Single molecule network analysis identifies structural changes to caveolae and scaffolds due to mutation of the caveolin-1 scaffolding domain

Abstract

Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.

Figure 1

Generation of F10 sub-clone from parental MDA-MB-231. (A) Western blots of endogenous CAV1, Gal3 and β-actin and, separately, of pCAV1 and β-actin, in MDA-MB-231 sub-clones. (B) Quantification of the number of migrated cells in Transwell migration assays of sub clones and the parental MDA-MB-231 cells. Data represent mean ± SEM from at least three independent experiments (n > 3 for each clone). One-way analysis of variance (ANOVA) with Tukey post-test; ***p < 0.001. (C) Sub-clones fluorescently labelled with Alexa Fluor 488-Phallodin, showing differences in cell morphology. Scale bar, 30 µm. Cell area and circularity of F-actin labelled cells were quantified from three independent experiments (n = 3 with at least 40 cells quantified per sub-clone in each experiment; ANOVA with Tukey post-test; **p < 0.01). (D) Parental MDA-MB-231 and sub-clones A10 and F10 CAV1 and Gal3 siRNA knockdown. Bar graphs of mean ± SEM cells migrated in Transwell migration assays (n = 5; ANOVA with Tukey post-test; *p < 0.05; **p < 0.01). FRET efficiency quantified in focal adhesions of cells transfected with a vinculin FRET sensor (n = 3; ANOVA with Tukey post-test; ***p < 0.001).

Figure 2

CAV1 CRISPR knockout from F10 sub-clone. (A) Schematic of the gRNA designed to target the ATG start site of the CAV1 sequence. (B) Western blot of CAV1 in MC5 CRISPR/CAS9 knockout and F10 sub-clone used to generate MC5. (C) Bar graphs of mean ± SEM of cells migrated between parental F10 and MC5 quantified from Transwell migration assays (n = 5; two-tailed unpaired t test; ***p < 0.001). (D) Vinculin FRET efficiency of F10 parental line and MC5 rescued with CAV1 wildtype and the CSD mutant (n = 3; ANOVA with Tukey post-test; *p < 0.05; **p < 0.01). See Supp. Fig. 5 for complete blots that were cropped for Fig. 1A,D.

Figure 3

SMLM network analysis segments CAV1 clusters from SMLM data. (A) Representative TIRF wide-field imaging of CAV1 and SMLM GSD imaging of CAV1 in MC5 cells transfected with CAV1 WT or CSD mutant. 3D point clouds were processed with 3D SMLM Network Analysis using iterative merging at 20 nm, filtering and segmentation to identify individual CAV1 blobs. Magnified SMLM and network analysis images of the boxed region highlights network analysis clusters (blobs) after processing. Scale bar, 2.5 µm; zooms, 250 nm. (B) Overall changes in features. Quantification of blob’s localizations distribution, anisotropy, localization’s distance to centroid, and blob’s network features between MC5 cells transfected with CAV1 WT or CSD mutant (n = 26 CAV1 WT cells, n = 21 CSD cells from four independent experiments; two-tailed unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). The rest of the global CAV1 features are shown in Supp. Fig 3.

Figure 4

CAV1 domain class identification via similarity analysis using Euclidean distance, group matching, and fraction of CAV1 domains. (A) Representative images processed with blob identification by machine learning using 3D SMLM Network Analysis. (B) Euclidean distance between blobs’ average features of the different classes of CAV1 WT (WT1-4) and CSD (CSD1-4) mutant to PTRF-expressing PC3 (PP1-4) and HeLa (H1-4) cells^,. Bolded values correspond with (C) Identifying caveolae and S2, S1B and S1A scaffold blob groups from the shortest Euclidian distances. The classes are color coded based on the matched CAV1 domains across CAV1 WT, CSD, and the previously studied PC3-PTRF and HeLa SMLM data. Proportion of blob classes between CAV1 WT and CSD mutant (two-tailed unpaired t test; *p < 0.05; **p < 0.01).

Figure 5

Changes in features of each class of CAV1 blobs. Bar graphs depicting changes in blob size, number of nodes, and anisotropy with respect to each class of blobs in CAV1 WT and CSD mutant (two-tailed unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Percent change of the features between CSD mutant and CAV1 WT (ANOVA with Tukey post-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 6

Convex hull analysis of CAV1 blobs. (A) 3D representation of the PCA for top blob from CAV1 WT and CSD based on the average of 28 network analysis features for the four CAV1 blob classes. Overlay of the 2D X–Y representation from the top 10 blobs closes to the average of CAV1 WT and CSD mutant features. (B) Bar graphs of the average blob volume and volume variance in the top 10 blobs of each cell at shrink factors of 0 (the most convex), 0.5, and 1 (the most indented) (two-tailed unpaired t test; *p < 0.05; ***p < 0.001; ****p < 0.0001).

Figure 7

Schematic diagram of convex hull analysis of CAV1 WT and CSD mutant scaffold blobs. Increased variance of the convex hull boundary at high shrink factors (most indented) compared to low shrink factors (the most convex) is suggestive of the more variable distribution of N-terminal CAV1 labeling in CAV1 WT relative to CAV1 CSD mutant scaffold domains. CSD indicated by blue box and N-terminus of CAV1 by N. Membrane is in green and convex hull boundary of CAV1 point clouds at shrink factor 1 in blue and at shrink factor 0 in red. Representative CAV1 domains are shown containing 6 CAV1′s and do not correspond in size, number or scale to any known CAV1 scaffold. Adapted from.