Direct visualization of protein transport and processing in the living cell by microinjection of specific antibodies.
We have prepared polyclonal antibodies to the cytoplasmic portion of the envelope glycoprotein G of vesicular stomatitis virus (VSV) by using synthetic peptides corresponding to either the 22 or 11 ultimate carboxy-terminal residues of the G as immunogens. When antibodies to the 22 residue peptide are microinjected into monolayer baby hamster kidney cells before or shortly after infection with wild-type VSV, G protein accumulates in large intracellular patches and little G is observed in the Golgi complex or at the cell surface. In contrast, when antibodies to the 11 residue peptide are injected, no such patches are observed and G protein is seen colocalized with the injected antibody at the endoplasmic reticulum, in the Golgi complex, in transport vesicles, and at the plasma membrane. Microinjection of these antibodies does not disturb the pathway or kinetics of G-protein transport. In cells infected with a temperature-sensitive mutant of VSV, 045, the glycoprotein accumulates in the endoplasmic reticulum at 39.8 degrees C, but rapidly moves through the Golgi apparatus and then to the cell surface after a temperature shift-down to 32 degrees C. Using rhodamine-coupled antibodies to the 11 residue peptide, a microscope stage equipped for precise temperature control, and a silicon intensifier target video camera, we can visualize by video light microscopy the synchronized exocytotic transport of the G protein directly in the living cell.
D000818:Animals; D000906:Antibodies; D002460:Cell Line; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D007668:Kidney; D007700:Kinetics; D008845:Microinjections; D008854:Microscopy, Electron; D009154:Mutation; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014741:Video Recording; D014759:Viral Envelope Proteins / Q000032:analysis / Q000378:metabolism*
D000906:Antibodies; D014759:Viral Envelope Proteins