Biology subjects

Gilbert, H. T.

Publications and source records attributed to Gilbert, H. T..

4 recordsLinked to original sources

bioRxiv2022

Loss of cytoskeletal proteostasis links dysregulation of cell size and mechanotransduction in mesenchymal stem cell senescence

Tissues are maintained by homeostatic feedback mechanisms where cells respond to, but also modify, the chemical and mechanical properties of the surrounding extracellular matrix. Mesenchymal stem cells (MSCs) resident in the marrow niche experience a diverse mechanical environment, but ageing can affect the composition and quality of bone and marrow tissues. Here we quantified the effect of replication-induced senescence on MSC morphology and their ability to correctly respond to different substrate stiffnesses. The matrix proteome was found to be sensitive to substrate stiffness, but pharmacological inhibition of cellular contractility perturbed this response, decreasing levels of tenascin-C, fibulins and fibronectin. Similar decreases in these mechanosensitive proteins were observed in senescent cells, suggested a decoupling of mechanotransduction pathways. Intracellular proteomic and transcriptomic analyses confirmed a decrease in components of the cytoskeletal chaperone complex CCT/TRiC in senescent MSCs. Finally, pharmacological inhibition of CCT/TRiC was able to partially recapitulate senescence-associated morphological changes in non-senescent MSCs. These results demonstrate a senescence-mediated perturbation to cytoskeletal homeostasis, pathways of mechanotransduction and the secretion of matrix proteins required for tissue maintenance.

cell biology↗

bioRxiv2021

Senescence inhibits the chaperone response to thermal stress

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 heat shock, as an in vitro model of the ageing stress response. We found through proteomic analysis that the maintenance of homeostasis deteriorated in senescent cells. Time-resolved analysis of factors regulating heat shock protein 70 kDa (HSPA1A) revealed a lack of capacities for protein turnover and translation to be key factors in limiting the stress response during senescence. A kinetic model predicted a consequence of these reduced capacities to be the accumulation of misfolded protein, a hypothesis supported by evidence of systematic changes to protein fold state. These results thus further our understanding of the underlying mechanistic links between ageing and loss of protein homeostasis.

cell biology↗

bioRxiv2019

Stimulation of cardiac fibroblast Piezo1 channels opposes myofibroblast differentiation and induces IL-6 secretion via Ca2+-mediated p38 MAP kinase activation

Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however its role in the heart is poorly understood. Cardiac fibroblasts are responsible for preserving the structural integrity of the myocardium and play a key role in regulating its repair and remodeling following stress or injury. We investigated expression and function of Piezo1 in cultured human and mouse cardiac fibroblasts. RT-PCR studies confirmed expression of Piezo1 mRNA in cardiac fibroblasts at similar levels to endothelial cells. Fura-2 intracellular Ca2+ measurements validated Piezo1 as a functional ion channel that was activated by the Piezo1 agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium, ruthenium red) and the Ca2+ response was reduced proportionally by Piezo1 siRNA knockdown or in cells from Piezo1+/- mice. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation opposed cardiac fibroblast differentiation; data confirmed by functional collagen gel contraction assays. Piezo1 activation using Yoda1 or mechanical stretch also increased the expression of interleukin-6 (IL-6), a mechanosensitive pro-hypertrophic and pro-fibrotic cytokine, in a Piezo1-dependent manner. Multiplex kinase activity profiling combined with kinase inhibitor studies and phospho-specific western blotting, established that Piezo1 activation stimulated IL-6 secretion via a pathway involving p38 MAP kinase, downstream of Ca2+ entry. In summary, this study reveals that cardiac fibroblasts express functional Piezo1 channels coupled to reduced myofibroblast activation and increased secretion of paracrine signaling molecules that can modulate cardiac remodeling.

cell biology↗

bioRxiv2018

Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading

Our current understanding of cellular mechano-signalling is based on static models, which do not replicate the dynamics of living tissues. Here, we examined the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 hour, 4% CTS at 1 Hz) morphological changes mimicked responses to increased substrate stiffness. As the strain regime was intensified (frequency increased to 5 Hz), we characterised rapid establishment of a broad, structured and reversible protein-level response, even as transcription was apparently downregulated. Protein abundance was quantified coincident with changes to protein conformation and post transcriptional modification. Furthermore, we characterised changes within the linker of nucleo- and cytoskeleton (LINC) complex of proteins that bridges the nuclear envelope, and specifically to levels of SUN domain-containing protein 2 (SUN2). The result of this regulation was to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

biophysics↗