Mechanism of microtubule stabilization in neurites : a novel approach capable of directly observing and testing microtubule stability.

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During outgrowth and elongation of neurites, microtubules (MTs) not only increase in quantity but also become progressively more stabilized. To elucidate the molecular mechanism of stabilization, we developed a novel approach capable of directly observing the stability of neuritic MTs by video-enhanced differential interference contrast (DIC) microscopy combined with a flow cell technique.By dissolving the membrane with detergent perfusion, we found that the established neurites of dorsal root ganglion cells cultured for more than 5 days contained MTs which persisted outside the cell for more than 30 minutes (Tashiro et al.J.Neurosci.Res., 50,81-93,1997). These stable MTs were usually single and floating above the substratum, with only point attachments along the length.For further characterization, we transected the exposed MTs by laser microbeam irradiation and observed their length changes with video-enhanced DIC microscopy. MT fragments started to shorten on both sides of the transection site, more rapidly from the newly generated plus ends than from the minus ends. The rate and pattern of shortening correlated with the persistence time after membrane removal ; stable MTs shortened slowly with intermittent pauses, while the more labile MTs shortened continuously at higher rates. Transection also revealed the presence of specific points where stable MTs are anchored to the substratum or disassembly is transiently halted (Kurachi et al., submitted).MTs reassembled from purified tubulin and MT-associated protein tau were not resistant to dilution by perfusion. We also found that dephosphorylation of the native tau resulted in the selective decrease in its MT-nucleation activity (Morita-Fujimura et al., Biochem.Biophys.Res.Comm., 225,462-468,1996).These results show that protection at MT ends and at specific points must be considered in addition to stabilization along the length by MT-associated proteins.