Molecular building block for cell movement and infection

Many bacterial pathogens cause their host cells to take them to the inside of the cell during an infection. To do this, they specifically manipulate the protein backbone of the cells. In this way, pathogens can better adhere, penetrate or spread inside the cells. A key role in controlling these alterations of the cytoskeleton is played by the WAVE complex, to which small regulatory proteins, called GTPases, can bind. Researchers at the Helmholtz Center for Infection Research (HZI) have now elucidated the multi-stage molecular activation mechanism of this complex. The studies provide the basis for further investigations to better understand the mechanisms of movement and the processes of infection. Their findings were published in the journal Current Biology.

The cells have what is known as a cytoskeleton – a network of filaments that not only supports the plasma membrane and reshapes the cell, but also supports the correct position of organelles and provides vesicle transport pathways. The cytoskeleton of the cell consists of small microfilaments consisting of actin protein and woven together as in a mesh. It is only through the interaction of the branched actin filaments, one nanometer thick, with special proteins of myosin and the development of the resulting power, that cell motions become possible. All of this plays an important role in cellular processes such as phagocytosis – the controlled uptake of potentially dangerous extracellular particles, such as microorganisms, by professional phagocytes. The cytoskeleton also has a particular importance in the infection of host cells by pathogens.

Bacteria can affect cellular signaling pathways and use them to colonize or invade cells. For example, pathogens such as Salmonella, Shigella or Yersinia inject certain proteins directly into their host cells with the aid of molecular sprays to stimulate the restructuring of the actin skeleton. The complex called WAVE (in English, the regulatory complex WAVE – WRC) is a target factor of the bacteria. It controls the formation of actin filaments. The team of researchers led by Professor Klemens Rottner, head of the HZI working group "Molecular Cell Biology" and a department of the same name from the Institute of Zoology of the Technical University of Braunschweig, S & H Is designed to better understand this interaction of bacteria with the host cytoskeleton.

Detailed research has allowed researchers to determine what molecular mechanism in cells activates the WAVE complex, which plays a decisive role in the polymerization of actin structural protein. The crucial signal is a molecular switch, the small Rac GTPase, which, by binding to the high-energy nucleotide GTP (guanosine triphosphate) – after its cleavage of GDP (guanosine diphosphate) – between position changes, turned on and off. "The WAVE complex as one of the key control elements is regulated by the binding of the Rac central molecular switch to two independent binding sites," said Klemens Rottner. Until now, however, the functions of these independent 5-subunit WAVE complex binding sites were still unclear.

For the experiments largely funded by the German Research Foundation (PROCOMPAS research training group: https://www.tu-braunschweig.de/procompas), the researchers used different mouse cell lines in which one or two sites simultaneously used CRISPR / Cas9. The technology was extinct. "With these mutant cells we were able to decode the different functions of the two binding sites of the WAVE complex," says Klemens Rottner scientist Matthias Schaks. For the structural biological badysis of the binding mechanism, the scientists also cooperated closely with HZI's "Structure and Function of Proteins" department, headed by Professor Wulf Blankenfeldt.

"We have been able to show that the first binding site designated" site A "is the main binding site for so-called allosteric activation of the WAVE complex, where it binds Rac, bound to the GTP, which modifies the three-dimensional structure of the complex. "said Klemens Rottner. On the other hand, the second binding site, the D site, is essential for the activation of the WAVE complex, but it is crucial for the efficient maintenance of its activity and therefore of its function in the cells, namely the formation of membrane protuberances. called lamellipodia. The mechanism of regulation described by the two sites of connection, very old from the point of view of the evolution, that the researchers could now confirm with simple mushrooms of the genus Dictyostelium, in cooperation with colleagues of the CRUK Beatson Institute from Glasgow.

The cellular systems developed in this project, which have elucidated the mechanism described, also allow the use in infection experiments of a variety of bacterial agents using cytoskeletal manipulation. their host cells, such as Salmonella and Gram-negative Shigella. Listeria and staphylococci. These scientific works should not only understand the complex interactions with the host and the infection pathways in his cells, but also prevent them.

You will find press photos and videos of this press release on the HZI website at https://www.helmholtz-hzi.de/de/aktuelles/news/ansicht/article/complete/ molekularer_baukasten_fuer_zellbewegung_und_infektion /

The Helmholtz Center for Infection Research:
At the Helmholtz Center for Infection Research (HZI), scientists are studying the mechanisms of infection and their defense. What makes bacteria or viruses pathogenic: Understanding this is the key to developing new drugs and vaccines. HZI is a member of the German Infection Research Center (DZIF). www.helmholtz-hzi.de

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Original publication:
Matthias Schaks, Shashi Prakash Singh, Frieda Kage, Peter Thomason, Thomas Klünemann, Anika Steffen, Wulf Blankenfeldt, Stralis Theresia, Robert H. Insall and Klemens Rottner: Rac GTPase's distinct interaction sites with the WAVE regulatory complex Non-redundant In Vivo Functions, Current Biology (2018). https://doi.org/10.1016/j.cub.2018.10.002

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