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15 pages, 2505 KiB

Open AccessArticle

Recombinant Peptide Production Softens Escherichia coli Cells and Increases Their Size during C-Limited Fed-Batch Cultivation

by Andreas Weber, Martin Gibisch, Daniel Tyrakowski, Monika Cserjan-Puschmann, José L. Toca-Herrera and Gerald Striedner

Int. J. Mol. Sci. 2023, 24(3), 2641; https://doi.org/10.3390/ijms24032641 - 30 Jan 2023

Cited by 2 | Viewed by 1303

Abstract

Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. [...] Read more.

Stress-associated changes in the mechanical properties at the single-cell level of Escherichia coli (E. coli) cultures in bioreactors are still poorly investigated. In our study, we compared peptide-producing and non-producing BL21(DE3) cells in a fed-batch cultivation with tightly controlled process parameters. The cell growth, peptide content, and cell lysis were analysed, and changes in the mechanical properties were investigated using atomic force microscopy. Recombinant-tagged somatostatin-28 was expressed as soluble up to 197 ± 11 mg g⁻¹. The length of both cultivated strains increased throughout the cultivation by up to 17.6%, with nearly constant diameters. The peptide-producing cells were significantly softer than the non-producers throughout the cultivation, and respective Young’s moduli decreased by up to 57% over time. A minimum Young’s modulus of 1.6 MPa was observed after 23 h of the fed-batch. Furthermore, an analysis of the viscoelastic properties revealed that peptide-producing BL21(DE3) appeared more fluid-like and softer than the non-producing reference. For the first time, we provide evidence that the physical properties (i.e., the mechanical properties) on the single-cell level are significantly influenced by the metabolic burden imposed by the recombinant peptide expression and C-limitation in bioreactors. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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15 pages, 3593 KiB

Open AccessArticle

Advanced Glycation End Products Effects on Adipocyte Niche Stiffness and Cell Signaling

by Roza Izgilov, Alex Naftaly and Dafna Benayahu

Int. J. Mol. Sci. 2023, 24(3), 2261; https://doi.org/10.3390/ijms24032261 - 23 Jan 2023

Cited by 3 | Viewed by 1552

Abstract

Adipose tissue metabolism under hyperglycemia results in Type II diabetes (T2D). To better understand how the adipocytes function, we used a cell culture that was exposed to glycation by adding intermediate carbonyl products, which caused chemical cross-linking and led to the formation of [...] Read more.

Adipose tissue metabolism under hyperglycemia results in Type II diabetes (T2D). To better understand how the adipocytes function, we used a cell culture that was exposed to glycation by adding intermediate carbonyl products, which caused chemical cross-linking and led to the formation of advanced glycation end products (AGEs). The AGEs increased the cells and their niche stiffness and altered the rheological viscoelastic properties of the cultured cells leading to altered cell signaling. The AGEs formed concomitant with changes in protein structure, quantified by spectroscopy using the 8-ANS and Nile red probes. The AGE effects on adipocyte differentiation were viewed by imaging and evidenced in a reduction in cellular motility and membrane dynamics. Importantly, the alteration led to reduced adipogenesis, that is also measured by qPCR for expression of adipogenic genes and cell signaling. The evidence of alteration in the plasma membrane (PM) dynamics (measured by CTxB binding and NP endocytosis), also led to the impairment of signal transduction and a decrease in AKT phosphorylation, which hindered downstream insulin signaling. The study, therefore, presents a new interpretation of how AGEs affect the cell niche, PM stiffness, and cell signaling leading to an impairment of insulin signaling. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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12 pages, 1566 KiB

Open AccessArticle

Nutrition Alters the Stiffness of Adipose Tissue and Cell Signaling

by Alex Naftaly, Nadav Kislev, Roza Izgilov, Raizel Adler, Michal Silber, Ruth Shalgi and Dafna Benayahu

Int. J. Mol. Sci. 2022, 23(23), 15237; https://doi.org/10.3390/ijms232315237 - 03 Dec 2022

Cited by 3 | Viewed by 1687

Abstract

Adipose tissue is a complex organ composed of various cell types and an extracellular matrix (ECM). The visceral adipose tissue (VAT) is dynamically altered in response to nutritional regimens that lead to local cues affecting the cells and ECM. The adipocytes are in [...] Read more.

Adipose tissue is a complex organ composed of various cell types and an extracellular matrix (ECM). The visceral adipose tissue (VAT) is dynamically altered in response to nutritional regimens that lead to local cues affecting the cells and ECM. The adipocytes are in conjunction with the surrounding ECM that maintains the tissue’s niche, provides a scaffold for cells and modulates their signaling. In this study, we provide a better understanding of the crosstalk between nutritional regimens and the ECM’s stiffness. Histological analyses showed that the adipocytes in mice fed a high-fat diet (HFD) were increased in size, while the ECM was also altered with changes in mass and composition. HFD-fed mice exhibited a decrease in elastin and an increase in collagenous proteins. Rheometer measurements revealed a stiffer ECM in whole tissue (nECM) and decellularized (deECM) in HFD-fed animals. These alterations in the ECM regulate cellular activity and influence their metabolic function. HFD-fed mice expressed high levels of the receptor for advanced-glycation-end-products (RAGE), indicating that AGEs might play a role in these processes. The cells also exhibited an increase in phosphoserine³³² of IRS-1, a decrease in the GLUT4 transporter levels at the cells’ membrane, and a consequent reduction in insulin sensitivity. These results show how alterations in the stiffness of ECM proteins can affect the mechanical cues transferred to adipocytes and, thereby, influence the adipocytes’ functionality, leading to metabolic disorders. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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14 pages, 2423 KiB

Open AccessArticle

Eukaryotic CRFK Cells Motion Characterized with Atomic Force Microscopy

by María Zamora-Ceballos, Juan Bárcena and Johann Mertens

Int. J. Mol. Sci. 2022, 23(22), 14369; https://doi.org/10.3390/ijms232214369 - 19 Nov 2022

Viewed by 1101

Abstract

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in [...] Read more.

We performed a time-lapse imaging with atomic force microscopy (AFM) of the motion of eukaryotic CRFK (Crandell-Rees Feline Kidney) cells adhered onto a glass surface and anchored to other cells in culture medium at 37 °C. The main finding is a gradient in the spring constant of the actomyosin cortex along the cells axis. The rigidity increases at the rear of the cells during motion. This observation as well as a dramatic decrease of the volume suggests that cells may organize a dissymmetry in the skeleton network to expulse water and drive actively the rear edge. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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17 pages, 4503 KiB

Open AccessArticle

Actin Stress Fibers Response and Adaptation under Stretch

by Roberto Bernal, Milenka Van Hemelryck, Basile Gurchenkov and Damien Cuvelier

Int. J. Mol. Sci. 2022, 23(9), 5095; https://doi.org/10.3390/ijms23095095 - 03 May 2022

Cited by 4 | Viewed by 1994

Abstract

One of the many effects of soft tissues under mechanical solicitation in the cellular damage produced by highly localized strain. Here, we study the response of peripheral stress fibers (SFs) to external stretch in mammalian cells, plated onto deformable micropatterned substrates. A local [...] Read more.

One of the many effects of soft tissues under mechanical solicitation in the cellular damage produced by highly localized strain. Here, we study the response of peripheral stress fibers (SFs) to external stretch in mammalian cells, plated onto deformable micropatterned substrates. A local fluorescence analysis reveals that an adaptation response is observed at the vicinity of the focal adhesion sites (FAs) due to its mechanosensor function. The response depends on the type of mechanical stress, from a Maxwell-type material in compression to a complex scenario in extension, where a mechanotransduction and a self-healing process takes place in order to prevent the induced severing of the SF. A model is proposed to take into account the effect of the applied stretch on the mechanics of the SF, from which relevant parameters of the healing process are obtained. In contrast, the repair of the actin bundle occurs at the weak point of the SF and depends on the amount of applied strain. As a result, the SFs display strain-softening features due to the incorporation of new actin material into the bundle. In contrast, the response under compression shows a reorganization with a constant actin material suggesting a gliding process of the SFs by the myosin II motors. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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19 pages, 4149 KiB

Open AccessArticle

The Role of Cytoskeleton Revealed by Quartz Crystal Microbalance and Digital Holographic Microscopy

by Nicoletta Braidotti, Maria Augusta do R. B. F. Lima, Michele Zanetti, Alessandro Rubert, Catalin Ciubotaru, Marco Lazzarino, Orfeo Sbaizero and Dan Cojoc

Int. J. Mol. Sci. 2022, 23(8), 4108; https://doi.org/10.3390/ijms23084108 - 07 Apr 2022

Cited by 3 | Viewed by 2227

Abstract

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which [...] Read more.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell–substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell’s ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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23 pages, 3724 KiB

Open AccessArticle

Effect of Different Parameters of In Vitro Static Tensile Strain on Human Periodontal Ligament Cells Simulating the Tension Side of Orthodontic Tooth Movement

by Changyun Sun, Mila Janjic Rankovic, Matthias Folwaczny, Thomas Stocker, Sven Otto, Andrea Wichelhaus and Uwe Baumert

Int. J. Mol. Sci. 2022, 23(3), 1525; https://doi.org/10.3390/ijms23031525 - 28 Jan 2022

Cited by 6 | Viewed by 2161

Abstract

This study aimed to investigate the effects of different magnitudes and durations of static tensile strain on human periodontal ligament cells (hPDLCs), focusing on osteogenesis, mechanosensing and inflammation. Static tensile strain magnitudes of 0%, 3%, 6%, 10%, 15% and 20% were applied to [...] Read more.

This study aimed to investigate the effects of different magnitudes and durations of static tensile strain on human periodontal ligament cells (hPDLCs), focusing on osteogenesis, mechanosensing and inflammation. Static tensile strain magnitudes of 0%, 3%, 6%, 10%, 15% and 20% were applied to hPDLCs for 1, 2 and 3 days. Cell viability was confirmed via live/dead cell staining. Reference genes were tested by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and assessed. The expressions of TNFRSF11B, ALPL, RUNX2, BGLAP, SP7, FOS, IL6, PTGS2, TNF, IL1B, IL8, IL10 and PGE2 were analyzed by RT-qPCR and/or enzyme-linked immunosorbent assay (ELISA). ALPL and RUNX2 both peaked after 1 day, reaching their maximum at 3%, whereas BGLAP peaked after 3 days with its maximum at 10%. SP7 peaked after 1 day at 6%, 10% and 15%. FOS peaked after 3 days with its maximum at 3%, 6% and 15%. The expressions of IL6 and PTGS2 both peaked after 1 day, with their minimum at 10%. PGE2 peaked after 1 day (maximum at 20%). The ELISA of IL6 peaked after 3 days, with the minimum at 10%. In summary, the lower magnitudes promoted osteogenesis and caused less inflammation, while the higher magnitudes inhibited osteogenesis and enhanced inflammation. Among all magnitudes, 10% generally caused a lower level of inflammation with a higher level of osteogenesis. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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Review

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20 pages, 1358 KiB

Open AccessReview

Piezo1 Channel as a Potential Target for Hindering Cardiac Fibrotic Remodeling

by Nicoletta Braidotti, Suet Nee Chen, Carlin S. Long, Dan Cojoc and Orfeo Sbaizero

Int. J. Mol. Sci. 2022, 23(15), 8065; https://doi.org/10.3390/ijms23158065 - 22 Jul 2022

Cited by 10 | Viewed by 3581

Abstract

Fibrotic tissues share many common features with neoplasms where there is an increased stiffness of the extracellular matrix (ECM). In this review, we present recent discoveries related to the role of the mechanosensitive ion channel Piezo1 in several diseases, especially in regulating tumor [...] Read more.

Fibrotic tissues share many common features with neoplasms where there is an increased stiffness of the extracellular matrix (ECM). In this review, we present recent discoveries related to the role of the mechanosensitive ion channel Piezo1 in several diseases, especially in regulating tumor progression, and how this can be compared with cardiac mechanobiology. Based on recent findings, Piezo1 could be upregulated in cardiac fibroblasts as a consequence of the mechanical stress and pro-inflammatory stimuli that occurs after myocardial injury, and its increased activity could be responsible for a positive feedback loop that leads to fibrosis progression. The increased Piezo1-mediated calcium flow may play an important role in cytoskeleton reorganization since it induces actin stress fibers formation, a well-known characteristic of fibroblast transdifferentiation into the activated myofibroblast. Moreover, Piezo1 activity stimulates ECM and cytokines production, which in turn promotes the phenoconversion of adjacent fibroblasts into new myofibroblasts, enhancing the invasive character. Thus, by assuming the Piezo1 involvement in the activation of intrinsic fibroblasts, recruitment of new myofibroblasts, and uncontrolled excessive ECM production, a new approach to blocking the fibrotic progression can be predicted. Therefore, targeted therapies against Piezo1 could also be beneficial for cardiac fibrosis. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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25 pages, 3815 KiB

Open AccessReview

Atomic Force Microscopy (AFM) Applications in Arrhythmogenic Cardiomyopathy

by Brisa Peña, Mostafa Adbel-Hafiz, Maria Cavasin, Luisa Mestroni and Orfeo Sbaizero

Int. J. Mol. Sci. 2022, 23(7), 3700; https://doi.org/10.3390/ijms23073700 - 28 Mar 2022

Cited by 8 | Viewed by 3090

Abstract

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disorder characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ventricular dilatation, cardiac dysfunction, arrhythmias, and sudden cardiac death. Interest in molecular biomechanics for these disorders is constantly growing. Atomic force microscopy (AFM) is a [...] Read more.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart muscle disorder characterized by progressive replacement of cardiomyocytes by fibrofatty tissue, ventricular dilatation, cardiac dysfunction, arrhythmias, and sudden cardiac death. Interest in molecular biomechanics for these disorders is constantly growing. Atomic force microscopy (AFM) is a well-established technic to study the mechanobiology of biological samples under physiological and pathological conditions at the cellular scale. However, a review which described all the different data that can be obtained using the AFM (cell elasticity, adhesion behavior, viscoelasticity, beating force, and frequency) is still missing. In this review, we will discuss several techniques that highlight the potential of AFM to be used as a tool for assessing the biomechanics involved in ACM. Indeed, analysis of genetically mutated cells with AFM reveal abnormalities of the cytoskeleton, cell membrane structures, and defects of contractility. The higher the Young’s modulus, the stiffer the cell, and it is well known that abnormal tissue stiffness is symptomatic of a range of diseases. The cell beating force and frequency provide information during the depolarization and repolarization phases, complementary to cell electrophysiology (calcium imaging, MEA, patch clamp). In addition, original data is also presented to emphasize the unique potential of AFM as a tool to assess fibrosis in cardiac tissue. Full article

(This article belongs to the Special Issue Biophysics and Mechanical Properties of Cells)

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