Nanoscopy reveals integrin clustering reliant on kindlin-3 but not talin-1

Abstract

Background: Neutrophils are the most abundant leukocytes in human blood, and their recruitment is essential for innate immunity and inflammatory responses. The initial and critical step of neutrophil recruitment is their adhesion to vascular endothelium, which depends on G protein-coupled receptor (GPCR) triggered integrin inside-out signaling that induces β2 integrin activation and clustering on neutrophils. Kindlin-3 and talin-1 are essential regulators for the inside-out signaling induced β2 integrin activation. However, their contribution in the inside-out signaling induced β2 integrin clustering is unclear because conventional assays on integrin clustering are usually performed on adhered cells, where integrin-ligand binding concomitantly induces integrin outside-in signaling.

Methods: We used flow cytometry and quantitative super-resolution stochastic optical reconstruction microscopy (STORM) to quantify β2 integrin activation and clustering, respectively, in kindlin-3 and talin-1 knockout leukocytes. We also tested whether wildtype or Pleckstrin homology (PH) domain deleted kindlin-3 can rescue the kindlin-3 knockout phenotypes.

Results: GPCR-triggered inside-out signaling alone can induce β2 integrin clustering. As expected, both kindlin-3 and talin-1 knockout decreases integrin activation. Interestingly, only kindlin-3 but not talin-1 contributes to integrin clustering in the scenario of inside-out-signaling, wherein a critical role of the PH domain of kindlin-3 was highlighted.

Conclusions: Since talin was known to facilitate integrin clustering in outside-in-signaling-involved cells, our finding provides a paradigm shift by suggesting that the molecular mechanisms of integrin clustering upon inside-out signaling and outside-in signaling are different. Our data also contradict the conventional assumption that integrin activation and clustering are tightly inter-connected by showing separated regulation of the two during inside-out signaling. Our study provides a new mechanism that shows kindlin-3 regulates β2 integrin clustering and suggests that integrin clustering should be assessed independently, aside from integrin activation, when studying leukocyte adhesion in inflammatory diseases.

Keywords: Integrin clustering; Kindlin-3; Neutrophil adhesion; STORM; Talin-1; β2 integrin.

Fig. 1

Kindlin-3 and talin-1 are essential for β₂ integrin activation. Wildtype (WT), kindlin-3 knockout (K3-KO), talin-1 knockout (TLN1-KO), and β₂ integrin knockout (β₂-KO) CXCR2-expressing HL60 (HL60-2) cells were differentiated to neutrophil-like cells. A-D, β₂ integrin activation was quantified by flow cytometry using conformation-specific antibodies mAb24 (A, C) and KIM127 (B, D) with or without IL-8 (A-B) or fMLP (C-D) stimulation. E-F, Total β₂ integrin expression was quantified by flow cytometry using antibody TS1/18 with or without IL-8 (E) or fMLP (F) stimulation. G-H, Expression of CXCR2 and FPR1 on cells. MFI, median fluorescence intensity. Means ± SD, n = 12 repeats from 3 individual experiments in A-D and G-H, n = 16 repeats from 4 individual experiments in E-F. ns p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA in A-F and one-way ANOVA in G-H followed by Tukey’s multiple comparison test

Fig. 2

Kindlin-3 but not talin-1 is critical for α_Lβ₂ integrin (LFA-1) clustering. A-I, Representative STORM images of integrin α_L on wildtype (WT, A-C), kindlin-3 knockout (K3-KO, D-F), and talin-1 knockout (TLN1-KO, G-I) differentiated HL60-2 cells without (A, D, G) or with IL-8 (B, E, H) or fMLP (C, F, I) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered α_L are shown as gray dots. Scale bars are 1 μm. J-M, The number of clusters per WT, K3-KO (J-K), or TLN1-KO (L-M) HL60-2 cell without or with IL-8 (J, L) or fMLP (K, M) stimulation. Means ± SD (n ≥ 50 cells from 4 individual experiments). ns, p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001, by two-way ANOVA followed by Tukey’s multiple comparison test

Fig. 3

Kindlin-3 but not talin-1 is important for α_Mβ₂ integrin (Mac-1) clustering. A-I, Representative STORM images of integrin α_M on wildtype (WT, A-C), K3-KO (D-F), and TLN1-KO (G-I) differentiated HL60-2 cells without (A, D, G) or with IL-8 (B, E, H) or fMLP (C, F, I) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered α_M were shown as gray dots. Scale bars are 1 μm. J-M, The number of clusters per WT, K3-KO (J-K), or TLN1-KO (L-M) HL60-2 cell without or with IL-8 (J, L) or fMLP (K, M) stimulation. Means ± SD (n ≥ 50 cells from 3 individual experiments). ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test

Fig. 4

PH domain in kindlin-3 is required for α_Lβ₂ (LFA-1) clustering. A-F, Representative STORM images of integrin α_L on differentiated HL60-2 cells containing wildtype kindlin-3 (WT-K3, A-C) and PH domain-deleted kindlin-3 (ΔPH-K3, D-F) without (A, D) or with IL-8 (B, E) or fMLP (C, F) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered α_L are shown as gray dots. Scale bars are 1 μm. G-H, The number of clusters per WT-K3 or ΔPH-K3 HL60-2 cell without or with IL-8 (g) or fMLP (H) stimulation. Means ± SD (n ≥ 75 cells from 3 individual experiments). ns p > 0.05; ** p < 0.01; **** p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test

Fig. 5

PH domain in kindlin-3 is required for α_M β₂ integrin (Mac-1) clustering. A-C, Representative STORM images of integrin α_M on differentiated HL60-2 cells containing wild-type kindlin-3 (WT-K3, A-C) and PH domain-deleted kindlin-3 (ΔPH-K3, D-F) without (A, D) or with IL-8 (B, E) or fMLP (C, F) stimulation after clustering analysis using Voronoi diagrams. Adjacent clusters are distinguished by different colors. Non-clustered α_M are shown as gray dots. Scale bars are 1 μm. G-H, The number of clusters per WT-K3 or ΔPH-K3 HL60-2 cell without or with IL-8 (G) or fMLP (H) stimulation. Means ± SD (n ≥ 85 cells from 3 individual experiments). ns p > 0.05; * p < 0.05; ** p < 0.01; **** p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test