Biology subjects

Shaw, T. J.

Publications and source records attributed to Shaw, T. J..

6 recordsLinked to original sources

bioRxiv2025

Neuroblastoma-associated ALK variants have distinct cellular and biochemical activities

Mutations in anaplastic lymphoma kinase (ALK) are associated with high-risk neuroblastoma, a childhood cancer arising in trunk neural crest cells. The role of ALK in undifferentiated NC is still unknown; however, the presence of activating mutations in ALK correlates with migratory and invasive cell behaviours in neuroblastoma cell lines. Here, we show the functional consequences of ALK overexpression on neural crest cells, by comparing wildtype ALK (ALKWT) protein to ALK gain-of-function variants ALKF1174L and ALKR1275Q. Elevated ALK activity, independent of mutational status leads to increased migration velocity and loss of directionality, while ALKF1174L overexpression presents additional effects on cytoskeletal protrusions. These results correlate with increased binding of ALKF1174L to GSK3, which has previously been shown to regulate cytoskeletal dynamics in neural crest cells. Further, molecular dynamics simulations of the ALK-GSK3 complex show high flexibility, suggestive of enhanced allosteric regulation. Together, our data show that activating mutations in ALK drive migratory changes in trunk NC cells, potentially mediated by its novel interacting partner GSK3. Significance StatementAnaplastic lymphoma kinase (ALK)-associated neuroblastoma phenotypes arise in the neural crest lineage, but the effects on neural crest cell behaviours are not well-studied. Here, we use primary mouse neural crest cells to study the functional relevance of ALK-activating mutations on cell migration, which we then biochemically link to interactions with GSK3-isoforms. We also directly compare the activities of wild-type ALK with two gain-of-function variants associated with metastatic disease, ALKF1174L and ALKR1275Q, and find that ALKF1174L causes increased cytoskeletal protrusions and GSK3 binding. Our study provides insights into a signalling pathway in neural crest that drives cytoskeletal rearrangements and pathological migration in neuroblastoma.

biochemistry↗

bioRxiv2025

Single-cell RNA sequencing clarifies dermal fibroblast subset representation in vitro and reveals variable persistence of keloid disease-associated features

In vitro models of scarring and fibrosis are essential to improve our understanding of disease mechanisms and ultimately develop much-needed effective therapeutic strategies. This is particularly true for keloids, the example of pathological scarring exploited in this study, as there is no animal model. Our emerging appreciation of fibroblast heterogeneity from single cell RNA sequencing (scRNA-seq) information leaves a knowledge gap about what is represented in typical fibroblast cultures. Specifically, it is important to know whether quantitative differences in fibroblast subtypes observed in pathological tissues are represented and/or whether disease-associated molecular alterations of subtypes are maintained. This study performed scRNA-seq on patient-matched keloid and normal adjacent dermis immediately following surgical removal, which was compared to sc- and bulk-RNA-seq on primary dermal fibroblast cultures from the same samples after 4+ passages. Freshly dissociated tissue showed anticipated differences in cell proportions in keloid versus normal skin; however, comparably for both tissue types, there was an assimilation of fibroblast subtypes after culture. Cultured cells clustered conspicuously from the original populations, with evidence of only minor heterogeneity persisting. Cells displayed, to varying degrees, elements of each of the original subset signatures, with FAP+/SFRP2+ mesenchymal features the strongest. Pseudo-bulk analysis of mesenchymal subpopulations ex vivo showed cell-intrinsic keloid versus normal skin transcriptional differences consistent with current disease understanding; however, only a subset of these persisted in vitro. Cell-cell communication analysis provides potential strategies to maintain specific cell populations and their in vivo phenotypes. As an example, we report that culture with ascorbic acid (stimulating cell-derived extracellular matrix) enriched the mesenchymal signature. The data presented herein provide resources supporting greater understanding of, and strategies to refine, essential human fibroblast culture models.

cell biology↗

bioRxiv2023

Characterisation and correction of polarisation effects in fluorescently labelled fibres

Many biological structures take the form of fibres and filaments, and quantitative analysis of fibre organisation is important for understanding their functions in both normal physiological conditions and disease. In order to visualise these structures, fibres can be fluorescently labelled and imaged, with specialised image analysis methods available for quantifying the degree and strength of fibre alignment. Here we show that fluorescently labelled fibres can display polarised emission, with the strength of this effect varying depending on structure and fluorophore identity. This can bias automated analysis of fibre alignment and mask the true underlying structural organisation. We present a method for quantifying and correcting these polarisation effects without requiring polarisation resolved microscopy, and demonstrate its efficacy when applied to images of fluorescently labelled collagen gels, allowing for more reliable characterisation of fibre microarchitecture.

cell biology↗

bioRxiv2023

A biomechanical switch promotes lysosomal remodelling and exocytosis in keloid fibroblasts

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM remodelling, leading to constitutive collagen and fibronectin deposition. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

cell biology↗

bioRxiv2023

Multi-scale spatial mapping of cell populations across anatomical sites in healthy human skin and basal cell carcinoma

Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this we have created a multi-scale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single cell RNA sequencing, spatial global transcriptional profiling and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community. Significance statementSingle cells RNA sequencing has revolutionised cell biology, enabling high resolution analysis of cell types and states within human tissues. Here, we report a comprehensive spatial atlas of adult human skin across different anatomical sites and basal cell carcinoma (BCC) - the most common form of skin cancer - encompassing in vivo optical coherence tomography, single cell RNA sequencing, global spatial transcriptomic profiling and in situ sequencing. In combination these modalities have allowed us to assemble a comprehensive nuclear-resolution atlas of cellular identity in health and disease.

cell biology↗

bioRxiv2021

A Workflow for Rapid Unbiased Quantification of Fibrillar Feature Alignment in Biological Images

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT - Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

biophysics↗