Fig. 3: Characterization of insulin receptor clusters.

a Schematic of cell treatments (top). Representative images of IR puncta undergoing deformation (bottom left), fission (bottom center), and fusion (bottom right). Quantification of total IR signal intensity over puncta pre- and post-deformation, fission or fusion. Images were taken 0.2 s or 0.5 s apart. b Schematic of cell treatments (top). Representative tc-PALM images of cells expressing IR-Dendra2 stimulated acutely with (3 nM) or without (0 nM) insulin for 5 min (bottom left). Scale bars are indicated. Representative tc-PALM traces (bottom right). c Frequency of IR cluster lifetime in cells not acutely stimulated with insulin (0 nM insulin, light blue) and acutely stimulated with insulin for 5 min (3 nM insulin, dark blue). The average lifetime (τ_avg) of short-lived IR clusters + /− SEM is reported. The number of IR clusters analyzed: sensitive 0 nM insulin plasma membrane 385 puncta, cytoplasm 136 puncta, nucleus 15 puncta; sensitive 3 nM insulin plasma membrane 430 puncta, cytoplasm 231 puncta, nucleus 72 puncta. Unpaired two-sided t test was used for statistical analysis. d Number of IR clusters per cell in insulin-sensitive cells not acutely stimulated with insulin (light blue) and acutely stimulated with insulin (dark blue). Data are represented as mean + /− SEM. The number of cells analyzed: sensitive 0 nM insulin plasma membrane 22 cells, cytoplasm 22 cells, nucleus 22 cells; sensitive 3 nM insulin plasma membrane 30 cells, cytoplasm 30 cells, nucleus 30 cells. Unpaired two-sided t test was used for statistical analysis for the cytoplasm, and unpaired one-sided t test was used for the nucleus. e Number of IR-Dendra2 detections per IR cluster in insulin-sensitive cells not acutely stimulated with insulin (light blue) and acutely stimulated with insulin (dark blue). Average number of IR detections per IR cluster is reported on top of each histogram. Data are represented as mean + /− SEM. The number of clusters analyzed: sensitive 0 nM insulin plasma membrane 430 clusters, cytoplasm 499 clusters, nucleus 37 clusters; sensitive 3 nM insulin plasma membrane 573 clusters, cytoplasm 551 clusters, nucleus 57 clusters. Unpaired two-sided t test was used for statistical analysis. f Representative images of IR-GFP and phosphorylated IRS1 (pIRS1) in insulin-sensitive HepG2 cells stimulated acutely with (3 nM) or without (0 nM) insulin for 5 min (left). Quantification of pIRS1 signal in IR clusters in insulin-sensitive HepG2 cells stimulated acutely with (blue) or without (dark blue) insulin for 5 min (right). Data are represented as mean + /− SEM. The number of IR clusters analyzed: sensitive 0 nM insulin 7640 clusters; sensitive 3 nM insulin 10,979 clusters. Unpaired two-sided t test was used for statistical analysis. g, Representative images of insulin-sensitive cells stimulated with the reported concentrations of insulin for 5 min. h, Quantification of IR signal in clusters in the cytoplasm. Data are represented as mean + /− SEM. The number of IR clusters analyzed: 0 nM insulin 141 clusters, 0.1 nM insulin 164 clusters, 1 nM insulin 163 clusters, 10 nM insulin 101 clusters, 100 nM insulin 100 clusters. i Immunoblot and quantification of pIRS1 relative to total IRS1 in insulin-sensitive cells stimulated with the reported concentrations of insulin for 5 min. Data are represented as mean + /− SEM. The number of biologically independent samples analyzed: nine per condition. Source data are provided as a Source Data file.