Determination of absolute protein numbers in single synapses by a GFP-based calibration technique

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To build a quantitative model of molecular organization of neurons, it is essential to have information about the number of protein molecules at individual synapses. Here we developed a method to estimate absolute numbers of individual proteins at actual excitatory synapses by calibrating the fluorescence intensity of microspheres with single EGFP molecules. In cultured hippocampal neurons, we observed a monotonous increase of postsynaptic protein numbers per single synapse during neuronal differentiation and subsequent stabilization. At maturity we calculated that a single excitatory postsynaptic site contains 100–450 of individual postsynaptic proteins, such as PSD-95, GKAP, Shank and Homer. This narrow range of postsynaptic protein content suggests relatively simple stoichiometry of postsynaptic molecular organization. The EGFP-based calibration technique provides an unprecedented general method for estimating the amounts of proteins in macromolecular complexes.

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Figure 1: Imaging of single molecules using TIRFM.
Figure 2: Quantification of fluorescence from single molecules and single microspheres.
Figure 3: Estimation of nonfluorescent EGFP-fusion molecules.
Figure 4: Estimation of scaffolding protein numbers by EGFP-based calibration.
Figure 5: Developmental changes of scaffolding protein numbers in single postsynaptic sites.


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We thank M. Inui for PSD-95 antibody, T. Furuichi for pan-Homer antibody, T. Urushido for generating recombinant adenoviruses, A.M. Craig and R. Shigemoto for valuable advice. The work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Science and Technology Corporation.

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Correspondence to Shigeo Okabe.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Relative affinity of monoclonal and polyclonal anti-GFP antibodies to EGFP or EYFP-tagged scaffolding proteins. (PDF 153 kb)

Supplementary Fig. 2

Relative immunoreactivity of PSD scaffolding proteins. (PDF 667 kb)

Supplementary Fig. 3

Spatial overlap of individual scaffolding protein clusters and their association with excitatory presynaptic structure. (PDF 508 kb)

Supplementary Fig. 4

Relative fluorescence intensity of PSD protein clusters with or without excitatory presynaptic structure. (PDF 97 kb)

Supplementary Fig. 5

Fluorescence intensity measurement of EGFP-Shank2 clusters before and after fixation. (PDF 308 kb)

Supplementary Fig. 6

Distribution of scaffolding protein content in single synapses determined by multiple antibodies. (PDF 130 kb)

Supplementary Methods (PDF 37 kb)

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Sugiyama, Y., Kawabata, I., Sobue, K. et al. Determination of absolute protein numbers in single synapses by a GFP-based calibration technique. Nat Methods 2, 677–684 (2005) doi:10.1038/nmeth783

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