Microtubule self-organization is gravity-dependent

Abstract

Although weightlessness is known to affect living cells, the manner by which this occurs is unknown. Some reaction-diffusion processes have been theoretically predicted as being gravity-dependent. Microtubules, a major constituent of the cellular cytoskeleton, self-organize in vitro by way of reaction-diffusion processes. To investigate how self-organization depends on gravity, microtubules were assembled under low gravity conditions produced during space flight. Contrary to the samples formed on an in-flight 1 x g centrifuge, the samples prepared in microgravity showed almost no self-organization and were locally disordered.

Figure 1

Ground experiment using the space flight module and hardware. Samples contained 10 mg⋅ml⁻¹ of phosphocellullose purified tubulin and 2 mM GTP in buffer (10). Cells of 1-mm optical pathlength, at 7°C, were placed in the low gravity compartment. (Left) Upright. (Center). Flat. (Right) The cell was flat in the 1 × g centrifuge compartment. Microtubules were formed by rapidly warming the solution to 36°C. The centrifuge was on for the first 13 min only, this being the low gravity period of the subsequent space flight. Different macroscopic morphologies form depending on whether the sample container is horizontal, vertical, or spinning, at a critical moment 6 min after instigating microtubule assembly. Once formed after about 6 h, the structures are stationary and independent of the gravity direction. Samples were photographed through linear cross polarizers with a wavelength retardation plate. The blue interference color arises from microtubules oriented at about 45° and the yellow color from those at 135° (–15).

Figure 2

Microtubule structures as formed during space flight. Microtubules were assembled once microgravity conditions were obtained. (Left and Center) Shown are the self-organized morphologies that arise for samples placed on the on-board 1 × g centrifuge, with the centrifugal field along and perpendicular to the long axis of the sample cuvette. The centrifuge was stopped after 13 min, immediately before re-entry, and the samples were left under 1 × g conditions for a further 5 h while the structures developed. (Right) Almost no self-organization occurs for samples subject to weightlessness during the first 13 min.

Figure 3

On the ground, self-organization also occurs over smaller distance scales. This photograph is taken from a sample prepared in the laboratory and placed between crossed circular polarizers. In addition to the periodicity of about 400 μm (A), it also shows periodicities of about 100 μm (B), 20 μm (C), and 5 μm (D). These levels of self-organization did not arise for samples assembled under low g conditions.

Figure 4

Kinetics of the formation of microtubules. After warming to 35°C, the tubulin solution assembles into microtubules whose concentration is proportional to the optical density at 350 nm. The optical pathlength of the sample is 1 mm; the optical density would be 10 times greater for a 1-cm-thick sample. The kinetics show an over-shoot; microtubule assembly is at a maximum between 6 and 10 min after instigating microtubule formation. This demonstrates a chemical instability between the relative proportions of free tubulin and microtubules that coincide approximately with the bifurcation time at which the system is gravity-dependent. Microtubule disassembly produces macroscopic concentration fluctuations that interact with gravity and trigger self-organization.

Figure 5

A possible mechanism for the formation of the self-organized structure. Microtubules are chemically anisotropic, growing and shrinking along the direction of their long axis. This leads to the formation of chemical trails, comprised of regions of high and low local tubulin concentration from their shrinking and growing ends respectively. These concentration trails (density fluctuations) are oriented along the direction of the microtubule. Neighboring microtubules will preferentially grow into regions in which the local concentration of tubulin is highest. When molecular transport is isotropic, microtubules grow and shrink equally in all directions, and the tubulin trails retain an isotropic distribution. For self-organization to occur, this symmetry must be broken. Gravity can do this by introducing a molecular transport term that is faster in the up-down direction. This gives rise to a slight directional bias in the formation of chemical trails. Microtubules will subsequently grow and form preferentially along the direction of these tubulin trails. These processes progressively reinforce themselves, resulting in the development of the periodic changes in microtubule orientation and concentration observed. In A, microtubules have just formed from the tubulin solution. They are still in a growing phase and have an isotropic arrangement. In B, microtubule disassembly has started to occur at the bifurcation time. This produces trails of high tubulin concentration from the shrinking ends of the microtubules. These macroscopic concentration fluctuations interact with gravity, denser fluctuations drifting downward and lighter ones upwards, leading to an anisotropy in molecular transport. In C, microtubules are growing and forming preferentially where the concentration of active tubulin is highest. Anisotropic molecular transport at the bifurcation time privileges microtubule growth along specific directions. Once started, this process subsequently mutually reinforces itself with time and leads to self-organization. When gravity is absent, molecular transport remains isotropic, and self-organization is not triggered.