Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons |
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Authors: | Fukunaga Yuko Hirase Ai Kim Hyeji Wada Natsuko Nishino Yuri Miyazawa Atsuo |
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Affiliation: | Bio-multisome Research Team, RIKEN SPring8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan. |
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Abstract: | 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. |
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