Structure and isoform diversity of the Arp2/3 complex

The movement of cells plays a fundamental role in many physiological and pathological processes, such as the development of an organism, the immune response or cancer metastasis. The key player in the ability of cells to move is the so-called actin cytoskeleton and its vast number of associated factors. Considering the importance of the actin cytoskeleton in movement and many other cellular processes, it comes as no surprise that its deregulation has also been implicated in diverse pathological conditions, such as the above-mentioned cancer metastasis, developmental disorders or pathogen infections. Therefore, the actin cytoskeleton is the target of extensive research to understand fundamental mechanisms underlying both the regulation of its dynamic assembly and disassembly, and to develop therapeutic approaches targeting associated pathological conditions. Monomeric actin polymerizes into filaments to form diverse higher-ordered structures with varying actin filament arrangement. These structures provide the force for cells to move, but also allow them to sense and manipulate their environment. A key factor in regulating the actin cytoskeleton is the multi-protein Arp2/3 complex. Besides its role in defining processes in cell migration, the Arp2/3 complex is also exploited by various bacteria and viruses for infection and spread. It nucleates new actin filaments using pre-existing filaments as a template, by forming so-called branch junctions on their side. In order to generate actin filament networks, the Arp2/3 complex needs to undergo an activation process, which includes large structural arrangements, such as the reorientation of the seven Arp2/3 complex subunits, as well as the binding of an actin filament and the recruitment of new actin monomers. In order to understand how the activation of the Arp2/3 complex occurs and hence obtain insight into its exact function, detailed structural information on the branch junction (meaning the active complex in association with actin filaments) is required. Due to methodological limitations in conventional structure determination approaches, this detailed information has remained elusive. In our project we will use and develop novel cryo-electron microscopy and image processing approaches to visualize the structure of the Arp2/3 complex branch junction directly within cells at resolutions smaller than one nanometer. This will allow us to describe for the first time how the active Arp2/3 complex is arranged. In addition, we will employ a combination of molecular biology, light microscopy, electron microscopy and image processing to study the role of the individual subunits of the Arp2/3 complex, and how they regulate the stability and dynamics of the branch junction, as well as its interaction with additional co-factors. Our results will provide important new information on a major regulator of the actin cytoskeleton and its role in a variety of processes in health and disease. 1

The movement of cells plays a fundamental role in many physiological and pathological processes, such as the development of an organism, the immune response or cancer metastasis. The key player in the ability of cells to move is the so-called actin cytoskeleton and its vast number of associated factors. The actin cytoskeleton is the target of extensive research to understand fundamental mechanisms underlying both the regulation of its dynamic assembly and disassembly, and to develop therapeutic approaches targeting associated pathological conditions. Monomeric actin polymerizes into filaments to form diverse higher-order structures with varying actin filament arrangement. These structures provide the force for cells to move, but also allow them to sense and manipulate their environment. A key factor in regulating the actin cytoskeleton is the multi-protein Arp2/3 complex. Besides its role in defining processes in cell migration, the Arp2/3 complex is also exploited by various bacteria and viruses for infection and spread. It nucleates new actin filaments by forming so-called branch junctions on the side of pre-existing filaments. In order to generate actin filament networks, the Arp2/3 complex needs to undergo an activation process, which includes large structural arrangements. In order to understand how the activation of the Arp2/3 complex occurs and hence obtain insight into its exact function, detailed structural information on the branch junction (meaning the active complex in association with actin filaments) is required. Due to methodological limitations in conventional structural biology approaches, this detailed information has remained elusive for long time. In our project we have developed novel cryo-electron microscopy and image processing approaches to visualize the structure of the Arp2/3 complex branch junction directly within cells at resolutions smaller than one nanometer. This has allowed us to describe for the first time how the active Arp2/3 complex is arranged. In addition, we have developed a computational toolbox to quantitatively analyze cryo-electron microscopy data visualizing cellular actin networks. In combination with genetic engineering of migratory cells we have further elucidated the importance of one Arp2/3 complex subunit and its isoforms, via describing in an integrated cellular structural biology approach how Arp2/3 complex composition determines stability and dynamics of the branch junction, as well as the positioning of additional co-factors. In summary, our results have provided important new information on a major regulator of the actin cytoskeleton and its role in a variety of processes in health and disease.