Integrin Adhesion Research Articles

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

BackgroundThe dystrophin-glycoprotein complex (DGC) is a critical adhesion complex of the muscle cell membrane, providing a mechanical link between the extracellular matrix (ECM) and the cortical cytoskeleton that stabilizes the sarcolemma during repeated muscle contractions. One integral component of the DGC is the transmembrane protein, sarcospan (SSPN). Overexpression of SSPN in the skeletal muscle of mdx mice (murine model of DMD) restores muscle fiber attachment to the ECM in part through an associated increase in utrophin and integrin adhesion complexes at the cell membrane, protecting the muscle from contraction-induced injury. In this study, we utilized transcriptomic and ECM protein-optimized proteomics data sets from wild-type, mdx, and mdx transgenic (mdxTG) skeletal muscle tissues to identify pathways and proteins driving the compensatory action of SSPN overexpression.MethodsThe tibialis anterior and quadriceps muscles were isolated from wild-type, mdx, and mdxTG mice and subjected to bulk RNA-Seq and global proteomics analysis using methods to enhance capture of ECM proteins. Data sets were further analyzed through the ingenuity pathway analysis (QIAGEN) and integrative gene set enrichment to identify candidate networks, signaling pathways, and upstream regulators.ResultsThrough our multi-omics approach, we identified 3 classes of differentially expressed genes and proteins in mdxTG muscle, including those that were (1) unrestored (significantly different from wild type, but not from mdx), (2) restored (significantly different from mdx, but not from wild type), and (3) compensatory (significantly different from both wild type and mdx). We identified signaling pathways that may contribute to the rescue phenotype, most notably cytoskeleton and ECM organization pathways. ECM-optimized proteomics revealed an increased abundance of collagens II, V, and XI, along with β-spectrin in mdxTG samples. Using ingenuity pathway analysis, we identified upstream regulators that are computationally predicted to drive compensatory changes, revealing a possible mechanism of SSPN rescue through a rewiring of cell-ECM bidirectional communication. We found that SSPN overexpression results in upregulation of key signaling molecules associated with regulation of cytoskeleton organization and mechanotransduction, including Yap1, Sox9, Rho, RAC, and Wnt.ConclusionsOur findings indicate that SSPN overexpression rescues dystrophin deficiency partially through mechanotransduction signaling cascades mediated through components of the ECM and the cortical cytoskeleton.

Using Micropatterned Supported Lipid Bilayers to Probe the Mechanosensitivity of Signaling Receptors.

The ligand-receptor complexes in the cell-cell and cell-ECM interfaces mediate the mechanical coupling of cells to their microenvironment and transduce downstream signals to modulate cell functions. In this chapter I describe a microfabrication strategy to prepare a substrate of spatially segregated supported lipid bilayers and ECM components on which cells can form juxtacrine receptor signaling complexes and integrin adhesions simultaneously. This platform is specifically applicable for microscopically monitoring the signal transduction of each receptor, as well as the modulatory effects of receptor signaling on integrin adhesions and cell behaviors.

Integrating physicomechanical and biological strategies for BTE: biomaterials-induced osteogenic differentiation of MSCs.

Large bone defects are a major global health concern. Bone tissue engineering (BTE) is the most promising alternative to avoid the drawbacks of autograft and allograft bone. Nevertheless, how to precisely control stem cell osteogenic differentiation has been a long-standing puzzle. Compared with biochemical cues, physicomechanical stimuli have been widely studied for their biosafety and stability. The mechanical properties of various biomaterials (polymers, bioceramics, metal and alloys) become the main source of physicomechanical stimuli. By altering the stiffness, viscoelasticity, and topography of materials, mechanical stimuli with different strengths transmit into precise signals that mediate osteogenic differentiation. In addition, externally mechanical forces also play a critical role in promoting osteogenesis, such as compression stress, tensile stress, fluid shear stress and vibration, etc. When exposed to mechanical forces, mesenchymal stem cells (MSCs) differentiate into osteogenic lineages by sensing mechanical stimuli through mechanical sensors, including integrin and focal adhesions (FAs), cytoskeleton, primary cilium, ions channels, gap junction, and activating osteogenic-related mechanotransduction pathways, such as yes associated proteins (YAP)/TAZ, MAPK, Rho-GTPases, Wnt/β-catenin, TGFβ superfamily, Notch signaling. This review summarizes various biomaterials that transmit mechanical signals, physicomechanical stimuli that directly regulate MSCs differentiation, and the mechanical transduction mechanisms of MSCs. This review provides a deep and broad understanding of mechanical transduction mechanisms and discusses the challenges that remained in clinical translocation as well as the outlook for the future improvements.

Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.

Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional β1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image β1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous β1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal β1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.

Integrin αIIbβ3 intermediates: From molecular dynamics to adhesion assembly

The platelet integrin αIIbβ3 undergoes long-range conformational transitions associated with its functional conversion from inactive (low-affinity) to active (high-affinity) during hemostasis. Although new conformations that are intermediate between the well-characterized bent and extended states have been identified, their molecular dynamic properties and functions in the assembly of adhesions remain largely unexplored. In this study, we evaluated the properties of intermediate conformations of integrin αIIbβ3 and characterized their effects on the assembly of adhesions by combining all-atom simulations, principal component analysis, and mesoscale modeling. Our results show that in the low-affinity, bent conformation, the integrin ectodomain tends to pivot around the legs; in intermediate conformations, the headpiece becomes partially extended, away from the lower legs. In the fully open, active state, αIIbβ3 is flexible, and the motions between headpiece and lower legs are accompanied by fluctuations of the transmembrane helices. At the mesoscale, bent integrins form only unstable adhesions, but intermediate or open conformations stabilize the adhesions. These studies reveal a mechanism by which small variations in ligand binding affinity and enhancement of the ligand-bound lifetime in the presence of actin retrograde flow stabilize αIIbβ3 integrin adhesions.

An altered extracellular matrix-integrin interface contributes to Huntington's disease-associated CNS dysfunction in glial and vascular cells.

Astrocytes and brain endothelial cells are components of the neurovascular unit that comprises the blood-brain barrier (BBB) and their dysfunction contributes to pathogenesis in Huntington's disease (HD). Defining the contribution of these cells to disease can inform cell-type-specific effects and uncover new disease-modifying therapeutic targets. These cells express integrin (ITG) adhesion receptors that anchor the cells to the extracellular matrix (ECM) to maintain the integrity of the BBB. We used HD patient-derived induced pluripotent stem cell (iPSC) modeling to study the ECM-ITG interface in astrocytes and brain microvascular endothelial cells and found ECM-ITG dysregulation in human iPSC-derived cells that may contribute to the dysfunction of the BBB in HD. This disruption has functional consequences since reducing ITG expression in glia in an HD Drosophila model suppressed disease-associated CNS dysfunction. Since ITGs can be targeted therapeutically and manipulating ITG signaling prevents neurodegeneration in other diseases, defining the role of ITGs in HD may provide a novel strategy of intervention to slow CNS pathophysiology to treat HD.

Contribution of integrin adhesion to cytokinetic abscission and genomic integrity.

Recent research shows that integrin-mediated adhesion contributes to the regulation of cell division at two key steps: the formation of the mitotic spindle at the mitotic entry and the final cytokinetic abscission at the mitotic exit. Failure in either of these processes will have a direct impact on the other in each round of the cell cycle and on the genomic integrity. This review aims to present how integrin signals are involved at these cell cycle stages under normal conditions and some safety mechanisms that may counteract the generation of aneuploid cells in cases of defective integrin signals.

Deterministic Single Cell Encapsulation in Asymmetric Microenvironments to Direct Cell Polarity.

Various signals in tissue microenvironments are often unevenly distributed around cells. Cellular responses to asymmetric cell-matrix adhesion in a 3D space remain generally unclear and are to be studied at the single-cell resolution. Here, the authors developed a droplet-based microfluidic approach to manufacture a pure population of single cells in a microscale layer of compartmentalized 3D hydrogel matrices with a tunable spatial presentation of ligands at the subcellular level. Cells elongate with an asymmetric presentation of the integrin adhesion ligand Arg-Gly-Asp (RGD), while cells expand isotropically with a symmetric presentation of RGD. Membrane tension is higher on the side of single cells interacting with RGD than on the side without RGD. Finite element analysis shows that a non-uniform isotropic cell volume expansion model is sufficient to recapitulate the experimental results. At a longer timescale, asymmetric ligand presentation commits mesenchymal stem cells to the osteogenic lineage. Cdc42 is an essential mediator of cell polarization and lineage specification in response to asymmetric cell-matrix adhesion. This study highlights the utility of precisely controlling 3D ligand presentation around single cells to direct cell polarity for regenerative engineering and medicine.

Molecular basis for integrin adhesion receptor binding to p21-activated kinase 4 (PAK4)

Integrin adhesion receptors provide links between extracellular ligands and cytoplasmic signaling. Multiple kinases have been found to directly engage with integrin β tails, but the molecular basis for these interactions remain unknown. Here, we assess the interaction between the kinase domain of p21-activated kinase 4 (PAK4) and the cytoplasmic tail of integrin β5. We determine three crystal structures of PAK4-β5 integrin complexes and identify the PAK-binding site. This is a region in the membrane-proximal half of the β5 tail and confirmed by site-directed mutagenesis. The β5 tail engages the kinase substrate-binding groove and positions the non-phosphorylatable integrin residue Glu767 at the phosphoacceptor site. Consistent with this, integrin β5 is poorly phosphorylated by PAK4, and in keeping with its ability to occlude the substrate-binding site, weakly inhibits kinase activity. These findings demonstrate the molecular basis for β5 integrin-PAK4 interactions but suggest modifications in understanding the potential cellular role of this interaction.

The basement membrane in epidermal polarity, stemness, and regeneration.

The epidermis is a specialized epithelium that constitutes the outermost layer of the skin, and it provides a protective barrier against environmental assaults. Primarily consisting of multilayered keratinocytes, the epidermis is continuously renewed by proliferation of stem cells and the differentiation of their progeny, which undergo terminal differentiation as they leave the basal layer and move upward toward the surface, where they die and slough off. Basal keratinocytes rest on a basement membrane at the dermal-epidermal junction that is composed of specific extracellular matrix proteins organized into interactive and mechanically supportive networks. Firm attachment of basal keratinocytes, and their dynamic regulation via focal adhesions and hemidesmosomes, is essential for maintaining major skin processes, such as self-renewal, barrier function, and resistance to physical and chemical stresses. The adhesive integrin receptors expressed by epidermal cells serve structural, signaling, and mechanosensory roles that are critical for epidermal cell anchorage and tissue homeostasis. More specifically, the basement membrane components play key roles in preserving the stem cell pool, and establishing cell polarity cues enabling asymmetric cell divisions, which result in the transition from a proliferative basal cell layer to suprabasal cells committed to terminal differentiation. Finally, through a well-regulated sequence of synthesis and remodeling, the components of the dermal-epidermal junction play an essential role in regeneration of the epidermis during skin healing. Here too, they provide biological and mechanical signals that are essential to the restoration of barrier function.

Vinculin recruitment to α-catenin halts the differentiation and maturation of enterocyte progenitors to maintain homeostasis of the Drosophila intestine.

Mechanisms communicating changes in tissue stiffness and size are particularly relevant in the intestine because it is subject to constant mechanical stresses caused by peristalsis of its variable content. Using the Drosophila intestinal epithelium, we investigate the role of vinculin, one of the best characterised mechanoeffectors, which functions in both cadherin and integrin adhesion complexes. We discovered that vinculin regulates cell fate decisions, by preventing precocious activation and differentiation of intestinal progenitors into absorptive cells. It achieves this in concert with α-catenin at sites of cadherin adhesion, rather than as part of integrin function. Following asymmetric division of the stem cell into a stem cell and an enteroblast (EB), the two cells initially remain connected by adherens junctions, where vinculin is required, only on the EB side, to maintain the EB in a quiescent state and inhibit further divisions of the stem cell. By manipulating cell tension, we show that vinculin recruitment to adherens junction regulates EB activation and numbers. Consequently, removing vinculin results in an enlarged gut with improved resistance to starvation. Thus, mechanical regulation at the contact between stem cells and their progeny is used to control tissue cell number.

C-Met-integrin cooperation: Mechanisms, tumorigenic effects, and therapeutic relevance.

c-Met is a receptor tyrosine kinase which upon activation by its ligand, the hepatocyte growth factor, mediates many important signalling pathways that regulate cellular functions such as survival, proliferation, and migration. Its oncogenic and tumorigenic signalling mechanisms, greatly contributing to cancer development and progression, are well documented. Integrins, heterogeneous adhesion receptors which facilitate cell-extracellular matrix interactions, are important in biomechanically sensitive cell adhesion and motility but also modulate diverse cell behaviour. Here we review the studies which reported cooperation between c-Met and several integrins, particularly β1 and β4, in various cell models including many tumour cell types. From the various experimental models and results analysed, we propose that c-Met-integrin cooperation occurs via inside-out or outside-in signalling. Thus, either c-Met activation triggers integrin activation and cell adhesion or integrin adhesion to its extracellular ligand triggers c-Met activation. These two modes of cooperation require the adhesive function of integrins and mostly lead to cell migration and invasion. In a third, less conventional, mode of cooperation, the integrin plays the role of a signalling adaptor for c-Met, independently from its adhesive property, leading to anchorage independent survival. Recent studies have revealed the influence of endocytic trafficking in c-Met-integrin cooperation including the adaptor function of integrin occurring on endomembranes, triggering an inside-in signalling, believed to promote survival of metastatic cells. We present the evidence of the cooperation in vivo and in human tissues and highlight its therapeutic relevance. A better understanding of the mechanisms regulating c-Met-integrin cooperation in cancer progression could lead to the design of new therapies targeting this cooperation, providing more effective therapeutic approaches than c-Met or integrin inhibitors as monotherapies used in the clinic.

Cytoophidia coupling adipose architecture and metabolism.

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IVcollagen(Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. Our results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue.

Extracellular matrix regulates force transduction at VE-cadherin junctions.

Increased tension on VE-cadherin (VE-cad) complexes activates adaptive cell stiffening and local cytoskeletal reinforcement--two key signatures of intercellular mechanotransduction. Here we demonstrate that tugging on VE-cad receptors initiates a cascade that results in downstream integrin activation. The formation of new integrin adhesions potentiates vinculin and actin recruitment to mechanically reinforce stressed cadherin adhesions. This cascade differs from documented antagonistic effects of integrins on intercellular junctions. We identify focal adhesion kinase, Abl kinase, and RhoA GTPase as key components of the positive feedback loop. Results further show that a consequence of integrin involvement is the sensitization of intercellular force transduction to the extracellular matrix (ECM) not by regulating junctional tension but by altering signal cascades that reinforce cell-cell adhesions. On type 1 collagen or fibronectin substrates, integrin subtypes α2β1 and α5β1, respectively, differentially control actin remodeling at VE-cad adhesions. Specifically, ECM-dependent differences in VE-cad force transduction mirror differences in the rigidity sensing mechanisms of α2β1 and α5β1 integrins. The findings verify the role of integrins in VE-cad force transduction and uncover a previously unappreciated mechanism by which the ECM impacts the mechanical reinforcement of interendothelial junctions.

Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C.elegans gonad morphogenesis.

Development of the C.elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.

Extracellular Matrix Interactions Provide Tumor Cells With an Escape Mechanism for Commitment to Differentiation

Extracellular Matrix Interactions Provide Tumor Cells With an Escape Mechanism for Commitment to Differentiation

Abstract P1082: Activation And Phosphorylation Of Rap1b In The Human Megakaryocytic Leukemia Cell Line DAMI

In platelet-producing megakaryocytes, the signaling protein Rap1b mediates both activation of integrins for cell adhesion and activation of the transcription factor ERK leading to gene expression changes. As a small G-protein, Rap1b acts as a molecular switch with an inactive GDP-bound state and an active GTP-bound state. Additionally, the association of Rap1b within the inner side of the plasma membrane is dictated by cAMP-dependent protein kinase (PKA) phosphorylation. In an unphosphorylated state, Rap1b is attached to the plasma membrane via a lipid anchor. Upon phosphorylation, Rap1b detaches from the membrane and is cytosolic. We hypothesize the location of active Rap1b, dictated by the guanine nucleotide bound (GDP/GTP) and its phosphorylation status, determines which signaling pathways Rap1b contributes to in megakaryocytes. To evaluate this hypothesis, we first need to optimize methods to detect Rap1b activation (GTP binding) and phosphorylation. Using the megakaryocyte cell line (DAMI), we over-expressed green fluorescent protein (GFP)-tagged Rap1b and treated with reagents to stimulate activation or phosphorylation and then use pull-down assays with the Rap binding domain (RBD) to capture activated Rap1b or pan phospho S/T antibodies, respectively. In addition, the activation of downstream proteins like ERK were evaluated in DAMI cells overexpressing GFP-tagged Rap1b phosphorylation mutants. Overall, this work seeks to determine how the spatial distribution of Rap1b, dictated by phosphorylation influences cellular processes.

Talin variant P229S compromises integrin activation and associates with multifaceted clinical symptoms.

Adhesion of cells to the extracellular matrix (ECM) must be exquisitely coordinated to enable development and tissue homeostasis. Cell-ECM interactions are regulated by multiple signalling pathways that coordinate the activation state of the integrin family of ECM receptors. The protein talin is pivotal in this process, and talin's simultaneous interactions with the cytoplasmic tails of the integrins and the plasma membrane are essential to enable robust, dynamic control of integrin activation and cell-ECM adhesion. Here, we report the identification of a de novo heterozygous c.685C>T (p.Pro229Ser) variant in the TLN1 gene from a patient with a complex phenotype. The mutation is located in the talin head region at the interface between the F2 and F3 domains. The characterization of this novel p.P229S talin variant reveals the disruption of adhesion dynamics that result from disturbance of the F2-F3 domain interface in the talin head. Using biophysical, computational and cell biological techniques, we find that the variant perturbs the synergy between the integrin-binding F3 and the membrane-binding F2 domains, compromising integrin activation, adhesion and cell migration. Whilst this remains a variant of uncertain significance, it is probable that the dysregulation of adhesion dynamics we observe in cells contributes to the multifaceted clinical symptoms of the patient and may provide insight into the multitude of cellular processes dependent on talin-mediated adhesion dynamics.

Adhesion-independent topography-based leukocyte migration.

Cells need to couple intracellular actin flows with the substrate to generate forward movement. This has traditionally been studied in the context of specific transmembrane receptors, particularly integrin adhesion receptors, which link extracellular adhesive molecules to the actin cytoskeleton. However, leukocytes and other cells can also migrate using integrin-independent strategies both in vivo and in vitro, though the cellular and environmental requirements for this mode are not fully understood. In seminal recent work, Reversat et al.1 develop a range of innovative 2D and 3D engineered microdevices and probe the biophysical mechanisms underlying T lymphocytes and dendritic cells in conditions of limited substrate adhesion. They identify a physical principle of mechano-coupling between retrograde actin flow and irregular extracellular confinement, which allows the cell to generate mechanical resistance and move in the absence of receptor-mediated adhesion. Through the combined use of experiments and theoretical modeling, this work resolves a long-standing question in cell biology and establishes mechanical interaction with an irregular-shaped 3D environment which may be relevant to cell migration in a range of tissue contexts.

Structure modifications of 2-phenylquinoline by Aspergillus genera produce novel derivatives with potent leishmanicidal and anti-inflammatory properties

Quinolines are heterocyclic molecules with a wide range of pharmacological purposes. The quinolinic system has been used for the synthesis of derivatives, producing molecules with significant improvement of the original activities. In this work, the alkaloid 2-phenylquinoline, the main substance of the Bolivian tree Galipea longiflora, traditionally employed for treating leishmaniasis, served as a substrate for biotransformation to obtain compounds for leishmaniasis applications. Using Aspergillus fumigatus, the novel and for the first-time reported 5,6-dihydro-5,6-dihydroxy-2-phenylquinoline diol was produced. Its biotransformation by A. flavus resulted in 4′‑hydroxy-2-phenylquinoline. When tested against Leishmania amazonensis, L. infantum, and L. braziliensis promastigotes, both showed time-related leishmanicidal effects significantly higher than those observed for the parental molecule. Transmission electron microscopy revealed that the derivatives affected several cellular structures including the nucleus. When assayed for biological properties within an inflammatory context usually present in leishmaniasis, they were neither toxic for macrophages and fibroblasts nor affected the behavior of immune cells or the expression levels of some adhesion integrins, but significantly altered the chemotaxis of neutrophils, suggesting additional properties in enhancing some important steps necessary for the defense and healing processes that develop just after the sand fly-infected saliva injection that triggers the infection. Our results indicate that monitored biotransformation of 2-phenylquinoline by Aspergillus genera can be a viable route to obtain highly active leishmanicidal substances with additional anti-inflammatory properties not reported before, as particularly observed for the new diol, which may be efficient in controlling leishmaniasis and its parasite burden.