Molecular Mechanisms of Calcium-Regulated alpha-Actinin

Cytoskeleton is a complex supramolecular structure within cells that helps them to maintain shape, stability and internal organization, as well as to perform various dynamic functions like movement. In this way it is analogous to the skeleton of complex multicellular organisms, however at a much smaller scale. The cytoskeleton is made up of different kinds of protein filaments and accessory proteins, which associate, dissociate and rearrange on-demand and in response to intra- and extracellular signals. Understanding its structure, dynamics, regulation and function is important both in fundamental science as well as in medicine where abnormal processes and mutations lead to cytoskeletal malfunctions and in turn to various diseases. We focus on the the most dynamic type of cytoskeletonthe actin cytoskeleton, where actin filaments are organized into 3D-networks or tightly packed bundles held together by ubiquitous proteins a-actinin-1 and -4, which self-associate to form homo-dimeric crosslinking units. In particular, we are interested how the crosslinking mode of these two a-actinin is regulated. The main aspect appears to be calcium-binding to a specific region within a-actinin, and we hypothesize that a-actinin phosphorylation and association of both species to form heterogeneous dimeric complexes further adds to the complexity of this regulatory mechanism. By studying these events at the detailed molecular level, we aim to answer how a-actinin-1 and -4 conformational plasticity is modulated by calcium binding. This will in turn help to understand the regulation of adhesion contacts, cell protrusions and stress fibers, which are structures pivotal for cell adhesion and movement. The experimental part of the project involves diverse methods to study proteins both in isolated environment as well as in complex cellular setup. Advanced methods of structural biology, including protein crystallography and cryo-electron microscopy, and cell biology approaches will be employed and supplemented with assays aimed to study the actin filament bundling activity of a-actinin-1 and -4. Project will be jointly run at University of Vienna, Austria, and University of Ljubljana, Slovenia, where the two young and dynamic collaborating groups led by Kristina Djinovic-Carugo (Austria) and Brigita Lenarcic (Slovenia) will join forces to tackle the project challenges. The results will deepen the understanding of structure and function of fundamental proteins a-actinin-1 and -4, pave the way for better understanding of the molecular basis of actinin-associated disease, and contribute to development of novel cell-based technologies.