Showing papers on "Role of cell adhesions in neural development published in 2016"

Integrin-mediated mechanotransduction.

Abstract: Cells can detect and react to the biophysical properties of the extracellular environment through integrin-based adhesion sites and adapt to the extracellular milieu in a process called mechanotransduction. At these adhesion sites, integrins connect the extracellular matrix (ECM) with the F-actin cytoskeleton and transduce mechanical forces generated by the actin retrograde flow and myosin II to the ECM through mechanosensitive focal adhesion proteins that are collectively termed the “molecular clutch.” The transmission of forces across integrin-based adhesions establishes a mechanical reciprocity between the viscoelasticity of the ECM and the cellular tension. During mechanotransduction, force allosterically alters the functions of mechanosensitive proteins within adhesions to elicit biochemical signals that regulate both rapid responses in cellular mechanics and long-term changes in gene expression. Integrin-mediated mechanotransduction plays important roles in development and tissue homeostasis, and its dysregulation is often associated with diseases.

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

Abstract: The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7 Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules

Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale

Abstract: Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds β integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to β integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin β1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome.

Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

Abstract: Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or “clutch” myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo , suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. SIGNIFICANCE STATEMENT Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and spatially linked to attenuation of RF at sites of forward membrane protrusion. Importantly, clutching of RF is modulated by extracellular matrix (ECM) proteins and soluble axon guidance cues, suggesting that it may regulate axon guidance in vivo . Consistent with this notion, we found that RF rates of spinal neuron growth cones were slower in vivo than what was observed in vitro . Together, our study provides the best evidence that growth cone–ECM adhesions clutch RF locally to guide axons in vivo .

ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions.

Abstract: ADAMTS10 and ADAMTS6 are homologous metalloproteinases with ill-defined roles. ADAMTS10 mutations cause Weill-Marchesani syndrome (WMS), implicating it in fibrillin microfibril biology since some fibrillin-1 mutations also cause WMS. However little is known about ADAMTS6 function. ADAMTS10 is resistant to furin cleavage, however we show that ADAMTS6 is effectively processed and active. Using siRNA, over-expression and mutagenesis, it was found ADAMTS6 inhibits and ADAMTS10 is required for focal adhesions, epithelial cell-cell junction formation, and microfibril deposition. Either knockdown of ADAMTS6, or disruption of its furin processing or catalytic sites restores focal adhesions, implicating its enzyme activity acts on targets in the focal adhesion complex. In ADAMTS10-depleted cultures, expression of syndecan-4 rescues focal adhesions and cell-cell junctions. Recombinant C-termini of ADAMTS10 and ADAMTS6, both of which induce focal adhesions, bind heparin and syndecan-4. However, cells overexpressing full-length ADAMTS6 lack heparan sulphate and focal adhesions, whilst depletion of ADAMTS6 induces a prominent glycocalyx. Thus ADAMTS10 and ADAMTS6 oppositely affect heparan sulphate-rich interfaces including focal adhesions. We previously showed that microfibril deposition requires fibronectin-induced focal adhesions, and cell-cell junctions in epithelial cultures. Here we reveal that ADAMTS6 causes a reduction in heparan sulphate-rich interfaces, and its expression is regulated by ADAMTS10.

In vivo epidermal migration requires focal adhesion targeting of ACF7

Abstract: Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.

Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction

Abstract: Epithelial monolayers undergo self-healing when wounded. During healing, cells collectively migrate into the wound site, and the converging tissue fronts collide and form a stable interface. To heal, migrating tissues must form cell-cell adhesions and reorganize from the front-rear polarity characteristic of cell migration to the apical-basal polarity of an epithelium. However, identifying the "stop signal" that induces colliding tissues to cease migrating and heal remains an open question. Epithelial cells form integrin-based adhesions to the basal extracellular matrix (ECM) and E-cadherin-mediated cell-cell adhesions on the orthogonal, lateral surfaces between cells. Current biological tools have been unable to probe this multicellular 3D interface to determine the stop signal. We addressed this problem by developing a unique biointerface that mimicked the 3D organization of epithelial cell adhesions. This "minimal tissue mimic" (MTM) comprised a basal ECM substrate and a vertical surface coated with purified extracellular domain of E-cadherin, and was designed for collision with the healing edge of an epithelial monolayer. Three-dimensional imaging showed that adhesions formed between cells, and the E-cadherin-coated MTM resembled the morphology and dynamics of native epithelial cell-cell junctions and induced the same polarity transition that occurs during epithelial self-healing. These results indicate that E-cadherin presented in the proper 3D context constitutes a minimum essential stop signal to induce self-healing. That the Ecad:Fc MTM stably integrated into an epithelial tissue and reduced migration at the interface suggests that this biointerface is a complimentary approach to existing tissue-material interfaces.

Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.

Abstract: Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

Nectin spot: a novel type of nectin-mediated cell adhesion apparatus

Abstract: Nectins are Ca 2+ -independent immunoglobulin (Ig) superfamily cell adhesion molecules constituting a family with four members, all of which have three Ig-like loops at their extracellular regions. Nectins play roles in the formation of a variety of cell–cell adhesion apparatuses. There are at least three types of nectin-mediated cell adhesions: afadin- and cadherin-dependent, afadin-dependent and cadherin-independent, and afadin- and cadherin-independent. In addition, nectins trans -interact with nectin-like molecules (Necls) with three Ig-like loops and other Ig-like molecules with one to three Ig-like loops. Furthermore, nectins and Necls cis -interact with membrane receptors and integrins, some of which are associated with the nectin-mediated cell adhesions, and play roles in the regulation of many cellular functions, such as cell polarization, movement, proliferation, differentiation, and survival, co-operatively with these cell surface proteins. The nectin-mediated cell adhesions are implicated in a variety of diseases, including genetic disorders, neural disorders, and cancers. Of the three types of nectin-mediated cell adhesions, the afadin- and cadherin-dependent apparatus has been most extensively investigated, but the examples of the third type of apparatus independent of afadin and cadherin are recently increasing and its morphological and functional properties have been well characterized. We review here recent advances in research on this type of nectin-mediated cell adhesion apparatus, which is named nectin spot.

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

Abstract: Animal cells can move in the body, for example to heal a wound, by protruding a leading edge forwards, attaching it to the surroundings and then pulling against these new attachments while disassembling the older ones. Mechanical forces regulate the assembly and disassembly of these attachments, known as focal adhesions, and so do signals from outside the cell that are transmitted to the adhesions via specialized proteins. However, it was not clear how the assembly and disassembly of adhesions is coordinated. CRL5 is a ubiquitin ligase, an enzyme that can mark other proteins for destruction. Cells migrate more quickly if CRL5 is inhibited, and so Teckchandani and Cooper set out to uncover whether CRL5 affects the assembly and disassembly of focal adhesions. The experiments showed that human cells lacking a crucial component of the CRL5 complex, SOCS6, disassemble adhesions faster than normal cells, but only at their leading edge and not at the rear. Teckchandani and Cooper also found that SOCS6 localizes to the leading edge by binding to a focal adhesion protein called Cas. Shortly after the attachments assemble, the Cas protein becomes tagged with a phosphate group and then acts to promote the adhesion to disassemble. Further experiments indicated that Cas was marked by the CRL5 complex and possibly destroyed while in or very close to the leading edge adhesions, slowing their disassembly. Together, these findings suggest that by binding Cas, SOCS6 regulates the turnover of adhesions, specifically by inhibiting disassembly and allowing adhesions to grow at the leading edge. Since SOCS6 is not present in adhesions outside of the leading edge, this may help explain how the older adhesions are disassembled. Future studies could next focus on the exact sequence of events that occur in focal adhesions after the CRL5 complex binds to Cas as the cell migrates.

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1

Abstract: Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

Association between tensin 1 and p130Cas at focal adhesions links actin inward flux to cell migration

Abstract: Cell migration is a highly dynamic process that plays pivotal roles in both physiological and pathological processes. We have previously reported that p130Cas supports cell migration through the binding to Src as well as phosphorylation-dependent association with actin retrograde flow at focal adhesions. However, it remains elusive how phosphorylated Cas interacts with actin cytoskeletons. We observe that the actin-binding protein, tensin 1, co-localizes with Cas, but not with its phosphorylation-defective mutant, at focal adhesions in leading regions of migrating cells. While a truncation mutant of tensin 1 that lacks the phosphotyrosine-binding PTB and SH2 domains (tensin 1-SH2PTB) poorly co-localizes or co-immunoprecitates with Cas, bacterially expressed recombinant tensin 1-SH2PTB protein binds to Cas in vitro in a Cas phosphorylation-dependent manner. Furthermore, exogenous expression of tensin 1-SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions. Taken together, our results show that tensin 1 links inwardly moving actin cytoskeletons to phosphorylated Cas at focal adhesions, thereby driving cell migration.

Cell Adhesion to the Extracellular Matrix

Abstract: Living cells within multicellular organisms interact with the surrounding extracellular matrix (ECM) via integrin receptors at specialized, cytoskeleton-rich sites. These adhesions display two characteristic functional and structural features: they are robust, enabling the creation of long-range, long-term tissue scaffolding, and they act as ‘environmental sensors,’ responding to differences in ECM properties such as composition, rigidity, micro-topography, and deformability. In this article, we will review the main characteristics of integrin adhesions, in intact animals and in cultured cells, addressing their complex nano-architecture, molecular heterogeneity, and dynamic reorganization.

Measuring mechanical tension across the focal adhesion protein talin-1

Abstract: Cell adhesion is an essential mechanism involved in many cellular processes, such as migration, proliferation and differentiation. The mechanical linkage between extracellular matrix and the f-actin cytoskeleton is mediated in specialized protein complexes, called focal adhesions. Key components of these cellular compartments are members of the integrin protein family; transmembrane proteins that connect to ligands in the extracellular matrix and recruit intracellular focal adhesion proteins. However, integrins have no catalytic function and cannot bind cytoskeletal components. The association with f-actin is mediated by intracellular adaptor molecules that link integrin tails and the cytoskeleton. These adhesion complexes not only mediate association with the extracellular matrix, but also serve as the mechanosensitive units of the cell. It has been known for some time that mechanical stimuli – as for example tissue rigidity – are epigenetic factors, regulating processes like organ development and stem cell differentiation. However, even though single components of the adhesion complex have been demonstrated to be involved in mechanosensitive processes, central mechanisms in mechanosensing through focal adhesions remained unknown. One of the major components responsible for the integrin-f-actin connection is the focal adhesion protein talin-1. Talin-1 directly binds intracellular integrin tails but also carries three f-actin binding sites and thus directly mediates the connection between extracellular Matrix and the cytoskeleton. Besides ist important role as integrin activator – and thus important promotor of integrin mediated adhesion – talin-1 has long been suspected as a mechanosensitive component in focal adhesions. Still, evidence of a regulatory role of talin-1 in mechanosensing in adhesive cells is still missing due to the lack of appropriate techniques. Using two single-molecule-calibrated FRET (Forster resonance energy transfer) based tension sensors, it could be demonstrated in this work that talin-1 is indeed subject to low-piconewton (pN) forces in integrin mediated adhesion processes. When localized in focal adhesion talin-1 bears forces of 7-10 pN. Regulation of talin-1 forces occurs through association with f-actin, either direct or indirect via binding of vinculin, a talin-1 interactor that strengthens the connection of talin-1 with the actin cytoskeleton. Disturbing the mechanical linkage of integrins to f-actin via talin-1 does not prevent integrin activation, but leads to defects in cell spreading and focal adhesion reinforcement. Furthermore, it could be shown that mechanical resilient linkages through talin-1 in focal adhesions are important for extracellular rigidity sensing.Taken together this work provides strong evidence that talin-1 mediated mechanical linkage between the extracellular matrix and the actin cytoskeleton is essential in mechanosignaling processes. For the first time it could be shown that talin-1 is subject to pN forces in living cells and that the force transmission is indeed dependent on f-actin and vinculin association with talin-1.

Caveolin-1 and membrane domain regulation of focal adhesions and tumor cell migration

Abstract: .................................................................................................................... ii PREFACE ...................................................................................................................... iv TABLE OF CONTENTS ................................................................................................ vi LIST OF FIGURES ........................................................................................................ ix ACKNOWLEDGEMENTS .............................................................................................. x CHAPTER

Molecular Mechanisms of Mechanosensitivity in Focal Adhesions

Abstract: Physical environment guides tissue regeneration and morphology in both health and disease In the past three decades, several experiments illustrated that mechanical cues are captured and transduced to biochemical signals in the cellular level (mechanotransduction) mediated by cell adhesion Cells adhere to their microenvironment through large protein assemblies known as focal adhesions that directly couple intra- and extra-cellular matrices and play a critical role in many vital cell functions including proliferation, differentiation and cell fate It is inherently difficult to investigate the molecular basis of focal adhesion formation and growth using current experimental methodologies due to the fine time- and length-scales of protein-protein interactions Here, I used molecular dynamics simulations to investigate the underlying molecular mechanisms of focal adhesion formation and maturation with atomic resolution Integrins are key focal adhesion receptors that reside on the cell membrane and mediate bi-directional signaling between cell cytoskeleton and ECM Focal adhesions are a mixture of integrin-associated protein complexes known as integrin modules that forms the basic adhesion units Integrin module formation is initiated by talin binding to the integrin tail, which is shown to be sufficient for integrin activation A few other focal adhesion proteins can also directly engage with integrin’s cytoplasmic tail and link it to the actin cytoskeleton It is not yet clear how simultaneous (cooperative) versus sequential (competitive) binding of focal adhesion proteins to integrin with respect to talin result in different functionalities of integrin modules In the first part of this study, competitive versus cooperative integrin binding between two important focal adhesion proteins –filamin and α-actinin– with talin were studied The purpose of this aim was to gain insight on integrin module formation that eventually determines its functional properties Maturation of focal adhesions follows an increase in local forces A well-established hypothesis on force transmission across focal adhesion complexes is the presence of mechanosensitive elements that change their conformations in response to force In the second part of this study, we investigated and argued force-induced conformational changes of two important focal adhesion proteins –vinculin and α-actinin– in order to shed light on their role in transmission of forces across focal adhesions leading to adhesion maturation and growth In conclusion, this study unravels the structural basis of mechanosensitivity of key focal adhesions Furthermore, important molecular interactions that give rise to mechanosensitivite characteristics of focal adhesions were studies Important impacts of the current study include but are not limited to the following: 1) our results was used to complement previous experimental studies and also construct new hypotheses for future experiments; 2) Understanding regulatory mechanisms of focal adhesions is critical for developing novel therapeutics for many diseases involving cell adhesion including cancer as it enhances target recognition and the accuracy of drug delivery systems 3) In addition, performing extensive simulations on protein complexes will contribute to improving the accuracy of various aspects of computational methods including empirical force fields that is indicative of our understanding of fundamental physical and chemical principles governing protein-protein interactions 4) And most importantly, this work provides a fundamental insight into the relation between structure and function of mechanosensitive proteins in focal adhesions