A kinetic analysis of the assembly of microtubules in vitro

Abstract

1. Introduction Microtubules have many different biological func- tions in which their dynamic properties play a central role. A detailed knowledge of the kinetics of assembly is therefore necessary for the understanding of their mechanism of action. In this study the kinetics of polymerisation, as followed by turbidity measurements, is analysed. As previously shown, [ 1,2] two steps have to be con- sidered: the nucleation and the propagation step. The turbidity measurements are correlated with electron microscopy which allows a quantitative analysis of the propagation kinetics. Our results correspond well with the data of Bryan [3] obtained with sonicated frag- ments. Information about the process of nucleation is also obtained. The role of the ring-oligomers, present in these preparations, is discussed both in the process of nucleation and propagation. 2. Materials and methods Tubulin is isolated from pig brains, in the first polymerisation cycle according to the method of Shelanski [4]. The two subsequent cycles, and the polymerisation studies are done in MES-buffer of pH 6.4 (50 mM MES, 70 mM KCl, 1 .O mM GTP, 0.5 mM MgClz, 1 mM EGTA and 1 mM NaNa). The protein is stored without glycerol in liquid nitrogen. Protein concentration is determined by the method of Lowry [5] , using bovine serum albumine (Serva) as standard. (S-)Tubulin prepared in this way, con- tains about 10% of microtubule-associated-proteins (MAPS) which induce the formation of ringlike oligomers at 4°C. Pure (PC-)tubulin protomers are prepared by phosphocellulose chromatography [6] . A mixture of protomers and MAPS is obtained by dissociation of the rings at high ionic strength (0.8 M KCl) and dilution back to 0.1 [7] . The same results were obtained by reaction with diamide at 3 mM, followed by the addition of excess dithiothreitol [8] . This mixture reassociates slowly. The ratio MAPS/ tubulin is decreased by the addition of PC-tubulin. For electron microscopy, solutions are diluted to 0.05 mg/ml with polymerisation buffer containing 50% ethylene glycol to prevent dissociation. The solutions are then applied to grids and stained with uranyl acetate. Polymerisation is initiated by large temperature jumps from 4-30°C in a cell constructed in the laboratory. Temperature changes exponentially with a half-life of 1.2 sec. The polymerisation reaction is followed by turbidity measurements at 350 nm. Turbidity was shown to be a measure of the weight concentration of the polymer [l] .