(A) Illustration of a zebrafish embryo highlighting the somites (blue) and presomitic mesoderm (yellow). (B–E) Confocal images of integrin α5-RFP (B and C) and αV-GFP (D and E) in wild-type (WT) embryos. As the somite boundary (SB) forms, α5 clusters to the basal side (dashed lines) of the anterior (A) and posterior (P) boundary cells (arrows). The white cross in (E) denotes a mesenchymal cell (MC) within a somite. Scale bars, 20 μm. (F) Basal/apical ratio of integrin intensity in anterior (SB/A, solid line) and posterior (SB/P, dashed line) boundary cells. Data are mean ± SD from n = 15 cell pairs in six embryos. (G–L) Integrin α5-Aquamarine (Aqm) and αV-Aqm co-expressed with different integrin β subunits tagged with mCitrine (mCit) in developing somites of MZα5−/− embryos. (G) α5β1, (H) αVβ1, (I) αVβ1-BiFC (bimolecular fluorescence complementation, used to increase heterodimer stability), (J) αVβ3, (K) αVβ5, and (L) αVβ6. Arrows in (H) indicate clustering on the somite border. Scale bars, 30 μm. (M) Clustering quantification via the SB/MC intensity ratio. Details of ROI selection shown in Figure S1A. α5β1, n = 18 measurements (12 embryos); αVβ1-BiFC, n = 21 (13 embryos); αVβ3, n = 18 (8 embryos); αVβ5, n = 16 (14 embryos); αVβ6, n = 19 (9 embryos). Data are mean ± SD. ∗∗∗p < 0.0001; n.s., not significant (two-sided t test). See also Figure S1.

(A) Illustration of the FRET assay for the integrin conformation. The integrin α subunit cytoplasmic tail was tagged with Aqm as a FRET donor, and the β subunit was tagged with mCit as a FRET acceptor. When the cytoplasmic tails separate during integrin activation, FRET should be reduced. The locations of the αGAAXR mutations (orange star) and the β3NIN333T mutation (purple star) are indicated. (B) Heatmap of the fluorescence lifetime of integrin α5-Aqm co-expressed with β1-mCit (denoted α5β1). The raw image and lifetime distribution are shown in Figures S1C and S1D. Warm colors on the SB represent longer donor lifetimes, indicating weaker FRET and the active conformation. (C) FRET efficiency (EFRET) of different integrin heterodimers. Sample size is the same as in Figure 1M, except αVβ1, n = 20 measurements (12 embryos). (D–K) Activatable integrin alleles: α5GAAKR-Aqm co-expressed with β1-mCit (D), α5GAAKRβ1-BiFC (E), αVGAANR-Aqm co-expressed with β3-mCit (F), αVGAANRβ3-BiFC (G), αV-Aqm co-expressed with N-glycan wedge allele β3NIN333T-mCit (H), αVGAANR-Aqm co-expressed with β5-mCit (I), αVGAANR-Aqm co-expressed with β6-mCit (J), and αVGAANRβ6-BiFC (K). White arrows in (D), (F), and (J) indicate clustering on the somite border. Scale bars, 30 μm. (L) Clustering quantification of activatable integrin alleles by the SB/MC intensity ratio. αVGAANRβ3-BiFC, n = 16 (9 embryos); αVβ3NIN333T, n = 21 (15 embryos); αVGAANRβ5, n = 15 (8 embryos); αVGAANRβ6-BiFC, n = 17 (8 embryos). α5GAAKRβ1-BiFC clustering cannot be measured because of the poor membrane expression in the mesenchyme. (M) EFRET of activatable integrin alleles. Sample size is the same as in (L), except α5GAAKRβ1, n = 18 (9 embryos); αVGAANRβ3, n = 14 (9 embryos); and αVGAANRβ6, n = 15 (7 embryos). EFRET of αVβ1 (C), α5GAAKRβ1, αVGAANRβ3, and αVGAANRβ6 (M) cannot be measured in the MC because of the poor membrane expression. (C and M) Data are mean ± SD. ∗∗∗p < 0.0001, two-sided t test. All experiments are in MZα5−/− embryos.

(A) Integrins and ECM proteins co-immunoprecipitated with integrins α5, αV, or αVβ3 identified via mass spectroscopy. The intensity-based absolute quantification (iBAQ) from each replicate is color coded to show relative protein abundance. Hierarchical cluster analysis is shown as the dendrogram (see Tables S2 and S3 for protein names). Note that basement membrane ligand laminins (lama1, lamb1a, lamc1) are roughly equal in all three datasets, while thrombospondins (thbs3b, thbs4b) and cartilage oligomeric matrix protein (comp/thbs5) are found exclusively in the αV dataset. ctl, control (FLAG-tagged myristoylated membrane-anchored GFP [mem-GFP]). (B and C) Integrin β subunits (B) and Fn (C) quantification using median-normalized iBAQ (miBAQ). Bar indicates mean ± SD, n = 3. (D–G) Somite localization of integrin α5β1 (D) and αVβ1-BiFC (E) in Fn double-mutant Fn−/− (fn1a−/−;fn1b−/−) embryos, ligand binding-deficient α5FYLDDβ1 in MZα5−/− embryos (F), and αVβ3 in heat shock promoter-driven Fn1a-mKikumi transgenic (hsp70:fn1a) embryos (G). Scale bars, 30 μm. (H and I) Clustering quantification (H) and EFRET (I) of α5β1 in the absence of Fn, α5FYLDD β1 in MZα5−/− embryos, and αVβ3 exposed to extra Fn1a. Fn−/−: α5β1, n = 20 measurements (9 embryos); Fn−/−: αVβ1-BiFC, n = 19 (11 embryos); MZα5−/−: α5FYLDD β1, n = 16 (8 embryos); hsp70:fn1a; αVβ3, n = 15 (7 embryos). Data are mean ± SD.

(A) Illustration of fluorescence cross-correlation spectroscopy (FCCS) measurements. The integrin α subunit cytoplasmic tail was tagged with RFP and the β subunit was tagged with GFP. When the two subunits move together through the confocal volume (upper panel), the green and red intensity fluctuations correlate, leading to a high cross-correlation curve (arrow in B); conversely, when the heterodimer subunits dissociate (lower panel), there is a lower cross-correlation curve (arrow in C). (B and C) FCCS measurements of the positive control (pos), which is a mem-GFP-RFP tandem fusion (B) and FCCS measure of αVGAANRβ3 (C). The auto-correlation functions (ACFs) for each channel are shown in red and green while the cross-correlation between the two channels is in blue. Data fitting is shown in black. Measurements were performed on the cell surface in somite MCs (white cross in Figure 1E). (D) Fcross of different integrin heterodimers calculated from FCCS. A lower Fcross indicates a weaker intra-heterodimer association. pos, positive control, mem-GFP-RFP tandem; neg, negative control, co-expression of mem-GFP and mem-RFP. Data are mean ± SD. ∗∗∗p < 0.0001, n.s., not significant (two-sided t test).