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Journal of Electron Microscopy Advance Access published online on October 23, 2007
Journal of Electron Microscopy, doi:10.1093/jmicro/dfm027
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons

Yuko Fukunaga1,2,, Ai Hirase1,2,, Hyeji Kim1, Natsuko Wada1, Yuri Nishino1,2, and Atsuo Miyazawa1,2,*

1 Bio-multisome Research Team, RIKEN SPring8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan
2 CREST, JST

* To whom correspondence should be addressed. E-mail: atsuo{at}spring8.or.jp

The subcellular localization of biomolecules at high resolution has traditionally been investigated by combining transmission electron microscopy (TEM) and chemical staining with heavy metals or immuno-based labeling with gold-conjugated antibodies. Here, we employ genetically encoded tags to examine the localization of proteins in transfected cultured cells by TEM. We purified a fusion protein of postsynaptic density-95 (PSD-95) coupled to three tandem repeats of metallothionein (MT) (PDS-95–3MT) from COS7 cells grown in the presence of Cd2+. PSD-95–3MT was detected as black particles by TEM. To visualize the subcellular localization of PSD-95–3MT, expression constructs encoding this fusion protein were transfected into primary hippocampal neurons cultured in medium containing Cd2+. The subcellular accumulation of PSD-95–3MT and Cd2+ provided excellent contrast in TEM micrographs. To address if genetically encoded tags affect the function of the target proteins, we found that the conjugation of 3MT to PSD-95 did not alter its association with known binding partners. These results demonstrate that 3MT coordinating Cd2+ is a valuable genetically encoded tag to study the localization of proteins by TEM.

Keywords     transmission electron microscopy, metallothionein, genetically encoded tag, PSD-95

Received     29 January 2007, accepted 13 September 2007


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