Fluorescently-labelled microtubules growing and shrinking in Xenopus laevis egg extracts Image courtesy of N. Le Bot.
Following the description by Inoue of the dynamic nature of spindle microtubules (see Milestone 5), researchers in the field actively sought to understand how tubulin polymerization could produce this behaviour. It was initially thought that microtubule polymerization by GTP-linked tubulin subunits followed a treadmilling model, according to which microtubule length would result from GTP-tubulin being added to one end and GDP-tubulin dissociating from the other. However, it was possible to test this model only after Weisenberg and Borisy achieved efficient microtubule polymerization in vitro in 1972 (see Milestone 6). This assay provided a starting point for the purification of microtubule-associated proteins, which were later shown to influence polymer dynamics and organization, and for the isolation of molecular motors (see also Milestone 15). But the key insight into microtubule polymerization properties came when Mitchison and Kirschner combined biochemical assays with microscopy to uncover the coexistence of growing and shrinking microtubules in vitro, in a state of 'dynamic instability'.
Previous studies had inferred the behaviour of individual microtubules from the biochemical properties of the bulk polymer. Mitchison and Kirschner used microtubule seeds incubated in solutions of various tubulin concentrations and, in addition to assessing these properties, they visualized the microtubules at fixed time points using microscopy. They observed that although the total polymer mass reached a plateau and remained constant, the microtubule population did not consist of a fixed number of microtubules of the same length. Instead, the number of microtubules decreased with time and their mean length increased. This demonstrated the coexistence of growing and shrinking microtubules, with the latter depolymerizing rapidly to provide new subunits for growth. Given that long microtubules eventually disappeared, they concluded that transitions between polymerization and depolymerization were probably rare. The authors coined the term 'dynamic instability' to describe these properties of microtubule polymerization.
The main differences between the dynamic instability and treadmilling models are the transitions from growth to shrinkage (catastrophe) or from shrinkage to growth (rescue) at the same end of the microtubule. To explain these transitions, Mitchison and Kirschner postulated that GTP-bound tubulin subunits were added during polymerization and that GTP then hydrolysed to GDP, resulting in the presence of a GTP-tubulin cap at the end of a growing microtubule. They surmised that this cap was more stable than the GDP-tubulin lattice; therefore, at low concentrations of GTP-tubulin subunits, the polymerization rate and the GTP cap length decreased, leading to the exposure of GDP-tubulin close to the end and increasing the probability of catastrophes.
This work was an instant classic, defining the dynamic properties of microtubules.
Erika Holzbaur
Live observations of individual microtubules using dark-field microscopy soon confirmed the existence of dynamic instability. In a review published 2 years after their initial study, Mitchison and Kirschner envisioned that dynamic instability might be crucial for the role of microtubules in mitosis and morphogenesis. They proposed that microtubules emanating from centrosomes transiently grow and shrink, probing the intracellular space in 'search' of mitotic chromosomes, which they 'capture' by selective stabilization, thereby participating in their alignment on the metaphase plate prior to chromosome segregation. Mitchison and Kirschner also suggested that a random search driven by microtubule dynamics followed by selective capture at specific cortical sites could explain how microtubules influence cell polarity and shape. Since then, the search for the molecules responsible for the selective capture has not ceased.
