Assembly dynamics of microtubules at molecular level
Microtubules – small tubes of protein – act as pillars and conveyers of cells in our body. They constantly grow and shrink, but up till now scientists had only a statistical image of their dynamic behaviour: stagnant pictures through electron microscopes. Researchers of the FOM-Institute for Atomic and Molecular Physics (AMOLF) in Amsterdam have developed a technology to investigate the growth and shrinkage of microtubules up to a scale of separately growing and falling molecules to these tubes. This leaves the way to a detailed insight in the action of microtubules in our cells. The researchers have published their results in the Nature issue of the Advanced Online Publication of 25 June 2006.
The cells of all organisms that have nuclei (yeast, plants, animals, people) are structured in a way that cells keep their shapes: the cytoskeleton. This cytoskeleton exists of microtubules and other filaments, such as actin filaments: long sequences of protein molecules. Along microtubules, for instance, organelles (a kind of small organs with specific functions) are conveyed through the cell and a protein like kinesin passes by. Microtubules usually consist of 13 or 14 filaments of the protein tubulin, together making a solid hollow tube with a diameter of about 25 nanometers. The tubes constantly grow and shrink by way of either attaching or breaking off tubulin-dimers (a dimer is a molecule consisting of two components). Observations from electron microscopes show the structure of the tip of the microtubules. At that point the growth and shrinkage take place. It also shows that at growth the tip of a microtubule is shaped by a ‘skin’ of convex tubulin filaments and that at shrinkage the tip consists of even more convex, separate tubulin filaments. The pictures that are taken with electron microscopes only yet provide statistical instantaneous exposure. On the progress of growth and shrinkage itself, there is only average data known about some thousands of tubulin molecules together, obtained by light microscopy. Researchers like to know how the dynamic behaviour of microtubules exactly run, because they play such an important role in the cell.
Having grow microtubules in the lab
Researchers* at the FOM-Institute for Atomic and Molecular Physics (AMOLF) have developed an experimental setting, in which the events can be followed that are taking place at the tip of a growing or shrinking microtubule up to the level of separate tubulin-dimers (8 nanometers long). In previous research Marileen Dogterom and her researchgroup at AMOLF have developed the technology to measure the growing power of a microtubule by securing a small tube with a so-called optical pair of tweezers and having grow the loose tip of the tube towards a solid obstacle. The tube will press the optical tweezers off its place due to the counter force of the obstacle. So, the force that is needed to keep the optical tweezers in its place will indicate the power that the growing microtubule is wielding. Thereupon, the growth of the microtubule can be deduced very accurately. The researchers put the experimental set-up in a cell through which they are able to having flow a tubulin-solution.
In this setting the researchers have now been investigating the growing and shrinking of a microtubule. The length of the tubulin-dimer is known (namely 8 nanometers) and one thing that can be expected is a growth or shrinkage in little steps of 8 nanometers – per tubulin-dimer on or off. It appears that a microtubule that has a sufficient concentration of tubulin available will almost immediately grow, sometimes in steps of 20 to 30 nanometers. In cooperation with the Max-Planck-Institut für molekulare Zellbiologie und Genetik in Dresden the researchers have also investigated the influence of the protein XMAP215. It is known that this protein stimulates the growth of microtubules. This appeared to be the case very clearly in the experiments. The microtubules now grew in steps of 40 to 60 nanometers. The growth also went faster in the latter case.
Cause of large growing steps
The question now to ask is where exactly these large steps do come from. Do loose tubulin-dimers join firstly and then suddenly the convex 'skin' of tubulin fragments at the tip of the microtubule becomes straightened? Or do (long) loose tubulin–oligomers (oligomers consist of a number of dimers) form firstly and then attach to the tip of the microtubule? The latter step seems to occur, so the researchers write. Also by adding XMAP215 the second process seems to come in action: XMAP215 stimulates loose tubulin-dimers to growing together, after which XMAP215 and tubulin-oligomers as a whole seem to become attached to the microtubule at once. If in that case the microtubule shrinks, this again is going to happen in steps. Obviously the XMAP215 has to fall out before the tubulin-dimers can crumble off.
The researchers write that their measuring method leaves the way for also examining the progress of the interaction between microtubules and other proteins and between microtubules and other cell parts, like the kinetochore (the material body that attaches microtubules to chromosomes during fission of cells). This is fundamental in order to be able to see into the behaviour of the microtubules.
*Jacob Kerssemakers, Laura Munteanu, Liedewij Laan, Tim Noetzel (Dresden), Marcel Janson and Marileen Dogterom
For more information contact Prof.dr Marileen Dogterom, phone +31 (0)20 608 12 49 or +31(0)20 608 12 34.