Dissociated bovine brain microtubule protein has been shown to reassemble at 0 degrees C in the presence of the drug taxol. Tubulin polymerization was monitored both by electron microscopy of the polymeric structures and by incorporation of tritiated GTP into filterable polymeric structures. Most of the labeled guanine nucleotide uptake into tubulin polymeric structures occurred in the first 30 minutes of incubation with the drug. The initial polymerization event results in the formation of protofilamentous tubulin ribbons. The first microtubules were noted after 1 hour of incubation with the drug. After 20 hours of incubation at 0 degrees C with taxol, the bulk of the polymerized tubulin appeared to be in the form of microtubules. Cold-stable tubulin rings with a mean diameter of 34 nm were present in the reaction mixture before the addition of taxol and throughout the 20-hour incubation. Most of the rings were apparently not involved in the taxol-induced microtubule assembly. The results are consistent with a model whereby taxol induces an initial formation of protofilamentous ribbon structures, mostly from free tubulin dimers, and a slower subsequent folding of the ribbon structures into microtubules.

Alkaloids↗

PubMed1980

Calcium ion induces endwise depolymerization of bovine brain microtubules.

Calcium ion induces rapid depolymerization of microtubules, and the mechanism of interaction of Ca2+ with cytoskeletal components may be metabolically significant. The sufficiency of an endwise depolymerization model for describing calcium-induced disassembly was demonstrated by three criteria: (a) correlation of the observed time course of disassembly with the initial polymer length distribution using a kinetic model described elsewhere (Kristofferson, D., Karr, T. L., and Purich, D. L. (1980) J. Biol. Chem. 255, 8567-8572); (b) observation that the initial rate of calcium-induced disassembly parallels the microtubule number of concentration which is experimentally manipulated by mechanical shearing; and (c) determination of the average polymer length, microtubule number concentration, and extent of depolymerization at various levels of added calcium ion to achieve successive partial disassembly extents. The rate constant for protomer release and other aspects of the disassembly mechanisms are also presented. Critical concentration measurements at low calcium ion levels are also consistent with endwise interactions.

Animals↗

PubMed1980

Assembly and disassembly properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate, and 5'-guanylyl methylenediphosphate.

We have examined the properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate (GMPP(NH)P), and 5'-guanylyl methylenediphosphate (GMPP(CH2)P) to identify features of the assembly or disassembly reactions uniquely related to hydrolysis. The assembly of microtubules with GTP or GMPP(NH)P was similar in terms of rates and extents of assembly, the length distributions, and podophyllotoxin-induced depolymerization. The greater rapidity of GMPP(CH2)P-supported assembly, however, resulted in shorter, more numerous microtubules and the rate of podophyllotoxin-induced depolymerization was consistent with an increased number of concentration of microtubules. Experiments with GTP or analogue incorporation and release indicated that GTP-tubule turnover corresponded to a rate of about 8% of the microtubule protein taken up or released per h. With GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of label uptake by unlabeled microtubules were considerably lower than observed with guanosine triphosphate. We suggest that exchange experiments can reflect contributions from head-to-tail polymerization and polymer length redistribution, but it is not as yet possible to evaluate the relative contributions of each process.

Animals↗

PubMed1980

Dynamics of linear protein polymer disassembly.

This report presents a kinetic model for endwise depolymerization of linear, self-assembling protein systems. We develop a reliable method for predicting the shape of depolymerization curves (remaining polymer weight versus time) on the basis of the initial polymer length distribution. Computer simulations are used to illustrate changes in the polymer length distribution and average polymer weight during disassembly. In addition, our method provides an accurate determination of the microscopic rate constant for subunit release. Application of this analysis to dilution-induced and cold induced disassembly of microtubules is illustrated in the preceding paper (Karr, T.L., Kristofferson, D., and Purich, D.L. (1980) J. Biol. Chem. 255, 8560-8566). A survey of other possible applications of this treatment to microtubules, flagella, F-actin, and tobacco mosaic virus protein is included.

Actins↗

PubMed1980

Mechanism of microtubule depolymerization. Correlation of rapid induced disassembly experiments with a kinetic model for endwise depolymerization.

Microtubule disassembly has been studied using a rapid dilution technique (Karr, T.L., and Purich, D.L. (1979) J. Biol. Chem. 254, 10885-10888). Disassembly curves, generated by computer from the solution of series first order differential equations (see following paper), were fit to experimental data with excellent agreement when the diluted microtubules contained no microtubule-associated proteins. The rate constant for dimer release from microtubules was found to be 154 s-1. Assuming a critical tubulin concentration of 8 to 9 x 10(-6) M, the apparent bimolecular rate constant (2 x 10(7) M-1 s-1) for assembly is near the diffusion limit. It was also possible to use the rapid dilution technique for quantitatively correlating the disassembly rate to the number concentration of microtubule ends. These findings suggest that the dynamics of tubulin interactions with microtubules may be characterized in terms of an endwise depolymerization model. A re-evaluation of cold induced depolymerization kinetics (see miniprint supplement) is also fully consistent with our analysis of disassembly dynamics.

Kinetics↗

PubMed1980