End-to-end annealing of microtubules in vitro.

Abstract

Abstract. Mixtures of pre-formed microtubules, polymerized from chicken erythrocyte and brain tubulin, rapidly anneal end-to-end in vitro in standard microtubule assembly buffer. The erythrocyte tubulin segments in annealed heteropolymers can be distinguished by an immunoelectron microscopic assay that uses an antibody specific for chicken erythrocyte betatubulin. An annealing process is consistent with the following observations: (a) Microtubule number decreases while the polymer mass remains constant. (b) As the total number of microtubules declines, the number of heteropolymers, and the number of segments contained in each heteropolymer, increases. (c) The size of the segments determined after annealing and antibody labeling is the same as the original microtubule polymers. (d) Points of discontinuity in the annealing heteropolymers can be observed directly by electron microscopy, and correspond to type-specific polymer domains. The junctions probably represent initial contact points during the annealing process. Microtubule annealing occurs rapidly in vitro and may be significant for determining properties of microtubule dynamics in vivo.I T is generally accepted that the dynamic properties of microtubules, such as the redistribution of microtubule lengths at steady state, can be explained in terms of the association of tubulin dimers with microtubule ends (1-4) rather than by the fragmentation and reannealing of microtubule polymers. Carlier and co-workers (5, 6) proposed a mechanism for length redistribution based on the appreciable differences in the rate constants for the association and dissociation of GTP-tubulin vs. GDP-tubulin with the ends of microtubules. Once the GDP-tubulin cores of small polymers are exposed to solvent, they depolymerize quickly (5, 7), and, due to the low rate of association of GTP-tubulin subunits with microtubules containing GDP-tubulin ends, the conversion back to growing polymers containing GTP-tubulin caps occurs infrequently (8). As the microtubule number decreases, the remaining microtubules with GTP-tubulin caps can lengthen because of an increase in the concentration of the tubulin dimer pool. The result of this activity is fewer but longer polymers. Although several investigators have observed microtubules to undergo changes in length (4, 9), the observations on length redistribution are also consistent with a redistribution mechanism based on microtubule annealing.In studies of the kinetics of actin polymerization, the significance of fragmentation and annealing has long been an area of interest. Experiments by Kawamura and Maruyama (10), Nakaoka and Kasai (11), and Kondo and Ishiwata (12) suggest that small F-actin fragments associate end-to-end to yield longer filaments in a concentration-dependent fashion, and recent models for actin polymerization (13-15) were found to fit the observed assembly kinetics better when terms for fragmentation and annealing of F-actin were included. Nevertheless, annealing of actin filaments or microtubuleshas not yet been directl...

Direct demonstration of actin filament annealing in vitro.

“…In the case of microtubules, for example, we observed that imperfections in the microtubule wall at the site of joining quickly become filled in or repaired, suggesting a possible requirement for the rearrangement of monomers in the polymer wall or the incorporation of exogenous tubulin monomers (Rothwell et al, 1986). However, our experiments showed that filament annealing was not affected in an obvious way by the addition of actin monomers, even at relatively high molar ratios of monomers to filament ends.…”

“…While monomer exchange at polymer ends is clearly an important factor in determining the growth and stability of polymers of actin and tubulin, it is likely that polymer fragmentation and annealing are also involved in determining the lengths and numbers of polymers at steady state. In recent studies of microtubule assembly, we demonstrated that microtubule polymers can rapidly join end-to-end (anneal) (Rothwell et al, 1986) and that, under certain conditions, mechanisms of polymer annealing and monomer exchange both play important roles in determining microtubule dynamics in vitro (Rothwell et al, 1987). In the present paper, we examine the role of annealing in the assembly of actin filaments.…”

Competitive Inhibition of High-Affinity Oryzalin Binding to Plant Tubulin by the Phosphoric Amide Herbicide Amiprophos-Methyl

“…2), the most likely explanation for polymer length redistribution is that many short microtubules annealed end-to-end to form fewer but longer microtubules. Endwise annealing of animal microtubules in vitro was described previously by Rothwell et al (1986). Further evidence for tobacco microtubule annealing was seen in the form of structural discontinuities along polymer walls, where the ends of different microtubules appeared to have joined.…”

“…Because a low APM concentration produced fewer but significantly longer microtubules without a large reduction in total polymer mass ( Fig. 2; Table I), long microtubules probably arose from an end-to-end annealing of short microtubules, a phenomenon described previously with animal microtubules in vitro (Rothwell et al, 1986). Apparently, annealing of tobacco microtubules occurred as a result of an APM-induced enhancement of dynamics at microtubule ends.…”

How the transition frequencies of microtubule dynamic instability (nucleation, catastrophe, and rescue) regulate microtubule dynamics in interphase and mitosis: analysis using a Monte Carlo computer simulation.

“…The above reasoning yielded a value for the tubulin association rate constant in the elongation (Walker et al, 1988;Simon et al, 1992) and in vivo (Sammak and Borisy, 1988;Shelden and Wadsworth, 1993) but was rare in interphase newt cells (Cassimeris et al, 1988) and in interphase sea urchin egg cytoplasmic extracts (Gliksman et al, 1992). Our simulation also neglected MT annealing (Rothwell et al, 1986), MT severing (Vale, 1991), nonnucleated MT self-assembly, and any minus-end polymerization or depolymerization (Mitchison, 1989;Sawin and Mitchison, 1991;Mitchison and Salmon, 1992) at the nucleation sites. Given our current state of knowledge about MT assembly in the cell, nucleation, and plus-end dynamic instability appear to be the dominate factors in regulating changes in MT assembly in the cell cycle.…”