title: Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.
abstract: In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.
authors.affiliation: Physical Chemistry of Biosystems, Institute of Physical Chemistry, University of Heidelberg, D69120 Heidelberg, Germany. s.kaufmann@uni-heidelberg.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Kaufmann
authors.forename: Ingrid M
authors.initials: IM
authors.lastname: Weiss
authors.forename: Volker
authors.initials: V
authors.lastname: Eckstein
authors.forename: Motomu
authors.initials: M
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018031:Connexin 43 / Q000096:biosynthesis* / Q000235:genetics; D004023:Dictyostelium / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018031:Connexin 43; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D002118:Calcium