The Critical Role of Kindlin-3 in β2 Integrin Clustering

Academic Background

Neutrophils are the most abundant type of white blood cells in human blood, and their recruitment is essential for innate immunity and inflammatory responses. The initial and critical step in neutrophil recruitment is their adhesion to vascular endothelial cells, which depends on G protein-coupled receptor (GPCR)-triggered integrin inside-out signaling, leading to the activation and clustering of β2 integrins. Although Kindlin-3 and Talin-1 are known to play important roles in integrin inside-out signaling, their specific contributions to β2 integrin clustering remain unclear. Traditional research methods are typically conducted on adherent cells, where integrin-ligand binding simultaneously triggers outside-in signaling, making it difficult to distinguish the effects of the two signaling pathways on integrin clustering.

To address this issue, researchers designed a study to observe and quantify β2 integrin clustering under inside-out signaling using high-resolution microscopy techniques such as STORM, and to explore the roles of Kindlin-3 and Talin-1 in this process.

Source of the Paper

This study was conducted by Yuanyuan Wu, Ziming Cao, and other researchers, with the primary authors affiliated with institutions such as the University of Connecticut School of Medicine and the University of Nevada School of Medicine. The paper was published in 2025 in the journal Cell Communication and Signaling, titled “Kindlin-3 but not talin-1 contributes to integrin inside-out signaling induced β2 integrin clustering.”

Research Process and Results

Research Process

1. Cell Model Establishment
   Researchers used CRISPR-Cas9 technology to knock out Kindlin-3 (K3-KO) and Talin-1 (TLN1-KO) in CXCR2-expressing HL60 cells, and differentiated them into neutrophil-like cells by treating them with 1.3% DMSO for 7 days. β2 integrin knockout (β2-KO) HL60 cells served as a negative control.

2. Integrin Activation Experiments
   Using flow cytometry, researchers detected the activation state of β2 integrins using specific antibodies such as Mab24 and Kim127. Mab24 was used to detect the high-affinity (H+) conformation, while Kim127 was used to detect the extended (E+) conformation. The results showed that IL-8 and fMLP stimulation significantly increased Mab24 binding in wild-type (WT) cells, but almost no Mab24 binding was detected in K3-KO and TLN1-KO cells, indicating that Kindlin-3 and Talin-1 are essential for β2 integrin activation.

3. Integrin Clustering Experiments
   To study the effect of inside-out signaling on β2 integrin clustering, researchers used STORM microscopy to image suspended HL60 cells. The results showed that IL-8 and fMLP stimulation significantly increased the clustering of LFA-1 and Mac-1 in WT cells, but no increase in clustering was observed in K3-KO cells. In contrast, the clustering of LFA-1 and Mac-1 significantly increased in TLN1-KO cells, indicating that Talin-1 is not involved in integrin clustering under inside-out signaling.

4. Functional Validation of the Kindlin-3 PH Domain
   To further investigate the mechanism of Kindlin-3 in integrin clustering, researchers transduced PH domain-deleted Kindlin-3 mutant (ΔPH-K3) or wild-type Kindlin-3 (WT-K3) into K3-KO cells. The results showed that WT-K3 transduction restored IL-8 and fMLP-induced clustering of LFA-1 and Mac-1, while ΔPH-K3 transduction failed to restore clustering, indicating that the PH domain of Kindlin-3 plays a critical role in integrin clustering.

Key Results

• Contribution of Kindlin-3 and Talin-1 to β2 Integrin Activation: The activation of β2 integrins was significantly reduced in K3-KO and TLN1-KO cells, indicating that both are indispensable for integrin activation.
• Critical Role of Kindlin-3 in Integrin Clustering: No IL-8 or fMLP-induced integrin clustering was observed in K3-KO cells, while clustering significantly increased in TLN1-KO cells, indicating that Kindlin-3 plays a dominant role in inside-out signaling.
• Importance of the Kindlin-3 PH Domain: The PH domain-deleted Kindlin-3 mutant failed to restore integrin clustering, indicating that the PH domain is crucial for Kindlin-3-mediated integrin clustering.

Conclusions and Significance

This study reveals a new mechanism by which Kindlin-3 regulates β2 integrin clustering under inside-out signaling and shows that Talin-1 does not play a major role in this process. This finding challenges the traditional view that integrin activation and clustering are tightly linked processes, suggesting that they may be regulated by different molecular mechanisms during inside-out signaling. Additionally, the study highlights the importance of the PH domain of Kindlin-3 in integrin clustering, providing new insights into the role of leukocyte adhesion in inflammatory diseases.

Research Highlights

• Application of High-Resolution Imaging Techniques: Using STORM microscopy, researchers observed and quantified β2 integrin clustering induced by inside-out signaling at the nanoscale for the first time.
• Distinguishing the Functions of Kindlin-3 and Talin-1: The study clarified the dominant role of Kindlin-3 in integrin clustering, while Talin-1 is only involved in integrin activation.
• Functional Validation of the PH Domain: The study revealed the critical role of the PH domain of Kindlin-3 in integrin clustering, providing a new potential target for the treatment of inflammatory diseases.

Additional Valuable Information

This study also provides new explanations for the pathological mechanisms of leukocyte adhesion deficiency (LAD-III), showing that the loss of Kindlin-3 not only affects integrin activation but also integrin clustering. This finding may offer new approaches for the treatment of LAD-III.

This research not only deepens our understanding of integrin signaling mechanisms but also provides new directions for the treatment of inflammatory diseases.