Amoeba proteus, one of the oldest eukaryotic unicellular organisms, for over a century has been widely used as a model to study cell motility. However, very little is known about molecular mechanism of its migration. Its length may reach up to 700 µm and the speed of migration up to several µm/s, which make A. proteus one of the largest and fastest eukaryotic cell. Data gathered so far indicate that amoeba motility depends only on dynamics of actin-based cytoskeleton. To date, only three cytoskeletal proteins have been cloned, one of them is amebin, recently discovered in our lab [Sobczak et al. (2007) Biochem Cell Biol 85:1-11].
Here, we have characterized amebin, and more precisely its C-terminal fragment expressed in bacteria as GST-fused protein; noteworthy this is the first ever recombinant protein produced on the base of A. proteus cDNA. C-amebin (molecular weight of the fusion protein is about 75 kDa) binds both to monomeric and filamentous actin it the Ca2+-independent manner, promoting filament bundling. We showed that a positively charged C-terminal segment of amebin with KLASMWEQ sequence, similar to that of caldesmon (a protein regulating acto-myosin interaction in vertebrates). We demonstrated that C-amebin inhibited the actin-dependent MgATPase of myosin thus implying that its interaction with actin was of physiological relevance. We have confirmed that by showing that microinjection of amoebae with the specific antibody against C-terminus of amebin significantly affected amoebae morphology and blocked migration. The data indicate that in A. proteus amebin may be a key regulator of the actin-cytoskeleton dynamics and actin-dependent motility. Also, they may contribute to the knowledge of evolution of actin-binding proteins.