Integrin Clustering Research Articles

Regulation of cell adhesion: a collaborative effort of integrins, their ligands, cytoplasmic actors, and phosphorylation.

Integrins are large heterodimeric type 1 membrane proteins expressed in all nucleated mammalian cells. Eighteen α-chains and eight β-chains can combine to form 24 different integrins. They are cell adhesion proteins, which bind to a large variety of cellular and extracellular ligands. Integrins are required for cell migration, hemostasis, translocation of cells out from the blood stream and further movement into tissues, but also for the immune response and tissue morphogenesis. Importantly, integrins are not usually active as such, but need activation to become adhesive. Integrins are activated by outside-in activation through integrin ligand binding, or by inside-out activation through intracellular signaling. An important question is how integrin activity is regulated, and this topic has recently drawn much attention. Changes in integrin affinity for ligand binding are due to allosteric structural alterations, but equally important are avidity changes due to integrin clustering in the plane of the plasma membrane. Recent studies have partially solved how integrin cell surface structures change during activation. The integrin cytoplasmic domains are relatively short, but by interacting with a variety of cytoplasmic proteins in a regulated manner, the integrins acquire a number of properties important not only for cell adhesion and movement, but also for cellular signaling. Recent work has shown that specific integrin phosphorylations play pivotal roles in the regulation of integrin activity. Our purpose in this review is to integrate the present knowledge to enable an understanding of how cell adhesion is dynamically regulated.

Spatial mapping of affinity changes for the integrin LFA-1 during cell migration using clusters identified based on local density.

Localization microscopy methods like Stochastic Optical Reconstruction Microscopy (STORM) are very well suited for exploring clustering of proteins, as the data inherently provide a list of molecular coordinates. Here we use state-of-art cluster analysis algorithms (DBSCAN) to explore the clustering behaviour of different affinity forms of the integrin LFA-1. It has been suggested that LFA-1 may form clusters, in order to increase the avidity to ICAM-1. However, this hypothesis still seems to be controversial. In this study, we found, variations in clustering behaviour among the different affinity forms of LFA-1 in migrating T-cells. We found that panLFA-1 is located in clusters throughout the polarised cell on ICAM-1, with an increased density of molecules and clusters in the mid area and rear of the cell, whereas the intermediate and high affinity form of LFA-1 showed an increased number in the mid area of a migrating cell and the high affinity form of LFA-1 in the front and rear. Together, these data suggest that, in addition to LFA-1 conformation, protein clustering might play a role in controlling cell-substrate adhesion on ICAM-1.By applying the cluster analysis algorithm DBSCAN to localization microscopy data, integrin clusters could be identified and different cluster parameters could be quantified.

Distinct Mechanosensing of Human Neural Stem Cells on Extremely Limited Anisotropic Cellular Contact.

Human neural stem cells (hNSCs) can alter their fate choice in response to the biophysical cues provided during development. In particular, it has been reported that the differentiation of neural stem cells (NSCs) is enhanced by anisotropic contact, which facilitates focal adhesion (FA) formation and cytoskeletal organization. However, a biomolecular mechanism governing how the cells process the biophysical cues from these anisotropic geometries to their fate commitment is still poorly understood due to the limited availability of geometrical diversities (contact width above 50 nm) applicable to cell studies. Here, we firstly demonstrate that the biomolecular mechanism for enhanced neurogenesis on an anisotropic nanostructure is critically dependent on the resolution of a contact feature. We observed a totally different cellular response to anisotropic geometries by first utilizing a high-resolution nanogroove (HRN) structure with an extremely narrow contact width (15 nm). The width scale is sufficiently low to suppress the integrin clustering and enable us to elucidate how the contact area influences the neurogenesis of hNSCs at an aligned state. Both the HRN and control nanogroove (CN) pattern with a contact width of 1 μm induced the spontaneous topographic alignment of hNSCs. However, intriguingly, the focal adhesion (FA) formation and cytoskeletal reorganization were substantially limited on the HRN, although the cells on the CN showed enhanced FA formation compared with flat surfaces. In particular, the hNSCs on the HRN surface exhibited a strikingly lower fraction of nuclear yes-associated protein (YAP) than on the CN surface, which was turned out to be regulated by Rho GTPase in the same way as the cells sense the mechanical properties of the environment. Considering the previously reported role of YAP on neurogenesis, our finding newly substantiates that YAP and Rho GTPase also can be transducers of hNSCs to process topographical alternation to fate decision. Furthermore, this study with the unprecedented high-resolution nanostructure suggests a novel geometry sensing model where the functional crosstalk between YAP signaling and Rho GTPase integrally regulate the fate commitment of the hNSCs.

PO-205 Characterisation of the integrin alpha v-dependent adhesome in mda-mb-435s melanoma cells

IntroductionIntegrins are heterodimeric glycoproteins that bind cells to extracellular matrix proteins. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) form that facilitate the linkage between integrins and the actin cytoskeleton and permit bidirectional signalling. The αV integrin is expressed in most tumour cells where it regulates an array of cellular functions and plays a role in anti-tumour drug resistance. The aim of our work was to assess αV-dependent changes in IAC composition in MDA-MB-435S melanoma cells in order to better understand the increased sensitivity to paclitaxel and vincristine upon integrin αV knockdown.Material and methodsIntegrin αV-specific shRNA was cloned into pSUPER.puro, transfected into MDA-MB-435S cells using Lipofectamine, and cell clones were selected using puromycin. The sensitivity of cells to antitumor drugs was determined using an MTT assay. Cell migration was monitored using a Transwell assay. IACs were isolated following crosslinking and their molecular composition analysed using mass spectrometry (MS)–based proteomics.Results and discussionsIn two MDA-MB-435S-derived cell clones with decreased expression of integrin αV, expressing 15% (2αV) or 5% (3αV) of the control cells amount, increased sensitivity to paclitaxel and vincristine, decreased sensitivity to cisplatin, and decreased migration were observed in line with previous results obtained following transient transfection with integrin αV siRNA. In cell clones 2αV and 3αV, which were smaller than control cells and lacked stress fibres, the number of focal adhesions was shown to be significantly lower as observed by interference reflection microscopy and immunofluorescence detection of phospho-paxillin, phospho-FAK and phospho-Src. MS analysis of isolated IACs from control MDA-MB-435S, 2αV and 3αV cells identified 282 proteins, including 36 out of 60 consensus adhesome proteins. As expected, in clones 2αV and 3αV, integrins αV, β3 and β5 were detected at much lower levels compared with control cells. In addition, lower levels of talin-1 and −2, vinculin, alpha-actinin-4, tensin-3, filamin-A and -B, liprin β1 and plectin were detected.ConclusionThese data will enable follow-up analyses of the mechanisms of signalling by integrins αVβ3/β5 and therefore represent a valuable resource to improve our understanding of the mechanisms involved in adhesion control of cell sensitivity to antitumor drugs and metastatic potential.

Integrin α6 and EGFR signaling converge at mechanosensitive calpain 2

Cells sense and respond to mechanical cues from the extracellular matrix (ECM) via integrins. ECM stiffness is known to enhance integrin clustering and response to epidermal growth factor (EGF), but we lack information on when or if these mechanosensitive growth factor receptors and integrins converge intracellularly. Towards closing this knowledge gap, we combined a biomaterial platform with transcriptomics, molecular biology, and functional assays to link integrin-mediated mechanosensing and epidermal growth factor receptor (EGFR) signaling. We found that high integrin α6 expression controlled breast cancer cell adhesion and motility on soft, laminin-coated substrates, and this mimicked the response of cells to EGF stimulation. The mechanisms that drove both mechanosensitive cell adhesion and motility converged on calpain 2, an intracellular protease important for talin cleavage and focal adhesion turnover. EGF stimulation enhanced adhesion and motility on soft substrates, but required integrin α6 and calpain 2 signaling. In sum, we identified a new role for integrin α6 mechanosensing in breast cancer, wherein cell adhesion to laminin on soft substrates mimicked EGF stimulation. We identified calpain 2, downstream of both integrin α6 engagement and EGFR phosphorylation, as a common intracellular signaling node, and implicate integrin α6 and calpain 2 as potential targets to inhibit the migration of cancer cells in stiff tumor environments.

Multivalent Ligands: Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin‐Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration (Adv. Healthcare Mater. 12/2018)

Multivalent Ligands: Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin‐Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration (Adv. Healthcare Mater. 12/2018)

Regulatory actions of 3',5'-cyclic adenosine monophosphate on osteoclast function: possible roles of Epac-mediated signaling.

Alterations in cellular levels of the second messenger 3',5'-cyclic adenosine monophosphate ([cAMP]i ) regulate a wide range of physiologically important cellular signaling processes in numerous cell types. Osteoclasts are terminally differentiated, multinucleated cells specialized for bone resorption. Their systemic regulator, calcitonin, triggers morphometrically and pharmacologically distinct retraction (R) and quiescence (Q) effects on cell-spread area and protrusion-retraction motility, respectively, paralleling its inhibition of bone resorption. Q effects were reproduced by cholera toxin-mediated Gs -protein activation known to increase [cAMP]i , unaccompanied by the [Ca2+ ]i changes contrastingly associated with R effects. We explore a hypothesis implicating cAMP signaling involving guanine nucleotide-exchange activation of the small GTPase Ras-proximate-1 (Rap1) by exchange proteins directly activated by cAMP (Epac). Rap1 activates integrin clustering, cell adhesion to bone matrix, associated cytoskeletal modifications and signaling processes, and transmembrane transduction functions. Epac activation enhanced, whereas Epac inhibition or shRNA-mediated knockdown compromised, the appearance of markers for osteoclast differentiation and motility following stimulation by receptor activator of nuclear factor kappa-Β ligand (RANKL). Deficiencies in talin and Rap1 compromised in vivo bone resorption, producing osteopetrotic phenotypes in genetically modified murine models. Translational implications of an Epac-Rap1 signaling hypothesis in relationship to N-bisphosphonate actions on prenylation and membrane localization of small GTPases are discussed.

Ionizing Radiation Induces Morphological Changes and Immunological Modulation of Jurkat Cells.

Impairment or stimulation of the immune system by ionizing radiation (IR) impacts on immune surveillance of tumor cells and non-malignant cells and can either foster therapy response or side effects/toxicities of radiation therapy. For a better understanding of the mechanisms by which IR modulates T-cell activation and alters functional properties of these immune cells, we exposed human immortalized Jurkat cells and peripheral blood lymphocytes (PBL) to X-ray doses between 0.1 and 5 Gy. This resulted in cellular responses, which are typically observed also in naïve T-lymphocytes in response of T-cell receptor immune stimulation or mitogens. These responses include oscillations of cytosolic Ca2+, an upregulation of CD25 surface expression, interleukin-2 and interferon-γ synthesis, elevated expression of Ca2+ sensitive K+ channels and an increase in cell diameter. The latter was sensitive to inhibition by the immunosuppressant cyclosporine A, Ca2+ buffer BAPTA-AM, and the CDK1-inhibitor RO3306, indicating the involvement of Ca2+-dependent immune activation and radiation-induced cell cycle arrest. Furthermore, on a functional level, Jurkat and PBL cell adhesion to endothelial cells was increased upon radiation exposure and was highly dependent on an upregulation of integrin beta-1 expression and clustering. In conclusion, we here report that IR impacts on immune activation and functional properties of T-lymphocytes that may have implications in both toxic effects and treatment response to combined radiation and immune therapy in cancer patients.

Integrin Clustering Matters: A Review of Biomaterials Functionalized with Multivalent Integrin-Binding Ligands to Improve Cell Adhesion, Migration, Differentiation, Angiogenesis, and Biomedical Device Integration.

Material systems that exhibit tailored interactions with cells are a cornerstone of biomaterial and tissue engineering technologies. One method of achieving these tailored interactions is to biofunctionalize materials with peptide ligands that bind integrin receptors present on the cell surface. However, cell biology research has illustrated that both integrin binding and integrin clustering are required to achieve a full adhesion response. This biophysical knowledge has motivated researchers to develop material systems biofunctionalized with nanoscale clusters of ligands that promote both integrin occupancy and clustering of the receptors. These materials have improved a wide variety of biological interactions in vitro including cell adhesion, proliferation, migration speed, gene expression, and stem cell differentiation; and improved in vivo outcomes including increased angiogenesis, tissue healing, and biomedical device integration. This review first introduces the techniques that enable the fabrication of these nanopatterned materials, describes the improved biological effects that have been achieved, and lastly discusses the current limitations of the technology and where future advances may occur. Although this technology is still in its nascency, it will undoubtedly play an important role in the future development of biomaterials and tissue engineering scaffolds for both in vitro and in vivo applications.

Nanoscale Resolution 3D Printing with Pin-Modified Electrified Inkjets for Tailorable Nano/Macrohybrid Constructs for Tissue Engineering.

Cells respond to their microenvironment, which is of a size comparable to that of the cells. The macroscale features of three-dimensional (3D) printing struts typically result in whole cell contact guidance (CCG). In contrast, at the nanoscale, where features are of a size similar to that of receptors of cells, the response of cells is more complex. The cell-nanotopography interaction involves nanoscale adhesion localized structures, which include cell adhesion-related particles that change in response to the clustering of integrin. For this reason, it is necessary to develop a technique for manufacturing tailorable nano/macrohybrid constructs capable of freely controlling the cellular activity. In this study, a hierarchical 3D nano- to microscale hybrid structure was fabricated by combinational processing of 3D printing and electrified inkjet spinning via pin motions. This method overcomes the disadvantages of conventional 3D printing, providing a novel combinatory technique for the fabrication of 3D hybrid constructs with excellent cell proliferation. Through a pin-modified electrified inkjet spinning, we have successfully fabricated customizable nano-/microscale hybrid constructs in a fibrous or mesh form, which can control the cell fate. We have conducted this study of cell-topography interactions from the fabrication approach to accelerate the development of next-generation 3D scaffolds.

The spatial molecular pattern of integrin recognition sites and their immobilization to colloidal nanobeads determine α2β1 integrin-dependent platelet activation

Collagen, a strong platelet activator, is recognized by integrin α2β1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2β1 integrin binding site is a tool to specifically study how α2β1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2β1 integrin binding sites trigger signaling through dot-like clusters of α2β1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2β1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt.

Phosphorylated immunoreceptor tyrosine-based activation motifs and integrin cytoplasmic domains activate spleen tyrosine kinase via distinct mechanisms

Spleen tyrosine kinase (Syk) is involved in cellular adhesion and also in the activation and development of hematopoietic cells. Syk activation induced by genomic rearrangement has been linked to certain T-cell lymphomas, and Syk inhibitors have been shown to prolong survival of patients with B-cell lineage malignancies. Syk is activated either by its interaction with a double-phosphorylated immunoreceptor tyrosine-based activation motif (pITAM), which induces rearrangements in the Syk structure, or by the phosphorylation of specific tyrosine residues. In addition to its immunoreceptor function, Syk is activated downstream of integrin pathways, and integrins bind to the same region in Syk as does pITAM. However, it is unknown whether integrins and pITAM use the same mechanism to activate Syk. Here, using purified Syk protein and fluorescence-based enzyme assay we investigated whether interaction of the integrin β3 cytoplasmic domain with the Syk regulatory domain causes changes in Syk activity similar to those induced by pITAM peptides. We observed no direct Syk activation by soluble integrin peptide, and integrin did not compete with pITAM-induced activation even though at high concentrations, the integrin cytoplasmic domain peptide competed with Syk's substrate. However, clustered integrin peptides induced Syk activation, presumably via a transphosphorylation mechanism. Moreover, the clustered integrins also activated a Syk variant in which tyrosines were replaced with phenylalanine (Y348F/Y352F), indicating that clustered integrin-induced Syk activation involved other phosphorylation sites. In conclusion, integrin cytoplasmic domains do not directly induce Syk conformational changes and do not activate Syk via the same mechanism as pITAM.

Toxoplasma gondii disrupts β1 integrin signaling and focal adhesion formation during monocyte hypermotility

The motility of blood monocytes is orchestrated by the activity of cell-surface integrins, which translate extracellular signals into cytoskeletal changes to mediate adhesion and migration. Toxoplasma gondii is an intracellular parasite that infects migratory cells and enhances their motility, but the mechanisms underlying T. gondii-induced hypermotility are incompletely understood. We investigated the molecular basis for the hypermotility of primary human peripheral blood monocytes and THP-1 cells infected with T. gondii Compared with uninfected monocytes, T. gondii infection of monocytes reduced cell spreading and the number of activated β1 integrin clusters in contact with fibronectin during settling, an effect not observed in monocytes treated with lipopolysaccharide (LPS) or Escherichia coli Furthermore, T. gondii infection disrupted the phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 (Tyr-397) and Tyr-925 and of the related protein proline-rich tyrosine kinase (Pyk2) at Tyr-402. The localization of paxillin, FAK, and vinculin to focal adhesions and the colocalization of these proteins with activated β1 integrins were also impaired in T. gondii-infected monocytes. Using time-lapse confocal microscopy of THP-1 cells expressing enhanced GFP (eGFP)-FAK during settling on fibronectin, we found that T. gondii-induced monocyte hypermotility was characterized by a reduced number of enhanced GFP-FAK-containing clusters over time compared with uninfected cells. This study demonstrates an integrin conformation-independent regulation of the β1 integrin adhesion pathway, providing further insight into the molecular mechanism of T. gondii-induced monocyte hypermotility.

TGF\u03b21 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness

Here we report exquisitely distinct material properties of primary vascular smooth muscle (VSM) cells isolated from the thoracic aorta of adult (8 months) vs. aged (30 months) F344XBN rats. Individual VSM cells derived from the aged animals showed a tense internal network of the actin cytoskeleton (CSK), exhibiting increased stiffness (elastic) and frictional (loss) moduli than those derived from the adult animals over a wide frequency range of the imposed oscillatory deformation. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic transforming growth factor β1 (TGFβ1) emerged as a specific modifier of age-associated VSM stiffening in vitro. TGFβ1 reinforced the mechanical phenotype of arterial aging in VSM cells on multiple time and length scales through clustering of mechanosensitive α5β1 and αvβ3 integrins. Taken together, these studies identify a novel nodal point for the long-range regulation of VSM stiffness and serve as a proof-of-concept that the broad-based inhibition of TGFβ1 expression, or TGFβ1 signal transduction in VSM, may be a useful therapeutic approach to mitigate the pathologic progression of central arterial wall stiffening associated with aging.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Effects of host cell sterol composition upon internalization of Yersinia pseudotuberculosis and clustered β1 integrin

Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δinv, ΔyadA, and ΔinvΔyadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The ΔinvΔyadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis.

Ubc9 Binds to ADAP and Is Required for Rap1 Membrane Recruitment, Rac1 Activation, and Integrin-Mediated T Cell Adhesion.

Although the immune adaptor adhesion and degranulation-promoting adaptor protein (ADAP) acts as a key mediator of integrin inside-out signaling leading to T cell adhesion, the regulation of this adaptor during integrin activation and clustering remains unclear. We now identify Ubc9, the sole small ubiquitin-related modifier E2 conjugase, as an essential regulator of ADAP where it is required for TCR-induced membrane recruitment of the small GTPase Rap1 and its effector protein RapL and for activation of the small GTPase Rac1 in T cell adhesion. We show that Ubc9 interacted directly with ADAP in vitro and in vivo, and the association was increased in response to anti-CD3 stimulation. The Ubc9-binding domain on ADAP was mapped to a nuclear localization sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by short hairpin RNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCR-induced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, although it had little effect on the TCR proximal signaling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 and RapL, but not Rap1-interacting adaptor molecule. Taken together, our data demonstrate for the first time, to our knowledge, that Ubc9 acts as a functional binding partner of ADAP and plays a selective role in integrin-mediated T cell adhesion via modulation of Rap1-RapL membrane recruitment and Rac1 activation.

Hyperbranched poly(ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells.

Unlike the fibroblast-like cells formed upon monolayer culture of human mesenchymal stem cells, the natural stem cell niche of the bone marrow and other types of tissues favours the formation of 3-dimensional (3D) cell clusters. The structuring and biological activity of these clusters are regulated by the contacts established by cells with both the basement membrane and neighbour cells and results in their asymmetric division and the consequent maintenance of both a stem population and a committed progeny. The present work demonstrates the potential of a synthetic substrate to mimic the stem cell niche in vitro. The side amino groups of a linear Poly-L-lysine were modified with hyperbranched poly-(ϵ-lysine) peptides, named as dendrons, tethered with the laminin-mimicking sequence, YIGSR. These dendrons presented the YIGSR sequence at the uppermost molecular branching ensuring a controlled spacing of the bioligand. When used to coat the surface of tissue culture plates in a serum-free in vitro cell culture system, the substrate was able to mimic the most relevant features of the basement membrane of the stem cell niche, i.e. the mesh structure of Collagen Type IV and the availability of laminin bioligands relevant to integrin biorecognition. The substrate biomimetic properties were tested for their ability to support the formation of human bone marrow mesenchymal stem cells (hMSCs) 3D spheroids similar to those observed in the natural stem cell niches and their ability to maintain stem cell pluripotency markers. These features were related to the substrate-specific expression and localisation of (i) cell adhesion receptors (i.e. β-integrin and N-cadherin), (ii) transcription factors of pluripotency markers and cytoskeleton protein and (iii) regulators of cell migration throughout cell culture passages 2 to 4. The results clearly demonstrate the formation of 3D spheroids starting from the asymmetric division of substrate-adhering spread cells, the clustering of relevant integrins and the expression of specific intracellular pathways controlling cytoskeleton formation suggesting their potential use as a substrate for the handling of stem cells prior to transplantation procedures.

Force loading explains spatial sensing of ligands by cells.

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking 'adaptor' protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds-the molecular clutches-respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

Quantitative proteomics reveal the anti-tumour mechanism of the carbohydrate recognition domain of Galectin-3 in Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a serious threat to human health. The carbohydrate recognition domain of Galectin-3 (Gal3C) has been reported to be an anti-tumour molecule. In this study, we aim to explore effects of Gal3C in HCC and its possible molecular mechanism with quantitative proteomics approach. We found that rGal3C stimulation could inhibit cell viability, migration and invasion of HepG2. After rGal3C stimulating, 190 proteins were differentially expressed. Eighty up-regulated proteins located mainly in extracellular exosome and involved in cell adhesion and metabolism, and 110 down-regulated proteins located in mitochondria and extracellular exosome, and related to processes of metabolism and oxidation-reduction. Of the differentially expressed proteins, CLU, NDRG1, CD166, S100A11 and Galectin-1 were carcinoma-related proteins affected by rGal3C. Potential receptors of rGal3C were explored by an UV cross-linking capture strategy. We showed that rGal3C could induce dephosphorylating of FAK/SRC. Blocking of the FAK/SRC pathway resulted in down-regulation of NDRG1. Immunofluorescence suggested that rGal3C could disrupt integrin clustering. Our study provides valuable insight into the anti-tumour mechanism of rGal3C in HCC on a proteomics level and is the first to reveal the possible mechanism involving integrin/FAK/SRC pathway and NDRG1. These results provide useful guidance of developing new therapies for HCC.