Fig. 6: Identification of the transmembrane and juxtamembrane domains as minimal essential domains of mouse and human CD99.

a Schematics of the TM-SR and TM-LR mutant CD99. b Confocal images of recombinant CD99-YFP, F-actin, and microtubules, including their colocalization (white). Plots of microtubule colocalization with actin in MTOC-proximal (n = 8 cells per group) and -distal (n = 10 cells per group) regions in transduced Cd99^−/−-cells after 15 min of anti-CD3 stimulation. c Alignment of CD99 amino acid sequences from different organisms using Clustal Omega⁷⁵. Identical sequences are highlighted in red; fully conserved sequences are marked with an asterisk, strongly conserved sequences with a colon, and weakly conserved sequences with a period. Jux, juxtamembrane region. d Functional restoration by TJ-CD99 introduced into Cd99^−/−-cells. Schematic of TJ-CD99. Confocal images of recombinant CD99-YFP, F-actin, microtubules, including their colocalization (white) in transduced Cd99^−/−-cells. pMT numbers (n = 16 cells per group) and microtubule colocalization with actin (n = 8 cells per group) are plotted. e Cytoskeletal derangements in CD99 knocked-down (KD-HuCD99) Jurkat T cells. Schematic of the DNA construct for human CD99 (sh1-HuCD99) knockdown. Confocal images of actin or microtubules (cyan) in transfected (mCherry-positive) Jurkat T cells after 15 min of anti-CD3 stimulation. Plots of F-actin (n = 12, 15 cells) and microtubule (n = 20, 11 cells) intensities. f Restored IS-cytoskeletal network following HuTJ-CD99 expression in KD-HuCD99-Jurkat cells. Schematic of HuTJ-CD99, mock, and KD (sh1-HuCD99) plasmids. Ms murine CD99, Hu human CD99. Confocal images of HuTJ-CD99 or mock expression (YFP, green) with F-actin or microtubules (red and cyan), and their colocalization (yellow) in KD-HuCD99-Jurkat cells. Plots of lamellipodia (LP) width (n = 21, 25 cells) and pMT numbers (n = 10 cells per group). Data represent three (b, d) or two (e, f) independent experiments. Data b, d–f 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.