Integrin Engagement Research Articles

The Combined Influence of Viscoelastic and Adhesive Cues on Fibroblast Spreading and Focal Adhesion Organization.

Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately impairs organ functionality. Cells bind to the ECM through integrins, where αv integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for in vitro hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior. We developed hyaluronic acid hydrogels with independently tunable cell-instructive properties (stiffness, viscoelasticity, ligand presentation) to address this challenge. Hydrogels with mechanics matching normal or fibrotic lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune viscoelasticity. Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via αvβ3 or α5β1. On fibrosis-mimicking stiff elastic hydrogels, preferential αvβ3 engagement promoted increased spreading, actin stress fiber organization, and focal adhesion maturation as indicated by paxillin organization in human lung fibroblasts. In contrast, preferential α5β1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, largely curtailed fibroblast spreading and focal adhesion organization independent of adhesive ligand type, highlighting its role in reducing fibroblast-activating behaviors. Together, these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors. The online version contains supplementary material available at 10.1007/s12195-021-00672-1.

Differential Engagement Of The Integrin Binding Domain Of Fibronectin Impacts Fibroblast Behavior

The switch between α5β1 and αvβ3 integrin engagement due to unfolding of the integrin binding domain (IBD) of fibronectin (Fn) has been theorized for a long time but was observed in vitro and murine models only recently. Fn is largely present in the extra cellular matrix (ECM) during wound healing and fibroproliferative diseases like idiopathic pulmonary fibrosis or cancer. Thus, further understanding the downstream effects of this differential integrin binding could elucidate several diseases’ mechanisms. Specifically, integrin α5β1 requires the regular, closed conformation of Fn's IBD, while αvβ3 can also bind the unfolded, open conformation. Moreover, cells can generate forces sufficient to cause such unfolding. We hypothesized that this change in Fn conformation affects lung fibroblasts by guiding integrin enrichment, pushing them towards a secretory and disease-relevant phenotype. We tested this premise by engineering Fn fragments containing the IBD site that mimicked either the open or closed conformation. Additionally, these fragments were designed to be covalently bound via thiol chemistry to a variety of substrates in order to preserve their structural integrity. Glass dishes coated with either Fn fragment were used as substrates for Single Cell Force Spectroscopy. By using fibroblasts expressing only α5β1, only αvβ3, and naïve murine lung fibroblasts we observed the need of proper α5β1 engagement to generate high adhesion forces even at short time points (<120s). Plating naïve fibroblasts on similarly functionalized dishes for up to 30 minutes enabled Mass Spec analysis of the adhesome and of the phosphorylation pathways emerging from the differential integrin engagement. Particularly, collagen XIIa and tubulin beta3 appeared to be enriched upon increased αvβ3 binding. Even on soft substrates (4kPa representing healthy lung tissue), lung fibroblasts plated on the open IBD Fn fragment engaged αvβ3 and display increased nuclear translocation of transcription factors associated with a secretory and contractile phenotype, after a 24h incubation. Fn (with Extra Domain A), Tropomyosin, Vimentin, Serpine1, and α integrins were among the genes upregulated on the open Fn fragment, compared to the closed fragment. These results were confirmed by qPCR and then bulk RNAseq assays. In conclusion, these data support that differential integrin engagement due to Fn IBD unfolding appears to affect cell phenotype in both the short and long run. Current efforts are focused on evaluating the efficacy of a novel αvβ3 inhibiting antibody on the progression of murine lung fibrosis. This work contributes to defining the link between changes in the ECM and cell behavior in the context of fibroproliferative diseases.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation.

While the mechanisms by which chemical signals control cell fate have been well studied, the impact of mechanical inputs on cell fate decisions is not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through linker of nucleoskeleton and cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

GSK3β, a Master Kinase in the Regulation of Adult Stem Cell Behavior.

In adult stem cells, Glycogen Synthase Kinase 3β (GSK3β) is at the crossroad of signaling pathways controlling survival, proliferation, adhesion and differentiation. The microenvironment plays a key role in the regulation of these cell functions and we have demonstrated that the GSK3β activity is strongly dependent on the engagement of integrins and protease-activated receptors (PARs). Downstream of the integrin α5β1 or PAR2 activation, a molecular complex is organized around the scaffolding proteins RACK1 and β-arrestin-2 respectively, containing the phosphatase PP2A responsible for GSK3β activation. As a consequence, a quiescent stem cell phenotype is established with high capacities to face apoptotic and metabolic stresses. A protective role of GSK3β has been found for hematopoietic and intestinal stem cells. Latters survived to de-adhesion through PAR2 activation, whereas formers were protected from cytotoxicity through α5β1 engagement. However, a prolonged activation of GSK3β promoted a defect in epithelial regeneration and a resistance to chemotherapy of leukemic cells, paving the way to chronic inflammatory diseases and to cancer resurgence, respectively. In both cases, a sexual dimorphism was measured in GSK3β-dependent cellular functions. GSK3β activity is a key marker for inflammatory and cancer diseases allowing adjusted therapy to sex, age and metabolic status of patients.

Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition

Of the many origins of pulmonary myofibroblasts, microvascular pericytes are a known source. Prior literature has established the ability of pericytes to transition into myofibroblasts, but provide limited insight into molecular cues that drive this process during lung injury repair and fibrosis. Fibronectin and RGD-binding integrins have long been considered pro-fibrotic factors in myofibroblast biology, and here we test the hypothesis that these known myofibroblast cues coordinate pericyte-to-myofibroblast transitions. Specifically, we hypothesized that αvβ3 integrin engagement on fibronectin induces pericyte transition into myofibroblastic phenotypes in the murine bleomycin lung injury model. Myosin Heavy Chain 11 (Myh11)-CreERT2 lineage tracing in transgenic mice allows identification of cells of pericyte origin and provides a robust tool for isolating pericytes from tissues for further evaluation. We used this murine model to track and characterize pericyte behaviors during tissue repair. The majority of Myh11 lineage-positive cells are positive for the pericyte surface markers, PDGFRβ (55%) and CD146 (69%), and display typical pericyte morphology with spatial apposition to microvascular networks. After intratracheal bleomycin treatment of mice, Myh11 lineage-positive cells showed significantly increased contractile and secretory markers, as well as αv integrin expression. According to RNASeq measurements, many disease and tissue-remodeling genesets were upregulated in Myh11 lineage-positive cells in response to bleomycin-induced lung injury. In vitro, blocking αvβ3 binding through cycloRGDfK prevented expression of the myofibroblastic marker αSMA relative to controls. In response to RGD-containing provisional matrix proteins present in lung injury, pericytes may alter their integrin profile.

Functional Bioinformatics Analyses of the Matrisome and Integrin Adhesome.

The extracellular matrix (ECM) is the noncellular compartment of living organisms and is formed of a complex network of cross-linked proteins, which is collectively known as the matrisome. Apart from providing the structure for an organism, cells interact and thereby communicate with the ECM. Cells interact with their surrounding ECM using cell-surface receptors, such as integrins. Upon integrin engagement with the ECM, cytoskeletal proteins are recruited to integrins and form a molecular protein complex known as the integrin adhesome. Global descriptions of the matrisome and integrin adhesome have been proposed using in silico bioinformatics approaches, as well as through biochemical enrichment of matrisome and adhesome fractions coupled with mass spectrometry-based proteomic analyses, providing inventories of their compositions in different contexts. Here, methods are described for the computational downstream analyses of matrisome and adhesome mass spectrometry datasets that are accessible to wet lab biologists, which include comparing datasets to in silico descriptions, generating interaction networks and performing functional ontological analyses.

Collective cancer cell invasion requires RNA accumulation at the invasive front

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

The mechano-sensitive response of β1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein

Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, β1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.

HCMV-induced signaling through gB–EGFR engagement is required for viral trafficking and nuclear translocation in primary human monocytes

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.

Biomaterials functionalized with nanoclusters of integrin- and syndecan-binding ligands improve cell adhesion and mechanosensing under shear flow conditions.

We have engineered biomaterials that display nanoclusters of ligands that bind both integrin and syndecan-4 cell receptors. These surfaces regulate cell behaviors under static conditions including adhesion, spreading, actin stress fiber formation, and migration. The syndecan-4 receptors are also critical mediators of cellular mechanotransduction. In this contribution we assess whether this novel class of materials can regulate the response of cells to applied mechanical stimulation, using the shear stress imparted by laminar fluid flow as a model stimulus. Specifically, we assess endothelial cell detachment due to flow, cell alignment due to flow, and cell adhesion from the flowing fluid. A high degree of cell retention was observed on surfaces containing integrin-binding ligands or a mixed population of integrin- and syndecan-binding ligands. However, the presence of both ligand types was necessary for the cells to align in the direction of flow. These results imply that integrin engagement is necessary for adhesion strength, but engagement of both receptor types aids in appropriate mechanotransduction. Additionally, it was found that surfaces functionalized with both ligand types were able to scavenge a larger number of cells from flow, and to do so at a faster rate, compared to surfaces functionalized with only integrin- or syndecan-binding ligands. These results show that interfaces functionalized with both integrin- and syndecan-binding ligands regulate a significant range of biophysical cell behaviors in response to shear stress.

Mechanical strength determines Ca2+ transients triggered by the engagement of β2 integrins to their ligands

Lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) are key adhesion receptors to mediate neutrophil (PMN) recruitment and intracellular calcium (Ca2+) signaling. Binding of LFA-1 and Mac-1 to their ligands is essential in triggering Ca2+ transients and activating Ca2+-dependent kinases involved in cytoskeletal remodeling and migratory function. While mechanical forces are critical in regulating integrin-mediated Ca2+ transients, it is still unclear how the bond strength of β2-integrin-ligand pair affects Ca2+ responses. Here three typical ligands with known mechanical features with LFA-1 and Mac-1 in our previous work were adopted to quantify their capabilities in inducing Ca2+ transients in adherent PMNs under shear flow. Data indicated that LFA-1 dominates Ca2+ transients in PMNs on intercellular adhesive molecule 1 (ICAM-1) and junctional adhesion molecule-A (JAM-A), while Mac-1 mediates Ca2+ transients induced by receptor for advanced glycation end products (RAGE), consistent with their corresponding bond strengths. These results link β2 integrin-ligand bond strength with Ca2+ transients in PMNs, suggesting high bond strength gives rise to strong Ca2+ response especially under physiological-like shear flow. The outcomes provide a new insight in understanding the mechanical regulatory mechanisms of PMN recruitment.

Integrin-Mediated Adhesion to Extracellular Matrix Protein Fibronectin Drives Megakaryocytosis in JAK2V617F+ Primary Myelofibrosis

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in Primary Myelofibrosis (PMF). Myelofibrosis was long regarded as a bystander of disease in PMF. However, as cells have the ability to sense the surrounding ECM through integrin receptors, we examined the hypothesis that abnormal ECM in myelofibrosis, mediated by integrin activation, contributes to BM megakaryocytosis in JAK2V617F+ PMF. Due to its complex genetic landscape, modeling PMF in animals is challenging. While there is no ideal model to replicate the human disease, Vav1-hJAK2V617F transgenic mice (JAK2V617F+) (Xing S et al. Blood. 2008; 111:5109-5117) are most appropriate for our studies because they express the most frequent mutation in PMF and show megakaryocytosis in BM and a predictable development of myelofibrosis throughout the animal's life. Fibronectin (FN) is a dimeric ECM glycoprotein produced by a variety of cells in BM. FN secretion by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Analysis of myelofibrotic BM from JAK2V617F+ mice revealed elevated levels of FN in BM ECM. Integrins are heterodimeric cell adhesion receptors of α and β subunits that connect the cell cytoskeleton to ECM. Integrin engagement and subsequent signaling affect cell differentiation, proliferation, migration and survival. Integrins α5β1, α4β1, αIIbβ3, and αυβ3 are FN-binding integrins known to be expressed in megakaryocytes. Expression analysis detected significantly elevated expression of α5, αIIb, αυ and β3 subunits in CD41+ megakaryocytes fromJAK2V617F+ mice in vitro and in vivo. Expression of β1 integrin was unchanged in megakaryocytes fromJAK2V617F+ mice, but expression of 9EG7, an antibody clone that detects the high-affinity conformation of β1 integrin, was elevated in JAK2V617F+ CD41+ megakaryocytes. Levels of the high-affinity form of β1 integrin decrease in response to inhibition of α5 integrin function (using Hmα5-1 antibody), indicating a high degree of correlation between affinity to FN and β1 activation in megakaryocytes fromJAK2V617F+ mice. As suggested by expression studies, in-vitro differentiated JAK2V617F+ megakaryocytes showed increased affinity to FN in adhesion assays. To ascertain the contribution of adhesion to FN in development of megakaryocytosis, megakaryocytes were differentiated in vitro on FN, with and without the presence of α5 integrin inhibitory antibody Hmα5-1. Culture on FN had a positive effect on the number of CD41+ megakaryocytes after four days of culture, especially in JAK2V617F+ BM. Importantly, inhibition of α5 integrin decreased the number of CD41+ megakaryocytes in JAK2V617F+ BM to nearly wild-type levels. To determine the effect of in vivo inhibition of α5 integrin on megakaryocyte numbers, the antibody clone 5H10-27, or MFR-5, previously reported to be effective in vivo, was used. Effective targeting in vivo of BM megakaryocytes by the 5H10-27 antibody was confirmed using a fluorochrome-labelled secondary antibody specific to the isotype of the injected clone (Rat IgG2a, κ). The same method was used to determine minimum required dose and interval of administration. JAK2V617F+ mice were treated with three doses of 5H10-27 or isotype control antibody (1mg/Kg IV every 48 hours). Analysis of treated animals on day 5 detected a decrease in megakaryocyte α5 integrin expression in animals treated with 5H10-27 antibody relative to control isotype antibody-treated animals. Platelet counts in peripheral blood and percentage of BM CD41+ megakaryocytes were consistently, albeit non-significantly, lower in 5H10-27 antibody-treated than in isotype antibody-treated animals. Corroborating our findings in mice, analysis of megakaryocytes from patients carrying the JAK2V617F mutation revealed elevated cell surface expression of α5 integrin subunit and increased adhesion to FN, which was dampened by an anti-α5 integrin antibody (Sam-1). Our results uncovered a global de-regulation of FN-binding integrin expression in megakaryocytes carrying the JAK2V617F mutation and the central role of FN-α5 integrin in development of megakaryocytosis in PMF. These results challenge the current paradigm by bringing ECM and myelofibrosis from a bystander position to center stage in the pathology of PMF. Disclosures No relevant conflicts of interest to declare.

Fibg390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. FibrinogenAEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to FibWT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, FibAEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation. Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells. In addition to disrupting fibrin-αMβ2 interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ390-396A limits metastatic potential. Disclosures Palumbo: Ionis Pharmaceuticals: Research Funding.

L-selectin shedding affects bacterial clearance in the lung: a new regulatory pathway for integrin outside-in signaling

AbstractPneumonia induced by Gram-negative bacteria is a common and serious disease associated with high morbidity and mortality. Elimination of bacterial pathogens relies on the recruitment and functions of neutrophils. The adhesion molecule L-selectin has recently been implicated in integrin activation in neutrophils (inside-out signaling). However, the molecular mechanism by which L-selectin participates in host defense against Klebsiella pneumoniae–induced pulmonary inflammation is unknown. We demonstrate that L-selectin–deficient mice are prone to pulmonary infection compared with wild-type controls. Mechanistically, L-selectin cleavage from the neutrophil surface triggered by integrin engagement is involved in neutrophil recruitment into the lung and bacterial clearance. Downstream of integrin ligation, the metalloproteinase A disintegrin and metalloproteinase 17 (ADAM17) sheds L-selectin from the neutrophil surface in an IRhom2-dependent manner. L-selectin cleavage enhances integrin-mediated outside-in signaling, resulting in increased neutrophil effector functions. Thus, we identify a novel regulatory mechanism in neutrophils required for an adequate immune response triggered by integrin engagement during K pneumoniae–induced pulmonary inflammation.

Altering integrin engagement regulates membrane localization of Kir2.1 channels.

How ion channels localize and distribute on the cell membrane remains incompletely understood. We show that interventions that vary cell adhesion proteins and cell size also affect the membrane current density of inward-rectifier K+ channels (Kir2.1; encoded by KCNJ2) and profoundly alter the action potential shape of excitable cells. By using micropatterning to manipulate the localization and size of focal adhesions (FAs) in single HEK293 cells engineered to stably express Kir2.1 channels or in neonatal rat cardiomyocytes, we establish a robust linear correlation between FA coverage and the amplitude of Kir2.1 current at both the local and whole-cell levels. Confocal microscopy showed that Kir2.1 channels accumulate in membrane proximal to FAs. Selective pharmacological inhibition of key mediators of protein trafficking and the spatially dependent alterations in the dynamics of Kir2.1 fluorescent recovery after photobleaching revealed that the Kir2.1 channels are transported to the cell membrane uniformly, but are preferentially internalized by endocytosis at sites that are distal from FAs. Based on these results, we propose adhesion-regulated membrane localization of ion channels as a fundamental mechanism of controlling cellular electrophysiology via mechanochemical signals, independent of the direct ion channel mechanogating.

Focal adhesion kinase–dependent activation of the early endocytic protein Rab5 is associated with cell migration

Focal adhesion kinase (FAK) is a central regulator of integrin-dependent cell adhesion and migration and has recently been shown to co-localize with endosomal proteins. The early endocytic protein Rab5 controls integrin trafficking, focal adhesion disassembly, and cell migration and has been shown to be activated upon integrin engagement by mechanisms that remain unclear. Because FAK is a critical regulator of integrin-dependent signaling and Rab5 recapitulates FAK-mediated effects, we evaluated the possibility that FAK activates Rab5 and contributes to cell migration. Pulldown assays revealed that Rab5-GTP levels are decreased upon treatment with a pharmacological inhibitor of FAK, PF562,271, in resting A549 cells. These events were associated with decreased peripheral Rab5 puncta and a reduced number of early endosome antigen 1 (EEA1)-positive early endosomes. Accordingly, as indicated by FAK inhibition experiments and in FAK-null fibroblasts, adhesion-induced FAK activity increased Rab5-GTP levels. In fact, expression of WT FAK and FAK/Y180A/M183A (open conformation), but not FAK/Arg454 (kinase-dead), augmented Rab5-GTP levels in FAK-null fibroblasts and A549 cells. Moreover, expression of a GDP-bound Rab5 mutant (Rab5/S34N) or shRNA-mediated knockdown of endogenous Rab5 prevented FAK-induced A549 cell migration, whereas expression of WT or GTP-bound Rab5 (Rab5/Q79L), but not Rab5/S34N, promoted cell migration in FAK-null fibroblasts. Mechanistically, FAK co-immunoprecipitated with the GTPase-activating protein p85α in a phosphorylation (Tyr397)-dependent manner, preventing Rab5-GTP loading, as shown by knockdown and transfection recovery experiments. Taken together, these results reveal that FAK activates Rab5, leading to cell migration.

Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression, and TGF-β1 secretion

Silver nanoparticles (AgNPs) are widely used in consumer and pharmaceutical products due to their antipathogenic properties. However, safety concerns have been raised due to their bioactive properties. While reports have demonstrated AgNPs can embed within the extracellular matrix, their effects on basement membrane (BM) production, integrin engagement, and tissue-integrity are not well-defined. This study analyzed the effects of AgNPs on BM production, composition and integrin/focal adhesion interactions in representative lung, esophageal, breast and colorectal epithelia models. A multidisciplinary approach including focused proteomics, QPCR arrays, pathway analyses, and immune-based, structural and functional assays was used to identify molecular and physiological changes in cell adhesions and the BM induced by acute and chronic AgNP exposure. Dysregulated targets included CD44 and transforming growth factor-beta, two proteins frequently altered during pathogenesis. Results indicate AgNP exposure interferes with BM and cell adhesion dynamics, and provide insight into the mechanisms of AgNP-induced disruption of epithelial physiology.

Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma

Originating in the brain, glioblastoma (GBM) is a highly lethal and virtually incurable cancer, in large part because it readily develops resistance to treatments. While numerous studies have investigated mechanisms enabling GBM cells to evade chemotherapy-induced apoptosis, few have addressed how their surrounding extracellular matrix (ECM) acts to promote their survival. Here, we employed a biomaterial-based, 3D culture platform to investigate systematically how interactions between patient-derived GBM cells and the brain ECM promote resistance to alkylating chemotherapies — including temozolomide, which is used routinely in clinical practice. Scaffolds for 3D culture were fabricated from hyaluronic acid (HA) — a major structural and bioactive component of the brain ECM — and functionalized with the RGD (arginine-glycine-aspartic acid) tripeptide to provide sites for integrin engagement. Data demonstrate that cooperative engagement of CD44, through HA, and integrin αV, through RGD, facilitates resistance to alkylating chemotherapies through co-activation of Src, which inhibited downstream expression of BCL-2 family pro-apoptotic factors. In sum, a bioengineered, 3D culture platform was used to gain new mechanistic insights into how ECM in the brain tumor microenvironment promotes resistance to chemotherapy and suggests potential avenues for the development of novel, matrix-targeted combination therapies designed to suppress chemotherapy resistance in GBM.

Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.