EhLimA, a novel LIM protein, localizes to the plasma membrane in Entamoeba histolytica.
The parasitic protozoan Entamoeba histolytica relies on a very dynamic cytoskeleton in order to invade and survive in host tissues. Identification of cytoskeletal elements is key to understanding these processes. Here we present the characterization of EhLimA, the first LIM protein of E. histolytica. EhLimA consists of a single LIM domain at its N terminus and exhibits the highest degree of homology with DdLimE from Dictyostelium discoideum. Immunofluorescence localization of EhLimA using anti-EhLimA antibodies revealed that EhLimA is highly concentrated at the plasma membrane of cells. Silencing or overexpression of the EhLimA gene did not have a significant effect on the growth or morphology of the parasite. EhLimA associates with the cytoskeleton as demonstrated by the enrichment of the protein in cytoskeleton fractions as well as in pull-down assays that revealed that cytoskeleton association involves interaction with actin. EhLimA binding to actin was shown to be dependent on the N-terminal LIM domain of EhLimA, as removal of even half of the LIM domain resulted in almost complete inhibition of the binding to actin. We also found that a portion of EhLimA floats to the lower-density regions of a sucrose gradient together with portions of the Gal-lectin light subunit and actin. Treatment of cells with the cholesterol-sequestering agent digitonin resulted in increased solubility of EhLimA. These results indicate that in addition to cytoskeletal association, EhLimA may also associate with lipid rafts in the parasite plasma membrane and suggest that EhLimA may be part of the molecular system connecting the actin cytoskeleton to membrane rafts.
Wender
Nomy
N
Department of Biological Chemistry, Weizmann Institute of Science, P.O. Box 26, Rehovot 76100, Israel.
Villalobo
Eduardo
E
Mirelman
David
D
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism; D004748:Entamoeba histolytica / Q000378:metabolism*; D020868:Gene Silencing; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D015800:Protozoan Proteins