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Diversity is strength in cells

Researchers from the Netherlands, France and Austria have engineered a minimal model of the cell’s cytoskeleton, an interpenetrating network of different protein filaments that controls cell deformability. To identify the role of filament interactions, they created a model consisting of two different protein filaments, vimentin and actin, connected by custom-designed cytolinker proteins. They showed that the two filaments together form stiffer networks than each filament alone. This mechanical synergy may explain how cells can tune their stiffness and strength to the demands of the human body.

Actin-vimentin bundles induced with the cytolinker, imaged by electron microscopy

Many studies on the components of the cytoskeleton but few on their interactions

Without the cytoskeleton, our cells would be shapeless bags of chemicals. The cytoskeleton forms a filamentous scaffold that controls the overall shape, internal organisation and also motility of the cell. The scaffold consists of three different types of protein filaments, each playing important roles in cell deformability: intermediate filaments, actin filaments and microtubules [1].

Although the properties of these three filaments have been extensively studied, the interactions between them remain poorly understood. Researchers do know, however, that these interactions are essential to the normal functioning of cells. In particular, previous research had shown that cells rely on specific proteins, commonly called cytolinkers, to glue the three types of filament together.

A new study on cytoskeletal interactions reveals more about the ability of cells to tune their stiffness

In a recent study published in the European Journal of Cell Biology, the Koenderink Lab (Delft University of Technology, Netherlands), in collaboration with researchers from France and Austria, developed a new minimal model system of the cytoskeleton to study how interactions between cytoskeletal filaments via cytolinkers control cell mechanics. They purified actin filaments and a special type of intermediate filament called vimentin and connected them via a custom-designed cytolinker protein called ACTIF. They designed the ACTIF protein using the actin- and vimentin-binding parts of plectin, an essential cytolinker protein in human cells.

Schematic of sequential steps in the TIRF experiments to probe ACTIF-mediated binding of F-actin to vimentin filaments (left). First ACTIF (5 nM in F-buffer) was flushed into the channel containing surface-bound vimentin filaments for 15 min. Next pre-formed F-actin (0.1 µM in F-buffer) was flushed in. Images (right) show snapshots after each step with a merged image of the final situation on the right. 

After establishing the functionality of their model system of the cytoskeleton, the researchers then tested its mechanical properties. They discovered that composite networks combining actin and vimentin are stiffer than expected from the sum of their parts. This mechanical synergy may explain how cells can dynamically tune their stiffness, which is important, for instance, when they have to migrate.

Collaboration with Austrian and French researchers to build a minimal model system from the bottom up

Irene Istúriz Petitjean

“It is very difficult to understand the role of cytoskeletal interactions in cells because cells are very complex. This is why we have used a bottom-up approach to reconstitute a minimal model system containing only the parts of the cell we are interested in. By studying the interactions between actin and vimentin, we can better understand the mechanical synergy between these two different cytoskeletal filaments, which is of crucial importance for cell mechanics and migration,” explained Irene Istúriz Petitjean, doctoral candidate in Gijsje Koenderink‘s team and first co-author of the study.

“Building this minimal model of the cytoskeleton was a real challenge, and we encountered many technical hurdles during the project. For instance, we had to design a shorter, minimal version of plectin, the natural cytolinker that connects actin and vimentin filaments in cells, because plectin is such a giant protein that it cannot be expressed and purified from bacteria,” said Irene. To address this hurdle, the Dutch team benefited from the support of Gerhard Wiche, from Max Perutz Laboratories, a joint venture between the University of Vienna and the Medical University of Vienna in Austria.

“Gerhard shared his expertise on plectin with us. He sent us an essential component to build ACTIF and helped us verify that our design provides a realistic minimal model system for actin and vimentin cross-linking.”

To prove the cross-linking activity of ACTIF, the researchers collaborated with Cécile Leduc and Quang D. Tran, from the Institut Jacques Monod (Université Paris Cité / CNRS) in France.“Cécile and Quang helped us at several stages. They helped us overcome bottlenecks in vimentin purification and thanks to their expertise in single-molecule imaging, we could prove by fluorescence imaging that ACTIF was indeed able to cross-link actin and vimentin filaments.”

Next steps and future collaborations

Gijsje Koenderink’s team has therefore succeeded in designing a minimal model system for cytoskeletal interactions in cells. The researchers are now studying the mechanical synergy between these two types of filaments in more depth, to understand how cells modify their mechanical properties by tuning cytoskeletal interactions. “We have also started a new collaboration with a team in Austria to express this construct in mammalian cells, so that we can directly compare observations in the synthetic model system with those in cells,” said Irene.

In the future, the team aims to encapsulate the system in cell-sized membrane containers to create synthetic cells with life-like mechanical properties.

References

Irene Istúriz Petitjean, Quang D. Tran, Angeliki Goutou, Zima Kabir, Gerhard Wiche, Cécile Leduc, Gijsje H. Koenderink, Reconstitution of cytolinker-mediated crosstalk between actin and vimentin,
European Journal of Cell Biology, 2024, 151403, ISSN 0171-9335

[1] Microtubules and Filaments

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