120 research outputs found
A beginner's guide to atomic force microscopy probing for cell mechanics
European Commission . Grant Number: CIG14-2013-631011
Dunhill Medical Trust . Grant Number: R454/111
Combined strategies for optimal detection of the contact point in AFM force-indentation curves obtained on thin samples and adherent cells
This work was supported in part by a Marie Curie CIG grant (PCIG14-GA-2013-631011 CSKFingerprints)
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
Frequency-modulated atomic force microscopy localises viscoelastic remodelling in the ageing sheep aorta
We gratefully acknowledge funding from the Royal Society for the provision of an International Travel Grant for Collaboration (R112205) to RA, and Wellcome Trust Value in People Award to RA and MJS. MJS and BD gratefully acknowledge the support of the Medical Research Council (www.mrc.ac.uk: grant reference G1001398)
Extracellular fluid viscosity enhances liver cancer cell mechanosensing and migration
The extracellular fluid (ECF) is a crowded environment containing macromolecules that determine its characteristic density, osmotic pressure, and viscosity, which greatly differ between tissues. Precursors and products of degradation of biomaterials enhance ECF crowding and often increase its viscosity. Also, increases in ECF viscosity are related to mucin-producing adenocarcinomas. However, the effect of ECF viscosity on cells remains largely unexplored. Here we show that viscosity-enhancing polymer solutions promote mesenchymal-like cell migration in liver cancer cell lines. Also, we demonstrate that viscosity enhances integrin-dependent cell spreading rate and causes actin cytoskeleton re-arrangements leading to larger cell area, nuclear flattening, and nuclear translocation of YAP and β-catenin, proteins involved in mechanotransduction. Finally, we describe a relationship between ECF viscosity and substrate stiffness in determining cell area, traction force generation and mechanotransduction, effects that are actin-dependent only on ≤ 40 kPa substrates. These findings reveal that enhancing ECF viscosity can induce major biological responses including cell migration and substrate mechanosensing
Assessment of the nano-mechanical properties of healthy and atherosclerotic coronary arteries by atomic force microscopy.
Nano-indentation techniques might be better equipped to assess the heterogeneous material properties of plaques than macroscopic methods but there are no bespoke protocols for this kind of material testing for coronary arteries. Therefore, we developed a measurement protocol to extract mechanical properties from healthy and atherosclerotic coronary artery tissue sections. Young's modulus was derived from force-indentation data. Metrics of collagen fibre density were extracted from the same tissue, and the local material properties were co-registered to the local collagen microstructure with a robust framework. The locations of the indentation were retrospectively classified by histological category (healthy, plaque, lipid-rich, fibrous cap) according to Picrosirius Red stain and adjacent Hematoxylin & Eosin and Oil-Red-O stains. Plaque tissue was softer (p < 0.001) than the healthy coronary wall. Areas rich in collagen within the plaque (fibrous cap) were significantly (p < 0.001) stiffer than areas poor in collagen/lipid-rich, but less than half as stiff as the healthy coronary media. Young's moduli correlated (Pearson's ρ = 0.53, p < 0.05) with collagen content. Atomic force microscopy (AFM) is capable of detecting tissue stiffness changes related to collagen density in healthy and diseased cardiovascular tissue. Mechanical characterization of atherosclerotic plaques with nano-indentation techniques could refine constitutive models for computational modelling
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
New Bioengineering Breakthroughs and Enabling Tools in Regenerative Medicine.
PURPOSE OF REVIEW: In this review, we provide a general overview of recent bioengineering breakthroughs and enabling tools that are transforming the field of regenerative medicine (RM). We focus on five key areas that are evolving and increasingly interacting including mechanobiology, biomaterials and scaffolds, intracellular delivery strategies, imaging techniques, and computational and mathematical modeling. RECENT FINDINGS: Mechanobiology plays an increasingly important role in tissue regeneration and design of therapies. This knowledge is aiding the design of more precise and effective biomaterials and scaffolds. Likewise, this enhanced precision is enabling ways to communicate with and stimulate cells down to their genome. Novel imaging technologies are permitting visualization and monitoring of all these events with increasing resolution from the research stages up to the clinic. Finally, algorithmic mining of data and soft matter physics and engineering are creating growing opportunities to predict biological scenarios, device performance, and therapeutic outcomes. SUMMARY: We have found that the development of these areas is not only leading to revolutionary technological advances but also enabling a conceptual leap focused on targeting regenerative strategies in a holistic manner. This approach is bringing us ever more closer to the reality of personalized and precise RM
Enhancement of photoactivity and cellular uptake of (Bu4N)2[Mo6I8(CH3COO)6] complex by loading on porous MCM-41 support. Photodynamic studies as an anticancer agent
The incorporation by ionic assembly of the hexanuclear molybdenum cluster (Bu4N)2[Mo6I8(CH3CO2)6] (1) in amino-decorated mesoporous silica nanoparticles MCM-41, has yielded the new molybdenum-based hybrid photosensitizer 1@MCM-41. The new photoactive material presents a high porosity, due to the intrinsic high specific surface area of MCM-41 nanoparticles (989 m2 g-1) which is responsible for the good dispersion of the hexamolybdenum clusters on the nanoparticles surface, as observed by STEM analysis. The hybrid photosensitizer can generate efficiently singlet oxygen, which was demonstrated by using the benchmark photooxygenation reaction of 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) in water. The photodynamic therapy activity has been tested using LED light as an irradiation source (λirr ~ 400-700 nm; 15.6 mW/cm2). The results show a good activity of the hybrid photosensitizer against human cervical cancer (HeLa) cells, reducing up to 70 % their viability after 20 min of irradiation, whereas low cytotoxicity is detected in the darkness. The main finding of this research is that the incorporation of molybdenum complexes at porous MCM-41 supports enhances their photoactivity and improves cellular uptake, compared to free clusters.Ministerio de Ciencia, Innovación y Universidades of Spain (grant RTI2018-101675-B-I00) is acknowledged. F.G. thanks Universitat Jaume I (grant UJI-B2021-51) for the financial support. R.M.-M. laboratory members thank the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). The research was supported by the Ministry of Science and Higher Education of the Russian Federation (M.N.S.). C.T. acknowledges the Generalitat Valenciana for her postdoctoral fellowship (APOSTD/2019/121). R.G. thanks Universitat Jaume I for a postdoctoral fellowship (POSTDOC-B/2018/09). We would like to thank Prof. Iván Mora-Seró (INAM-UJI) for the singlet oxygen phosphorescence measurements. The help of Jean Colombari in the final phase of this work is also recognized. SCIC-UJI is acknowledged for the technical support
Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration
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