Quantitative localization of the plasma membrane molecules by SDS-digested freeze-fracture replica labeling

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The glutamatergic synaptic transmission is a major excitatory input in the central nervous system, and the effects of glutamate are mediated primarily by ionotropic glutamate receptors accumulated at postsynaptic specialization in the neuronal plasma membrane. Change in quantity of these receptors expressed in the membrane has been suggested as a critical determinant for the synaptic efficacy, however anatomical evidence has not been provided.

In 1995, Fujimoto developed an epoch-making technique for localization of plasma membrane molecules, termed SDS-digested freeze-fracture replica labeling (SDS-FRL), by which distribution of these molecules can be visualized in a two-dimensional manner with high resolution. In collaboration with Fujimoto, we have applied this technique to brain tissue with some modifications, and proved its high sensitivity and quantitative capability in visualizing molecules in the neuronal plasma membrane. Here, we introduce our current protocol of the SDS-FRL with recent results on quantitative localization of ionotropic glutamate receptors in the hippocampal neurons and their dynamic change after potentiation of the synaptic transmission.

One of the questions to be answered in the era of post genome, when most of the protein members constituting living organisms are identified, would be protein species and quantity of each protein expressed in particular cell types and in their subcellular domains. Such information is indispensable for understanding mechanisms involved in each of life phenomena. In 1995, Fujimoto developed an epoch-making technique for localization of plasma membrane molecules, termed SDS-digested freeze-fracture replica labeling (SDS-FRL), by which molecules on the plasma membrane can be visualized in a two-dimensional manner with high sensitivity and high resolution. In collaboration with Fujimoto, we have applied this technique to brain tissue with some modifications, and proved its high sensitivity and quantitative capability in visualizing molecules in the plasma membrane (Tanaka et al., 2005, Hagiwara et al., 2005). Here, we introduce our current protocol of SDS-FRL together with several results obtained with this technique, which could be further modified for various purposes and other tissues to bring out the full potential of this powerful technique.

Protein species and quantity of each protein expressed in particular cell types and in their subcellular domains regulate each of life phenomena.