Determination of absolute protein numbers in single synapses by a GFP-based calibration technique.
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.
Sugiyama
Yoshiko
Y
Department of Cell Biology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
Kawabata
Izumi
I
Sobue
Kenji
K
Okabe
Shigeo
S
D000818:Animals; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology*
D009419:Nerve Tissue Proteins; D049452:Green Fluorescent Proteins