Figure 2.
Comparative characteristics of endogenous and overexpressed AMPAR diffusion in live and fixed neurons. A, Specificity of sptPALM localization. Contribution of the intracellular staining to the signal detected in sptPALM images was analyzed by comparing the histograms of instantaneous diffusion coefficients on cells where extracellular Eos coupled to GluA1 was cleaved by the TEV protease (protease) to that of control cells incubated with protease inactivated by boiling (boiled protease). B, Specificity of the antibodies binding in uPAINT. Comparison of the intensity of GluA2 endogenous labeling with uPAINT technique on wild-type neurons (wild-type) and neurons from GluA2 knock-out mice. These experiments reveal a nonspecific labeling of ∼15%. Instantaneous diffusion histograms are normalized to trajectories/μm2. C, Comparison of AMPAR mobility in live and fixed cells with different antibodies. Two different antibodies yield comparable mobility histograms in live cells. Performing uPAINT on fixed cells results in a histogram shifted toward immobile fractions. D, Different antibodies against GluA2 used for uPAINT provide comparable confinement kinetics. The confinement value of fixed molecules is twofold smaller than the confined fraction of mobile AMPAR in the synapse, indicating that the accuracy of localization does not limit the measurement of nanodomain confinement properties in live neurons. E, F, Diffusion coefficient histograms of various AMPAR subunits in multiple cells using sptPALM (E) and uPAINT (F). Illustration of the variability of instantaneous diffusion coefficient histograms from whole dendritic segments of neurons transfected with Eos::GluA1, Eos::GluA2, Eos::GluA2 + untagged GluA1, and endogenous surface AMPAR. For each construct, the distribution of six different cells is provided together with the mean distribution computed from all cells (rightmost histograms). Eos::GluA2 mobility distribution presents a narrow distribution enriched in highly mobile fraction, whereas Eos::GluA1 shows a broad bimodal distribution with a fast and slow or immobile fraction. Surface AMPA receptors labeled by antibody against GluA2 exhibit a broad distribution similar to that of Eos::GluA1.