A Perspective on the Experimental Techniques for Studying Lamins

3Lamins are type V intermediate filaments that collectively form a meshwork underneath the inner nuclear membrane, called nuclear lamina. Furthermore, they are also present in the nucleoplasm. Lamins are experiencing a growing interest, since a wide range of diseases are induced by mutations in the gene coding for A-type lamins, globally known as laminopathies. Moreover, it has been demonstrated that lamins are involved in other pathological conditions, like cancer. The role of lamins has been studied from several perspectives, exploiting different techniques and procedures. This multidisciplinary approach has contributed to resolving the unique features of lamins and has provided a thorough insight in their role in living organisms. Yet, there are still many unanswered questions, which constantly generate research in the field. The present work is aimed to review some interesting experimental techniques performed so far to study lamins. Scientists can take advantage of this collection for their novel investigations, being aware of the already pursued and consolidated methodologies. Hopefully, advances in these research directions will provide insights to achieve better diagnostic procedures and effective therapeutic options.openopenPecorari, Ilaria; Borin, Daniele; Sbaizero, OrfeoPecorari, Ilaria; Borin, Daniele; Sbaizero, Orfe

An engineering insight into the relationship of selective cytoskeletal impairment and biomechanics of HeLa cells

It is widely accepted that the pathological state of cells is characterized by a modification of mechanical properties, affecting cellular shape and viscoelasticity as well as adhesion behaviour and motility. Thus, assessing these parameters could represent an interesting tool to monitor disease development and progression, but also the effects of drug treatments. Since biomechanical properties of cells are strongly related to cytoskeletal architecture, in this work we extensively studied the effects of selective impairments of actin microfilaments and microtubules on HeLa cells through force-deformation curves and stress relaxation tests with atomic force microscopy. Confocal microscopy was also used to display the effects of the used drugs on the cytoskeletal structure. In synergy with the aforementioned methods, stress relaxation data were used to assess the storage and loss moduli, as a complementary way to describe the influence of cytoskeletal components on cellular viscoelasticity. Our results indicate that F-actin and microtubules play a complementary role in the cell stiffness and viscoelasticity, and both are fundamental for the adhesion properties. Our data support also the application of biomechanics as a tool to study diseases and their treatments

The local mechanosensitive response of primary cardiac fibroblasts is influenced by the microenvironment mechanics

Cardiac fibroblasts (CFs) are essential for preserving myocardial integrity and function. They can detect variations in cardiac tissue stiffness using various cellular mechanosensors, including the Ca2+ permeable mechanosensitive channel Piezo1. Nevertheless, how CFs adapt the mechanosensitive response to stiffness changes remains unclear. In this work we adopted a multimodal approach, combining the local mechanical stimulation (from 10 pN to 350 nN) with variations of culture substrate stiffness. We found that primary rat CFs cultured on stiff (GPa) substrates showed a broad Piezo1 distribution in the cell with particular accumulation at the mitochondria membrane. CFs displayed a force-dependent behavior in both calcium uptake and channel activation probability, showing a threshold at 300 nN, which involves both cytosolic and mitochondrial Ca2+ mobilization. This trend decreases as the myofibroblast phenotype within the cell population increases, following a possible Piezo1 accumulation at focal adhesion sites. In contrast, the inhibition of fibroblasts to myofibroblasts transition with soft substrates (kPa) considerably reduces both mechanically- and chemically-induced Piezo1 activation and expression. Our findings shed light on how Piezo1 function and expression are regulated by the substrate stiffness and highlight its involvement in the environment-mediated modulation of CFs mechanosensitivity

Cellular biomechanics impairment in keratinocytes is associated with a C-terminal truncated desmoplakin: An atomic force microscopy investigation.

In a tissue continuously challenged by mechanical stresses, such as the skin or the heart, cells perceive information about their microenvironment through several adhesive protein complexes and activate cell-signaling events to maintain tissue cohesion. Consequently, alteration of cell adhesion components leads to aberrant assembly of the associated cytoplasmic scaffolding and signaling pathways, which may reflect changes to the tissue physiology and mechanical resistance. Desmoplakin is an essential component of the cell-cell junction, anchoring the desmosomal protein complex to the intermediate filaments (IFs). Inherited mutations in desmoplakin are associated with both heart and skin disease (cardiocutaneous syndrome). In this study, we investigated the mechanical properties of human keratinocytes harboring a cardiocutaneous-associated homozygous C-terminal truncation in desmoplakin (JD-1) compared to a control keratinocyte line (K1). Using Single Cell Force Spectroscopy (SCFS) AFM-based measurements, JD-1 keratinocytes displayed an overall alteration in morphology, elasticity, adhesion capabilities and viscoelastic properties, highlighting the profound interconnection between the adhesome pathways and the IF scaffold.Fondation Leducq, Transatlantic Network of Excellence (14-CVD 03)

The effect of artificial weathering on PP coextruded tape and laminate

The aim of this work was to analyse the influence of artificial weathering on polypropylene (PP) selfreinforced composite both as fabric constituent (tape) and as laminate. Particular attention has been focused on the interaction between radiation and material microstructural characteristics, especially for the selective action that the former causes in PP amorphous regions. The evaluation of carbonyl index performed by Fourier Transform Infrared (FT-IR) spectroscopy has highlighted that tapes are more prone to degradation since their drawn structure induces internal stress. Differential scanning calorimetry (DSC) measurements have denoted a shift towards lower temperature of PP melting peak. While crystallinity determination performed by DSC and FT-IR spectroscopy has underlined an increasing trend for laminate over exposure time due to the higher amount of amorphous phase, Raman spectroscopy has revealed that photo-degradation induces a rise of the isomeric defect fraction, limiting chemicrystallisation both for tapes and laminates

Biomechanical defects and rescue of cardiomyocytes expressing pathologic nuclear lamins

Given the clinical impact of LMNA cardiomyopathies, understanding lamin function will fulfill a clinical need and will lead to advancement in the treatment of heart failure. A multidisciplinary approach combining cell biology, atomic force microscopy (AFM) and molecular modeling was used to analyze the biomechanical properties of human lamin A/C gene (LMNA) mutations (E161K, D192G, N195K) using an in vitro neonatal rat ventricular myocyte (NRVM) model

Cellular Biomechanic Impairment in Cardiomyocytes Carrying the Progeria Mutation: An Atomic Force Microscopy Investigation

Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations

Bimaterial Composites via Colloidal Rolling Techniques: II, Sintering Behavior and Thermal Stresses

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65987/1/j.1151-2916.1999.tb02260.x.pd

Deoxynivalenol induces structural alterations in epidermoid carcinoma cells A431 and impairs the response to biomechanical stimulation

Morphology together with the capability to respond to surrounding stimuli are key elements governing the spatial interaction of living cells with the environment. In this respect, biomechanical stimulation can trigger significant physiological cascades that can potentially modulate toxicity. Deoxynivalenol (DON, vomitoxin) is one of the most prevalent mycotoxins produced by Fusarium spp. and it was used to explore the delicate interaction between biomechanical stimulation and cytotoxicity in A431 cells. In fact, in addition of being a food contaminant, DON is a relevant toxin for several organ systems. The combination between biomechanical stimulation and the mycotoxin revealed how DON can impair crucial functions affecting cellular morphology, tubulin and lysosomes at concentrations even below those known to be cytotoxic in routine toxicity studies. Sub-toxic concentrations of DON (0.1\u20131 \u3bcM) impaired the capability of A431 cells to respond to a biomechanical stimulation that normally sustains trophic effects in these cells. Moreover, the effects of DON (0.1\u201310 \u3bcM) were partially modulated by the application of uniaxial stretching (0.5 Hz, 24 h, 15% deformation). Ultimately, proteomic analysis revealed the potential of DON to alter several proteins necessary for cell adhesion and cytoskeletal modulation suggesting a molecular link between biomechanics and the cytotoxic potential of the mycotoxin

Origin of Hysteresis Observed During Fatigue of Ceramic-Matrix Composites

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66076/1/j.1151-2916.1990.tb05239.x.pd