Panel A shows merged CD36WT, beta 1 integrin, and CD9 fluorescence with center, periphery, and boxed region. Panel B shows the CD36WT detection channel with boxed region and boundary outlines. Panel C shows the beta 1 integrin detection channel with boxed region and boundary outlines. Panel D shows the CD9 detection channel with boxed region and boundary outlines. Panel E shows an overlay of detected signals with colored circles, boundaries, and detection legend. Panel F shows the CD36WT center interaction network linking beta 1 integrin, CD9, CD81, and CD151. Panel G shows the CD36WT periphery interaction network linking beta 1 integrin, CD9, CD81, and CD151. Panel H shows merged CD36G12V, beta 1 integrin, and CD9 fluorescence with center, periphery, and boxed region. Panel I shows the CD36G12V detection channel with boxed region and boundary outlines. Panel J shows the beta 1 integrin detection channel with boxed region and boundary outlines. Panel K shows the CD9 detection channel with boxed region and boundary outlines. Panel L shows an overlay of detected signals with colored circles, boundaries, and detection legend. Panel M shows the CD36G12V center interaction network linking beta 1 integrin, CD9, CD81, and CD151. Panel N shows the CD36G12V periphery interaction network linking beta 1 integrin, CD9, CD81, and CD151
G12V mutation weakens CD36 colocalization with β 1 -integrin and CD9 and alters CD36 colocalization with CD81 and CD151 based on subcellular region (center vs. periphery). (A) Representative three-color fixed-cell image of CD36 (magenta), β1-integrin (cyan), and CD9 (yellow) on the surface of a TIME cell expressing Halo-tagged CD36WT and imaged via TIRFM. Scale bar, 5 μm. (B–D) Particle detections (shown as dots) overlaid on the individual channels. Scale bar, 5 μm. (E) Overlay of the three-channel detections for the boxed area in A–D, with color-coding following that in A–D. Circles highlight colocalization events between the different molecules, following color-coding in the legend at the bottom of the panel. Scale bar, 1 μm. In A–E, solid lines show the segmented cell edge; dashed lines specify the boundary between the periphery and the center. (F and G) Representation of the colocalization networks of CD36WT, β1-integrin, CD9, CD81, and CD151 in the center (F) and periphery (G) regions. Network arrows involving/not involving CD36 are shown in red/gray, for visual clarity. Arrows point from target molecule to reference molecule and represent the extent of target colocalization with reference. Solid arrows indicate significant target colocalization with reference (P value ≤ 0.05), with arrow thickness proportional to level of significance (i.e., extent of colocalization). “+ molecule name” next to arrow represents condition molecule, and indicates that target colocalization with reference is significantly enhanced for subset of CD36 colocalized with condition. An exception is CD36 colocalization with β1-integrin in the periphery region (indicated by * in G), which is significantly enhanced for the subset of CD36 colocalized with CD9 and also for the subset of β1-integrin colocalized with CD9. Font size of condition name reflects level of significance (i.e., extent) of enhancement. Dashed arrows indicate that target colocalization with reference is only significant for the subset of CD36 colocalized with the condition. Molecules with no connecting arrow do not exhibit significant colocalization. (H–N) Same as A–G but for CD36G12V. See Table S1 for the significance P values from which these graphs were generated. The images shown in B, C, D, and I are, respectively, the same as the images shown in Fig. S1, C, D, E, and H. See Table 4 for the number of cells, the number of experimental repeats, and the number of objects per channel per ROI used for analysis.