Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm

• Published in: Journal of structural biology 2005-12, Vol.152 (3), p.157-168
• Main Authors: Schoenenberger, C.-A., Buchmeier, S., Boerries, M., Sütterlin, R., Aebi, U., Jockusch, B.M.
• Format: Article
• Language: English
• Subjects: Actin antibody; Actin conformation; Actin Cytoskeleton - chemistry; Actin Cytoskeleton - immunology; Actins - chemistry; Actins - genetics; Actins - immunology; Active Transport, Cell Nucleus - drug effects; Amino Acid Sequence; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibody Specificity - immunology; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Cell Line; Cell Nucleus - chemistry; Cell Nucleus - metabolism; Cytoplasm - chemistry; Cytoplasm - metabolism; Epitopes - genetics; Epitopes - immunology; Fibroblasts - chemistry; Fibroblasts - drug effects; Fibroblasts - metabolism; HeLa Cells; Humans; Latrunculin; Marine Toxins - pharmacology; Mice; Microscopy, Fluorescence; Models, Molecular; Molecular Sequence Data; Nucleocytoplasmic transport; Nucleus; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; Rabbits; Rats; Thiazoles - pharmacology; Thiazolidines; Vaccination
• ISSN: 1047-8477; 1095-8657
• DOI: 10.1016/j.jsb.2005.09.003

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked ‘lower dimer’ (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.