Biology subjects

Humphries, J. D.

Publications and source records attributed to Humphries, J. D..

12 recordsLinked to original sources

bioRxiv2026

PDLIM5 Modulates YAP1 Localisation and Fibrogenic Gene Expression in Hepatic Stellate Cells

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

cell biology↗

bioRxiv2025

Regulation of reticular adhesions by KANK2 and talin2 in two melanoma cell lines

Integrins bind to extracellular matrix proteins and, upon clustering, form multimolecular integrin adhesion complexes (IACs) that connect to and regulate the cell cytoskeleton, influencing various aspects of normal and tumour cell behaviour. Alongside well-characterized nascent adhesions, focal adhesions (FAs), fibrillar adhesions (FBs) and hemidesmosomes, a new class of IACs, reticular adhesions (RAs), have been identified. RAs, initially described as flat clathrin lattices formed by integrin V{beta}5, lack association with actin and are devoid of FAs markers. The physiological role of RAs in normal and tumor cells is still incompletely understood and requires further investigation. Previously, we analysed IACs of two melanoma cell lines, MDA-MB-435S and RPMI-7951, grown under long term culture conditions, and demonstrated that both cell lines preferentially use integrin V{beta}5 for adhesion. Here we present a comprehensive analysis of RAs in these two melanoma cell lines that differ in their ability to form FBs. To determine RAs composition, we treated cells with actin polymerisation inhibitor cytochalasin D (CytoD) which disrupts FAs, allowing isolation of RAs, which were analysed by MS-based proteomics, Western blotting and immunofluorescence. Known RA-associated proteins, including the AP-2 adaptor complex, disabled homolog 2 (DAB2) and Numb were identified in both lines, along with talin2. Notably, we also detected the presence of KN motif and ankyrin repeat domains protein (KANK2) in RA isolates. Proximity ligation analysis following CytoD-induced actin disruption confirmed the proximity of KANK2 and talin2 in RAs. We then investigated the effect of talin2 or KANK2 knockdown on RAs composition. While both talin2 and KANK2 are located in RAs, neither is essential for RA formation. Talin2 knockdown led to a reduction in RA components abundance in both cell lines. In MDA-MB-435S cell line, KANK2 produced a similar effect, mirroring the functional interaction of talin2 and KANK2 in FAs. However, in RPMI-7951 cells, KANK2 knockdown had no significant effect on RA components abundance. This discrepancy likely reflects the preferential localization of KANK2 in FBs and underscores the differing roles of talin2 and KANK2 in V{beta}5-mediated FAs across the two cell lines. These findings underscore the complexity of adhesion signalling and highlight the importance of adhesion crosstalk in regulating cellular function.

cell biology↗

bioRxiv2025

Hypoxia-induced extracellular matrix changes are conserved in cancer and directly impact radiotherapy benefit

Our overarching aim was to determine how hypoxia affects the extracellular matrix (ECM). Transcriptomic analysis (21,941 patients; 10 cancer types) identified ECM remodelling as the predominant pathway affected by hypoxia. Multi-omics confirmed that hypoxia impacts ECM organisation and collagen degradation; 53 ECM genes were affected, of which 74% were HIF1/HIF2-regulated. Spatial transcriptomics highlighted different hypoxia remodelling processes in tumour and stroma. Five ECM genes commonly affected in tumour and in vitro constituted a signature. This signature was independently prognostic and independently predictive of radiotherapy benefit in multiple malignancies. Patients within either high or low hypoxic-ECM score tertiles benefited from radiotherapy versus surgery. Hypoxic ECMs generated in vitro increased adhesion and decreased migration of cancer cells, an effect enhanced by irradiation. Immunofluorescence demonstrated that hypoxia decreased collagen fibre number, and irradiation decreased cell-ECM interactions. Taken together, these findings demonstrate hypoxia induces pan-cancer ECM changes, directly impacting radiotherapy benefit.

cancer biology↗

bioRxiv2025

A TRPV4-dependent calcium signaling axis governs lamellipodial actin architecture to promote cell migration

Cell migration is crucial for development and tissue homeostasis, while its dysregulation leads to severe pathologies. Cell migration is driven by the extension of actin-based lamellipodia protrusions, powered by actin polymerization, which is tightly regulated by signaling pathways, including Rho GTPases and Ca2+ signaling. While the importance of Ca2+ signaling in lamellipodia protrusions has been established, the molecular mechanisms linking Ca2+ to lamellipodia assembly are unknown. Here, we identify a novel Ca2+ signaling axis involving the mechano-gated channel TRPV4, which regulates lamellipodia protrusions in various cell types. Using Ca2+ and FRET imaging, we demonstrate that TRPV4-mediated Ca2+ influx upregulates RhoA activity within lamellipodia, which then facilitates formin-mediated actin assembly. Mechanistically, we identify CaMKII and TEM4 as key mediators relaying the TRPV4-mediated Ca2+ signal to RhoA. These data define a molecular pathway by which Ca2+ influx regulates small GTPase activity within a specific cellular domain - lamellipodia - and demonstrate the critical role in organizing the actin machinery and promoting cell migration in diverse biological contexts.

cell biology↗

bioRxiv2025

Localization of KANK2 to Focal or Fibrillar Adhesions Influences Migration in Melanoma Cell Lines

BackgroundIntegrins form focal adhesions (FAs) at the cell edge and fibrillar adhesions (FBs) located centrally. Talin 1 is essential to FAs, while talin2 is found in FAs and FBs. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics and are recruited to adhesions through interaction with talins. Previously, we showed in MDA-MB-435S melanoma cells that KANK2 is part of integrin V{beta}5 FAs, that its interaction with talin2 regulates actin-microtubule (MT) crosstalk and that KANK2 knockdown mimics the effect of integrin V or {beta}5 knockdown by reducing cell migration. Here, in another melanoma cell line RPMI-7951 we observed that KANK2 is part of FAs and FBs and that KANK2 knockdown increases cell migration. Therefore, we analyse integrin adhesion complexes in RPMI-7951 cells, explore the localization and role of KANK2 in FAs and FBs. MethodsKnockdown in human melanoma RPMI-7951 cells was achieved by transfection with gene-specific siRNAs. Integrin adhesion complexes were isolated and analysed by mass spectrometry. Immunofluorescence analysis, live cell imaging and the proximity ligation assays were done using confocal microscopy. Cell migration was assessed using Transwell Cell Culture Inserts. MTT assays were performed to determine cell sensitivity to paclitaxel. Data were statistically evaluated using one-way or two-way ANOVA or unpaired Students t-tests in GraphPad Software. ResultsWe demonstrate that RPMI-7951 melanoma cells use integrin V{beta}5 FAs and integrin 5{beta}1 FBs for adhesion, and that KANK2 is part of both structures. KANK2 is predominantly in proximity to talin1 at the cell edge (FAs) and in proximity to talin2 in the cell center (FBs). KANK2 in FAs functionally interacts with talin1 to maintain FAs, and with talin2 to regulate their dynamics. KANK2 is a component of FBs, and its knockdown mimics integrin 5 knockdown by increasing MT-dependent cell migration. ConclusionsOur study reveals the distinct roles of KANK2 in FAs and FBs. We show that KANK2 is a component of FBs, linking them to MTs and promoting their stabilisation. Loss of integrin 5 or KANK2 from FBs increases cell migration, a process that relies on the MT cytoskeleton. Plain English summaryCells adhere to their surroundings using specialized structures called adhesions, which are formed by different types of integrins and have different cell localisations: focal adhesions at the cell edge and fibrillar adhesions in the cell center. Talin1 is essential for focal adhesions, while talin2 is present in focal and fibrillar adhesions. KANK (kidney ankyrin repeat-containing) family proteins regulate adhesion dynamics by connecting talins to the microtubule cytoskeleton. Our study explored the different roles of KANK2 in focal or fibrillar adhesions, by focusing on its interactions with integrins and talins. We show that in RPMI-7951 cells, which form V{beta}5 focal and 5{beta}1 fibrillar adhesions, KANK2 is present in both of these adhesions. KANK2 supports V{beta}5 focal adhesion maintenance via talin1 and regulates their dynamics via talin2. KANK2 is also a component of 5{beta}1 fibrillar adhesions, and its depletion mimics integrin 5 knockdown and enhances cell migration, a process dependent on the microtubule cytoskeleton. In conclusion, our study demonstrates that KANK2 plays distinct roles in different adhesion structures, contributing to both focal and fibrillar adhesion function.

cell biology↗

bioRxiv2022

Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry

IntroductionVasculogenic mimicry (VM), the process of tumor cell trans-differentiation to endow endothelial-like characteristics supporting de novo vessel formation, is associated with poor prognosis in several tumor types, including small cell lung cancer (SCLC). In genetically engineered mouse models (GEMMs) of SCLC, NOTCH and MYC co-operate to drive a neuroendocrine (NE) to non-NE phenotypic switch and co-operation between NE and non-NE cells is required for metastasis. Here, we define the phenotype of VM-competent cells and molecular mechanisms underpinning SCLC VM using circulating tumor cell-derived explant (CDX) models and GEMMs. MethodsWe analysed perfusion within VM vessels and their association with NE and non-NE phenotypes using multiplex immunohistochemistry in CDX and GEMMs. VM-proficient cell subpopulations in ex vivo cultures were molecularly profiled by RNA sequencing and mass spectrometry. We evaluated their 3D structure and defined collagen-integrin interactions. ResultsWe show that VM vessels are present in 23/25 CDX models and in 2 GEMMs. Perfused VM vessels support tumor growth and only Notch-active non-NE cells are VM-competent in vivo and ex vivo, expressing pseudohypoxia, blood vessel development and extracellular matrix (ECM) organization signatures. On Matrigel, VM-primed non-NE cells re-model ECM into hollow tubules in an integrin {beta}1-dependent process. ConclusionsWe identify VM as an exemplar of functional heterogeneity and plasticity in SCLC and these findings take significant steps towards understanding the molecular events that enable VM. These results support therapeutic co-targeting of both NE and non-NE cells to curtail SCLC progression and to improve SCLC patient outcomes in future.

cancer biology↗

bioRxiv2021

Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin 6{beta}4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin {beta}4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for 6{beta}4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

cancer biology↗

bioRxiv2021

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms. SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.

cell biology↗

bioRxiv2020

A SNAI2-PEAK1 stromal axis drives progression and lapatinib resistance in HER2-positive breast cancer by supporting a cytokine expression profile that converges on PI3K/Akt signaling

Intercellular mechanisms by which the stromal microenvironment contributes to solid tumor progression and targeted therapy resistance remain poorly understood, presenting significant clinical hurdles. PEAK1 (Pseudopodium-Enriched Atypical Kinase One) is an actin cytoskeleton- and focal adhesion-associated pseudokinase that promotes cell state plasticity and cancer metastasis by mediating growth factor-integrin signaling crosstalk. Here, we determined that stromal PEAK1 expression predicts poor outcomes in HER2-positive breast cancers high in SNAI2 expression and enriched for MSC content. Notably, we identified that mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) express high PEAK1 protein levels and MSCs require PEAK1 to potentiate tumorigenesis, lapatinib resistance and metastasis of HER2-positive breast cancer cells. Analysis of PEAK1-dependent secreted factors from MSCs revealed a CCL4-, INHBA- and GDF5-focused network that converged on PI3K/Akt signaling. In this regard, we observed that MSC expression of PEAK1 is required for sustained Akt phosphorylation in neighboring HER2-positive breast cancer cells following lapatinib treatment. Finally, we uncovered a significant correlation between INHBA and PEAK1 expression levels in breast cancer, and that INHBA is an excellent predictor of disease relapse and decreased survival in HER2-positive tumors enriched for PEAK1 expression and MSC content. Importantly, we provide the first evidence that PEAK1 promotes tumorigenic phenotypes through a previously unrecognized SNAI2-PEAK1-INHBA-PI3K/Akt stromal to tumor cell signaling axis. These results establish a new, targetable intercellular mechanism that may be leveraged to improve targeted therapy responses and patient outcomes in breast cancer and other stroma-rich malignancies.

cancer biology↗

bioRxiv2020

Definition of the fibroblast adhesome using multiplexed proximity biotinylation

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry and topological organisation of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.

cell biology↗

bioRxiv2018

Eps8 is a convergence point integrating EGFR and integrin trafficking and crosstalk

Crosstalk between adhesion and growth factor receptors plays a critical role in tissue morphogenesis and repair, and aberrations contribute substantially to neoplastic disease. However, the mechanisms by which adhesion and growth factor receptor signalling are integrated, spatially and temporally, are unclear.\n\nWe used adhesion complex enrichment coupled with quantitative proteomic analysis to identify rapid changes to adhesion complex composition and signalling following growth factor stimulation. Bioinformatic network and ontological analyses revealed a substantial decrease in the abundance of adhesion regulatory proteins and co-ordinators of endocytosis within 5 minutes of EGF stimulation. Together these data suggested a mechanism of EGF-induced receptor endocytosis and adhesion complex turnover.\n\nCombinatorial interrogation of the networks allowed a global and dynamic view of adhesion and growth factor receptor crosstalk to be assembled. By interrogating network topology we identified Eps8 as a putative node integrating 5{beta}1 integrin and EGFR functions. Importantly, EGF stimulation promoted internalisation of both 5{beta}1 and EGFR. However, perturbation of Eps8 increased constitutive internalisation of 5{beta}1 and EGFR; suggesting that Eps8 constrains 5{beta}1 and EGFR endocytosis in the absence of EGF stimulation. Consistent with this, Eps8 regulated Rab5 activity and was required for maintenance of adhesion complex organisation and for EGF-dependent adhesion complex disassembly. Thus, by co-ordinating 5{beta}1 and EGFR trafficking mechanisms, Eps8 is able to control adhesion receptor and growth factor receptor bioavailability and cellular contractility.\n\nWe propose that during tissue morphogenesis and repair, Eps8 functions to spatially and temporally constrain endocytosis, and engagement, of 5{beta}1 and EGFR in order to precisely co-ordinate adhesion disassembly, cytoskeletal dynamics and cell migration.

cell biology↗

bioRxiv2017

Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies.

The ability to provide an unbiased qualitative and quantitative description of the global changes to proteins in a cell or an organism would permit the systems-wide study of complex biological systems. Label-free quantitative shotgun proteomic strategies (including LC-MS ion intensity quantification and spectral counting) are attractive because of their relatively low cost, ease of implementation, and the lack of multiplexing restrictions when comparing multiple samples. Owing to improvements in the resolution and sensitivity of mass spectrometers, and the availability of analytical software packages, protein quantification by LC-MS ion intensity has increased in popularity. Here, we have addressed the importance of chromatographic alignment on protein quantification, and then assessed how spectral counting compares to ion intensity-based proteomic quantification. Using a spiked-in protein strategy, we analysed two situations that commonly arise in the application of proteomics to cell biology: (i) samples with a small number of proteins of differential abundance in a larger non-changing background, and (ii) samples with a larger number of proteins of differential abundance. To perform these assessments on biologically relevant samples, we used isolated integrin adhesion complexes (IACs). Technical replicate analysis of isolated IACs resulted in a range of alignment scores using the Progenesis QI software package and demonstrated that higher LC-MS chromatographic alignment scores increased the precision of protein quantification. Furthermore, implementation of a simple sample batch-running strategy enabled good chromatographic alignment for hundreds of samples over multiple batches. Finally, we applied the sample batch-running strategy and compared quantification by LC-MS ion intensity to spectral counting and found that quantification by LC-MS ion intensity was more accurate and precise. In summary, these results demonstrate that chromatographic alignment is important for precise and accurate protein quantification based on LC-MS ion intensity and accordingly we present a simple sample re-ordering strategy to facilitate improved alignment. These findings are not only relevant to label-free quantification using Progenesis QI but may be useful to the wide range of MS-based quantification strategies that rely on chromatographic alignment.

bioinformatics↗