Vimentin Plays a Crucial Role in Fibroblast Ageing by Regulating Biophysical Properties and Cell Migration

Ageing is the result of changes in biochemical and biophysical processes at the cellular level that lead to progressive organ decline. Here we focus on the biophysical changes that impair cellular function of human dermal fibroblasts using donors of increasing age. We find that cell motility is impaired in cells from older donors, which is associated with increased Young’s modulus, viscosity, and adhesion. Cellular morphology also displays parallel increases in spread area and cytoskeletal assembly, with a threefold increase in vimentin filaments alongside a decrease in its remodelling rate. Treatments with withaferin A or acrylamide show that cell motility can be modulated by regulating vimentin assembly. Crucially, decreasing vimentin amount in cells from older individuals to levels displayed by the neonatal donor rescues their motility. Our results suggest that increased vimentin assembly may underlay the aberrant biophysical properties progressively observed at the cellular level in the course of human ageing and propose vimentin as a potential therapeutic target for ageing-related diseases

Lifeact-GFP alters F-actin organization, cellular morphology and biophysical behaviour

Live-imaging techniques are at the forefront of biology research to explore behaviour and function from sub-cellular to whole organism scales. These methods rely on intracellular fluorescent probes to label specific proteins, which are commonly assumed to only introduce artefacts at concentrations far-exceeding routine use. Lifeact, a small peptide with affinity for actin microfilaments has become a gold standard in live cell imaging of the cytoskeleton. Nevertheless, recent reports have raised concerns on Lifeact-associated artefacts at the molecular and whole organism level. We show here that Lifeact induces dose-response artefacts at the cellular level, impacting stress fibre dynamics and actin cytoskeleton architecture. These effects extend to the microtubule and intermediate filament networks as well as the nucleus, and ultimately lead to altered subcellular localization of YAP, reduced cell migration and abnormal mechanical properties. Our results suggest that reduced binding of cofilin to actin filaments may be the underlying cause of the observed Lifeact-induced cellular artefacts

The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransduction

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalize with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells

Experimental Verification of the Elastic Formula for the Aspirated Length of a Single Cell Considering the Size and Compressibility of Cell During Micropipette Aspiration

In this study, an aspiration system for elastic spheres was developed to verify the approximate elastic formula for the aspirated length of a single solid-like cell undergoing micropipette aspiration (MPA), which was obtained in our previous study by theoretical analysis and numerical simulation. Using this system, foam silicone rubber spheres with different diameters and mechanical properties were aspirated in a manner similar to the MPA of single cells. Comparisons between the approximate elastic formula and aspiration experiments of spheres indicated that the predictions of the formula agreed with the experimental results. Additionally, combined with the MPA data of rabbit chondrocytes, differences in terms of the elastic parameters derived from the half-space model, incompressible sphere model, and compressible sphere model were explored. The results demonstrated that the parameter ξ (ξ = R/a, where R is the radius of the cell and a is the inner radius of the micropipette) and Poisson’s ratio significantly influenced the determination of the elastic modulus and bulk modulus of the cell. This work developed for the first time an aspiration system of elastic spheres to study the elastic responses of the MPA of a single cell and provided new evidence supporting the use of the approximate elastic formula to determine cellular elastic parameters from the MPA data

Chondrocyte dedifferentiation increases cell stiffness by strengthening membrane-actin adhesion

Funded by Engineering and Physical Sciences Research Counci