Mechanism od Filamin Action in Response to Mechanical Stimuli

Abstract

Molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation, which is critical in homeostasis and many diseases, are not well understood. Dictyostelium discoideum cells exposed to a brief burst of shear flow show rapid and transient activation of multiple components of the signal transduction network, a response that requires an intact actin cytoskeleton of the cell. However, exactly what aspect of the actin cytoskeleton network is responsible for sensing and/or transmitting the signal is unclear. Previous data from our laboratory suggested that actin crosslinking protein filamin is involved in the ability of cells to respond to shear flow. In this study we further characterized the mechanism of filamin action in this response. Filamin itself showed rapid and transient relocalization from the cytosol to the cortex following 2 sec stimulation with shear flow. To detect activation of the signal transduction network in the presence or absence of this actin binding protein, we used fluorescently tagged Ras binding domain biosensor that detects active Ras and was previously shown to relocalize to the cortex following mechanical stimulation. Reduced responsiveness of the network to stimulation with shear flow in the absence of filamin was specific to mechanical stimuli since response to global stimulation with a chemoattractant was comparable between cells with or without filamin. To understand how filamin might be regulating shear flow induced responses we generated truncation constructs of filamin lacking either the actin binding domain or the dimerization domain. Studies are underway to determine whether these truncation constructs are able to rescue the reduced response of filamin null cells to brief stimulation with shear flow, which will offer insight into the molecular mechanism of filamin action in this context.