Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms.
The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-beta antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-alpha, but by diverse mechanisms. The translocating antibody blocked the association with importin-alpha, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-alpha binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives.
Marg
Andreas
A
Abteilung Molekulare Muskelphysiologie, Max-Delbrück-Centrum für Molekulare Medizin, 13125 Berlin, Germany.
Meyer
Thomas
T
Vigneron
Marc
M
Vinkemeier
Uwe
U
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008845:Microinjections; D050794:STAT1 Transcription Factor / Q000276:immunology / Q000378:metabolism; D028901:alpha Karyopherins / Q000276:immunology / Q000378:metabolism; D028961:beta Karyopherins / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D028901:alpha Karyopherins; D028961:beta Karyopherins; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma