Fig. 4

Heteromer lifetimes are longer than those of homomers. Typical single fluorescent-molecule image sequences, showing that ACP-GluA1 (magenta spots) and Halo7-GluA2 (green spots) form transient heterodimers (a), heterotrimers (b), and heterotetramers (c) in the HEK293-PM (typical results from 128 or more independent movies, respectively). Magenta and green arrowheads indicate ACP-GluA1 and Halo7-GluA2 monomers, respectively, cyan arrowheads indicate heterodimers, yellow arrowheads show heterotrimers (b, c), and orange arrowheads indicate heterotetramers (c). d The distribution of the durations of GluA1–GluA2-heterodimers (magenta), shown together with those of homodimers of GluA1–GluA1, GluA2–GluA2, and TM–TM, obtained by simultaneous two-color imaging using ACP- and Halo7-tags. Here, the colocalizations of a protein of interest using different tag proteins (ACP and Halo7 proteins) are called homo (rather than hetero)-dimers, trimers, and tetramers because ACP and Halo7 by themselves did not exhibit any sign of homo and hetero interactions³⁹, and in two-color experiments, only homodimers of different colors were included in the colocaization duration histograms. Each histogram could be fitted as those in Fig. 3d, e. Since virtually the same lifetimes were obtained from the histograms for GluA1–GluA2 heterodimers (and heterotetramers in e) when the tag proteins were exchanged (Supplementary Fig. 9c, d), the two histograms were averaged in this figure (and also in e). To avoid excessive complexity of the figure, only the best-fit functions are shown without histograms for the homodimers of GluA1–GluA1 and GluA2–GluA2 and for the incidental colocalization of ACP-TM and Halo7-TM (see Supplementary Fig. 9a, b for the histograms). The homodimer lifetimes obtained by two-color experiments were virtually the same as those obtained by single-color experiments (Fig. 3d; Supplementary Figs. 5b and 8d, e; Table 1). e The distribution of the durations of heterotetramers, including 1:3, 2:2, and 3:1 GluA1/A2 heterotetramers (any heterotetramers detected with two colors), shown together with those of the homotetramers of GluA1 and GluA2. Note that the distributions for the homotetramers were obtained by single-color experiments. For the duration distributions of homotetramers of GluA1 and GluA2, see Fig. 3e and Supplementary Fig. 8d. For statistical parameters, see Supplementary Table 4