Supplementary Data for PhD Thesis: Myeloid cell ageing and wound healing: analysing ageing-related changes to myeloid cells and their impact on cutaneous wound healing

Single-cell RNA sequencing was peformed in young and mouse skin and Day 3 wounds to characterise the effects of ageing on healing progression. Differential expression gene lists for all cell populations charaterised in the thesis: Myeloid cell ageing and wound healing: analysing ageing-related changes to myeloid cells and their impact on cutaneous wound healing are included in this metadat

MicroCT of stained bovine IVD quarter segments after 2 weeks incubation

Visualising the 3D microstructure of stained and native intervertebral discs using X-ray microtomography C.M. Disney 1,2, K. Madi 3, A.J. Bodey 4, P.D. Lee 3, J.A. Hoyland 2,5, M.J. Sherratt 2 1 Centre for Doctoral Training in Regenerative Medicine, 2 Division of Cell Matrix Biology and Regenerative Medicine, 3 School of Materials, University of Manchester, UK. 4 Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire, UK. 5 NIHR Manchester Biomedical Research Centre, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, UK. A comparison of three stains; iodine potassium iodide (I2KI), phosphomolybdic acid (PMA) and phosphotungstic acid (PTA). Chemically fixed (10% formal saline) quarter segments of bovine tail intervertebral disc were incubated for two weeks before being imaged by laboratory microCT. Reconstruction software (Phoenix dato s|x2 reconstruction) was used to generate 3D datasets from the projections. This reconstructed data relates to figure 3 and is saved as 8-bit .raw with image dimensions given in the file name

Mesenchymal stem cell integrin-associated complexes

The composition and stiffness of the extracellular matrix (ECM) environment that surrounds Multipotent mesenchymal stem cells (MSCs) stem cells dictates transcriptional programming, thereby affecting stem cell lineage decision-making. Cells sense force between themselves and their microenvironment controlling the capability of MSCs to differentiate into adipocytes, osteocytes and chondrocytes. Force sensing is transmitted by integrin receptors and their associated adhesion signalling complexes. To identify regulators of MSC force sensing we sought to catalogue MSC adhesion complex composition. Therefore we isolated integrin-associated adhesion complexes formed in MSCs plated on the ECM ligand fibronectin. We identifed proteins using mass spectrometry that define a MSC specific subset of adhesion complex proteins consisting of key linkages to the actin cytoskeleton together with integrin signalling and force sensing components

PXD025280

Cells respond to stress by synthesising chaperone proteins that correct protein misfolding to maintain function. However, protein homeostasis is lost in ageing, leading to aggregates characteristic of protein-folding diseases. Whilst much is known about how these diseases progress, discovering what causes protein-folding to deteriorate could be key to their prevention. Here, we examined primary human mesenchymal stem cells (hMSCs), cultured to a point of replicative senescence and subjected to heatshock, as an in vitro model of the ageing stress response. We found through proteomic analysis that the maintenance of homeostasis deteriorated in senescent cells, coincident with lowered levels of a functional module of chaperone proteins associated with heat shock protein 70 kDa (HSPA1A). Further analysis of the temporal dynamics of the proteomic and transcriptomic stress response revealed a lack of translational capacity to be a limiting factor in the capacity of senescent cells to mitigate proteotoxic stress

Primary Human MSCs subjected to 1 hr intense cyclic tensile strain with/without 24 hr recovery

Cells resident in tissues must be resilient to the physical demands of their surroundings. Our current understanding of cellular mechano-signalling is largely based on static systems, but these models do not reproduce the dynamic nature of living tissue. Here, we examined the time-resolved response of primary human mesenchymal stem cells (hMSCs) to periods of cyclic tensile strain (CTS). We observed parallels between morphological changes following low-intensity strain (1 hour, 4% CTS at 1 Hz) and responses to increased substrate stiffness. However, as the strain regime was intensified (CTS at ≥ 2 Hz), we characterised a broad, structured and reversible protein-level response, even as transcription was apparently shut down. Regulation of the linker of nucleo- and cytoskeleton (LINC) complex proteins, and specifically of SUN domain-containing protein 2 (SUN2), was found to decouple mechano-transmission within the cell and hence isolate the nucleus from cellular deformation

U2OS Adhesion Isolations +/- CytochalasinD and Neomycin

U2OS cells preferentially utilise integrin alphaV beta5 in adhesion complexes when grown under standard cell culture conditions for 3 days. AlphaV beta5 can be found in both classical 'Focal' adhesions and in novel 'Reticular' adhesions that do not associate with F-actin or other known adhesion protein components. In order to identify components of reticular adhesions, cells were treated with cytochalasinD in order to disrupt F-actin and promote loss of focal adhesions. Remaining reticular adhesion complexes were stabilised with DTBP crosslinking reagent before adhesion complexes were isolated. Preliminary data suggested that depletion of PIP2 by Neomycin treatment would lead to disruption of reticular adhesions preferentially over focal adhesions and so this manipulation was included in these experiments

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. 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 on a global level