1,721,071 research outputs found
Clutching at Guidance Cues: The Integrin–FAK Axis Steers Axon Outgrowth
Get PDFIntegrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin–FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury
Investigating the interactions between paxillin, vinculin and talin in regulating the dynamics of cell-extracellular matrix adhesions
Get PDFFocal adhesions provide a bi-directional, highly dynamic interface between the extracellular and intracellular environments. The abundance and diversity of proteins recruited to the adhesion sites enable focal adhesions to act as highly efficient mechanosensitive signalling hubs that regulate multiple aspects of cell behaviour including cell movement. Paxillin is one of the proteins identified to localise to focal adhesions. This study explored the interaction of paxillin with the focal dhesionassociated proteins talin, vinculin and focal adhesion kinase, and how such interactions are regulated. Biochemical characterisation using a suite of assays, including fluorescence polarisation, microscale thermophoresis, nuclear magnetic resonance, size exclusion chromatography and pulldowns, identified two novel interactions: 1) the paxillin LIM domains bind talin R9-R12, and 2) multiple paxillin LD motifs cooperatively bind to the head and talin domains of vinculin. We propose a model whereby talin facilitates the localisation of paxillin in proximity of vinculin at adhesions. Once recruited, paxillin functions to control the activation state of vinculin and thus regulate adhesion dynamics, influencing a whole host of cellular activities including cell migration and apopotosis
Advancements in antimicrobial nanoscale materials and self-assembling systems
No full textAntimicrobial resistance is directly responsible for more deaths per year than either HIV/AIDS or malaria and is predicted to incur a cumulative societal financial burden of at least $100 trillion between 2014 and 2050. Already heralded as one of the greatest threats to human health, the onset of the coronavirus pandemic has accelerated the prevalence of antimicrobial resistant bacterial infections due to factors including increased global antibiotic/antimicrobial use. Thus an urgent need for novel therapeutics to combat what some have termed the ‘silent pandemic’ is evident. This review acts as a repository of research and an overview of the novel therapeutic strategies being developed to overcome antimicrobial resistance, with a focus on self-assembling systems and nanoscale materials. The fundamental mechanisms of action, as well as the key advantages and disadvantages of each system are discussed, and attention is drawn to key examples within each field. As a result, this review provides a guide to the further design and development of antimicrobial systems, and outlines the interdisciplinary techniques required to translate this fundamental research towards the clinic
Characterising a Novel Interaction between Rap1b and Rhea sheds light on new Mechanisms for Focal Adhesion Assembly
Get PDFFor the first time, we reveal a direct interaction between Rap1b and the fly homolog of talin, Rhea. Using a combination of biochemical and biophysical techniques, the Rap1 binding site on Rhea has been successfully mapped. Additionally, we reveal that an acidic-to-basic K17E substitution, on Rhea, completely abolishes Rap1 binding. Our collaborators have shown that this mutation results in non-viable embryos and our data links the Rap1:Rhea interaction to this lethal phenotype. The implications of our findings support currently proposed mechanisms of RIAM-independent integrin activation, that would challenge our understanding of focal adhesion formation.
Furthermore, we propose a double-dependent Rap1 integrin-activation pathway, involving Rap1 directly interacting with the FERM domain, alongside the known Rap1-dependent recruitment of talin.
Optimisations have allowed us to express both the wild-type and mutant Rhea F0 domain in E.coli BL21(DE3) cells. Efficient purification via Ni-NTA-based affinity chromatography results in yields of ~50-60 mg/litre being obtained. Using circular dichroism, it is shown that substitution of the K17 residue does not interfere with the structural integrity of Rhea; both proteins have identical full spectrum measurements and Tm values.
Optimal expression of the conserved G-domain of mouse Rap1b was achieved in the CK600K cell line. This region is highly conserved to that in fly (90% identical). NMR was used to show direct interaction between drosophila Rhea F0 and Rap1b; whilst additionally confirming that Rap1b was unable to induce chemical shifts in the F0-K17E mutant. Triple resonance NMR experiments revealed the location of the Rap1 binding site on the wild-type Rhea F0, with V15, K17, T18, K37 and E40 being highlighted at the centre of this interaction. Structural models of Rap1:Rhea F0 binding agree with our findings, with the 5 highlighted residues seen to make close contact with the Rap1 switch I domain.
Together this work confirms a direct interaction between Rhea and Rap1 whilst providing biochemical validation for the lethal phenotypes observed in mutant flies. It also provides further insight into new mechanisms of focal adhesion formation and integrin activation
Interaction of Talin with Proteins Containing LD-Motifs
Get PDFTalin is a 250 kDa cytoplasmic protein that activates integrins and provides a link to cytoskeletal actin, thus producing the necessary force to stabilise adhesions. Talin is classically defined as an integrin-activator, but here we show that talin also couples adhesion assemblies to cortical microtubules via the Cortical Microtubule Stabilising Complex (CMSC) and plays a role in the mechano-regulation of the cell cycle.
Cross talk between cortical microtubules and focal adhesions (FA) plays a critical role in cell polarity and migration. Microtubules regulate the turnover of adhesions and, equally, FAs help capture and stabilise microtubules in their vicinity. The molecular basis for this mechanism was unknown and remained a key question within the field.
Here, I describe biochemical and biophysical evidence that the interaction between KANK and talin is the crucial link between the macromolecular assemblies FAs and CMSC. Fluorescence polarisation (FP) and Nuclear Magnetic Resonance (NMR) data show that the conserved KN domain in KANK1 binds to the talin rod domain R7 via a LD talin-binding motif. Through the design of point mutants in both the KANK1 KN domain and talin R7 domain this interaction could be perturbed. Our data show that the KANK1 KN domain binds to talin through a helix addition mechanism. Immunofluorescence work in HeLa cells corroborates our findings on the importance of this interaction, and that a single talin point mutation (G1404L) is enough to abrogate the association of FAs with the CMSC and, in turn, disrupts microtubule dynamics at the cell edge. The discovery of KANK1 as a binding partner of talin provides the missing link for how microtubules are targeted to FAs.
The talin:KANK interaction gave a new insight on LD talin-binding motifs and allowed us to develop a novel pipeline for identifying talin-binding partners. After designing a LD talin-binding search motif, we identified cyclin dependent kinase1 (CDK1) as a talin-binding protein. CDK1 is the master regulator of the cell cycle, helping to drive cells from G2 phase into mitosis. Using similar biochemical and biophysical techniques I characterise the interaction between talin and CDK1. FP and NMR show that CDK1 binds to the talin R8 domain via helices 32 and 33 and based on structural modelling, we propose that this interaction also occurs through helix addition.
Biochemical data combined with phosphoproteomics shows that the CDK1-cyclinA complex phosphorylates talin1 and talin2 isoforms at two unique sites (Ser1589 in talin1 and Ser1489 in talin2). Interestingly, in talin2 this novel phosphorylation site is in the talin actin-binding site (ABS2). We postulate that this could be a mechanism to regulate the coupling/uncoupling of actin to talin. Talin phosphorylation could perturb actin binding, thus reducing tension across the adhesions and leading to their disassembly. This interaction gives an insight into how adhesions and the cell cycle are entwined and poses many questions regarding how adhesion formation and disassembly regulates or occurs within the cell cycle.
My thesis describes the discovery of two novel talin-binding partners KANK and CDK1 which reveals talin is a crucial player in both coupling FAs to microtubules and gives new evidence into how adhesions can be involved in the regulation of the cell cycle
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Developing an innovative approach to identify novel talin-binding ligands in the brain
No full textCell adhesion, a process underlying the maintenance and development of multicellularity, is fundamentally dependent on the activity of talin as a mechanosensitive signalling nexus. Talin couples integrin-mediated cell-to-matrix adhesions to the actin cytoskeleton, facilitating the formation of integrin adhesion complexes, which act as sophisticated information processing centres. Variation of talin rod domain conformation, following adhesion, promotes different talin-mediated recruitment and signalling outputs, enabling the modification of a cell's internal programming. The recently published MeshCODE theory describes how information in the brain may be stored binarily via the mechanicalistic alteration of talin rod binary switches, highly enriched at every synapse. To validate this theory and discover a connection between neuronal activity and talin-mediated mechanical signalling, an innovative method, termed the talin 'fishing' rod experiment, was developed to identify novel talin-binding ligands in murine neuronal extract. Many proteins were identified as promising neuronal talin-binding ligands, including dynamin-1, synaptojanin-1, and myosin-Va, all of which exhibit particular significance to many aspects of synaptic transmission, particularly in the regulation of synaptic vesicle endo- and exocytosis. A bioinformatics pipeline and subsequent scoring system was also developed to accompany this experimental approach, enabling the identification of probable talin-binding sites in known synaptic vesicle proteins through I/LD motif evaluation. Whilst only weak binding was demonstrated between several talin rod domains and the proteins selected through bioinformatics analysis, it implies that both the bioinformatics pipeline and scoring system is effective in identifying promising talin-binding I/LD motifs. The identification of adhesome proteins, prior to this project, was restricted to cell lines and fibroblasts, limiting our knowledge of talin-binding ligands outside of these cell types. However, the talin 'fishing' rod experiment, developed in this project, can be utilised to help fill this current knowledge gap. Validation of the promising interactors, identified in this study, will support the concepts presented in the MeshCODE theory, potentially enabling the scientific community to achieve understanding regarding the enigma of memory storage at long last
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
