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Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies
Published on: March 19, 2014
Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin
Marina Theodosiou1, Moritz Widmaier1, Ralph T Böttcher1
1Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany.
Abstract:
Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn(2+). Despite compromised integrin activation and adhesion, Mn(2+) enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner.
Insights
Talin and kindlin proteins cooperatively activate integrins for cell adhesion. Kindlin also independently initiates cell spreading by binding paxillin and focal adhesion kinase (FAK).
Area of Science:
- Cell biology
- Molecular and cell biology
- Integrin signaling
Background:
- Integrin activation is crucial for ligand binding and cellular signaling.
- The roles of talin and kindlin in integrin activation in non-hematopoietic cells remain unclear.
Purpose of the Study:
- To investigate the distinct and cooperative roles of talin and kindlin in integrin activation and cell adhesion in fibroblasts.
- To elucidate the mechanism by which kindlin promotes cell spreading independently of talin.
Main Methods:
- Fibroblast cell culture and gene deletion (talin or kindlin knockout).
- Assessment of β1 integrin activation, fibronectin (FN) adhesion, and cell spreading.
- Analysis of protein-protein interactions (kindlin-paxillin, paxillin-FAK) and focal adhesion kinase (FAK) activation.
Main Results:
- Fibroblasts lacking talin or kindlin showed impaired β1 integrin activation and adhesion to FN.
- While both proteins are required for integrin activation, Mn(2+) induced cell spreading in talin-deficient cells but not kindlin-deficient cells.
- Kindlin directly bound paxillin, leading to FAK activation and lamellipodia formation, facilitating spreading in a talin-independent manner.
Conclusions:
- Talin and kindlin work together to activate integrins, enabling fibronectin binding and cell adhesion.
- Kindlin plays a critical role in assembling a signaling complex at adhesion sites, independent of talin, to promote cell spreading.
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