Figures and data
Development and characterization of anti-GluA2 Fab conjugated to AuNP.
(A) Conjugation strategy for covalent linkage of AuNP to anti-GluA2 Fab. (B) Test-scale conjugations of Fab and AuNP at various Fab:AuNP ratios, run on native 12% acrylamide/10% glycerol gel. (C) Normalized FSEC traces of GFP-tagged GluA2 mixed with 1-2 µL of Fab-AuNP conjugate, measured at an excitation/emission of 480/510 nm (top; corresponding to GFP-tagged GluA2) or an absorbance at 500 nm (bottom; corresponding to AuNP). (D) Snapshot from single particle cryo-electron microscopy micrograph image showing two individual Fab-AuNP bound native mouse hippocampus AMPARs next to models depicting possible orientational views seen in image (PDB: 7LDD) (left). Model of anti-GluA2 Fab-AuNP bound to AMPAR with GluA2 in the B and D positions (right).
Development and characterization of transgenic mouse line for synaptic targeting.
(A) Red fluorescent protein, mScarlet, was inserted at the C-terminus of vGlut1, which is present within synaptic vesicles at excitatory presynaptic terminals. In another mouse line, green fluorescent protein, EGFP, was inserted at the C-terminus of PSD95, which is highly expressed at excitatory postsynaptic sites. Homozygous vGlut1-mScarlet and PSD95-EGFP mice were crossed to generate a mouse line expressing both vGlut1-mScarlet and PSD95-EGFP to facilitate synapse identification. The white circle represents the hippocampus region. (B) Assessment of vGlut1-mScarlet and PSD95-EGFP in solubilized mouse whole brain tissue by FSEC confirms expression of each fluorescent fusion protein.
Hippocampus brain slice preparation and FIB milling of lamella.
(A) Ultra-thin brain slice made from a vGlut1-mScarlet and PSD95-EGFP mouse. The red dashed circle indicates the hippocampus. Scale bar: 2 mm (B) A wide field image of excised hippocampus brain slice. The image also illustrates CA3 axons projecting to CA1 apical dendrites in the Schaffer collateral (arrow). Fluorescent images show vGlut1-mScarlet (top) and PSD95-EGFP (bottom) from the same hippocampus slice on the left. The dashed line (red or white) area indicates the region of interest for FIB milling (CA1 region of hippocampus), which is further trimmed with a scalpel knife. S.R: Stratum Radiatum, and S.L-M: Stratum lacunosum-moleculare. Scale bar: 500 µm (C) Overlay image of two trimmed hippocampus brain slices on EM grid, before high pressure freezing. The white dashed line indicates the stratum radiatum, the area with brightest fluorescence in the CA1 region and where the Schaffer collateral projects. Scale bar: 500 µm (D) A wide field green fluorescence image of a sample grid after high-pressure freezing. (E) Cryo-confocal image from a sample grid showing both vGlut1-mScarlet (red) and PSD95-EGFP (green) puncta. (F) Zoomed image of area indicated in E. White arrows point to colocalization of red and green fluorescent signals and indicate potential synapses. (G) SEM image of polished lamella. (H) FIB image of same lamella in G. (I) Cryo-confocal image of lamella. White arrows at fluorescence colocalization points indicate examples of regions targeted in tomography data collection.
Cryo-electron tomography imaging of glutamatergic synapses within brain tissue.
(A) Medium montage map image of lamella. (B I-III) Enlarged images of lamella indicated in A. (C i-iii) Slices from SIRT filtered tomograms collected at squares indicated in B. The synaptic cleft in each tomogram is indicated with a black arrow. Mit: mitochondria and MT: microtubule side views. One gold fiducial is visible in C iii. Scale bars are 100 nm. Each slice shown is about 10 nm thick. (D) Example image of myelin membranes (D1), top down view of microtubule (D2), synaptic vesicles (D3), and part of a mitochondria (D4). Scale bars are 20 nm. Each slice shown is 10 nm thick.
Anti-GluA2 Fab-AuNP labeling defines AMPAR position in single particle cryo-electron and cryo-electron tomography data.
(A) Snapshot from low defocus single particle cryo-electron microscopy dataset showing four pairs of Fab-AuNPs bound to native mouse hippocampus AMPAR. (B) Quantitation of nearest neighbor AuNP projection distances from full ∼1000 micrograph dataset from (A), overlayed with Gaussian fit of data. Distance between structured C-termini of Fabs in hippocampal AMPAR structure (PDB: 7LDD) shown for reference. (C) Segmentation (left) and slice through tomogram (right; denoised using IsoNet) of mouse hippocampus CA1 tissue stained with anti-GluA2 Fab-AuNP, with AuNPs visible in-between the pre- and postsynaptic membranes. Quantitation and Gaussian fits of nearest neighbor AuNP distances (D) and distance to the closest point on the pre- or postsynaptic membrane (E) from five Fab-AuNP labeled synapses. Distance between structured C-terminus of Fab and the putative membrane location in hippocampal AMPAR structure (PDB: 7LDD) shown for reference.
