Visualization of proteins in intact cells with a clonable tag for electron microscopy.
Identification 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 RecA-MT1 in the nucleoid. The electron-dense tag was easily visualized by electron tomography and in frozen-hydrated cells.
Diestra
Elia
E
Cell Structure Lab, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas-CSIC, Darwin 3, Campus de Cantoblanco, Madrid 28049, Spain.
Fontana
Juan
J
Guichard
Paul
P
Marco
Sergio
S
Risco
Cristina
C
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D020285:Cryoelectron Microscopy / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D017612:Gold Compounds / Q000737:chemistry; D006651:Histocytochemistry / Q000379:methods*; D058619:Maltose-Binding Proteins; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics; D008854:Microscopy, Electron / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009113:Muramidase / Q000737:chemistry; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
D002352:Carrier Proteins; D017612:Gold Compounds; D058619:Maltose-Binding Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C038016:gold chloride; D008668:Metallothionein; D011938:Rec A Recombinases; D009113:Muramidase; D009238:N-Acetylmuramoyl-L-alanine Amidase