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Abstract

In the brain, the strength of each individual synapse is defined by the complement of proteins present or the "local proteome." Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

Keywords: DNA-point accumulation for imaging in nanoscale topography; molecular quantification; super-resolution imaging; synaptic proteins.

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Figures

Fig. 1

Fig. 1

DNA-PAINT imaging of synaptic proteins:…

Fig. 1

DNA-PAINT imaging of synaptic proteins: (a) scheme of protein labeling for DNA-PAINT using…

Fig. 1 DNA-PAINT imaging of synaptic proteins: (a) scheme of protein labeling for DNA-PAINT using antibodies. A secondary antibody is decorated with a docking strand, in which a fluorophore-labeled complementary imager strand binds transiently and generates the single-molecule signal, (b) super-resolved DNA-PAINT image of GluA2-containing AMPAR with single synapses assigned manually (scale bar 500 nm), (c) the number of single-molecule localizations in a DNA-PAINT experiment is constant over long acquisition times, and (d) local protein copy numbers are determined from the association rate of the imager strand binding to the target strand (kon), which is the inverse of the time between binding events (dark time, τd). The number of antibody-labeled proteins in a synapse is related to 1/τd.
Fig. 2

Fig. 2

Simulation of DNA-PAINT data and…

Fig. 2

Simulation of DNA-PAINT data and qPAINT analysis. (a), (b) DNA origami patterns containing…

Fig. 2 Simulation of DNA-PAINT data and qPAINT analysis. (a), (b) DNA origami patterns containing 20 target sites spaced (a) 40 nm and (b) 15 nm apart were simulated as synthetic clusters of synaptic proteins. A dark time analysis of single docking strands [yellow circle in (a)] yielded τd,single. Copy numbers of single synthetic clusters (orange circles and numbers) and of assemblies of three clusters (blue circles and numbers) were determined by extracting τd,cluster and calibrating with τd,single. Using simulation parameters that match the expected range of AMPAR clustering, a detection efficiency of 0.84 was determined (scale bar 500 nm).
Fig. 3

Fig. 3

Quantitative PAINT imaging of GluA2-containing…

Fig. 3

Quantitative PAINT imaging of GluA2-containing AMPAR in single synapses: (a) large images (i)…

Fig. 3 Quantitative PAINT imaging of GluA2-containing AMPAR in single synapses: (a) large images (i) are generated by tiling multiple single confocal images, (ii) using immunostained MAP2 as a dendritic marker. Insets show (iii) co-staining of synapses with PSD95, and (iv) a zoom-in of the DNA-PAINT image of GluA2-containing AMPARs with numbers indicating detected AMPARs; (b) copy numbers of GluA2-containing AMPAR in single synapses along two single dendrites highlighted in (ii) [yellow circles in (ii) mark the location of single synapses analyzed], yielding 24 (±16  s.d., n=13 synapses; dendrite 1) and 21 (±15  s.d., n=10 synapses; dendrite 2); (c) the analysis of 56 synapses in 4 dendrites from two neurons (2 dendrites per neuron) yielded an average number of 23 (±15  s.d.) GluA2-containing AMPA receptors per synapse (scale bars 80  μm and 500 nm).
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