Stable isotope labeling by amino acids in cell culture-based liquid chromatography-mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

摘要

Microtubules (MTs) are highly dynamic polymers composed of alpha- and beta-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 [C-13(6)]leucine-labeled alpha-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [C-13(6)]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 alpha-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled alpha-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. (C) 2014 Elsevier Inc. All rights reserved.