Fig. 4: Identification of IQGAP1 and Myosin IIA as domain-mediated interaction partners of CD99.

a Venn diagram of representative CD99 interactors in the actomyosin and microtubule systems shown in Supplementary Fig. 4a, b, and Supplementary Data 1. b, c Levels of cytoskeletal components in the Cyt-Mut-CD99 or TM-Mut-CD99 interactome, compared to the levels in the rWT interactome. Plots of the log₂ fold change (FC) in the values of the majority of the interactors (except for myosin and IQGAP1), myosin (MyoII) proteins, and IQGAP1, as detected in the Cyt-Mut-CD99 and TM-Mut-CD99 interactomes relative to the rWT interactome (n = 2 samples per group) (b). Schematic illustration of the binding of MyoII and IQGAP1 to different recombinant CD99 molecules (c), based on the log₂FC values in Supplementary Fig. 4d. d, e Immunoblots of MyoIIA or IQGAP1 in coimmunoprecipitates of rWT-CD99, Cyt-Mut-CD99, and TM-Mut-CD99 (using anti-myc antibody) from the corresponding cells (d), and MyoIIA, IQGAP1, actin, and α-tubulin in coimmunoprecipitates of CD99 (using anti-CD99 antibody) from WT-cells after anti-CD3-stimulation (e). f–h Colocalization analysis in rWT and mutant cells. Images of two-way (green) and three-way (white) molecular colocalization (based on Supplementary Fig. 4e) (f). Image of three-way colocalization (rWT-CD99, IQGAP1, and MyoIIA) overlaid on DIC image of rWT-cells, and plot of three-way colocalization signal intensities measured along the line (cyan) across the MTOC (red asterisk) (g). Plots of two-way colocalizations (n = 9 cells per group) (h). i, j Interaction between IQGAP1 and MyoIIA in in WT- or Cd99^-/--cells. Confocal images of colocalization (white) between IQGAP1 and MyoIIA on DIC image background of WT- and Cd99^−/−-cells. Plots of the IQGAP1 and MyoIIA signal intensities measured along the MTOC-crossing line, and the colocalization (n = 10 cells per group) (i). Immunoblot of MyoIIA in coprecipitates of IQGAP1 (j). k, l Interaction of microtubules with IQGAP1 or MyoIIA in WT- or Cd99^−/−-cells. Confocal images of microtubules with IQGAP1 or MyoIIA, and colocalization (yellow). Plots of colocalization of microtubules with IQGAP1 or MyoIIA (n = 9 cells per group) (k). Immunoblot of actin, MyoIIA, and IQGAP1 in coprecipitates of α-tubulin (l). The tubulin and actin immunoblots were reused from Fig. 2e. m CD99 domain-mediated interaction of IQGAP1, MyoIIA, actin, and microtubules. Data (a, b, d–l) are representative of two (a, b, j) or three (d–i, k, l) independent experiments. Data (h, i, k) are shown as mean ± SEM. Statistical significance was assessed using unpaired two-tailed t-test. Exact p-values are presented in the figure. Source data are provided as a Source Data file.