Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Abstract

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-b 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-a, but by diverse mechanisms. The translocating antibody blocked the association with importin-a, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-a 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. ' 2008 International Society for Advancement of Cytometry Key terms antibody; native protein; microinjection; nucleocytoplasmic shuttling; import; export; GFP TRANSPORT of proteins and macromolecules between the nucleus and the cytoplasm of eukaryotic cells is an important mechanism for cytoplasmic regulation of nuclear activities. Nucleocytoplasmic exchange occurs through specialized channels called ''nuclear pore complexes'' (NPCs), which perforate the double layer of the nuclear envelope (1). The NPCs function as selectivity filters allowing passive diffusion of molecules up to $40 kDa, whereas components of higher molecular weight are usually restricted from autonomous exchange (2,3). A well-characterized pathway for nuclear transport of proteins relies on the GTPase Ran and the transport factors (also called carriers or karyopherins) importin-a and importin-b or CRM1 for import or export, respectively (4,5). In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or i...

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