PubMedGoogle Scholar
  22. 22.
    Bandrowski A, Brush M, Grethe JS, Haendel MA, Kennedy DN, Hill S, Hof PR, Martone ME, Pols M, Tan SC, Washington N, Zudilova-Seinstra E, Vasilevsky N (2016) The Resource identification initiative: a cultural shift in publishing. Brain Behav 6:1–14CrossRefGoogle Scholar
  23. 23.
    Kurien BT, Scofield RH (2015) Western blotting of high and low molecular weight proteins using heat. Methods Mol Biol 1312:247–255CrossRefPubMedGoogle Scholar
  24. 24.
    Pau G, Fuchs F, Sklyar O, Boutros M, Huber W (2010) EBImage-an R package for image processing with applications to cellular phenotypes. Bioinformatics 26:979–981CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Dunn KW, Kamocka MM, McDonald JH (2011) A practical guide to evaluating colocalization in biological microscopy. AJP Cell Physiol 300:C723–C742CrossRefGoogle Scholar
  26. 26.
    Wickham H (2009) ggplot2: elegant graphics for data analysis. Springer, New YorkCrossRefGoogle Scholar
  27. 27.
    Arnold JB (2016) ggthemes: extra themes, scales and geoms for “ggplot2,”. Retrieved from http://cran.r-project.org/package=ggthemes
  28. 28.
    Landis SC, Amara SG, Asadullah K, Austin CP, Blumenstein R, Bradley EW, Crystal RG, Darnell RB, Ferrante RJ, Fillit H, Finkelstein R, Fisher M, Gendelman HE, Golub RM, Goudreau JL, Gross RA, Gubitz AK, Hesterlee SE, Howells DW, Huguenard J, Kelner K, Koroshetz W, Krainc D, Lazic SE, Levine MS, Macleod MR, McCall JM, Moxley RT, Narasimhan K, Noble LJ, Perrin S, Porter JD, Steward O, Unger E, Utz U, Silberberg SD (2012) A call for transparent reporting to optimize the predictive value of preclinical research. Nature 490:187–191CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Gagnon C, White D, Cosson J, Huitorel P, Eddé B, Desbruyères E, Paturle-Lafanechère L, Multigner L, Job D, Cibert C (1996) The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility. J Cell Sci 109(Pt 6):1545–1553PubMedGoogle Scholar
  30. 30.
    Werner SR, Dotzlaf JE, Smith RC (2008) MMP-28 as a regulator of myelination. BMC Neurosci 9:83CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ikegami K, Setou M (2010) Unique post-translational modifications in specialized microtubule architecture. Cell Struct Funct 35:15–22CrossRefPubMedGoogle Scholar
  32. 32.
    Kalebic N, Sorrentino S, Perlas E, Bolasco G, Martinez C, Heppenstall PA (2013) aTAT1 is the major a-tubulin acetyltransferase in mice. Nat Commun 4:1962CrossRefPubMedGoogle Scholar
  33. 33.
    Matyash V, Kettenmann H (2010) Heterogeneity in astrocyte morphology and physiology. Brain Res Rev 63:2–10CrossRefPubMedGoogle Scholar
  34. 34.
    Oberheim NA, Takano T, Han X, He W, Lin JH, Wang F, Xu Q, Wyatt JD, Pilcher W, Ojemann JG, Ransom BR, Goldman SA, Nedergaard M (2009) Uniquely hominid features of adult human astrocytes. J Neurosci 29:3276–3287CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Nedergaard M, Ransom B, Goldman SA (2003) New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci 26:523–530CrossRefPubMedGoogle Scholar
  36. 36.
    Stipursky J, De Sampaio e Spohr TCL, Sousa VO, Gomes FCA (2012) Neuron-astroglial interactions in cell-fate commitment and maturation in the central nervous system. Neurochem Res 37:2402–2418CrossRefPubMedGoogle Scholar
  37. 37.
    Katsetos CD, Reginato MJ, Baas PW, D’Agostino L, Legido A, Tuszyn Ski JA, Dráberová E, Dráber P (2015) Emerging microtubule targets in glioma therapy. Semin Pediatr Neurol 22:49–72CrossRefPubMedGoogle Scholar
  38. 38.
    Azizi SA, Krynska B (2013) Derivation of neuronal cells from fetal normal human astrocytes (NHA). Methods Mol Biol 1078:89–96CrossRefPubMedGoogle Scholar
  39. 39.
    Creppe C, Malinouskaya L, Volvert ML, Gillard M, Close P, Malaise O, Laguesse S, Cornez I, Rahmouni S, Ormenese S, Belachew S, Malgrange B, Chapelle JP, Siebenlist U, Moonen G, Chariot A, Nguyen L (2009) Elongator controls the migration and differentiation of cortical neurons through acetylation of α-tubulin. Cell 136:551–564CrossRefPubMedGoogle Scholar
  40. 40.
    Rivieccio AM, Brochier C, Willis DE, Walker AB, D’Annibale AM, McLaughlin K, Siddiq A, Kozikowski AP, Jaffrey SR, Twiss JL, Ratan RR, Langley B (2009) HDAC6 is a target for protection and regeneration following injury in the nervous system. Proc Natl Acad Sci USA 106:19599–19604CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Mar FM, Simoes AR, Leite S, Morgado MM, Santos TE, Rodrigo IS, Teixeira CA, Misgeld T, Sousa MM (2014) CNS axons globally increase axonal transport after peripheral conditioning. J Neurosci 34:5965–5970CrossRefPubMedGoogle Scholar
  42. 42.
    Noack M, Leyk J, Richter-Landsberg C (2014) HDAC6 inhibition results in tau acetylation and modulates tau phosphorylation and degradation in oligodendrocytes. Glia 62:535–547CrossRefPubMedGoogle Scholar
  43. 43.
    Gadau SD (2015) Detection, distribution and amount of posttranslational α-tubulin modifications in immortalized rat schwann cells. Arch Ital Biol 153:255–265PubMedGoogle Scholar
  44. 44.
    Southwood CM, Peppi M, Dryden S, Tainsky MA, Gow A (2007) Microtubule deacetylases, SirT2 and HDAC6, in the nervous system. Neurochem Res 32:187–195CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of BiologyNew Mexico State UniversityLas CrucesUSA

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