Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses
- Howard Hughes Medical Institute, Oregon Health and Science University, United States
- Vollum Institute, Oregon Health and Science University, United States
- RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Howard Hughes Medical Institute, United States
- Howard Hughes Medical Institute, Janelia Research Institute, United States
Figures
Figure 1 with 2 supplements
Development and characterization of anti-GluA2 Fab conjugated to gold nanoparticle (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 fluorescence-detection size-exclusion chromatography (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).
-
Figure 1—source data 1
PDF file containing original gels for Figure 1B, indicating relevant bands.
- https://cdn.elifesciences.org/articles/98458/elife-98458-fig1-data1-v1.pdf
-
Figure 1—source data 2
Original files for gel analysis are displayed in Figure 1B.
- https://cdn.elifesciences.org/articles/98458/elife-98458-fig1-data2-v1.zip
Figure 1—figure supplement 1
Preparation of PEGylated AuNP-15F1 Fab conjugate.
(A) Design of anti-GluA2 15F1 Fab construct for gold nanoparticle (AuNP) conjugation. The light chain (LC) is included without modification, while the heavy chain contains an extension of the constant domain 1 to include a single hinge cysteine for AuNP conjugation, followed by a 3 C protease cleavage site and Twin-Strep tag. (B) Strategy for covalent conjugation and subsequent PEGylation of anti-GluA2 15F1 Fab and 3-MBA-protected AuNPs. (C) Full-scale conjugation of 15F1 Fab and AuNP using 2:1 Fab:AuNP ratio, with entire reaction run on native 12% acrylamide/10% glycerol gels (representative shown). The gel bands cut out for subsequent purification are denoted by the dashed red box. (D) Assessment of AuNP PEGylation with 0.5 or 5 mM PEG550-SH by size-exclusion chromatography (SEC), using HPLC measurement of tryptophan fluorescence (top) and AuNP absorbance at 500 nm (bottom).
-
Figure 1—figure supplement 1—source data 1
PDF file containing original gels for Figure 1—figure supplement 1C, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/98458/elife-98458-fig1-figsupp1-data1-v1.pdf
-
Figure 1—figure supplement 1—source data 2
Original files for gel analysis are displayed in Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/98458/elife-98458-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Single particle cryo-electron microscopy (cryo-EM) of Fab-gold nanoparticle (AuNP) bound to native mouse hippocampal AMPAR.
Example single particle cryo-EM micrographs of native mouse hippocampal AMPAR bound to anti-GluA2 15F1 Fab-AuNP taken at (A) ‘higher’ (−4.5 to –5.0 µm) and (B) ‘lower’ (−1.0 to –1.2 µm) defocus ranges. Example automated bead-picking hits from low-defocus micrographs denoted with green circles.
Figure 2
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, the 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 fluorescence-detection size-exclusion chromatography (FSEC) confirms expression of each fluorescent fusion protein.
Figure 3 with 2 supplements
Hippocampus brain slice preparation and focused ion beam (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 electron microscopy (EM) grid, before high-pressure freezing. The white dashed line indicates the stratum radiatum, the area with the 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) Scanning electron microscopy (SEM) image of the 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.
Figure 3—figure supplement 1
Pre-soaking tissue with 15F1 Fab-gold nanoparticle (AuNP) blocks the majority of available GluA2 binding sites.
Blocked staining experiment in which mouse hippocampus tissue slices were either left in buffer (top) or presoaked with 15F1 Fab-AuNP (bottom) before subsequent staining with Janelia Fluor 646 (JF646)-labeled 15F1 Fab. Representative images from each sample are shown. Scale bars are 20 µm.
Figure 3—figure supplement 2
Cryo-correlative light and electron microscopy (Cryo-CLEM) guides synaptic targeting on lamella.
(A) Cryo-confocal image of lamella showing vGlut1-mScarlet (red) and PSD95-EGFP (green) signals. A reflection image (gold) was used to identify the lamella shape and location. (B) Cryo-CLEM image of the lamella. Medium montage map image of lamella is aligned with and overlayed on the cryo-confocal image from A. Black arrows indicate the colocalization of fluorescent signals. (C) Zoomed images of areas indicated in B (i-iii). Cryo-confocal (left) and TEM image (right) are from the same location. Synaptic vesicle-containing compartments are well-aligned with presynaptic red fluorescence signal (asterisk). Scale bars are 500 nm.
Figure 4 with 1 supplement
Cryo-electron tomography imaging of glutamatergic synapses within brain tissue.
(A) Medium montage map image of lamella. (B I-III) Enlarged images of lamella are indicated in A. (C i-iii) Slices from SIRT-filtered tomograms were 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.
Figure 4—figure supplement 1
Power spectra and contrast transfer function (CTF) fitting of tilt series.
IMOD calculation of Fast Fourier Transform, or FFT (left), and Ctfplotter fits (right) for three tilts from a single representative tilt series.
Figure 5 with 2 supplements
Anti-GluA2 Fab-gold nanoparticle (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.
Figure 5—figure supplement 1
Analysis of spatial constraints of 15F1 Fab-gold nanoparticle (AuNP) conjugation.
(A) Structure of native hippocampal AMPAR bound to 15F1 Fab (PDB: 7LDD), with the position and relative distance between the last structured 15F1 Fab heavy chain residues, K216, on each Fab and between K216 and the membrane denoted. (B) Spatial representation of possible AuNP positioning in the 15F1Fab-AuNP:AMPAR complex, restraining the AuNP to a distance of 35 Å from K216, with ranges of inter-AuNP and AuNP-membrane distances denoted.
Figure 5—video 1
Representative tomogram and segmentation.
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Cryo-electron tomographic investigation of native hippocampal glutamatergic synapses
eLife 13:RP98458.
https://doi.org/10.7554/eLife.98458.3
