Nascent Adhesion Clustering: Integrin-Integrin and Integrin-Substrate Interactions

Nascent adhesions (NAs) are a general precursor to the formation of focal adhesions (FAs) that provide a fundamental mechanism for cell adhesion that is, in turn, involved in cell proliferation, migration, and mechanotransduction. Nascent adhesions form when cells come into contact with substrates at all rigidities and generally involve the clustering of ligated integrins that may recruit un-ligated integrins. Nascent adhesions tend to take on characteristic sizes in the range of O(100nm–150nm) in diameter and tend to contain integrin numbers of O(20–60). The flexible, adaptable model we present provides and clear explanation of how these conserved cluster features come about. Our model is based on the interaction among ligated and un-ligated integrins that arise due to deformations that are induced in the cell membrane-cell glycocalyx and substrate system due to integrin activation and ligation. This model produces a clearly based interaction potential, and from it an explicit interaction force among integrins, that our stochastic diffusion-interaction simulations then show will produce nascent clusters with experimentally observed characteristics. Our simulations reveal effects of various key parameters related to integrin activation and ligation as well as some unexpected and previously unappreciated effects of parameters including integrin mobility and substrate rigidity. Moreover, the model’s structure is such that refinements are readily incorporated and specific suggestions are made as to what is required for further progress in understanding nascent clustering and the development of mature focal adhesions in a truly predictive manner.

Role of 12-LOX in the Platelet Storage Lesion

Background: 12-lipoxygenase (12-LOX) is an enzyme abundant in platelets which can contribute to the platelet storage lesion by oxidizing polyunsaturated fatty acids (PUFAs) released from phospholipid membranes. We and others have shown that the PUFA arachidonic acid (AA) and its lipid oxidation products, such as 12-hydroxyeicosatetraenoic acid (12-HETE), accumulate during storage and have inhibitory effects on platelet recovery, survival, and function. However, several PUFAs are substrates for 12-LOX, and their resulting oxylipins may have different effects. We used targeted metabolomics to quantify PUFAs and oxylipins and platelet function assays to characterize function of fresh and stored wild-type (WT) and 12-LOX -/- platelets. Methods: Blood from WT and 12-LOX -/- mice was collected by retro-orbital bleeding. Platelet-rich plasma (PRP) was generated from whole blood. After fresh samples were aliquoted, the remaining PRP was separated in two groups. One group was stored at room temperature with agitation (RT) for 24 hours, and the other for 48 hours. Metabolites were extracted from samples and quantified by targeted metabolomics as described previously. We assessed platelet function by αIIbβ3 integrin activation by flow cytometry. In vivo recovery of function was measured by transfusing stored platelets into UBiC-GFP mice and stimulating platelets with agonists, followed by gating for transfused (GFP-negative) platelets by flow cytometry. For recovery and survival, we traced biotinylated fresh, 24h, or 48h-stored platelets after transfusion in vivo. Results: We quantified metabolites present in platelets by targeted metabolomics to monitor their changes in concentration over storage time. Among the 10 PUFAs and 28 related oxylipins we analyzed, 15 of 38 analytes showed a significant difference in PRP from WT and 12-LOX-/- mouse samples. The major metabolites of 12-LOX include 12-HETE, 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA), from AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). 12-HETE, 12-HEPE, and 14-HDHA were only detected at <8 nmol/L levels in fresh PRP from 12-LOX -/- mice compared to 668 ± 409nM, 149 ± 85nM, and 295 ± 154nM from WT mice, respectively. After 24 hours of storage at RT, 12-HETE, 12-HEPE, and 14-HDHA dramatically increased to 29.0±4.2µM, 3.7±1.1µM, and 6.3±0.8µM in PRP from WT mice, respectively. As expected, these same metabolites remained at low nmol/L levels in 12-LOX-/- samples during storage accompanied by a significant increase of their precursors AA, EPA, and DHA due to lack of 12-LOX activity. Interestingly, there was also a significant reduction in 15-HETE, 17-HDHA, and 13-hydroxyoctadecadienoic acid (13-HODE) in the 12-LOX -/- mice compared to the WT mice, which are primarily produced by the 15-LOX enzyme. Additionally, we observed a significant decrease of metabolites mediated via the cyclooxygenase (COX) pathway in PRP from 12-LOX-/- mice, including prostaglandin E2 (PGE2), PGD2, thromboxane B2, and 12-hydroxyheptadecatrienoic acid (12-HHTrE). Function-wise, fresh 12-LOX -/- platelets were less responsive to agonists compared to WT platelets. Surprisingly, after transfusion of fresh 12-LOX -/- platelets, we found comparable αIIbβ3-integrin activation results after 1, 4, and 24h of circulation time. In contrast, 24h and 48h of storage of 12-LOX -/- platelets led to significantly lower pre-activation at baseline and a significantly lower activation response than WT platelets after 1h and 4h of circulation time. No significant differences were observed after 24h of circulation time. We observed a clear trend for longer survival after 24 and 48h of storage. Conclusions: We found many metabolic changes between 12-LOX -/- and WT mice during storage. While the 12-LOX -/- mouse model highlights the primary metabolic differences that occur without 12-LOX activity, other changes, such as differences in COX or additional LOX isoform activity, may attenuate oxylipin production. Functionally, we observed less pre-activation and better survival in functional studies, but this may be due to a combined effect of each of these individual metabolites. Future studies will have to determine the roles of individual oxylipins. Disclosures Stolla: Cerus: Research Funding.

Integrin activation is an essential component of SARS-CoV-2 infection

SARS-CoV-2 infection depends on binding its spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The S protein expresses an RGD motif, suggesting that integrins may be co-receptors. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating cell entry and productive infection. We used flow cytometry and confocal microscopy to show that SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which induces integrin extension, enhances cell entry of SARS-CoV-2R18. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activation status. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand-binding function of integrins via a talin-dependent mechanism, and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13. Using cell-permeable peptide inhibitors of talin and Gα13 binding to the cytoplasmic tail of an integrin's β subunit, we demonstrate that talin-mediated signaling is essential for productive infection.

Phostensin Enables Lymphocyte Integrin Activation and Population of Peripheral Lymphoid Organs

Rap1 GTPase drives assembly of the Mig10/RIAM/lamellipodin–Integrin–Talin (MIT) complex that enables integrin dependent lymphocyte functions. Here we used tandem affinity tag based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (PTSN), a regulatory subunit of protein phosphatase 1, is a component of the complex. PTSN mediates dephosphorylation of Rap1 thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes PTSN, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18 null mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable to defective activation of integrins. Ppp1r18 null T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, PTSN enables lymphocyte integrin mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of PTSN ameliorates T cell mediated colitis.

The connection between Rap1 and Talin1 in CD4+ T Lymphocytes

Agonist induced increase in integrin affinity for ligands (activation) plays a pivotal role in T cell trafficking and functions. Activation requires Rap1 GTPase-mediated recruitment of talin1 to the integrins in the plasma membrane. Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that serves this function in T cells. In addition, Rap1 directly binds to talin1 to enable integrin activation in platelets. Here, we assessed the relative contributions of the Rap1-talin1 interaction and RIAM and provide a complete accounting of the connections between Rap1 and talin1 that support integrin activation in conventional CD4+ (Tconv) and CD25HiFoxp3+CD4+ regulatory T (Treg) cells. Disruption of both Rap1 binding sites in talin1 (talin1 (R35E,R118E)) causes a partial defect in αLβ2, α4β1 and α4β7 integrin activation in both Tconv and Treg cells with resulting defects in T cell homing and functions. Over-expression of RIAM bypasses the integrin activation defect in Tconv cells expressing talin1 (R35E,R118E), indicating that RIAM can substitute for Rap1 binding to talin in integrin activation. Conversely, deletion of RIAM in talin1 (R35E,R118E) Tconv cells abrogates activation of αLβ2, α4β1 and α4β7. RIAM and lamellipodin (LPD) are mammalian members of the MRL protein family; LPD plays a more important role than RIAM in Treg cell integrin activation. Nevertheless, loss of RIAM profoundly exacerbates the defects in Treg cell function caused by the talin1 (R35E,R118E) mutation. Most importantly, deleting both MRL proteins combined with talin1 (R35E,R118E) phenocopies the complete lack of integrin activation observed in Rap1a/b null Treg cells. In sum, these data reveal the functionally significant connections between Rap1 and talin1 that enable αLβ2, α4β1 and α4β7 integrin activation in T cells.

The solute carrier MFSD1 decreases β1 integrins activation status and thus tumor metastasis

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier MFSD1 in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in a mouse model. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turn-over, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, down-regulation of MFSD1 expression was observed during early steps of tumorigenesis and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor spread.

Integrin activation is an essential component of SARS-CoV-2 infection

Cellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18 particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18 in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18 with basal state integrins, but is ineffective against Mn2+-activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18 binding regardless of integrin activity state. Integrins transmit signals bidirectionally: 'inside-out' signaling primes the ligand binding function of integrins via a talin dependent mechanism and 'outside-in' signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13 and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα13 binding to the cytoplasmic tail of an integrin's β subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.

Kindlin-3 disrupts an intersubunit association in the integrin LFA1 to trigger positive feedback activation by Rap1 and talin1

Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLβ2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the β2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the β2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/β clasp) and unbending of the ectodomains of the α/β heterodimer. These conformational changes promoted high-affinity talin1 binding to the β2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.