A large number of actin regulatory proteins have been studied, but in general, they are from different organisms, making it difficult to know what fraction of these proteins might be necessary for the functioning of actin in a single type of cell. The long term objective of this proposal is to completely characterize all of the actin-regulatory proteins from a single cell type, Dictyostelium discoideum, including both soluble and membrane-associated proteins. The first soluble protein to be characterized is a 30,000 dalton protein which crosslinks actin in a calcium-independent fashion. This protein will be compared with the previously described actin-crosslinking proteins by immunological cross-reactivity and peptide mapping, to look for relatedness (which might explain why there are several proteins of overlapping function in this organism). Interactions among the various crosslinking proteins will also be looked for, and evidence that they are under separate regulation will be sought. The next step is to proceed with a systematic search for more soluble actin-regulatory proteins, using effects on the low shear viscosity of actin as an assay. Subfractionation of each fraction will continue until a single peak of activity is obtained. The first approach to be used with membranes is to make antibodies vs. whole membranes, and look for the ability of this antibody to block the effect of membranes on the low shear viscosity of actin. If blocking activity is obtained, the antibody will be fractionated by adsorbing it to portions of a lane of Dictyostelium membrane proteins run on a gel. If blocking activity is not obtained, several alternate approaches to the question of membrane actin-binding proteins are proposed.
| Haarer, B K; Lillie, S H; Adams, A E et al. (1990) Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells. J Cell Biol 110:105-14 |
| Brown, S S; Petzold, A S (1987) Using antibodies against Dictyostelium membranes to identify an actin-binding membrane protein. J Cell Biol 104:513-8 |
