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Visualization of proteins in intact cells with a clonable tag for electron microscopy

AuthorsDiestra, Elia; Fontana, Juan; Guichard, Paul; Marco, Sergio; Risco, Cristina
KeywordsClonable gold tags
Electron microscopy
Electron tomography
E. coli proteins
Maltose binding protein
Issue Date2009
CitationJournal of Structural Biology (2009), doi:10.1016/j.jsb.2008.11.009 (in press)
AbstractIdentification of proteins in 3D maps of cells is a main challenge in structural cell biology. For light microscopy (LM) clonable reagents such as green fluorescent protein represented a real revolution and equivalent reagents for transmission electron microscopy (TEM) have been pursued for a long time. To test the viability of the metal-binding protein metallothionein (MT) as a tag for TEM in cells we have studied three MT-fusion proteins in Escherichia coli: AmiC, a component of the division ring, RecA, a DNA-binding protein, and a truncated cytoplasmic form of maltose-binding protein (MBP). Proteins fused to MT were expressed in E. coli. live cells treated with gold salts were processed by fast-freezing and freeze-substitution. Small electron-dense particles were detected in sections of bacteria expressing the MT-fusion proteins and immunogold labelling confirmed that these particles were associated to the fusion proteins. The distribution of the particles correlated with the functional locations of these proteins: MBP–MT3 concentrated in the cytoplasm, AmiC-MT1 in the bacterial division ring and RecAMT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.
Description12 pages, 9 figures.-- PMID: 19114107 [PubMed].-- Supplementary information (Suppl. movies S1-S3) available at: http://dx.doi.org/10.1016/j.jsb.2008.11.009
Available online Dec 10, 2008 [Epub ahead of print].
Publisher version (URL)http://dx.doi.org/10.1016/j.jsb.2008.11.009
Appears in Collections:(CNB) Artículos
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