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Moving on transient tracks



8 September 2016 – Microtubule research is as multifaceted as the structures themselves. Scientists in the field study the molecule’s structure and dynamics, its function in cell division, cell migration or axon growth, its role in evolution or disease development. “The tubulin field has become so broad you can hardly call it a field anymore,” said Carsten Janke, who co-organized a large EMBL | EMBO symposium on microtubules in May this year to bring the different communities together and to celebrate tubulin’s 50th anniversary.


Discovering the building blocks

The history of molecular microtubule research began in the 1960s, when Gary Borisy in the lab of Edwin Taylor embarked on a risky PhD project – isolating the components of the mitotic spindle to understand its function. In a very elegant approach, he used colchicine, which was thought to block mitosis by interfering with the spindle, as an entry point.


But after Borisy succeeded in isolating the “colchicine-binding protein”, he found, to his surprise, that it was also present in non-dividing cells, particularly in brain. In fact, it could be isolated from a number of different sources that all had one thing in common: They contained microtubules. Borisy and Taylor thus suggested that the “colchicine-binding protein” – today known as tubulin – was the microtubule subunit. It was the beginning of a long story full of exciting discoveries.


Counting individuals

For many years thereafter, researchers were puzzled about how microtubules form and disappear. They suspected some sort of “dynamic equilibrium” between tubulin molecules and polymers, but the details were unknown until Timothy Mitchison in the lab of Marc Kirschner discovered them as a side project in the 1980s.


The goal of Mitchison’s PhD thesis was to purify centrioles, the structures that nucleate mitotic spindles, using an assay of growing microtubules to test the purity of his fractions. But he couldn’t isolate enough protein to do anything useful, so he started taking a closer look at his assay instead. “I counted the number of nucleated microtubules as a function of tubulin concentration and realized this strange behavior where microtubules were just disappearing,” he recalled. The researchers coined the term “dynamic instability” to describe this behavior: microtubules switch between phases of growth and rapid shrinkage, called “catastrophe”, whereby growth and catastrophe were generated by different mechanisms. “All prior work was done on microtubules in bulk and averaging them,” said Mitchison. He discovered a new phenomenon by looking at individuals and counting their numbers.


The discovery of dynamic instability explained a lot. Growth and sudden depolymerization could do mechanical work, like pulling chromosomes. Mitchison and Kirschner also proposed that microtubules would display an “exploratory behavior”, where they would grow until they either attach to something or collapse. This “search and capture” mechanism could explain, for example, how microtubules catch hold of chromosomes.


Looking for myosin and finding kinesin

Discoveries are not always straightforward. When Ronald Vale discovered kinesin, he was really looking for a non-muscle myosin. Vale was interested in how material is transported along axons and suspected some form of myosin, since myosin was known to move along actin cables in muscle cells. But eventually, using a combination of in vitro biochemistry and electron microscopy, he noted that organelles in axons were actually moving along microtubules. When he managed to isolate the corresponding motor protein, he called it kinesin.


“The roots of cell biology come from studying muscle and cilia, where myosin and dynein had been discovered. But those were considered very specialized tissue. It really took the discovery of kinesin to realize the sliding mechanism in cilia can be generalized to the motor for transport in neurons and other cells,” said Mitchison.


Kinesin moves along microtubules – but how? Does it jump, flip, walk, or slide? Understanding the mechanism required looking at single molecules. Joe Howard, working with Ron Vale, looked hard for conditions for single molecule motility – and found them. “Single molecules had thus far only been studied in the ion channel field,” Howard explained, referring to work by Erwin Neher and Bert Sakmann that was later awarded the Nobel Prize. Howard's assay for kinesin was the first single molecule assay outside the ion channel field. Thanks to this technology, we now know that it walks, we know its step size and how it generates force.


From atoms to complex systems

Microtubule research was driven by a love of detail and attention to small things – single molecules – to explain general principles, and this work is ongoing. “One of the open questions in the field still is catastrophe,” said Howard. It is known that microtubules are protected in the growth phase by a “GTP cap” and losing the cap results in microtubule teardown. But how is the cap lost, what controls the length of microtubules? Many labs are working on this problem, using single molecule techniques and advanced microscopy.


But there is another movement going on, which is alluded to in the EMBL | EMBO symposium title: Microtubules: From Atoms to complex systems. “The next challenge is to look at microtubules in the context of cells and organisms” said Janke. This approach may, eventually, also lead to clinical applications. Taxol, for example, binds microtubules and is used to treat cancer. It causes side effects, particularly in neuronal tissues, but just the fact that it is somewhat specific to cancer cells is curious enough. “How can there be any selectivity between tissues, if the drug’s target protein is in virtually every cell?” asked Mitchison. “If we understood how Taxol killed cancer cells, that would help us develop better drugs,” he said.


Similarly, genetic phenotypes that affect microtubules are often selective to certain cell types, despite the fact that the respective proteins are expressed ubiquitously. Different tubulin gene variants and post-translational modifications may generate subtle differences in microtubule behaviour. “For a long time the significance of these small differences was a puzzle. But we now know that they manifest in situations where processes need to be precisely controlled, like in neuronal development,” said Janke. Indeed, many mutations that cause neurodevelopmental disorders are either in a tubulin gene or a gene for microtubule-associated proteins. Understanding the consequences of these small changes in the context of the whole organism is still an open field. “I expect that the number of people interested in microtubules will increase exponentially. And neuronal development is really one of the most important aspects,” said Howard.


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Tilmann KießlingTilmann Kießling
Head, Communications
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