Integrin intra-heterodimer affinity inversely correlates with integrin activatability

Abstract

Integrins are heterodimeric cell surface receptors composed of an α and β subunit that mediate cell adhesion to extracellular matrix proteins such as fibronectin. We previously studied integrin α5β1 activation during zebrafish somitogenesis, and in the present study, we characterize the integrin αV fibronectin receptors. Integrins are activated via a conformational change, and we perform single-molecule biophysical measurements of both integrin activation via fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) and integrin intra-heterodimer stability via fluorescence cross-correlation spectroscopy (FCCS) in living embryos. We find that integrin heterodimers that exhibit robust cell surface expression, including αVβ3, αVβ5, and αVβ6, are never activated in this in vivo context, even in the presence of fibronectin matrix. In contrast, activatable integrins, such as integrin αVβ1, and alleles of αVβ3, αVβ5, αVβ6 that are biased to the active conformation exhibit poor cell surface expression and have a higher intra-heterodimer dissociation constant (K_D). These observations suggest that a weak integrin intra-heterodimer affinity decreases integrin cell surface stability and increases integrin activatability.

Keywords: cell adhesion; fibronectin; fluorescence cross-correlation spectroscopy; fluorescence lifetime imaging; fluorescence resonance energy transfer; integrin; intra-heterodimer stability; molecular dynamics; somitogenesis; zebrafish.

Figure 1.. Integrins α5β1 and αVβ1, but not αVβ3, αVβ5, and αVβ6, cluster along somite boundaries

(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.

Figure 2.. FRET-FLIM reveals heterodimer-specific activation on the somite boundary

(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 α^GAAXR mutations (orange star) and the β3^NIN333T 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 (E_FRET) 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: α5^GAAKR-Aqm co-expressed with β1-mCit (D), α5^GAAKRβ1-BiFC (E), αV^GAANR-Aqm co-expressed with β3-mCit (F), αV^GAANRβ3-BiFC (G), αV-Aqm co-expressed with N-glycan wedge allele β3^NIN333T-mCit (H), αV^GAANR-Aqm co-expressed with β5-mCit (I), αV^GAANR-Aqm co-expressed with β6-mCit (J), and αV^GAANRβ6-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. αV^GAANRβ3-BiFC, n = 16 (9 embryos); αVβ3^NIN333T, n = 21 (15 embryos); αV^GAANRβ5, n = 15 (8 embryos); αV^GAANRβ6-BiFC, n = 17 (8 embryos). α5^GAAKRβ1-BiFC clustering cannot be measured because of the poor membrane expression in the mesenchyme. (M) E_FRET of activatable integrin alleles. Sample size is the same as in (L), except α5^GAAKRβ1, n = 18 (9 embryos); αV^GAANRβ3, n = 14 (9 embryos); and αV^GAANRβ6, n = 15 (7 embryos). E_FRET of αVβ1 (C), α5^GAAKRβ1, αV^GAANRβ3, and αV^GAANRβ6 (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. See also Figure S1 and Table S1.

Figure 3.. Integrins α5β1 and αVβ1 are the functional fibronectin (Fn) receptors during zebrafish somitogenesis

(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 α5^FYLDDβ1 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 E_FRET (I) of α5β1 in the absence of Fn, α5^FYLDD 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^−/−: α5^FYLDD β1, n = 16 (8 embryos); hsp70:fn1a; αVβ3, n = 15 (7 embryos). Data are mean ± SD. See also Tables S1, S2, and S3 and Figure S2.

Figure 4.. Integrin intra-heterodimer affinity inversely correlates with integrin activatability

(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 αV^GAANRβ3 (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) F_cross of different integrin heterodimers calculated from FCCS. A lower F_cross 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). See also Table 1 and Figure S3.