title: Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag.
abstract: Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm.
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Hirabayashi
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan RSC-University of Hyogo Leading Program Center, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukunaga
authors.affiliation: Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan RSC-University of Hyogo Leading Program Center, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan atsuo@sci.u-hyogo.ac.jp.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Miyazawa
mesh_terms: D000818:Animals; D020285:Cryoelectron Microscopy / Q000379:methods*; D000076122:Disks Large Homolog 4 Protein; D055032:Electron Microscope Tomography / Q000379:methods*; D006624:Hippocampus / Q000166:cytology; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000648:ultrastructure*; D008668:Metallothionein / Q000235:genetics; D008867:Microtomy; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020080:Tandem Repeat Sequences
chemical_list: D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D008668:Metallothionein
keywords: CEMOVIS; PSD-95; electron tomography; genetically encoded tag; metallothionein; primary cultured hippocampal neurons