title: Electron microscopic analysis of a fusion protein of postsynaptic density-95 and metallothionein in cultured hippocampal neurons.
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.
authors.affiliation: Bio-multisome Research Team, RIKEN SPring8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukunaga
authors.forename: Ai
authors.initials: A
authors.lastname: Hirase
authors.forename: Hyeji
authors.initials: H
authors.lastname: Kim
authors.forename: Natsuko
authors.initials: N
authors.lastname: Wada
authors.forename: Yuri
authors.initials: Y
authors.lastname: Nishino
authors.forename: Atsuo
authors.initials: A
authors.lastname: Miyazawa
mesh_terms: D000818:Animals; D019556:COS Cells; D002104:Cadmium / Q000737:chemistry / Q000633:toxicity; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D000076122:Disks Large Homolog 4 Protein; D051528:Guanylate Kinases; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D020080:Tandem Repeat Sequences; D014162:Transfection
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein; D051528:Guanylate Kinases