Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin–membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 μm wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young’s Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. Septin knock-out animals have established that septins are essential for animal physiology, but their molecular function remains elusive. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether actin-decorating septins organize as filaments and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay for probing the presence and composition of septin filaments in situ in cells, we show that all septins decorating actin stress fibers are present as filaments whose integrity depends on octameric septin protomers. Atomic force microscopy nanoindentation measurements on cells confirmed that cell stiffness depends on the presence of octamer-containing septin filaments. Super-resolution structured illumination microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that actin-associated septin filaments are membrane-bound and largely immobilized. Finally, reconstitution assays on supported lipid bilayers showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin fibers at the plasma membrane.

Mechanical biomarkers distinguish health conditions through quantitative mechanical measurements. The emergence and establishment of nanotechnology in the last decades have provided new tools to obtain mechanical biomarkers at the nanoscale.Mechanical measurements are reproducible, label-free, start to be applied in vivo can be high throughput, and require small samples. Mechanical protocols in clinical practice at the macro scale like palpation or blood pressure measurement are routinely used by medical doctors. Nanotechnology brought mechanical sensing to the next scale, where cells, tissues, and proteins can be probed and linked to medical conditions. Mechanical changes in cells and tissues may be detected before other markers, such as protein expression, providing an important advantage as biomarkers. In the present review, we explore the biomarker's historical evolution, describe mechanical biomarkers on various diseases and novel discoveries in the nanomechanical field for their characterization. We conclude that mechanical biomarkers are establishing novel hallmarks in diseases, in several cases for early diagnostics of diseases and discovery of drug targets in the proteins involved in the mechanical changes, while advances in instrumentation are bringing commercial products into the clinical practice. Mechanical biomarkers along with clinical testing are establishing an important niche in the market, whose demand is increasing due to the expansion of personalized medicine and unmet needs in the clinics.

Coupled atomic force microscopy viscoelastic mapping and interference contrast microscopy revealed larger adhesion, stiffening and solidification during monocyte differentiation into macrophages.

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher-level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 μm wide microfluidic channel. The fluid shear stress induces large, near ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

Atomic force microscopy (AFM) has become indispensable for studying biological and medical samples. More than two decades of experiments have revealed that cancer cells are softer than healthy cells (for measured cells cultured on stiff substrates). The softness or, more precisely, the larger deformability of cancer cells, primarily independent of cancer types, could be used as a sensitive marker of pathological changes. The wide application of biomechanics in clinics would require designing instruments with specific calibration, data collection, and analysis procedures. For these reasons, such development is, at present, still very limited, hampering the clinical exploitation of mechanical measurements. Here, we propose a standardized operational protocol (SOP), developed within the EU ITN network Phys2BioMed, which allows the detection of the biomechanical properties of living cancer cells regardless of the nanoindentation instruments used (AFMs and other indenters) and the laboratory involved in the research. We standardized the cell cultures, AFM calibration, measurements, and data analysis. This effort resulted in a step-by-step SOP for cell cultures, instrument calibration, measurements, and data analysis, leading to the concordance of the results (Young's modulus) measured among the six EU laboratories involved. Our results highlight the importance of the SOP in obtaining a reproducible mechanical characterization of cancer cells and paving the way toward exploiting biomechanics for diagnostic purposes in clinics.