Microtubule Assembly in vitro. Purification of Assembly-Promoting Factors

Abstract

The role of microtubule-associated proteins in the assembly of tubulin to microtubules in vitro has been studied.1. It has been confirmed that pure tubulin obtained by phosphocellulose column chromatography does not significantly assemble in vitro in the absence of minor components which co-polymerize with tubulin. Although tubulin aggregates in a morpholino-ethanesulfonate buffer containing high Mg2 + concentrations, this process was neither inhibited by Ca2+ or colchicine, nor reversed by cold exposure.2. Microtubule-associated proteins were prepared, either by phosphocellulose column chromatography or by a direct method based on boiling reassembled microtubules in the presence of 2 mM dithiothreitol and 0.75 M NaCl. From each of these preparations two protein fractions were purified, either by Ultrogel ACAW chromatography or by sucrose gradient ultracentrifugation. The first one, with a high molecular weight, did not promote tubulin assembly; ageing of this material did not induce any activity. On the other hand, the second fraction, with an apparent molecular weight of 70000 (z protein), when almost completely purified, was active in promoting assembly.Thus a single specific protein is able to promote assembly of pure tubulin.Increasing evidence is available showing that in several systems where the microtubules are not stable as in cilia, specific signals induce a rapid massive polymerization of microtubules from a preexisting store of their basic subunit 6-S tubulin [l -41.For instance, in previous preliminary publications [5,6] we have shown that the amount of tubulin is not a limiting factor during rat brain development. However, the rate of polymerization in vitro and the overall extent of microtubule assembly is much lower with crude supernatants prepared from young rat brain than with control adult rat brain preparations. These results suggested that a 'factor' or 'signal' is needed for microtubule assembly and that the amount of this factor is limiting in the young brain.The nature of these signals or factors remains controversial.

Modulation of Microtubule Dynamics by Tau in Living Cells: Implications for Development and Neurodegeneration

“…We also found that tau decreases the rate and extent of microtubule growth in cells. These data are in contrast to the widely recognized ability of tau to promote microtubule growth and assembly under non-steady state conditions, both in vitro (Cleveland et al, 1977;Fellous et al, 1977) and in cells (Caceres and Kosik, 1990;Knops et al, 1991;Esmaeli-Azad et al, 1994; reviewed in Buee et al, 2000). They are also in contrast to the taumediated enhancement of the rate and extent of microtubule growth observed in dynamics studies in vitro, also under non-steady state conditions of net microtubule assembly (Drechsel et al, 1992;Trinczek et al, 1995).…”

Fractionation of Brain Microtubule‐Associated Proteins

“…After a final centrifugation step at 4 "C (100000 x g, 30 min), to remove some aggregated protein, the microtubule protein solution in buffer A was made 0.75 M in NaCl and 10 mM in 2-mercaptoethanol [21]. The final protein concentration was approximately 8 mg/ml.…”

“…We isolated heat-stable associated proteins from total microtubule protein in a manner similar to that used by Nunez and coworkers [21]. Microtubule protein, purified by three temperaturedependent cycles of polymerization/depolymerization in buffer A, was incubated for 4 min at 98 "C in the presence of 0.75 M NaCl and 10 mM 2-mercaptoethanol (see Materials and Methods).…”

Ca2+‐ and calmodulin‐dependent flip—flop mechanism in microtubule assembly—disassembly

“…While tau factor is essential for the tubulin to assemble [2][3][4][5][6], Ca 2+-dependent association of calmodulin with tau factor prevents the tan from interacting with tubulin thus inhibiting or reversing tubulin assembly. At decreased Ca2+levels, calmodulin-tau interaction ceases, leading to the interaction of tau-tubulin, with the consequent assemby-formation.…”

“…When the tubulin is freed of the microtubule-associated proteins by phosphocellulose chromatography, it can no longer assemble into the microtubules under standard polymerization conditions. Addition of the tau proteins back to the PC-tubulin fully restored its polymerizability [2][3][4][5][6]. In [3] it was concluded that tau is both necessary and sufficient for nucleation and elongation of microtubules from PC-tubulin.…”