Adhesion Assembly Research Articles

MYO1B enhances colorectal cancer metastasis by promoting the F-actin rearrangement and focal adhesion assembly via RhoA/ROCK/FAK signaling.

BackgroundColorectal cancer (CRC) has a high worldwide incidence and mortality. Tumor metastasis is one of the primary reasons for the poor prognosis of CRC patients. However, the mechanism underlying CRC metastasis is still unclear. Myosin 1B (MYO1B) is important for cell migration and motility and is part of the myosin superfamily that contains various myosins. Studies of prostate, cervical, and head and neck cancer have revealed preliminary findings concerning the effect of MYO1B on tumor metastasis. However, the role of MYO1B in CRC metastasis, as well as its underlying mechanism, remains unknown.MethodsQuantitative real-time PCR and immunohistochemical staining methods were used to analyze the expression of MYO1B in human CRC and normal mucosa tissues. Lentivirus vector-based MYO1B oligonucleotides and short hairpin RNA (shRNA) were used to examine the functional relevance of MYO1B in CRC cells. Co-immunoprecipitation, western blotting, and immunofluorescence assays were used to investigate the underlying mechanism of MYO1B-mediated cell migration.ResultsThe expression of MYO1B was increased in most CRC tissues and was positively associated with a greater risk of tumor metastasis and poor prognosis for patients. MYO1B was significantly associated with the migration and invasion properties of CRC cells in vitro and in vivo. MYO1B promoted F-actin rearrangement through the ROCK2/LIMK/Cofilin axis by enhancing the activation of RhoA. MYO1B also promoted the assembly of focal adhesions by targeting RhoA.ConclusionsMYO1B plays a vital role in CRC metastasis by promoting the activation of RhoA. MYO1B may not only be a valid biomarker for predicting the risk of metastasis and poor prognosis in CRC but may also be a potential therapeutic target for patients with a high risk of tumor metastasis.

Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell-substrate adhesion in cancer cells and mechanosensitivity of cell proliferation.

The actin cytoskeleton is the primary driver of cellular adhesion and mechanosensing due to its ability to generate force and sense the stiffness of the environment. At the cell's leading edge, severing of the protruding Arp2/3 actin network generates a specific actin/tropomyosin (Tpm) filament population that controls lamellipodial persistence. The interaction between these filaments and adhesion to the environment is unknown. Using cellular cryo-electron tomography we resolve the ultrastructure of the Tpm/actin copolymers and show that they specifically anchor to nascent adhesions and are essential for focal adhesion assembly. Re-expression of Tpm1.8/1.9 in transformed and cancer cells is sufficient to restore cell-substrate adhesions. We demonstrate that knock-out of Tpm1.8/1.9 disrupts the formation of dorsal actin bundles, hindering the recruitment of α-actinin and non-muscle myosin IIa, critical mechanosensors. This loss causes a force-generation and proliferation defect that is notably reversed when cells are grown on soft surfaces. We conclude that Tpm1.8/1.9 suppress the metastatic phenotype, which may explain why transformed cells naturally downregulate this Tpm subset during malignant transformation.

The cell adhesion molecule TMIGD1 binds to moesin and regulates tubulin acetylation and cell migration

BackgroundThe cell adhesion molecule transmembrane and immunoglobulin (Ig) domain containing1 (TMIGD1) is a novel tumor suppressor that plays important roles in regulating cell–cell adhesion, cell proliferation and cell cycle. However, the mechanisms of TMIGD1 signaling are not yet fully elucidated.ResultsTMIGD1 binds to the ERM family proteins moesin and ezrin, and an evolutionarily conserved RRKK motif on the carboxyl terminus of TMIGD1 mediates the interaction of TMIGD1 with the N-terminal ERM domains of moesin and ezrin. TMIGD1 governs the apical localization of moesin and ezrin, as the loss of TMIGD1 in mice altered apical localization of moesin and ezrin in epithelial cells. In cell culture, TMIGD1 inhibited moesin-induced filopodia-like protrusions and cell migration. More importantly, TMIGD1 stimulated the Lysine (K40) acetylation of α-tubulin and promoted mitotic spindle organization and CRISPR/Cas9-mediated knockout of moesin impaired the TMIGD1-mediated acetylation of α-tubulin and filamentous (F)-actin organization.ConclusionsTMIGD1 binds to moesin and ezrin, and regulates their cellular localization. Moesin plays critical roles in TMIGD1-dependent acetylation of α-tubulin, mitotic spindle organization and cell migration. Our findings offer a molecular framework for understanding the complex functional interplay between TMIGD1 and the ERM family proteins in the regulation of cell adhesion and mitotic spindle assembly, and have wide-ranging implications in physiological and pathological processes such as cancer progression.

\u03b12\u03b21 integrins spatially restrict Cdc42 activity to stabilise adherens junctions

BackgroundKeratinocytes form the main protective barrier in the skin to separate the underlying tissue from the external environment. In order to maintain this barrier, keratinocytes form robust junctions between neighbouring cells as well as with the underlying extracellular matrix. Cell–cell adhesions are mediated primarily through cadherin receptors, whereas the integrin family of transmembrane receptors is predominantly associated with assembly of matrix adhesions. Integrins have been shown to also localise to cell–cell adhesions, but their role at these sites remains unclear.ResultsHere we show that α2β1 integrins are enriched at mature keratinocyte cell–cell adhesions, where they play a crucial role in organising cytoskeletal networks to stabilize adherens junctions. Loss of α2β1 integrin has significant functional phenotypes associated with cell–cell adhesion destabilisation, including increased proliferation, reduced migration and impaired barrier function. Mechanistically, we show that α2β1 integrins suppress activity of Src and Shp2 at cell–cell adhesions leading to enhanced Cdc42–GDI interactions and stabilisation of junctions between neighbouring epithelial cells.ConclusionOur data reveals a new role for α2β1 integrins in controlling integrity of epithelial cell–cell adhesions.

MYH9-dependent polarization of ATG9B promotes colorectal cancer metastasis by accelerating focal adhesion assembly

Tumour metastasis is a major reason accounting for the poor prognosis of colorectal cancer (CRC), and the discovery of targets in the primary tumours that can predict the risk of CRC metastasis is now urgently needed. In this study, we identified autophagy-related protein 9B (ATG9B) as a key potential target gene for CRC metastasis. High expression of ATG9B in tumour significantly increased the risk of metastasis and poor prognosis of CRC. Mechanistically, we further find that ATG9B promoted CRC invasion mainly through autophagy-independent manner. MYH9 is the pivotal interacting protein for ATG9B functioning, which directly binds to cytoplasmic peptide segments aa368–411 of ATG9B by its head domain. Furthermore, the combination of ATG9B and MYH9 enhance the stability of each other by decreasing their binding to E3 ubiquitin ligase STUB1, therefore preventing them from ubiquitin-mediated degradation, which further amplified the effect of ATG9B and MYH9 in CRC cells. During CRC cell invasion, ATG9B is transported to the cell edge with the assistance of MYH9 and accelerates focal adhesion (FA) assembly through mediating the interaction of endocytosed integrin β1 and Talin-1, which facilitated to integrin β1 activation. Clinically, upregulated expression of ATG9B in human CRC tissue is always accompanied with highly elevated expression of MYH9 and associated with advanced CRC stage and poor prognosis. Taken together, this study highlighted the important role of ATG9B in CRC metastasis by promoting focal adhesion assembly, and ATG9B together with MYH9 can provide a pair of potential therapeutic targets for preventing CRC progression.

Hyaluronan (HA) Immobilized on Surfaces via Self-Assembled Monolayers of HA-Binding Peptide Modulates Endothelial Cell Spreading and Migration through Focal Adhesion.

The extracellular matrix (ECM) modulates a multitude of cell functions, and this regulation is provided by key ECM components forming a complex network. Hyaluronic acid (HA) is an abundant component of the ECM that binds to proteins and influences various activities of endothelial cells (ECs). Although the effect of soluble HA on cell spreading has been studied, the impact of peptide-bound HA has not yet been investigated in great detail. We aim to comprehensively study the roles of immobilized HA on the regulation of EC behavior compared to the more conventional use of soluble HA. A 2D model surface formed by self-assembled monolayers (SAMs) of a HA-binding peptide (Pep-1) is used as an anchor for HA immobilization. Mixed SAMs, consisting of thiolated Pep-1 and 1-octanethiol, are prepared and characterized by using ellipsometry and contact angle measurement. Full density Pep-1 SAMs are more hydrophilic and bind more HA than mixed SAMs. Cell spreading and migration are enhanced by immobilized low molecular weight (LMW) HA, which also facilitates cell alignment and elongation under laminar flow conditions and potentially drives directional migration. This effect is not mediated by the expression of CD44, and immobilized LMW HA is found to accelerate the assembly of focal adhesions. Such biomimetic surfaces provide new insights into the role of HA in regulating the spreading and phenotype of endothelial cells.

RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration.

The airway epithelium’s ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract’s normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

Iain Donald Campbell. 24 April 1941—5 March 2014

Iain Campbell defined the study of proteins by nuclear magnetic resonance spectroscopy (NMR) in the UK, and was a towering international figure in biophysics and structural cell biology. His scientific career spanned nearly 50 years, almost entirely spent at the University of Oxford. As a PhD student he recorded electron spin resonance spectra, then later became a pioneer in the application of NMR methodology to whole cells, determining the world's second and UK's first protein structure by NMR. He ended his career as one of the leading scientific lights in integrin adhesion and focal cell assembly. His scientific contributions are characterized by intellectual rigour and a desire to solve the problem by applying the most appropriate tools. All who knew Iain noted his incredible work ethic, his precision and in particular his wry humour. The co-workers trained by Iain form the backbone of protein NMR internationally today, a tribute to his mentorship. His loss was deeply felt by colleagues across the world and of course most of all by his loving family.

Molecular insight into the outside‐in activation of integrin αvβ3 by 24( S )‐hydroxycholesterol

Objective Oxysterols such as 25-hydroxycholesterol (25HC) are metabolites of cholesterol produced in the human body. Oxysterols are implicated in many physiological and pathological processes and are shown to regulate the immune system. Recently, we demonstrated that during virus infections, 25HC amplifies proinflammatory response by directly binding to αvβ3 and α5β1 integrins and by activating FAK-NFkB signaling pathway, resulting in the production of proinflammatory mediators such as TNFα and IL-6. Surprisingly, our combined in silico and in vitro studies revealed that 25HC binds to integrins at a novel binding site (site 2) that is distinct from the classical ‘RGD’ binding site, where many extracellular matrix proteins with Arg-Gly-Asp motif are known to bind. Integrins are ubiquitous and play an important role in cell adhesion, cell-cell interactions, cell signaling, and extracellular matrix assembly. Given the abundance of both integrins and endogenous oxysterols, we hypothesized that other oxysterols (other than 25HC) might also activate integrins in a similar fashion. In this study, using a combined computational and experimental approach, we show that 24(S)-hydroxycholesterol (24HC) binds to integrin αvβ3 with high-affinity and triggers proinflammatory response via integrin-FAK pathway. Methods Molecular docking and all-atom molecular dynamics (MD) simulations were used to obtain binding poses and to study the ligand stability and conformational changes, respectively. Induced fit docking of 24HC at site 2 of αVβ3 ectodomain (PDB ID 3IIJE) was performed using MOE software. All-atom MD simulations were performed with GROMACS v5.1.2 using CHARMM36 force field and TIP3P water model. MD simulations were run in triplicates each for 200 ns. The proinflammatory response was determined by the production of TNF using ELISA. Results Our studies reveal the preferred binding orientation of 24HC, which is distinct from that of 25HC. Binding of 24HC induced structural changes in the specificity determining loop (SDL) of the βI domain, leading to an increase in solvent-accessible surface area (SASA) of the RGD site, disruption of native SDL-βI and SDL-β-propeller interface contacts, possibly altering the conformation of the RGD binding site. Robust production of TNF analyzed by ELISA indicates activation of the inflammatory response by 24HC. Similar to 25HC, 24HC also requires integrin-FAK pathway for inflammatory response since, FAK inhibitor, PF-431396 abrogated 24HC mediated response. Conclusions: As the primary cholesterol metabolite with exclusive production in the brain, these findings may provide key insight into factors that influence the complexities of brain function. Our study, for the first time, offers useful insight into the structural and mechanistic bases for the potential proinflammatory response mediated by 24HC through integrins and paves the way for drug discovery efforts towards identifying therapeutics in the treatment of infectious, inflammatory, and immune disorders.

Podosome dissolution and focal adhesion assembly drive the enhanced migratory capacity of dendritic cells infected by ectromelia virus

Abstract Podosomes and focal adhesions (FAs) are both adhesive cytoskeletal structures engaged in cell-matrix adhesion of many different cell types, including dendritic cells (DCs). Despite sharing almost, the same proteins, e.g. vinculin, paxillin, talin and Src family proteins, podosomes and FAs show differences in their architecture, dynamics and function. While highly dynamic podosomes are organized in an actin-rich core surrounded by a ring of adhesive molecules with a perpendicular orientation to the extracellular matrix (ECM), more stable FAs have elongated structure and exhibit tangential orientation to the substrate surface. A switch from podosomes to FAs is often observed during TLR4-mediated DC maturation and is associated with an increased motility of these cells. Ectromelia virus (ECTV) is an orthopoxvirus that can control several different aspects of the cytoskeleton organization and dynamics in DCs and, thus, provides a useful model to study virus-cytoskeleton interactions and cell motility in these specialized immune cells, in which ECTV can productively replicate. Our results show that DCs infected with ECTV completely lose podosomes during early stages of infection, within the viral factory formation phase in the cytoplasm (4 hpi). Instead of podosomes, ECTV-infected DCs develop FAs, which are larger but less numerous than in uninfected control cells. FAs are predominantly observed within long-branched cellular projections formed extensively during later stages of infection (18–24 hpi). Loss of podosomes is associated with enhanced migration of infected DCs in vitro, suggesting that ECTV may potentially exploit DC migration to facilitate dissemination of the virus in vivo.

Vimentin regulates the assembly and function of matrix adhesions.

The intermediate filament protein vimentin is a widely used phenotypic marker for identifying cells of the mesenchymal linkage such as fibroblasts and myofibroblasts, but the full repertoire of vimentin's functional attributes has not been fully explored. Here we consider how vimentin, in addition to its contributions to mechanical stabilization of cell structure, also helps to control the assembly of cell adhesions and migration through collagen matrices. While the assembly and function of matrix adhesions are critical for the differentiation of myofibroblasts and many other types of adherent cells, a potential mechanism that explains how vimentin affects the recruitment and abundance of centrally important proteins in cell adhesions has been elusive. Here we review recent data indicating that vimentin plays a central regulatory role in the assembly of focal adhesions which form in response to the attachment to collagen. We show that in particular, vimentin is a key organizer of the β1 integrin adhesive machinery, which affects cell migration through collagen. This review provides a comprehensive picture of the surprisingly broad array of processes and molecules with which vimentin interacts to affect cell function in the context of fibroblast and myofibroblast adhesion and migration on collagen.

Role of Src/FAK in migration and invasion mediated by extracellular vesicles from MDA-MB-231 cells stimulated with linoleic acid.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid in occidental diets, which mediate a variety of processes in human breast cancer cells, including migration and invasion. Extracellular vesicles (EVs) are vesicles released from endosomes and plasma membrane that are composed of a variety of molecules, including proteins, nucleic acids and lipids. EVs from cancer cells promote processes related with cancer progression. In the present study, we demonstrate that treatment of MDA-MB-231 cells with EVs from MDA-MB-231 cells stimulated with LA (LA EVs) promote migration and invasion via Src activity. LA EVs induce activation of FAK via Src activity and of Src and Akt2. LA EVs also induce the assembly of focal adhesions and MMP-9 secretion. These findings demonstrate that LA EVs mediate an autocrine and/or paracrine Src/FAK signaling pathway to promote migration and invasion.

Protein tyrosine phosphatases in cell adhesion.

Adhesive structures between cells and with the surrounding matrix are essential for the development of multicellular organisms. In addition to providing mechanical integrity, they are key signalling centres providing feedback on the extracellular environment to the cell interior, and vice versa. During development, mitosis and repair, cell adhesions must undergo extensive remodelling. Post-translational modifications of proteins within these complexes serve as switches for activity. Tyrosine phosphorylation is an important modification in cell adhesion that is dynamically regulated by the protein tyrosine phosphatases (PTPs) and protein tyrosine kinases. Several PTPs are implicated in the assembly and maintenance of cell adhesions, however, their signalling functions remain poorly defined. The PTPs can act by directly dephosphorylating adhesive complex components or function as scaffolds. In this review, we will focus on human PTPs and discuss their individual roles in major adhesion complexes, as well as Hippo signalling. We have collated PTP interactome and cell adhesome datasets, which reveal extensive connections between PTPs and cell adhesions that are relatively unexplored. Finally, we reflect on the dysregulation of PTPs and cell adhesions in disease.

ECM-Mimetic Nylon Nanofiber Scaffolds for Neurite Growth Guidance.

Numerous nanostructured synthetic scaffolds mimicking the architecture of the natural extracellular matrix (ECM) have been described, but the polymeric nanofibers comprising the scaffold were substantially thicker than the natural collagen nanofibers of neural ECM. Here, we report neuron growth on electrospun scaffolds of nylon-4,6 fibers with an average diameter of 60 nm, which closely matches the diameter of collagen nanofibers of neural ECM, and compare their properties with the scaffolds of thicker 300 nm nanofibers. Previously unmodified nylon was not regarded as an independent nanostructured matrix for guided growth of neural cells; however, it is particularly useful for ultrathin nanofiber production. We demonstrate that, while both types of fibers stimulate directed growth of neuronal processes, ultrathin fibers are more efficient in promoting and accelerating neurite elongation. Both types of scaffolds also improved synaptogenesis and the formation of connections between hippocampal neurons; however, the mechanisms of interaction of neurites with the scaffolds were substantially different. While ultrathin fibers formed numerous weak immature β1-integrin-positive focal contacts localized over the entire cell surface, scaffolds of submicron fibers formed β1-integrin focal adhesions only on the cell soma. This indicates that the scaffold nanotopology can influence focal adhesion assembly involving various integrin subunits. The fabricated nanostructured scaffolds demonstrated high stability and resistance to biodegradation, as well as absence of toxic compound release after 1 month of incubation with live cells in vitro. Our results demonstrate the high potential of this novel type of nanofibers for clinical application as substrates facilitating regeneration of nervous tissue.

Abstract PS5-43: Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity

Abstract IntroductionClassically, cancer therapy approaches lead to tumor cell death or an alternative fate such as senescence. Senescent cells remain viable but without proliferation markers. Senescent cells secrete factors with diverse actions that influence surrounding cells, the extracellular matrix (ECM) and the immune system, a phenomenon termed the senescence-associated secretory phenotype (SASP). Cyclin-dependent kinase 4/6 inhibitors (CDKi) are game-changers in the therapy of metastatic hormone receptor-positive, HER2-negative breast cancer. A potential mechanism of the action of these agents is induction of senescence in breast tumor cells, going beyond cell cycle arrest. However, senescence can be studied mainly using invasive biopsies, and therefore the prevalence and importance of senescence in patients are largely unknown. Moreover, only few circulating biomarkers exist to predict activity or measure CDKi effects. In order to develop a correlative liquid biopsy for CDKi activity, we have analyzed epigenetic (methylation) changes following therapy-induced senescence in vitro. We sought to identify (un)methylated loci that will be affected by different approaches and later can be discovered in circulating cell-free DNA. MethodsWe have treated luminal breast cancer MCF-7 cells with Doxorubicin, CDKi (Palbociclib) or by irradiation. This protocol resulted in up to 80% of cells with senescence-related beta-galactosidase activity. DNA methylation was profiled using Illumina Infinium MethylationEPIC 850K BeadChip. Differentially methylated loci (mDNA) were identified using GenomeStudio and Minfi. Analyses were done using GREAT, CSGene, and Reactome databases. Methylation age was analyzed using the Horvath Methylation Calculator. Statistical significance was defined as p<0.05, q<0.1. ResultsSignificantly differentially methylated sites (in comparison to untreated cells) were revealed as following: 9111 sites in Doxorubicin treated cells, 3828 sites in Palbociclib treated cells, and 694 sites in irradiated cells. These loci comprise 1%, 0.45%, and 0.08% of analyzed methylation sites, respectively. 324 loci were similarly changed following the three treatment options ('common sites'). We found that the 'geographic' distribution of intragenic and intergenic methylation sites (5' UTR, gene body etc.) in all treated cells was similar. Gene set over-representation analysis revealed that sites associated with genes of the 'collagen metabolic process' set were significantly altered in drug-treated cells. Pathway investigation of the 324 'common sites' revealed that ECM-related 'focal adhesion assembly' is the most significant pathway involved. Specific analysis of senescence-related gene sets showed that 15.1% (76/503) of senescence genes changed after Doxorubicin treatment, 6.2% (31/503) after Palbociclib and 1% (5/503) after irradiation. Pathway analysis of these genes showed that drugs affected the 'oxidative stress-induced senescence' pathway, while irradiated cells had SASP-related genes affected.Despite the above mentioned methylation changes, age calculation based on methylation clock showed that all samples had similar age (6), regardless of manipulation. ConclusionSingle treatment of MCF-7 cells with known senescence inducers results in changes in methylation patterns. A significant number of common loci changed following all types of treatments, suggesting them as potential surrogate loci of senescence. Also, we have reproduced the well-known interplay between collagen and senescence/SASP.This preliminary data sheds light on epigenetic changes following treatment-induced senescence. Further studies are needed to validate whether these methylation changes can be found in vivo in tumors and in patients’ cell-free DNA following therapy with CDKi. Citation Format: Albert Grinshpun, Anatoli Kustanovich, Joshua Moss, Yael Gabai, Yuval Dor, Ittai Ben-Porath. Senescence-related methylation changes following therapy as potential biomarker for CDK4/6 inhibitor activity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-43.

Polymer Nanopillars Induce Increased Paxillin Adhesion Assembly and Promote Axon Growth in Primary Cortical Neurons

AbstractThe complexity of the extracellular matrix consists of micro‐ and nanoscale structures that influence neuronal development through contact guidance. Substrates with defined topographic cues mimic the complex extracellular environment and can improve the interface between cells and biomedical devices as well as potentially serve as tissue engineering scaffolds. This study investigates axon development and growth of primary cortical neurons on OrmoComp nanopillars of various dimensions. Neuronal somas and neurites form adhesions and F‐actin accumulations around the pillars indicating a close contact to the topography. In addition, higher pillars (400 nm) confine the growing neurites, resulting in greater neurite alignment to the topographical pattern compared to lower pillars (100 nm). A comprehensive analysis of growth cone dynamics during axon development shows that nanopillars induce earlier axon establishment and change the periodicity of growth cone dynamics by promoting elongation. This results in longer axons compared to the flat substrate. Finally, the increase in surface area available for growth cone coupling provided by nanopillar sidewalls is correlated with increased assembly of paxillin‐rich point contact adhesions and a reduction in actin retrograde flow rates allowing for accelerated and persistent neurite outgrowth.

Role of Actin Cytoskeleton in E-cadherin-Based Cell–Cell Adhesion Assembly and Maintenance

The cellular cytoskeleton consisting of microtubules, intermediate filaments and the actin filaments is a dynamic structure providing shape and structural stability to cells. Particularly, the actin cytoskeleton formed by a combination of polymerized actin molecules and several other actin binding proteins including myosin is key to sensing and development of mechanical forces in cells. Given this and other features, the actin cytoskeleton has been implicated in a variety of cellular process including cellular motility and migration, cytokinesis, phagocytosis, cytoplasmic streaming, organelle transport, cellular transformation and metastasis, cellular metabolism, cell–matrix adhesion, and cell–cell adhesion. The latter is mediated by E-cadherin in the epithelial tissue and is fundamental to tissue morphogenesis and normal development. Here we discuss the role of the actin cytoskeleton in the assembly and maintenance of E-cadherin-based cell–cell adhesion through the formation of cellular appendages such as filopodia and lamellipodia and thus, impinging on one of the fundamental features of multicellular organisms.

Molecular basis for Ras suppressor-1 binding to PINCH-1 in focal adhesion assembly

Ras suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating cell adhesion and is involved in such physiological and pathological processes as focal adhesion assembly and tumor metastasis. Rsu-1 interacts with zinc-finger type multi-LIM domain-containing adaptor protein PINCH-1, known to be involved in the integrin-mediated consensus adhesome, but not with its highly homologous family member PINCH-2. However, the structural basis for and regulatory mechanisms of this specific interaction remain unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture, with eight LRRs shielded by N- and C-terminal capping modules. We showed that the conserved concave surface of the Rsu-1 LRR domain binds and stabilizes the PINCH-1 LIM5 domain via salt bridge and hydrophobic interactions, while the C-terminal non-LIM region of PINCH-2 sterically disfavors Rsu-1 binding. We also showed that Rsu-1 can be assembled, via PINCH-1-binding, into a heteropentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2, which constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Our mutagenesis and cell biological data emphasize the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading, providing crucial molecular insights into Rsu-1-mediated cell adhesion with implications for disease development.

Initiation of focal adhesion assembly by talin and kindlin: A dynamic view.

Focal adhesions (FAs) are integrin-containing protein complexes regulated by a network of hundreds of protein-protein interactions. They are formed in a spatiotemporal manner upon the activation of integrin transmembrane receptors, which is crucial to trigger cell adhesion and many other cellular processes including cell migration, spreading and proliferation. Despite decades of studies, a detailed molecular level understanding on how FAs are organized and function is lacking due to their highly complex and dynamic nature. However, advances have been made on studying key integrin activators, talin and kindlin, and their associated proteins, which are major components of nascent FAs critical for initiating the assembly of mature FAs. This review will discuss the structural and functional findings of talin and kindlin and their immediate interaction network, which will shed light upon the architecture of nascent FAs and how they act as seeds for FA assembly to dynamically regulate diverse adhesion-dependent physiological and pathological responses.

Coordinated assembly and release of adhesions builds apical junctional belts during de novo polarisation of an epithelial tube.

ABSTRACTUsing the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Mpp5a- and Rab11a-mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose that these roles for both Mpp5a and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction-remodelling role of Mpp5a is not specific to dividing cells.