Rapid functional analysis in Xenopus oocytes of Po protein adhesive interactions.
We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site.
Yoshida
M
M
Department of Biochemistry and Molecular Biology, The Mount Sinai School of Medicine, New York, New York 10029, USA.
Colma
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D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics; D005260:Female; D049452:Green Fluorescent Proteins; D015417:Hereditary Sensory and Motor Neuropathy / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D018993:Myelin P0 Protein / Q000235:genetics / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D014018:Tissue Distribution; D014981:Xenopus
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D018993:Myelin P0 Protein; D049452:Green Fluorescent Proteins