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 identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.
Bricheux
G
G
Laboratoire de Biologie des Protistes, Université Blaise Pascal de Clermont-Ferrand, UPRESA CNRS, Aubière, France. bricheux@infobigen.fr
Coffe
G
G
Bayle
D
D
Brugerolle
G
G
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015139:Blotting, Southern; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D018076:DNA, Complementary / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D007150:Immunohistochemistry; D008840:Microfilament Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010587:Phagocytosis; D020033:Protein Isoforms; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014246:Trichomonas vaginalis / Q000378:metabolism*
D000199:Actins; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D008840:Microfilament Proteins; D020033:Protein Isoforms; C072061:coronin proteins