The effects of methylamine osmolytes on the cytoskeleton microtubule assembly

Tseng, Huang-Chun
Major Professor
Donald J. Graves
Committee Member
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Methylamine osmolytes are enriched in urea-rich cells. It is unclear, however, whether methylamines can promote microtubule assembly either in vitro or in cells. The osmolyte trimethylamine N-oxide (TMAO) at 200 mM increases the extent and the rate of formation of polymerized microtubule complex caused by microtubule-associated protein tau in vitro. Furthermore, TMAO counteracts the inhibition on the assembly caused by urea. Besides TMAO, betaine, another methylamine osmolyte, was found to have the same effects on MT as TMAO;Phosphorylation can affect the function of tau. Cyclic-AMP-dependent protein kinase (PKA)-phosphorylated tau does not promote tubulin assembly as judged by spectrophotometric and atomic force microscopy measurements, unless TMAO is;included in these assays. TMAO is also found to promote tubulin assembly with glycogen synthase kinase-3beta-phosphorylated tau. TMAO does not act by causing a chemical dephosphorylation of phosphorylated tau but it acts to overcome the functional deficit caused by phosphorylation. PKA-phosphorylated tau binds to tubulin in the presence of TMAO and lowers the critical concentration of tubulin needed for assembly. These results indicate that PKA-phosphorylated tau retains the ability to bind tubulin and promote tubulin assembly. TMAO is required, however, to sensitize the reaction;TMAO was examined its effects on the microtubule assembly of the XR1 glial cell line. The results of immunofluorescent staining showed that an increase in microtubule assembly occurred when culture media contained 200 mM TMAO. The change of microtubule assembly was correlated with the concentrations of intracellular TMAO, analyzed by 1H-NMR spectroscopy. The intracellular concentration of TMAO reached 30--50 mM after 6-hour incubation with 200 mM TMAO. During this time period microtubule assembly increased significantly. A spectrophotometric assay for microtubule assembly in vitro showed that 50 mM TMAO affected microtubule assembly, supporting the observations in cells. TMAO did not affect F-actin fibers as judged by fluorescent staining with phalloidin. Glycerol did not increase microtubule assembly of XR1 cells. These results indicate that the methylamine osmolytes, such as TMAO, can specifically enhance microtubule assembly in cells. Possible uses of TMAO in relation to studies of tubulin assembly in relation to neurodegenerative diseases, such as Alzheimer's disease, are presented in this dissertation.