Profilin:actin in cell motility : A search for profilin:actin binding proteins
Abstract: The profilin:actin complex is a major source of actin for actin filament growth in vivo. A number of proteins regulating either profilin or actin has been described since profilin:actin was isolated during the 1970s. Since then, profilin and actin and their binding partners have been intensively studied. The ability of profilin:actin to interact with the fast polymerizing end of actin filaments focus the interest to components that regulates this interaction; this is the theme in this thesis.A chemically cross-linked and therefore non-dissociable profilin:actin complex, called PxA, was used in these studies which led to development of a rapid screening method to search for proteins that bind to profilin:actin. The method allows a simultaneous detection of proteins that separately interact with profilin, actin and/or profilin:actin. Here the technique was used to screen cell and tissue extracts, before and after gel filtration, for components that showed a unique interaction with the profilin:actin complex. Mass spectrometry was then used for their identification. Furthermore it was demonstrated that profilin:actin binding components are present in RNA containing, large molecular weight complexes.Two different PxA immunizations generated two separate populations of affinity purified profilin and actin antibodies. The actin antibodies from these two populations showed significant differences in the staining pattern when used for fluorescence microscopy of tissue cultured cells. One of these appeared to bind monomeric actin while the other bound to filamentous actin. Both of the profilin antibody preparations stained cells in a dotted pattern. The distribution of epitopes recognized by the different actin antibody preparations was determined using a combination of protease digestion, gel electrophoresis and mass spectrometry. The result demonstrated partially different epitope recognition.The actin associated protein palladin contains sequence motifs typical for profilin-binding proteins suggesting that profilin may bind palladin. The potential profilin-palladin interaction was studied using a combination of biochemical and histochemical techniques. The interaction was observed in vitro, and the two proteins co-distributed in actin rich regions in tissue cultured cells. These results suggest that palladin recruits profilin and/or profilin:actin to sites of actin dynamics.
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