Passive partner beats rivals - New research tool to investigate actin, one of the most abundant and important proteins
Actin provides cells with stability, mobility and the ability to transport molecular cargo. It is also a key player for muscle contraction and cell division. These diverse functions make it one of the most abundant, most important and therefore most intensively studied proteins. One aspect of these studies is the role of actin in the development of cancer and in many other diseases. Marker molecules aimed at highlighting actin under the microscope have so far suffered from several restrictions. Researchers at the Max Planck Institutes of Biochemistry and Neurobiology in Martinsried, Germany, were now able to develop a novel actin marker from a naturally occurring protein that binds to actin in yeast cells. The researchers successfully introduced the so-called “Lifeact” into various types of cells and tissues without effects on cellular actin functions, making it superior to its competitors. The new marker could for the first time allow basic as well as biomedical research on actin without restrictions. (Nature Methods, June 8, 2008)
Actin is an essential part of the cytoskeleton, a flexible and highly dynamic network of filaments. Some of these thin fibres are made of actin molecules. “Actin filaments serve as mechanical support to maintain the cells’ shape” explains Dr. Roland Wedlich-Söldner, junior group leader at the Max Planck Institute of Biochemistry and one of the project leaders. “They’re also used as transport tracks: So-called motor proteins carry molecules and other cargo along actin to their respective cellular destinations.” Without actin cells would collapse and largely freeze to a standstill.
Most of the time actin filaments occur in large assemblies. “They can even build dynamic networks,” says Dr. Michael Sixt from the Max Planck Institute of Biochemistry and also responsible for the study. “This molecular mesh allows whole cells to flow forward.” This kind of cell motility still holds some surprises as Sixt, Wedlich-Söldner and colleagues discovered recently. The researchers found proof that white blood cells are able to move exclusively with the help of their actin networks. This unique ability among mobile cells makes leucocytes independent from their environment and enables them to patrol the body for pathogens.
Functions like cell motility require high flexibility and dynamics of the actin filaments, a characteristic they share with other fibres of the cytoskeleton. They’re being assembled on demand – and just as swiftly disassembled again. Most of the time actin filaments undergo a so-called treadmilling cycle: While new actin molecules are added to one end of the fibre, an equal number is removed from the other end. Therefore filaments appear to move through cells: growing and shrinking at the same time and rate.
Research tools need to account for this extraordinary dynamics. Methods that yield static snapshots from cells are often restricted to some types of questions. Actin filaments are therefore often observed in living cells with the help of microscopes and video cameras. For these kinds of investigation, though, scientists depend on markers which bind to actin or other target molecules in the cell. These markers are usually coupled to fluorescent dyes or proteins which upon illumination in the microscope will reveal the position of the dye and indirectly the position of the marker and its target.
A perfect marker resembles a quiet observer with endurance: It binds with high precision and strongly enough to be detected but without disturbing its target’s function. “Most currently available actin markers do not even come close to this ideal,” says Sixt. “These molecules are either complicated to handle or only recognize certain types of actin structures. The biggest drawback is that to some degree they all influence actin’s activity – and this could potentially render results useless.”
The team led by Wedlich-Söldner and Sixt has now developed a novel actin marker that manages to avoid this kind of disturbance. Starting point for their study was the protein Abp140, which binds to actin in yeast cells – and is one of hundreds of so-called actin binding proteins. “All members of this protein class are potential candidates for markers,” says Wedlich-Söldner. “And some have been put to the use already. Unfortunately, the size of these markers can already be a problem. Actin filaments are tightly decorated with all sorts of actin binding proteins and any marker will have to squeeze in between in sizable numbers to be visible at all.” The Max-Planck researchers therefore used truncated versions of Abp140 to determine the minimal region of the protein that conveys the ability to bind to actin. “To our surprise, we ended up with a fragment of only 17 amino acids or protein building blocks,” remembers Wedlich-Söldner. “This small piece of protein has then been subjected to a variety of tests.” And “Lifeact”, the novel marker, proved a success in all respects. In combination with fluorescent dyes the molecule showed its worth in all tested tissues and cell types from yeast and kidney cells to white blood cells and neurons – without any toxic side effects or influence on actin’s functions.
“We even found Lifeact to be more specific and sensitive compared to other available markers,” says Wedlich-Söldner. “The molecule is also cheap in production, easy in its application and can be adapted to special demands. It is hard to imagine anything closer to the ideal marker. This will open new options for basic and biomedical research to investigate the actin molecule in all of its functions.” With the help of the Max Planck Innovation GmbH the researchers have filed for a patent. Once a company is interested in “Lifeact” it will be easier to provide researchers with this novel and versatile marker. [SW]
Julia Riedl, Alvaro H Crevenna, Kai Kessenbrock, Jerry Haochen Yu, Dorothee Neukirchen, Michal Bista, Frank Bradke, Dieter Jenne, Tad A Holak, Zena Werb, Michael Sixt & Roland Wedlich-Söldner: Lifeact: a versatile marker to visualize F-actin. Nature Methods, 8. Juni 2008.
Further informationen can be obtained from:
Dr. Roland Wedlich-Söldner
Max Planck Institute of Biochemistry
Tel.: +49 89 8578 3410
Dr. Michael Sixt
Max Planck Institute of Biochemistry
Tel.: +49 89 8578 2849
Eva-Maria Diehl, Public Relations
Max Planck-Institute of Biochemistry
Tel.+49 89 8578 2824