Biology subjects

Ball, N. J.

Publications and source records attributed to Ball, N. J..

7 recordsLinked to original sources

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↗

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↗

bioRxiv2019

Btbd6-dependent Plzf recruitment to Cul3 E3 ligase complexes through BTB domain heterodimerization

The Cul3 adaptor Btbd6 plays crucial roles in neural development by driving the ubiquitin-dependent degradation of promyelocytic zinc finger transcription factor (Plzf). Btbd6 has conserved motifs, BTB-BACK-PHR, and by analogy with other BTB-BACK adaptors, might be expected to bind to Cul3 through the BTB-BACK domain, and to substrate through the PHR domain. However, we now present a mode of adaptor-substrate interaction through heterodimerisation between the normally homodimeric BTB domains of Btbd6 and Plzf. This heterodimerization appears to occur through monomer exchange that is detected only at or near physiological concentrations. The Btbd6-Plzf heterodimer thus formed assembles into a ternary complex with Cul3. In addition we show that the BTB and PHR domains of Btbd6 promote localisation in the nucleus and that the BACK domain contains a nuclear export signal. Our findings support a model whereby Btbd6 moves into and out of the nucleus, iteratively sweeping Plzf into the cytoplasm and enabling complex formation with Cul3 that presents Plzf for ubiquitination.\n\nHighlightsO_LIA general mechanism for recruitment of BTB domain-containing substrates by BTBdomain adaptors for the Cul3 E3 ligase complex\nC_LIO_LINuclear export of the Plzf/Btbd6 complex mediated by a NES within the Btbd6 BACK domain\nC_LIO_LICul3-dependent Plzf ubiquitylation through heterodimerisation of BTB domains on adaptor and substrate by monomer exchang\nC_LI

biochemistry↗