Characterization cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments, We have undertaken the ident ification of actin-associated proteins that regulate actin dynamics, A mono clonal antibody 4C12 raised against a cytoskeletal fraction of I: vaginalis labeled a protein doublet at circa 50 kDa, These two bands were recognized by the antibody against Dictyostelium discoideum coronin, During cell extr action and actin polymerization, T, vaginalis coronin cosedimented with F-a ctin, By two-dimensional gel electrophoresis, the protein doublet was separ ated into two sets of isoforms covering two Ip zones around 6 and 7, By scr eening a T, vaginalis library with 4C12, two clones Cor I and Cor 2 were is olated. This gene duplicity is a particularity among unicellular organisms examined, The complete sequence of the gene Cor I encodes a 435-residue pro tein with a calculated molecular mass of 48 kDa and Ip of 5.58, The incompl ete sequence Cor 2 was very similar but,vith a more basic calculated Ip tha n Cor 1 on the same region, T, vaginalis coronin had 50% similarity with th e coronin family, possessing the five WD-repeats and a leucine zipper in it s C-terminal part, Double immunofluorescence labeling showed that coronin m ainly colocalized with actin at the periphery of the adherent amoeboid cell s. However, coronin labeling displayed patches within a reticular array. Im munogold electron microscopy confirmed the coronin labeling in the actin-ri ch microfilamentous fringe beneath the plasma membrane, with accumulation i n phagocytic zones and pseudopodial extensions, In T, vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important r ole in actin dynamics and may be a downstream target of a signaling mechani sm for the cytoskeleton reorganization.