Light microscopic observations of individual microtubules reconstituted from brain tubulin

The course of polymerization of individual brain microtubules could be observed with a light microscope employing dark field illumination. Statistical analysis of the increase in microtubule length during the polymerization was in accordance with the time course of viscosity change of the tubulin solution. After a plateau level in viscosity was attained, there was no significant change in histograms showing length distribution. These observations were confirmed with fixed and stained microtubules, using a phase contrast microscope. Observations with dark field illumination revealed that reconstituted microtubules depolymerized and disappeared immediately upon exposure to buffer containing CaCl₂ or sulphydryl reagents such as p chloromercuriphenyl sulphonic acid (PCMPS) and p chloromercuribenzoic acid (PCMB). They were also cold labile. The growth of heterogeneous microtubules which were assembled by mixing purified tubulin dimers with ciliary outer fibres could also be followed with these optical systems.