167. THE ROLE OF SCAR/WAVE IN THE REGULATION OF CELL SHAPE AND TRACTION FORCES DURING AMOEBOID MIGRATION
Name: Effie E Bastounis
Grad Year: 2012
Cell migration is involved in important physiological processes (immune system, embryonic development, wound healing), as well as in disease processes (metastatic spreading of cancer, formation of atherosclerotic plaques in blood vessel). In particular, we are interested in amoeboid cell migration by using the amoeboid form of Dictyostelium. Dicty has similarities with neutrophils and macrophages and so can be used as a model for understanding the behavior of these more physiologically relevant cells.
Amoeboid motility is the result of the repetition of a set of periodic cell shape changes leading to oscillations of the cell length which compose the so called motility cycle: protrusion of pseudopods through actin polymerization and attachment to the substrate, contraction of the cell body, detachment and retraction of the back of the cell through contractile proteins (e.g. myosin II) and relaxation. This cycle is driven by the dynamics of actin filaments which are partially controlled by the activation of Arp2/3 through the SCAR complex. Our aim is to identify the way individual members of the SCAR complex affect the dynamic behavior of actin filaments by comparing wild type migrating Dictyostelium cells with cell lines carrying mutations in these proteins.
We acquire time-lapse images of Dictyostelium migrating towards a source of chemoattractant (cAMP). At each instant of time we acquire two images: one containing the DIC image and another one with a GFP reporter for F-actin (ABP-GFP).
For this comparison we intend to characterize the motility cycle quantitatively by extracting the dominant shape changes using Principal Component Analysis (PCA) of time-lapse images. This analysis will enable us to study the corresponding traction forces and distribution of fluorescence-labeled reporters of F-actin in migrating cells and their association to the regulated activity and localization of molecular markers.
Using wild-type cells (wt) as reference, we investigate the SCAR activity by studying different null cells (pirA- ,napA- ,scrA- ). We thus study what kind of shape changes they implement as well as the period they need to implement this repertoire
We also associate the shape changes with the distribution of F-actin which we are able to visualize by using the ABP-GFP reporter. Finally we are expanding our analysis to biochemical techniques that can reveal the role that disruption of a gene that encodes for a member of the SCAR complex can affect another member as well as the study of double knockout cells (scrA-/pirA-, scrA-/napA-).