Smallest Forces with Enormous Effect on Molecules Molecular regulation of changes in cell shape and cell movement measured and made visible
Movement processes of cells are made possible by dynamic macromolecules that can build up and also break down quickly in a targeted manner. These macromolecules are part of the cytoskeleton and are called actin filaments. The formation of these very thin filaments can exert thrust forces. The thrust forces of filament bundles in turn lead to the advancement of cellular elongation or protrusions of the cell envelope. These elongations are important for cell movement, for example, and also in feeding pathogens such as pathogenic bacteria. In muscle, actin and myosin filaments are used to pull the muscle cells together via tensile forces. This happens billions of times in the course of a human life when our heart pumps every second. Professor Klemens Rottner from the Technische Universität and his team, together with colleagues from the University of Bordeaux in France, have been able to show how the pushing forces are controlled at the molecular level. The results have now been published in the journal “Nature Cell Biology“.
The formation of actin filament networks, which control cell shape change and cell movement, are regulated by molecular machines (WAVE complex), some of which have a highly complex structure. Although these machines at the plasma membrane must theoretically be subjected to thrust and, in part, tensile forces, it has never been possible to directly visualise or measure the forces acting on them. The effect that these thrust forces could possibly have on the functioning of the molecular machines has also not been researched.
“In our work, we were able to show for the first time how individual components of the actin polymerisation machines move at the plasma membrane, and how their movement changes depending on active polymerisation of the filaments they regulate,” says Professor Klemens Rottner, a molecular biologist at Technische Universität Braunschweig and at the Helmholtz Centre for Infection Research (HZI) in Braunschweig. In addition, the scientists in Germany and France were able to measure thrust forces acting on individual molecular complexes in living cells for the first time. These thrust forces are in the piconewton range, i.e. in the range of a millionth of a millionth of a newton. This means that the molecules are subjected to extremely small forces that nevertheless have a large effect.
Feedback loop of basic biological processes
“We were not only able to measure the forces, but also to show how these forces work. The individual molecular complexes are quasi mechanically removed from the membrane and then replaced by fresh molecular complexes. But since we were also able to show that an experimental immobilisation of the actin polymerisation machines increases the activity and a shorter residence time of these complexes therefore conversely leads to reduced activity, this means that nature has built in a kind of mechanical regulator here,” says Professor Rottner. This compensatory function leads, on the one hand, to fast actin polymerisation being throttled by rapid exchange of the actin polymerisation machinery and, on the other hand, to too slow polymerisation being counteracted by slowing down the exchange of the machinery. This new mechanism of a mechanical feedback loop enables a special robustness of such fundamental processes in biology.
Thrust forces visible in cells
At the beginning of the research project, CRISPR/Cas9, a method that was awarded the Nobel Prize in Chemistry in 2020 was used. This was used to first genetically destroy various subunits of the actin polymerisation machines mentioned above and to insert new units. One of these machines is called the WAVE complex. This complex is crucial for the formation of actin networks, which in turn are needed for cell movement or phagocytosis.
In collaboration with Grégory Giannone’s team from the University of Bordeaux and scientists from the École Polytechnique in Paris, special imaging techniques were used to track the movement of individual WAVE complexes within these networks. This revealed particular patterns of movement that could only be explained by thrust forces generated by actin filaments. To prove this, the WAVE complexes observed in cells were modified so that they protruded through the cell surface and could now be grasped on the outside of the cell. This made it possible to measure the forces acting on them from the outside. In addition, these complexes could also be manipulated from the outside, for example, they could be pulled out of their current position or even united into larger groups. The latter led to the observation of a mechanical feedback mechanism.
Genetic investigations at TU Braunschweig
Die genetische Manipulation fand an der TU Braunschweig in der Arbeitsgruppe von Professor Klemens Rottner statt. Das Entfernen verschiedener Untereinheiten des WAVE-Komplexes sowie das Einbringen der für die Kraftmessungen benötigten mutierten WAVE-Komplexe nahmen die beiden ehemaligen Doktorand*innen Dr. Frieda Kage und Dr. Matthias Schaks vor. „Zudem war meine Arbeitsgruppe in diesem Projekt dafür zuständig, zellbiologisch durch Messung unterschiedlicher Parameter sicherzustellen, dass die in Zellen eingebrachten, veränderten WAVE-Komplexe ihre Funktionstüchtigkeit in der Regulation der Aktinpolymerisation trotz der Manipulationen nicht verloren haben“, sagt Prof. Rottner.
Die erzielten Ergebnisse geben neue Einblicke in das Wechselspiel zwischen Signalgebung, molekularer Regulation und mechanischer Belastung, das auf die Steuerungsmoleküle dieser Aktinpolymerisationsprozesse wirkt. Zudem führt die Berücksichtigung mechanischer Einflüsse auf die molekulare Regulation biologischer Prozesse zu neuen Ansätzen für die mögliche Entwicklung von Hemm- oder Wirkstoffen, die bei der Krebsbekämpfung (im Besonderen bei der Metastasierung) oder als Antiinfektiva eine Rolle spielen könnten.