Biology subjects

Goult, B. T.

Publications and source records attributed to Goult, B. T..

At least 19 recordsLinked to original sources

bioRxiv2026

Subcellular pharmacology of a novel cell-permeable fluorescent phosphodiesterase inhibitor using advanced fluorescence microscopy

Direct visualisation of drug-target engagement within living cells remains a major challenge. Here we develop a cell-permeable fluorogenic analogue of the phosphodiesterase 4 (PDE4) inhibitor rolipram (MAN193), generated by conjugation to fluorescein diacetate. Following intracellular activation, the probe functions as a near-neutral PDE4 antagonist and enables direct visualisation of endogenous PDE4 populations. Using advanced fluorescence spectroscopy imaging approaches, including molecular brightness analysis and fluorescence anisotropy imaging, we demonstrate rolipram-displaceable binding of the analogue to cytosolic PDE4 and resolve thesubcellular distribution of binding sites. We further show that the probe enables visualisation of untagged PDE4 at focal adhesion complexes in cardiomyocyte-like cells. Together these findings establish fluorogenic drug conjugation combined with quantitative spectroscopy imaging as a generalisable strategy to map intracellular drug-target engagement with subcellular resolution, providing an effective framework for interrogating the spatial pharmacology of small molecules in living systems.

biophysics↗

bioRxiv2026

An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

SHANK proteins form core postsynaptic density (PSD) scaffolds that organise synaptic signalling complexes through multiple protein-protein interaction domains, including PDZ domains that typically recognise C-terminal peptide motifs. Here, we identify an internal PDZ recognition mechanism that links the synaptic protein Densin-180 to SHANK and promotes SHANK scaffold assembly. We map SHANK binding to an internal PDZ-binding motif in Densin-180 (residues 843-863) and show by NMR spectroscopy and fluorescence polarisation that this motif binds SHANK1-3 PDZ domains with high affinity and specificity, competing with canonical C-terminal ligands. The crystal structure of the Densin-180-SHANK complex reveals that Phe858 inserts into the hydrophobic pocket of the PDZ domain despite the absence of a terminal carboxylate. In neurons, this interaction mediates Densin-180 recruitment to dendritic spines and drives activity-dependent reorganisation of postsynaptic SHANK3 assemblies, whereas mutation of Phe858 disrupts both processes. Disrupting the Densin-180-SHANK interaction using a Densin-180-derived peptide impairs activity-dependent structural plasticity of spines, accompanied by defects in PSD organisation and actin cytoskeleton remodelling. These findings define an internal mode of PDZ recognition and reveal how Densin-180 couples neuronal activity to the remodelling of postsynaptic SHANK scaffolds.

neuroscience↗

bioRxiv2026

The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules

KANK proteins link integrin adhesions to the cortical microtubule stabilising complex (CMSC) through interactions with the adhesion adaptor talin. However, how KANK proteins are regulated remains unclear. Here we show that the KN domain of KANK proteins contains separable regions that mediate talin binding and a conserved intramolecular interaction. Using fluorescence polarisation, NMR spectroscopy and structural analysis, we map an interaction between the N-terminal KN domain and the C-terminal ankyrin repeat domain and identify residues 60-68 of the KN domain as required for this intramolecular interaction. In contrast, the canonical LD motif within residues 30-60 mediates binding to talin. Deletion of residues 60-68 disrupts the intramolecular interaction while preserving talin binding, demonstrating that the KN domain contains distinct modules for talin engagement and intramolecular regulation. This regulatory architecture is conserved across the KANK family, although sequence variation modulates the strength of the intramolecular interaction. Together, these findings identify a modular organisation within the KANK KN domain that separates talin recognition from intramolecular regulation and is consistent with an autoinhibitory mechanism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/731086v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@e2dd22org.highwire.dtl.DTLVardef@240996org.highwire.dtl.DTLVardef@1f1926borg.highwire.dtl.DTLVardef@c3e523_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO The KN domain of KANK proteins contains separable talin-binding and intramolecular interaction modules.The LD motif (blue) mediates binding to the talin R7 domain, whereas residues 60-68 (yellow) are required for interaction with the C-terminal ankyrin repeat domain. An AlphaFold model is shown as a structural interpretation of the intramolecular KN-ankyrin repeat interaction identified in this study. C_FIG

biochemistry↗

bioRxiv2026

Talin controls the spatial distribution of vinculin tension in focal adhesions

Cells transmit force between the extracellular matrix and the actin cytoskeleton through integrin adhesion complexes centred on talin and vinculin. Vinculin binds talin through -helical vinculin-binding sites (VBS) that are exposed when talin rod domains unfold under force. Dissecting the significance of this interaction has relied heavily on the A50I mutation in vinculin, which has been widely used as a talin-binding-null mutant. Here we show that although the A50I mutation abolishes binding to the -catenin VBS, it retains nanomolar-affinity binding to multiple talin VBS. We therefore designed an improved mutant, I12K/A50I, that eliminates this residual talin binding. Biochemical assays and single-molecule stretching experiments demonstrate that I12K/A50I VD1 fails to bind talin even when VBS are exposed by force. Using vinculin tension and conformation sensors, we show that talin binding is required for efficient recruitment of vinculin to focal adhesions and for establishing spatial gradients of vinculin tension. However, vinculin can still experience mechanical load in the absence of talin binding. These results demonstrate that A50I is not a talin-binding-null and reveal that, while talin is not required for vinculin loading, it is essential for organising the spatial distribution of mechanical load within adhesion complexes.

cell biology↗

bioRxiv2026

An Aggregation Prone Region (APR) in talin controls talin self-interactions to regulate integrin adhesion complex dynamics

The tight regulation of the integrin family of extracellular matrix (ECM) receptors is essential for the coordination of most cellular processes. Talin regulates integrin function by binding to the cytoplasmic tail of the integrin beta subunit. Two integrin-binding sites (IBS) in talin have been reported, one in the talin head domain (IBS1), and a second in the C-terminal rod region of talin (IBS2), mapped to the R11-R12 domains. Whilst the structural details of integrin binding to IBS1 are well understood, IBS2s mode of binding integrin is less clear, and the biochemical details of this site have been elusive. Here we report that talin R11 contains a cryptic high affinity talin-binding site. We show that mechanical unfolding of R11 exposes an Aggregation Prone Region (APR) in Helix50 that oligomerizes with other talin R11s. Our data support a model whereby the process of mechanically unfolding one R11, exposes and maintains the APR in a high affinity conformation that drives unfolding of other R11 domains and oligomerisation. Atomic Force Microscopy confirms that the APR region alone forms micron long amyloid-like fibres, via a beta-sheet amyloid arrangement which can be abolished using a canonical "gatekeeper" point mutation, V2078K, that prevents APR interactions. Introducing this gatekeeper mutant in talin eliminates recruitment of talin IBS2 to integrin adhesion sites. The APR region in talin overlaps with a vinculin-binding site in Helix50, and we show a novel role for vinculin as a mechano-chaperone for talin, able to resolve the talin large species by binding to the VBS in Helix50. In light of this new information, we propose that the ability of R11-R12 to target to integrin adhesion complexes and enhance integrin activation can be better explained by a model where functional aggregation of talin serves to recruit and activate more talin molecules at the adhesion site leading to enhanced integrin activation. Whilst further work is required to fully exclude integrin binding to R11 we suggest that the name IBS2 might be a misnomer and propose the name Aggregation Prone Region 1 (APR1) for this talin oligomerisation motif.

cell biology↗

bioRxiv2025

Filopodome proteomics identifies CCT8 as a MYO10 interactor critical for filopodia functions

Cancer cells utilize filopodia to explore, adhere to, and invade their surrounding microenvironment, yet the protein networks that organize these protrusions remain incompletely defined. To uncover the molecular machinery underlying MYO10-positive filopodia, we targeted the fast biotin ligase TurboID to the filopo-dia tip-localized motor protein MYO10. Proximity biotinylation in two cell types revealed hundreds of potential MYO10 interactors. Surprisingly, there was limited overlap between the cell lines, indicating a previously unknown level of cell-type specificity in filopodia composition. A targeted microscopy and siRNA screen identified MINK1, SCRIB, CSNK1A1, and CCT8 as new regulators of filopodia formation. Focusing on one common interactor between cell lines, CCT8, known as a subunit of the chaperonin TRiC (TCP1 Ring Complex), we found that CCT8 associates with the MYO10 motor domain and regulates MYO10 filopodia independently of TRiC. Depleting CCT8 affected filopodia dynamics and impaired cell spreading, migration, and invasion in breast cancer cells. These findings establish CCT8 as a TRiC-independent regulator of MYO10 filopodia across different cancer cell types, highlight the surprising cell-type-specificity of filopodia composition, and provide a strategy and resource for studying filopodia in various biological contexts.

cell biology↗

bioRxiv2025

Cyclin-dependent kinase-like 5 (CDKL5) binds to talin and is anchored at the postsynaptic density via direct interaction with PDZ domains

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine kinase essential for brain development and function. Mutations in the CDKL5 gene cause CDKL5 deficiency disorder (CDD), a severe early-onset epileptic encephalopathy characterised by defects in synapse formation and function. Despite extensive research, the molecular mechanisms by which CDKL5 mutations disrupt synaptic function and lead to epilepsy remain unclear. Here, we report that the major neuronal isoform of CDKL5 contains a C-terminal PDZ domain-binding motif. We demonstrate that this motif mediates interactions with the PDZ domains of PSD-95 and SHANK proteins, facilitating the recruitment of CDKL5 to the postsynaptic density. Disruption of CDKL5s PDZ-binding motif results in its mislocalisation and impaired spine formation. Additionally, we show that CDKL5 directly interacts with the mechanosensitive synaptic scaffold protein talin, via the N-terminal kinase domain of CDKL5 and the R8 rod domain of talin. Our findings establish how CDKL5 is targeted to synapses and suggest that its activity may be spatially regulated through talin-mediated mechanical signalling. We propose that the spatial positioning of the CDKL5 kinase domain might be mechanically-operated and regulated by talin domain unfolding. As talin undergoes structural transitions in its force-dependent binary switch domains, the kinase domain bound to R8 would be moved up and down within the synaptic compartment as a function of the changing talin conformation. These insights enhance our understanding of the pathogenic mechanisms underlying CDKL5 variants with premature stop codons and highlight the need to re-evaluate studies that have used C-terminally tagged or the non-PDZ-binding isoform of CDKL5 to assess its neuronal function. O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/672337v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@a11d72org.highwire.dtl.DTLVardef@19428b7org.highwire.dtl.DTLVardef@1fd1693org.highwire.dtl.DTLVardef@d5299c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Cyclin-dependent kinase-like 5 (CDKL5) is a mechanically regulated enzyme. The C-terminal PDZ-binding motif in CDKL5 anchors CDKL5 at the postsynaptic density (PSD). (A) The large unstructured region serves to tether the CDKL5 kinase domain at the synapse. i) The kinase domain is restricted to the blue semi-circle, giving CDKL5 a zone of activity, with radius the length of the tether (>200 nm). ii) Epilepsy-causing mutations that introduce premature stop codons untether CDKL5. (B) The mechanosensitive synaptic scaffold protein talin is also tethered to the membrane by interaction with integrins and/or amyloid precursor protein (APP). The CDKL5-binding domain, R8 is shown in dark green. The force-dependent binary switch domains in the talin rod region open and close in response to force and change the length of the molecule and the location of the CDKL5-binding site. (C) As talin changes its conformation, the CDKL5-binding site on R8 is moved up and down within the synapse, as a result the kinase activity of CDKL5 is targeted to discrete locations within the synapse. Loss of this localisation or incorrect positioning of the kinase within the synapse would result in loss of synchronisation of the synapse in the context of the circuit it is part of and, we propose, result in re-entry circuits and epilepsy. See also Supplementary Movie 1. C_FIG

neuroscience↗

bioRxiv2025

Optogenetic control of mechanotransduction based on light-induced homodimerization of talin

Integrin-based cell adhesions (IACs) serve as primary sites where piconewton-scale actomyosin-generated mechanical forces are transmitted to the extracellular matrix (ECM), generating traction forces that drive cell-ECM responses including adhesion, migration, and mechano-signaling. Talin, a large (270 kDa) cytosolic adaptor protein, is the principal force-transmission protein in integrin-based adhesions, containing multiple mechanosensitive domains and protein-protein interaction sites that orchestrate molecular events in mechanosensing. As a highly modular multi-domain protein, talin has been identified as an effective target for chemogenetic and optogenetic manipulation of integrin-based mechanotransduction. However, a key limitation of previous approaches is the reliance on heterodimerization modules to control talin function, requiring the expression of two modified talin fragments. In practice, achieving precise expression levels in such a 2-component approach can be challenging, particularly when combined with other genetic tools. Since talin naturally contains a C-terminal dimerization domain that forms part of its actin-binding site, we reasoned that the molecularly engineered talin with a C-terminal optically-controlled homodimerizer could enable single-component optogenetic control of mechanotransduction. This approach would facilitate multiplexing with other molecular perturbations or experimental techniques. Here, we describe an opto-homodimerizable talin based on the pdDronpa1.2 optogenetic module, which enables optogenetic control of talin by a single construct. We demonstrate that light-induced talin dimerization promotes talin recruitment to IACs, adhesion formation, actin retrograde flow engagement, and downstream mechanotransduction signaling. Conversely, light-induced talin monomerization rapidly disassembles focal adhesions, disrupts talin-actin linkages, and accelerates actin retrograde flow, underscoring the critical roles of talin dimerization. Furthermore, our single-construct design allows facile multiplexing of optogenetic modulation of integrin-mediated mechanotransduction with super-resolution single-molecule tracking, revealing the essential role of talin dimerization for integrin v{beta}5 engagement.

cell biology↗

bioRxiv2024

The structure of an Amyloid Precursor Protein/talin complex indicates a mechanical basis of Alzheimer's Disease.

Misprocessing of Amyloid Precursor Protein (APP) is one of the major causes of Alzheimers disease. APP is a transmembrane protein comprising a large extracellular region, a single transmembrane helix and a short cytoplasmic tail containing an NPxY motif (normally referred to as the YENPTY motif). Talins are synaptic scaffold proteins that connect the cytoskeletal machinery to the plasma membrane via binding to one of two highly conserved NPxY motifs in the cytoplasmic tail of integrin transmembrane receptors. Here we report the crystal structure of an APP/talin1 complex identifying a new way to couple the cytoskeletal machinery to synaptic sites via APP. Proximity Ligation Assay (PLA) confirmed the close proximity of talin1 and APP in primary neurons, and we show that talin1 depletion has a dramatic effect on APP processing in cells. Structural modelling indicates that APP has the capacity to form an extracellular meshwork that mechanically couples the cytoskeletal meshworks of both the pre-, and post-synaptic compartments. In this context, we propose APP processing as a mechanical signalling pathway with similarities to Notch signalling, whereby the cleavage sites in APP represent mechanical sensors, with varying accessibility to cleavage by secretases. During synaptogenesis in healthy neurons, the APP/talin linkage would provide an exquisite mechanical coupling between synapses, with tightly controlled APP processing providing instructions to maintain this synchrony. Furthermore, APP directly coupling to the binary switches in talin indicates a role for APP in mechanical memory storage as postulated by the MeshCODE theory. The implication that APP is a regulator of mechanical signalling leads to a new hypothesis for Alzheimers disease, where mis-regulation of APP dynamics results in loss of mechanical integrity of the synapse, corruption and loss of mechanical binary data, and excessive generation of the toxic plaque-forming A{beta}42 peptide. Much needs to be done to experimentally validate this idea, but we present here a novel theory of Alzheimers Disease with a role for APP in the mechanically coded binary information storage in the synapse, which identifies a potential novel therapeutic strategy for treating Alzheimers Disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/583202v4_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@ba0768org.highwire.dtl.DTLVardef@13c4672org.highwire.dtl.DTLVardef@352178org.highwire.dtl.DTLVardef@288bf6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Graphical Abstract Legend: Amyloid Precursor Protein (APP) forms an extracellular meshwork that provides a coupling between the mechanical signalling machinery of the pre- and post-synaptic neurons.A) APP binds to talin indicating that APP will experience mechanical forces which leads us to propose that APP processing is a Notch-like signalling pathway that maintains mechanical homeostasis at the synapse. B) The crystal structure of APP bound to talin F2F3. C) APP is part of the adhesion complex that attaches talin to the membrane. D) The loss of adhesion integrity in the synapse, or other factors that lead to misprocessing of APP will perturb this coupling and contribute to Alzheimers Disease. See also Movie 1. C_FIG

neuroscience↗

bioRxiv2023

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is primarily expressed in the vasculature in vivo and that its depletion leads to vascular abnormalities in vivo and loss of barrier integrity in cultured endothelial cells. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct, high-affinity interaction with CCM2. Modeling and functional testing of TLNRD1 and CCM2 mutants reveal that their interaction is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the 4-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Notably, a CCM2 pathogenic mutation linked to vascular dementia in patients maps to the interface and disrupts the interaction. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity by controlling TLNRD1 bundling activity.

cell biology↗

bioRxiv2023

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

cell biology↗

bioRxiv2023

The focal adhesion protein talin is a mechanically-gated A-kinase anchoring protein (AKAP)

The cAMP-dependent protein kinase (Protein Kinase A; PKA) is a ubiquitous, promiscuous kinase whose activity is focused and specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to the extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), intracellular complexes coupling ECM-bound integrins to the actin cytoskeleton, suggesting the existence of one or more FA AKAPs. Using a combination of a promiscuous biotin ligase fused to PKA type-II regulatory (RII) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 -helices bundled into 13 rod domains, R1-R13. Direct binding assays and nuclear magnetic resonance spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Finally, single-molecule experiments with talin1 and PKA, and experiments in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. These observations identify the first mechanically-gated anchoring protein for PKA, a new force-dependent binding partner for talin1, and thus a new mechanism for coupling cellular tension and signal transduction.

cell biology↗

bioRxiv2023

The mechanical response of vinculin

Vinculin is a mechanosensitive adapter protein that links the actin network to cell-extracellular matrix adhesions and cell-cell adhesions. It is perhaps the best characterized mechanoeffector, as it is recruited to sites of adhesion in response to force on the mechanotransducers talin and alpha-catenin. Here we examined the mechanical properties of vinculin to assess its potential role as a mechanotransducer. We find that at physiological loading rates, the structural domains of vinculin unfold at forces in the 5-15 pN range and rapidly refold when forces are reduced back to 1 pN. Thus, vinculin domains also have the potential to act as force dependent molecular switches, akin to those in talin and alpha-catenin. As with the force dependent switches in talin, the unfolding of these domains in vinculin introduces large extension changes in the vinculin cytoskeletal linkage up to 150 nm with 20-30 nm steps of unfolding. Modelling of the tension-dependent interactions of the unstructured vinculin linker region with a model protein containing two SH3 domains indicated that even unstructured protein regions can mediate force-dependent interactions with ligands, where the binding of a dual-SH3 model protein is predicted to be significantly suppressed by forces greater than 10 pN. Together, these findings suggest that vinculin has a complex mechanical response with force-dependent interaction sites, suggesting it also acts as a mechanotransducer, recruiting partners in response to force.

biophysics↗

bioRxiv2023

The structural basis of the Talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions.

Adhesion between cells and the extracellular matrix (ECM) is mediated by heterodimeric ({beta}) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organises cytosolic signalling proteins into discrete complexes on {beta}-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we developed a novel crystallographic method to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN motif of KANK1 has a novel fold, where a {beta}-turn stabilises the -helical region, explaining its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localises throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.

biochemistry↗

bioRxiv2022

Next generation protein-based materials capture and preserve projectiles from supersonic impacts

Extreme energy dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation. To resolve these limitations we have turned to nature, utilising proteins that have evolved over millennia to enable effective energy dissipation. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in the production of the first reported example of a talin shock absorbing material (TSAM). When subjected to 1.5 km/s supersonic shots, TSAMs were shown not only to absorb the impact, but to capture/preserve the projectile, making TSAMs the first reported protein material to achieve this.

synthetic biology↗

bioRxiv2022

A novel cancer-associated cassette exon in TLN1 alters Talin 1 mechanosensitivity

Talin 1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b results in an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1-R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics. Finally, we show that the TGF-{beta}/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

cancer biology↗

bioRxiv2022

Myosin-X recruits lamellipodin to filopodia tips

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. Yet, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped RAPH1 interaction with adhesome components to its talinbinding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises an area with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not involved in regulating integrin activity in filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

cell biology↗

bioRxiv2022

The MeshCODE to scale - Visualising synaptic binary information.

The Mercator projection map of the world provides a useful, but distorted, view of the relative scale of countries. Current cellular models suffer from a similar distortion. Here, we undertook an in-depth structural analysis of the molecular dimensions in the cells computational machinery, the MeshCODE, that is assembled from a meshwork of binary switches in the scaffolding proteins talin and vinculin. Talin contains a series of force-dependent binary switches and each domain switching state introduces quantised step-changes in talin length on a micrometre scale. The average dendritic spine is 1 {micro}m in diameter so this analysis identifies a plausible Gearbox-like mechanism for dynamic regulation of synaptic function, whereby the positioning of enzymes and substrates relative to each other, mechanically-encoded by the MeshCODE switch patterns, might control synaptic transmission. Based on biophysical rules and experimentally derived distances, this analysis yields a novel perspective on biological digital information.

neuroscience↗