Periodic Reporting for period 4 - PoInt (Principles of Integrin Mechanics and Adhesion)
Reporting period: 2023-10-01 to 2025-03-31
In multicellular organisms, cells adhere to each other and to their extracellular matrix (ECM). The cell-cell and cell-ECM adhesion is crucial to form and maintain tissues. The cell-ECM adhesion processes are mediated by the family of integrin proteins. These are large molecules passing the cell membrane. Inside the cell their tail connects to the cytoskeleton via adaptor proteins, while the large extracellular portion anchors the cell to ECM substrates. During processes such as wound healing or tissue differentiation and growth, plasticity of the integrin adhesion process is of essential importance and flexibly modulated in response to signals from within and outside the cell. As can be expected from molecules with such essential functions, integrins have been implicated in various diseases, ranging from cardiovascular to inflammatory diseases and cancer growth and metastasis.
Signal dependent „integrin activation“ is induced by binding of the adaptor proteins talin and kindlin to integrin tails and the actomyosin cytoskeleton. This binding affects the shape/conformation of the entire molecule. In a second step, integrins cluster and assemble into gigantic signalling hubs, where they integrate biochemical and biophysical signals to achieve their full functional output. As of today, the key steps of integrin activation are largely unknown and the underlying physical principles still need to be identified.
The PoInt project utilizes a combination of quantitative single molecule measurements, reconstitution of minimal and cellular adhesion complexes as well as development of multicellular structures and organoids to investigate this process. PoInt has four major aims:
(1) unravel how forces are propagated through the talin-integrin tail bonds and how force-induced integrin shape changes affect signaling.
(2) use novel force spectrometers to determine energy landscapes and the high-resolution structure of fibronectin-integrin complexes.
(3) test how integrin tail-binding adaptors, cortical F-actin and specific domains of integrins induce integrin clustering in model membranes in vitro.
(4) unravel how integrins integrate chemical and biophysical signals during organ development.
A better understanding of these fundamental principles in cell adhesion biology and its signalling mechanisms will enable novel strategies to curb adhesion functions without completely blocking integrins, thus limiting the enormous side effects of current interventions.
Signal dependent „integrin activation“ is induced by binding of the adaptor proteins talin and kindlin to integrin tails and the actomyosin cytoskeleton. This binding affects the shape/conformation of the entire molecule. In a second step, integrins cluster and assemble into gigantic signalling hubs, where they integrate biochemical and biophysical signals to achieve their full functional output. As of today, the key steps of integrin activation are largely unknown and the underlying physical principles still need to be identified.
The PoInt project utilizes a combination of quantitative single molecule measurements, reconstitution of minimal and cellular adhesion complexes as well as development of multicellular structures and organoids to investigate this process. PoInt has four major aims:
(1) unravel how forces are propagated through the talin-integrin tail bonds and how force-induced integrin shape changes affect signaling.
(2) use novel force spectrometers to determine energy landscapes and the high-resolution structure of fibronectin-integrin complexes.
(3) test how integrin tail-binding adaptors, cortical F-actin and specific domains of integrins induce integrin clustering in model membranes in vitro.
(4) unravel how integrins integrate chemical and biophysical signals during organ development.
A better understanding of these fundamental principles in cell adhesion biology and its signalling mechanisms will enable novel strategies to curb adhesion functions without completely blocking integrins, thus limiting the enormous side effects of current interventions.
Force transmission by talin and kindlin at integrin tail domains
Fusion constructs between the integrin-binding domain 3 of talin and the beta integrin tail were designed and DNA handles added to the ends of this construct. Single molecule optical tweezers experiments showed that the talin-integrin bond is mechanically much weaker than expected in the current cell biological models. However, adding kindlin protects the talin-integrin bond and renders it force insensitive. This discovery points towards a mechanism where a tripartite complex between integrin, talin, and kindlin enhances the mechanical stability of the connection between cytoskeleton and extracellular matrix in the early events of adhesion formation. Further work with a number of variants of talin and integrin showed that this effect depends on the integrin isoform and is present in beta 1d but not in beta1a integrin subunit. This work has been published in PNAS (Bodescu et al., 2023).
Role of integrin-actomyosin linkage for kidney development
By combining NMR, biochemistry and cell biology, we discovered that talin and kindlin binding to the β1-integrin tail (β1-CT) can induce a conformational change in the β1-CT that increases talin and decreases kindlin affinity. We also discovered that this asymmetric affinity regulation is accompanied by a direct interaction between talin and kindlin, which can promote the simultaneous binding of talin and kindlin to β1-CTs. Disrupting either the allosteric communication between the binding sites of talin and kindlin or their direct interaction in cells severely compromised integrin functions. A revised manuscript describing the study has been resubmitted for publication.
Based on this finding, we used the results from the NMR line broadening experiment of our study to perform an alanine scan of the β1-CT in regions that were extraordinarily perturbed in ternary complex titrations to identify residues that might play a role in the allosteric communication between talin and kindlin. First, we measured the affinity of kindlin2 towards these mutants. We did not identify significant changes in affinity for kindlin suggesting that the allosteric coupling is activated upon simultaneous binding of talin and kindlin. Second, we measured the talin affinity for the β1-CT mutants in the absence and presence of kindlin2 and found that certain substitutions between the talin and kindlin NPxY motifs reduce talin affinity in the presence of kindlin. The most interesting pair of mutations that we discovered in this mutational analysis was β1-CT-S785A and -S785D. Whereas the apparent talin affinity for β1-CT-S785A was unaffected by the presence of kindlin2, the apparent THD1 affinity for β1-CT-S785D significantly decreased by the presence of kindlin2.
For a long time we assumed that β1-CT-S785 is bound and phosphorylated by an unknown kinase. However, crosslinking proteomics and an siRNA screen of all known kinases did not produce hits indicating that not phosphorylation but the conformational change of the tail by the β1-CT-S785D affects talin binding. To test the in vivo consequences of the talin affinity change for β1-CT-S785D, we generated a mouse model carrying the β1-S785D substitution in the germline. Whereas mice carrying the β1-S785A substitution are perfectly normal, mice carrying β1-S785D mice lack kidneys. Our preliminary data indicate that the ureteric bud outgrowth is severely compromised in β1-S785D mice. Importantly, we could confirm with a series of in vivo and ex vivo experiments that the kidney defect is caused by reduced talin affinity and not by integrin tail modification such as serine-785 phosphorylation. The studies are completed and we are in the process of finalizing a manuscript.
In parallel to the kidney model, other organoid systems were tested. Single cell derived organoid systems of human mammary gland and murine pancreatic tumor were identified to be the most effective way to move forward to establish the role of cell adhesion on the self organization of epithelial cell layers.
Fusion constructs between the integrin-binding domain 3 of talin and the beta integrin tail were designed and DNA handles added to the ends of this construct. Single molecule optical tweezers experiments showed that the talin-integrin bond is mechanically much weaker than expected in the current cell biological models. However, adding kindlin protects the talin-integrin bond and renders it force insensitive. This discovery points towards a mechanism where a tripartite complex between integrin, talin, and kindlin enhances the mechanical stability of the connection between cytoskeleton and extracellular matrix in the early events of adhesion formation. Further work with a number of variants of talin and integrin showed that this effect depends on the integrin isoform and is present in beta 1d but not in beta1a integrin subunit. This work has been published in PNAS (Bodescu et al., 2023).
Role of integrin-actomyosin linkage for kidney development
By combining NMR, biochemistry and cell biology, we discovered that talin and kindlin binding to the β1-integrin tail (β1-CT) can induce a conformational change in the β1-CT that increases talin and decreases kindlin affinity. We also discovered that this asymmetric affinity regulation is accompanied by a direct interaction between talin and kindlin, which can promote the simultaneous binding of talin and kindlin to β1-CTs. Disrupting either the allosteric communication between the binding sites of talin and kindlin or their direct interaction in cells severely compromised integrin functions. A revised manuscript describing the study has been resubmitted for publication.
Based on this finding, we used the results from the NMR line broadening experiment of our study to perform an alanine scan of the β1-CT in regions that were extraordinarily perturbed in ternary complex titrations to identify residues that might play a role in the allosteric communication between talin and kindlin. First, we measured the affinity of kindlin2 towards these mutants. We did not identify significant changes in affinity for kindlin suggesting that the allosteric coupling is activated upon simultaneous binding of talin and kindlin. Second, we measured the talin affinity for the β1-CT mutants in the absence and presence of kindlin2 and found that certain substitutions between the talin and kindlin NPxY motifs reduce talin affinity in the presence of kindlin. The most interesting pair of mutations that we discovered in this mutational analysis was β1-CT-S785A and -S785D. Whereas the apparent talin affinity for β1-CT-S785A was unaffected by the presence of kindlin2, the apparent THD1 affinity for β1-CT-S785D significantly decreased by the presence of kindlin2.
For a long time we assumed that β1-CT-S785 is bound and phosphorylated by an unknown kinase. However, crosslinking proteomics and an siRNA screen of all known kinases did not produce hits indicating that not phosphorylation but the conformational change of the tail by the β1-CT-S785D affects talin binding. To test the in vivo consequences of the talin affinity change for β1-CT-S785D, we generated a mouse model carrying the β1-S785D substitution in the germline. Whereas mice carrying the β1-S785A substitution are perfectly normal, mice carrying β1-S785D mice lack kidneys. Our preliminary data indicate that the ureteric bud outgrowth is severely compromised in β1-S785D mice. Importantly, we could confirm with a series of in vivo and ex vivo experiments that the kidney defect is caused by reduced talin affinity and not by integrin tail modification such as serine-785 phosphorylation. The studies are completed and we are in the process of finalizing a manuscript.
In parallel to the kidney model, other organoid systems were tested. Single cell derived organoid systems of human mammary gland and murine pancreatic tumor were identified to be the most effective way to move forward to establish the role of cell adhesion on the self organization of epithelial cell layers.
The multi-faceted approach combining physics, protein biochemistry and cell-biology will take the textbook knowledge on integrin-mediated adhesion to a new quantitative level. We expect to decipher for the first time the force signalling pathways from the extracellular matrix to the cytoskeleton. On the molecular scale, the combination of single molecule mechanics with fluorescence will shed light on a vital but hitherto not well-understood process. The obtained insights into the force feedback mechanisms steering the organoid structure formation are defining a new framework for our understanding of morphogenesis of organoids. A mechanistic understanding of integrin signalling will provide a basis for novel therapeutic concepts to modulate integrin activity in disease.