title: Stable isotope labeling by amino acids in cell culture-based liquid chromatography-mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures.
abstract: Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography-mass spectrometry (LC-MS) method to measure the fraction of [(13)C6]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [(13)C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.
authors.affiliation: Neuroscience Biology, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Polson
authors.affiliation: Pharmaceutical Candidate Optimization-Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: Cantone
authors.affiliation: Pharmaceutical Candidate Optimization-Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA.
authors.forename: Cong
authors.initials: C
authors.lastname: Wei
authors.affiliation: Pharmaceutical Candidate Optimization-Discovery Analytical Sciences, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA.
authors.forename: Dieter M
authors.initials: DM
authors.lastname: Drexler
authors.affiliation: Neuroscience Biology, Bristol-Myers Squibb Research and Development, Wallingford, CT 06492, USA. Electronic address: jere.meredith@bms.com.
authors.forename: Jere E
authors.initials: JE
authors.lastname: Meredith
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000166:cytology; D018929:Cell Culture Techniques; D002853:Chromatography, Liquid; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D008870:Microtubules / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D051381:Rats; D015203:Reproducibility of Results
chemical_list: D010455:Peptides
keywords: LC–MS; Microtubule; Neurons; SILAC; Tubulin