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Publication

Post-Translational Tubulin Modifications in Human Astrocyte Cultures.

V Bleu Knight | Elba E Serrano

Neurochemical research | 2017

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.

Pubmed ID: 28512712 RIS Download

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Associated grants

Agency: NIMHD NIH HHS, United States
Id: G12 MD007592

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This is a list of tools and resources that we have found mentioned in this publication.

Mouse Anti-Tubulin, polyglutamylated Monoclonal Antibody, Unconjugated, Clone B3 (antibody)

RRID:AB_297929

This monoclonal targets Tubulin

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Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (antibody)

RRID:AB_2631447

This polyclonal targets Mouse IgG - H&L

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Anti-beta III Tubulin antibody [EP1569Y] (Alexa Fluor® 555) ab202519 (antibody)

RRID:AB_2631274

This monoclonal targets beta tubulin

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Acetylated Tubulin (antibody)

RRID:AB_609894

This monoclonal targets derived from the hybridoma 6-11B-1 produced by the fusion of mouse myeloma cells and splenocytes from a mouse immunized with acetylated tubulin from the outer arm of Strongylocentrotus purpuratus (sea urchin).

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