Selective binding and lateral clustering of α5β1 and αvβ3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly

Coordination of the specific functions of α5β1 and αvβ3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvβ3 and α5β1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5β1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvβ3 ligand at 30 and 60 nm spacings. Analysis of αvβ3 and α5β1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5β1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvβ3 ligand. αvβ3 integrin clusters are more pronounced on αvβ3 ligand, though they can also be detected in cells adhering to α5β1 ligand. Furthermore, α5β1 integrin clusters are present in cells adhering to α5β1 ligand, and often colocalize with αvβ3 clusters. Taken together, these findings indicate that the activation of αvβ3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.

α5β1与αvβ3整合素（integrin）的特定功能协同调控，对于细胞黏附、铺展与迁移的精准调控至关重要，但不同整合素特异性串扰对这些过程的具体贡献仍未明确。为探明αvβ3与α5β1整合素的具体功能，我们采用了呈现固定化、整合素选择性肽模拟配体的金颗粒纳米阵列。该配体与整合素的结合具备高度选择性，细胞可在两种配体表面完成铺展；不过在α5β1配体表面，细胞铺展速度更快、投影细胞面积更大，且两种效应均依赖配体间距。对黏着斑（adhesion plaque）的定量分析显示，当配体间距为30 nm与60 nm时，黏附于αvβ3配体的细胞内黏着斑尺寸显著增大。对αvβ3与α5β1整合素簇的分析表明，黏附于α5β1配体的细胞中纤维状黏附结构更为突出，而黏附于αvβ3配体的细胞中，整合素簇主要定位于细胞边缘。αvβ3整合素簇在αvβ3配体表面表现更为显著，但在黏附于α5β1配体的细胞中也可检测到此类簇。此外，α5β1整合素簇存在于黏附于α5β1配体的细胞中，且常与αvβ3簇发生共定位。综上，本研究结果表明，配体结合介导的αvβ3整合素激活虽并非细胞初始黏附与铺展所必需，但对于稳定黏着斑的形成至关重要。