English

The Tip of a Growing Microtubule and its Implications for Polymer Dynamics

Background

The dynamics of microtubules (MTs) is important for the organization, motility, and morphogenesis of cells. Most tubulin subunits join and leave a MT at its plus tip, so the structure of this site is informative about intermediates in tubulin polymerization. Moreover, a dynamic MT is a machine that can push or pull on cellular objects, changing their position. The force a growing MT can exert is modest (~2pN) whereas shortening MTs can pull hard (~30 pN). Cells may use these capabilities to deform membranes, move organelles, and to organize then segregate the chromosomes in preparation for cell division.

Aims

We have studied the structure of the tips of growing and shortening MTs, both in cells and in test tubes, to learn about the changes in structure that tubulin experiences as it polymerizes and depolymerizes.

Methods

Cells were embedded in plastic, sectioned and imaged by electron tomography, a method that provides 3-D images of structures with resolutions of ~4 nm. We have also used this and related imaging methods to study MTs growing in vitro that were rapidly frozen, MTs that were fixed and then frozen, and MTs that were fixed or not, then negatively stained.

Results

The growing MTs in our images almost all displayed flared ends in which protofilaments (PFs, i.e. linear strands of tubulin) bent outward from the MT axis. Most PFs lay in planes that contained the MT axis. PF curvature was greatest at its tip and decreased linearly with approach to the MT wall. Tip PF curvature resembled the curvature of isolated tubulin as measured by several crystallographers. Analysis of the structural data by Nikita Gudimchuk and colleagues, using Brownian dynamics, has demonstrated that thermal fluctuations can provide sufficient movement to let the PFs straighten, find neighbors, and form lateral bonds to zip the PFs into the elongating MT wall. Model fitting to published data on rates of MT growth and the forces generated by dynamic MTs provide parameter values that are reasonable for both the strength and activation energies of all inter-tubulin bonds.

Conclusions

We conclude that MTs polymerize by the addition of curved tubulin dimers onto the tips of bent PFs, which straighten by thermal motions to allow MT elongation. This simple model solves several enigmas about tubulin dynamics and allows straightforward interpretation of the synergistic activity of factors that enhance the rate of MT growth. There are, however, many unsolved problems, such as how tubulin's structure changes during polymerization and how the subsequent hydrolysis of bound GTP alters the properties of tubulin and/or the bonds between tubulins in the MT lattice.