Exposure of biological preparations to radiofrequency electromagnetic fields under low gravity.

There is interest as to whether the electromagnetic fields used in mobile radiotelephony might affect biological processes. Other weak fields such as gravity intervene in a number of physical and biological processes. Under appropriate in vitro conditions, the macroscopic self-organization of microtubules, a major cellular component, is triggered by gravity. We wished to investigate whether self-organization might also be affected by radiotelephone electromagnetic fields. Detecting a possible effect requires removing the obscuring effects triggered by gravity. A simple manner of doing this is by rotating the sample about the horizontal. However, if the external field does not also rotate with the sample, its possible effect might also be averaged down by rotation. Here, we describe an apparatus in which both the sample and an applied radiofrequency electromagnetic field (1.8 GHz) are stationary with respect to one another while undergoing horizontal rotation. The electromagnetic field profile within the apparatus has been measured and the apparatus tested by reproducing the in vitro behavior of microtubule preparations under conditions of weightlessness. Specific adsorption rates of electromagnetic energy within a sample are measured from the initial temperature rise the incident field causes. The apparatus can be readily adapted to expose samples to various other external fields and factors under conditions of weightlessness.

Effect of weightlessness on colloidal particle transport and segregation in self-organising microtubule preparations.

Weightlessness is known to effect cellular functions by as yet undetermined processes. Many experiments indicate a role of the cytoskeleton and microtubules. Under appropriate conditions in vitro microtubule preparations behave as a complex system that self-organises by a combination of reaction and diffusion. This process also results in the collective transport and organisation of any colloidal particles present. In large centimetre-sized samples, self-organisation does not occur when samples are exposed to a brief early period of weightlessness. Here, we report both space-flight and ground-based (clinorotation) experiments on the effect of weightlessness on the transport and segregation of colloidal particles and chromosomes. In centimetre-sized containers, both methods show that a brief initial period of weightlessness strongly inhibits particle transport. In miniature cell-sized containers under normal gravity conditions, the particle transport that self-organisation causes results in their accumulation into segregated regions of high and low particle density. The gravity dependence of this behaviour is strongly shape dependent. In square wells, neither self-organisation nor particle transport and segregation occur under conditions of weightlessness. On the contrary, in rectangular canals, both phenomena are largely unaffected by weightlessness. These observations suggest, depending on factors such as cell and embryo shape, that major biological functions associated with microtubule driven particle transport and organisation might be strongly perturbed by weightlessness.

Historical and conceptual background of self-organization by reactive processes.

Order, form, pattern and organization are properties central to much living matter. The physicochemical processes by which an initially homogeneous solution of reacting chemicals or biochemicals might self-organize is hence a question of fundamental biological importance. In most cases, solutions of reacting chemicals in a test-tube do not self-organize. Because of this, for many years, it was not thought possible that reactive processes could result in self-organization. However, progressively over the last hundred years, it has been shown that this is not always the case, and under certain conditions, the combination of reaction with molecular diffusion can lead to macroscopic self-organization. In 'complex' systems comprised of populations of strongly coupled elements, new 'emergent' properties, such as self-organization, arise by way of the dynamics of the system. Self-organizing reaction-diffusion systems form a specific type of complex system. Here, I will give a personal overview of the conceptual and historical background to this approach with an emphasis on biological self-organization.

Microtubules viewed as molecular ant colonies.

Populations of ants and other social insects self-organize and develop 'emergent' properties through stigmergy in which individual ants communicate with one another via chemical trails of pheromones that attract or repulse other ants. In this way, sophisticated properties and functions develop. Under appropriate conditions, in vitro microtubule preparations, initially comprised of only tubulin and GTP, behave in a similar manner. They self-organize and develop other higher-level emergent phenomena by a process where individual microtubules are coupled together by the chemical trails they produce by their own reactive growing and shrinking. This behaviour is described and compared with the behaviour of ant colonies. Viewing microtubules as populations of molecular ants may provide new insights as to how the cytoskeleton may spontaneously develop high-level functions. It is plausible that such processes occur during the early stages of embryogenesis and in cells.

Ground-based methods reproduce space-flight experiments and show that weak vibrations trigger microtubule self-organisation.

The effect of weightlessness on physical and biological systems is frequently studied by experiments in space. However, on the ground, gravity effects may also be strongly attenuated using methods such as magnetic levitation and clinorotation. Under suitable conditions, in vitro preparations of microtubules, a major element of the cytoskeleton, self-organise by a process of reaction-diffusion: self-organisation is triggered by gravity and samples prepared in space do not self-organise. Here, we report experiments carried out with ground-based methods of clinorotation and magnetic levitation. The behaviour observed closely resembles that of the space-flight experiment and suggests that many space experiments could be carried out equally well on the ground. Using clinorotation, we find that weak vibrations also trigger microtubule self-organisation and have an effect similar to gravity. Thus, in some in vitro biological systems, vibrations are a countermeasure to weightlessness.

Microtubule self-organisation by reaction-diffusion processes in miniature cell-sized containers and phospholipid vesicles.

Under appropriate conditions, in vitro microtubule preparations self-organise over macroscopic distances by a process of reaction and diffusion. To investigate whether such self-organisation can also occur in objects as small as a cell or an embryo we carried out experiments in miniature containers of cellular dimension. When assembled under self-organising conditions in wells of 120-500 microm, microtubules developed organised structures. Self-organisation is strongly affected by shape, being highly favoured by elongated forms. In wells of more complex shape, geometrical factors may either oppose or strengthen one another and so inhibit or reinforce self-organisation. Microtubules were also assembled within phospholipid vesicles of 2-5 microm diameter. Under self-organising conditions, we observed large shape changes from spheroids to long tubes (50-100 microm) and intertwined coils. We conclude that self-organisation of microtubules by reaction-diffusion processes can occur in containers of cellular dimensions and is capable of strongly deforming the cellular membrane.

Brief exposure to high magnetic fields determines microtubule self-organisation by reaction-diffusion processes.

A frequent feature of microtubule organisation in living systems is that it can be triggered by a variety of biochemical or physical factors. Under appropriate conditions, in vitro microtubule preparations self-organise by a reaction-diffusion process in which self-organisation depends upon, and can be triggered by, weak external physical factors such as gravity. Here, we show that self-organisation is also strongly dependent upon the presence of a high magnetic field, for a brief critical period early in the process, and before any self-organised pattern is visible. These results provide evidence that external physical factors trigger self-organisation by way of an orientational bias that breaks the symmetry of the reaction-diffusion process. As microtubule organisation is central to many cell functions, this behaviour provides a mechanism by which strong magnetic fields can intervene in biological processes.

Microtubule self-organisation by reaction-diffusion processes causes collective transport and organisation of cellular particles.

BACKGROUND: The transport of intra-cellular particles by microtubules is a major biological function. Under appropriate in vitro conditions, microtubule preparations behave as a 'complex' system and show 'emergent' phenomena. In particular, they form dissipative structures that self-organise over macroscopic distances by a combination of reaction and diffusion. RESULTS: Here, we show that self-organisation also gives rise to a collective transport of colloidal particles along a specific direction. Particles, such as polystyrene beads, chromosomes, nuclei, and vesicles are carried at speeds of several microns per minute. The process also results in the macroscopic self-organisation of these particles. After self-organisation is completed, they show the same pattern of organisation as the microtubules. Numerical simulations of a population of growing and shrinking microtubules, incorporating experimentally realistic reaction dynamics, predict self-organisation. They forecast that during self-organisation, macroscopic parallel arrays of oriented microtubules form which cross the reaction space in successive waves. Such travelling waves are capable of transporting colloidal particles. The fact that in the simulations, the aligned arrays move along the same direction and at the same speed as the particles move, suggest that this process forms the underlying mechanism for the observed transport properties. CONCLUSIONS: This process constitutes a novel physical chemical mechanism by which chemical energy is converted into collective transport of colloidal particles along a given direction. Self-organisation of this type provides a new mechanism by which intra cellular particles such as chromosomes and vesicles can be displaced and simultaneously organised by microtubules. It is plausible that processes of this type occur in vivo.

Comparison of reaction-diffusion simulations with experiment in self-organised microtubule solutions.

This article deals with the physical chemical processes underlying biological self-organization by which an initially homogenous solution of reacting chemicals spontaneously self-organizes so as to give rise to a preparation of macroscopic order and form. Theoreticians have predicted that self-organization can arise from a coupling of reactive processes with molecular diffusion. In addition, the presence or absence of an external field, such as gravity, at a critical moment early in the self-organizing process may determine the morphology that subsequently develops. We have found that the formation in vitro of microtubules, a major element of the cellular skeleton, show this type of behaviour. The microtubule preparations spontaneously self-organise by way of reaction and diffusion, and the morphology of the state that forms depends on the presence of gravity at a critical moment early in the process. We have developed a numerical reaction-diffusion scheme, based on the chemical dynamics of a population of microtubules, which simulates the experimental self-organisation. In this article we outline the main features of these simulations and discuss the manner by which a permanent dialogue with experiment has helped develop a microscopic understanding of the collective behaviour.

Numerical simulations of microtubule self-organisation by reaction and diffusion.

This article addresses the physical chemical processes underlying biological self-organisation by which a homogenous solution of reacting chemicals spontaneously self-organises. Theoreticians have predicted that self-organisation can arise from a coupling of reactive processes with molecular diffusion. In addition, the presence of an external field, such as gravity, at a critical moment early in the process may determine the morphology that subsequently develops. The formation, in-vitro, of microtubules, a constituent of the cellular skeleton, shows this type of behaviour. The preparations spontaneously self-organise by reaction-diffusion and the morphology that develops depends upon the presence of gravity at a critical bifurcation time early in the process. Here, we present numerical simulations of a population of microtubules that reproduce this behaviour. Microtubules can grow from one end whilst shrinking from the other. The shrinking end leaves behind a chemical trail of high tubulin concentration. Neighbouring microtubules preferentially grow into these regions, whilst avoiding regions of low tubulin concentration. The chemical trails produced by individual microtubules thus activate and inhibit the formation of neighbouring microtubules and this progressively leads to self-organisation. Gravity acts by way of its directional interaction with the macroscopic density fluctuations present in the solution arising from microtubule disassembly.

Gravity dependence of microtubule preparations.

The mechanisms by which biological processes are effected by gravity are not understood. Theoreticians have proposed that gravitational effects could come about from the bifurcation properties of certain types of non-linear chemical reactions that self-organise by reaction and diffusion. We have found that in-vitro preparations of microtubules, an important element of the cellular skeleton, show this type of behaviour. They self-organise by reaction and diffusion and the morphology that arises depend upon the presence of gravity, at a critical moment or bifurcation time, early in the process. At a molecular level this behaviour results from an interaction of gravity with macroscopic concentration and density fluctuations created by microtubule contraction and elongation. Numerical simulations predict macroscopic self-organisation in qualitative agreement with experiment. It is plausible that microtubule organisation by these processes occurs in-vivo.

Microtubule self-organisation and its gravity dependence.

The molecular processes by which gravity affects biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical reactions do not depend upon gravity. It has been proposed that biological systems might depend on gravity by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. In such reactions, the initially homogenous solution spontaneously self-organises by way of a combination of reaction and diffusion. Theoreticians have predicted that the presence or absence of an external field, such as gravity, at a critical moment early in the self-organising process may determine the morphology that subsequently develops. We have found that the formation in vitro of microtubules, a major element of the cellular skeleton, shows this type of behaviour. The microtubule preparations spontaneously self-organise by way of reaction and diffusion, and the morphology of the state that forms depends upon gravity at a critical bifurcation time early in the process. Experiments carried out under low gravity conditions show that the presence of gravity at the bifurcation time actually triggers the self-organising process. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive. At a microscopic level the behaviour results from an interaction of gravity with the concentration and density fluctuations that arise from processes of microtubule shortening and elongation. We have developed a numerical reaction-diffusion scheme, based on the chemical dynamics of a population of microtubules, that simulate self-organisation. These simulations provide insight into how self-organisation occurs at a microscopic level and how gravity triggers this process. Recent experiments on cell lines cultured in space suggest that microtubule organisation may not occur properly under low gravity conditions. As microtubule organisation is essential to cellular function, it is quite plausible that the type of processes described in this article provide an underlying explanation for the gravity dependence of living systems at a cellular level.