Post-Translational Tubulin Modifications in Differentiated Human Neural Stem Cells

Abstract

ABSTRACTThe tubulin protein fulfills a variety of cellular functions that range from chromosomal separation to locomotion. Functional diversity is achieved through the expression of specific tubulin isotypes in different cell types or developmental time periods. Post-translational modifications (PTMs) of tubulin also are vital for specific intracellular tasks, such as binding and recruiting motor proteins. In neurons, the isotypic expression profile for tubulin is well characterized, and the importance of PTMs for proper neuronal function has gained recent attention due to their implication in neurodegenerative disorders. In contrast, the role of tubulin specializations in the specification of neural cell fate has received minimal attention and studies of tubulin PTMs and isotypes in neuroglia such as astrocytes are relatively few. To bridge this knowledge gap, we undertook an analysis of PTMs in neurons and astrocytes derived from the federally approved H9 hESC-derived human neural stem cell (hNSC) line. In hNSCs, basal cells can be directed to assume neural fate as neurons or astrocytes by specifying different media growth conditions. Immunocytochemical methods, fluorescent antibody probes, and confocal microscopy facilitated image acquisition of fluorescent signals from class III β-tubulin (βIII-tubulin), acetylated tubulin, and polyglutamylated tubulin. Fluorescent probe intensities were assessed with the ‘EBImage’ package for the statistical programming language R, and compared using Student’s t-tests. Qualitative analysis indicated that βIII-tubulin, acetylated tubulin, and polyglutamylated tubulin were expressed to some degree in basal hNSCs and their media-differentiated hNSC neuronal and astroglial progeny. In media-differentiated hNSC astrocyte progeny, quantification and statistical analysis of fluorescence probe intensity showed that acetylated tubulin/ βIII-tubulin ratios were greater than the ratio for polyglutamylated tubulin/ βIII-tubulin. These findings represent a snapshot of the dynamic and varied changes in the tubulin expression profile during the specification of neural cell fate. Results imply that investigations of tubulin PTMs have the potential to advance our understanding of the generation and regeneration of nervous tissue.