Cell Behavior and the Role of Profilin

Detta är en avhandling från Stockholm : Wenner-Grens institut för experimentell biologi

Sammanfattning: Profilin is a key regulator of the microfilament system. It binds to actin monomers in a 1:1 complex, forming the profilin:actin complex, which is the major precursor of actin for filament formation in vivo. The distribution of profilin has been studied in a variety of cells. It is present not only in the cytoplasm but also in the nucleus. In the cytoplasm, it is evenly distributed in a dotted pattern, which is concentrated at the edge of advancing lamellipodia and in the perinuclear region. In the nucleus, it is localized to Speckles and Cajal bodies. However, the distribution of the profilin:actin has not been possible to establish due to the lack of specific reagents. In this thesis I present the localization of the profilin:actin complex and demonstrate the importance of profilin during cell migration.The distribution of the profilin:actin complex was studied using affinity purified antibodies generated against a covalently coupled variant of profilin:actin in colocalization experiments with VASP and the Arp2/3 complex. In both cases, close co-distribution with profilin:actin was found. In order to study the role of profilin in vivo in migratory cells, I used the siRNA-technique to deplete profilin from motile mouse melanoma B16 cells. The particular cell line employed expressed actin fused to green fluorescent protein, which enabled imaging of live cells. Upon profilin-deficiency severe effects on cell behavior were observed, e.g. the cells lacked the ability to form characteristic broad lamellipodia at advancing edges, instead small protruding structures were generated and extended with a significantly reduced rate compared to control cells. Observations were also made suggesting that profilin regulates the expression of actin in mammalian cells.A new experimental system for studies of myoblast fusion and subsequent myotube formation in vitro was also established during these studies. This will facilitate systematic studies of molecular processes connected to muscle development.