Bridging the gap between simplified biomimetic systems and cell free extracts to understand actin dynamics. – Golden Gate

Cell shape changes are involved in many cellular processes including cell morphogenesis, establishment of cell polarity and cell motility (Pollard and Borisy, 2003). Cells change shape by exerting internal forces based on dynamical assembly of cytoskeletal proteins. In our project, we will focus on the assembly and dynamics of one cytoskeletal polymer, actin. Actin is a dynamic polymer that undergoes assembly and disassembly simultaneously. Complex cytoskeletal rearrangements and dynamics can be observed in vivo or in concentrated cell extracts. However, individual reactions are difficult to isolate in these conditions. There is a need of simplified systems in which actin dynamics can be reconstituted in the presence of actin associated factors in order to understand the mechanism of force generation. Ideally, we would like to measure each reaction on actin simultaneously and determine how rates depend on the concentrations and activities of each component of the system. Our project is based on existing results. First, the observation of single actin filament dynamics using a minimum set of proteins (Michelot et al., 2007) was achieved in the Blanchoin lab. Second, macroscopic movement of objects based on these actin dynamics were reconstituted in the Sykes lab using beads placed in purified proteins (Bernheim-Groswasser et al., 2002). These existing results prove that it is possible to reconstitute actin dynamics and actin-based movement in a much simpler system than a cell. However, whether or not these experiments accurately reproduce cytoplasmic events is the issue we want to address here. In fact, whereas actin dynamics can be reproduced in purified systems in some cases, the whole mechanism of cellular actin dynamics is far more complex and involves much more players. We propose to study in parallel single actin filament dynamics and actin-based movements both in pure protein assays that we will complexify with known actin associated factors, one by one and in cell free extracts using sophisticated imaging systems. This bottom-up approach should reveal if actin dynamics require other factors in cell cytoplasm. We will also reprogramme cells to prepare manipulable extracts in which actin filament dynamics can be monitored while undergoing steady-state actin filament turnover. Using the expertise of the Gautreau lab, we will simplify these extracts by removing, one by one, putative components involved in actin dynamics. This top-down approach will allow us to build a bridge between complex behaviors of actin filaments in a cellular context and over-simplified systems able to reconstitute actin dynamics. Actin dynamics occurs at the cell membranes mainly. Therefore, bulk experiments, where all actin associated factors freely diffuse in solution, will not be sufficient to address the mechanism of force generation and movement. Hence, we will place actin regulators at the surface of reconstituted membranes and monitor their deformations. Moreover, we will use the proteins in their native form, i.e. native multiprotein complexes when appropriate rather than isolated recombinant proteins. Single actin filament dynamics and movement will be then analyzed through theoretical models and simulations based on established parameters. From these approaches we will dissect the many layers of regulation necessary to organize actin filaments in dynamical larger ordered structures necessary for cellular function. We are confident that general principles about molecular mechanisms in the actin cytoskeleton will emerge from this work.