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. 2018 Jun 4;217(6):1929–1940. doi: 10.1083/jcb.201707075

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© 2018 Spiess et al.

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Figure 2. — Distinct organization of active and inactive β1 integrin nanoclusters within FAs. (A) Left: Representative STED image of an Hs578T cell labeled with antiactive β1 integrin mAb 9EG7 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (B) Left: Representative STED image of Hs578T cells labeled with antiinactive β1 integrin mAb AIIB2 (rhodamine). Right: Spatial distribution of detected intensity peaks within FAs. (C) Bars show FAs scored for linear versus unstructured patterns in labelings of mAbs K20 (n = 23 images), 9EG7 (n = 21 images), and AIIB2 (n = 18 images), displayed as mean percentages ± SEM. (D) Automated linearity analysis. Left: Means ± SD of fitted RANSAC lines needed to deplete all segmented clusters per FA for K20 (n = 13 cells), 9EG7 (n = 11 cells), and AIIB2 (n = 11 cells) labelings. Right: Mean ± SD number of clusters per fitted RANSAC line for the same experimental set. Kolmogorov-Smirnov test: *, P < 0.05; **, P < 0.01. (E) Bars show median NND (quartile distribution in boxes; decile distributions in whiskers) of STED-identified clusters for mAb 9EG7 (n = 19 images) and mAb AIIB2 (n = 19) labelings within FAs. The two distributions display different variances. (F and G) Left: Representative STORM image of Hs578T cells labeled with mAb 9EG7 (F) or mAb AIIB2 (G; Alexa Fluor 405 through Alexa Fluor 647). White boxes indicate zooms shown on right. Red circles show DBSCAN-identified clusters in zoomed areas. Bars: (A, B, F, and G,main images) 5 µm; (F and G, insets) 500 nm. (H) Error bars show median NNDs (quartile distribution in boxes; decile distribution in whiskers) of β1 integrin clusters in STORM images of mAb 9EG7 (n = 12 images) and mAb AIIB2 (n = 14 images) labeling. The two distributions display different variances. (I and J) The bars show median cluster size (I) and median number of cluster localizations (J) of STORM images (quartile distribution in boxes; decile distribution in whiskers) of mAb 9EG7 (n = 14 images) and mAb AIIB2 (n = 14) labeling within FAs. t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

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