Fig. 3

GluA1 monomers form metastable homooligomers with exponential lifetimes of <200 ms, and dissociate into monomers. Typical single-molecule fluorescence image sequences of two, three, and four diffusing ACP-GluA1 molecules undergoing transient dimerization, trimerization, and tetramerization in the HEK293-cell PM (from 236, 54, and 90 independent movies; a, b, and c, respectively). In the image sequence in a, two ACP-GluA1 molecules (green and magenta arrowheads) became colocalized in frame 23 (33 ms per frame, normal video rate) and diffused together for the next eight frames (for a 0.30-s total colocalization time; light blue arrowheads), until they became separated and diffused independently (blue and purple arrowheads). Colors of the subtrajectories are the same as those for the arrowheads (the circle and arrow show the locations at the start and at the time in the frame number on top, respectively). Since these experiments were conducted with a single color (ATTO594), after the dimerization event, which molecule corresponds to each one before dimer formation could not be determined (hence, different colors in the trajectory). b, c The spots with the intensities of monomers, dimers, trimers, and tetramers are indicated by magenta, blue, green, and orange arrowheads. d The distributions of the homodimer durations for ACP-GluA1, ACP-GluA1ΔNTD, and ACP-TM, obtained by measuring the durations of all of the observed homo-dimerization events. The distribution for ACP-TM was obtained at 22 °C, a temperature at which ACP-TM’s diffusion coefficient becomes practically the same as those of ACP-GluA1 and ACP-GluA1ΔNTD observed at 37 °C. Each histogram could be fitted well with a single exponential decay function (dotted curves), providing the dimer lifetime (given in parentheses after correction for the photobleaching lifetime of the fluorescent probe). Related data for Halo7-GluA1 and Halo7-TM are shown in Supplementary Fig. 5b. e The distributions of the homotetramer durations of ACP-GluA1 and ACP-GluA1ΔNTD. Each histogram could be fitted well with a single exponential decay function (solid curves), providing the tetramer lifetime (dotted curves for GluA1 homodimers). For statistical parameters, see Supplementary Table 4. Related GluA2 results are shown in Supplementary Fig. 8