Biology subjects

Radmacher, M.

Publications and source records attributed to Radmacher, M..

4 recordsLinked to original sources

bioRxiv2023

Characterizing the viscoelastic properties of different fibroblasts in 2D and 3D collagen gels

We assessed cell mechanical properties in both 2D and 3D environments employing compliant type I collagen matrices. Firstly, collagen gels of varying stiffness were prepared using a photocrosslinker to increase gel stiffness. Using methacrylic anhydride and UV light, a 10-fold increase in apparent Youngs modulus with respect to the soft collagen gel was achieved (0.2 kPa to 2 kPa). In addition, cells were plated onto the different collagen gels and hard Petri dishes (as a super stiff substrate) and their mechanical properties were evaluated. An increase in apparent Youngs modulus was observed in Dupuytren fibroblasts behavior when increasing substrate stiffness, supporting its myofibroblast phenotype (3.8 kPa to 5.2 kPa from soft collagen gels to hard Petri dishes). Secondly, gels mechanics, in which fibroblasts were embedded, were evaluated over time to assess cells contraction properties. Gels apparent Youngs modulus increased over time regardless of fibroblasts type and cells presented dendritic protrusions. Rheological properties of both cells and gels were extracted using AFM sweep frequency scheme and power law structural damping model for data analysis. As a summary, we have found that fibroblasts contractile properties, related to myofibroblast differentiation and development are highly influenced on the mechanical properties of the surrounding environment, being stiffer environments those that favor the increase in fibroblast mechanical tension.

biophysics↗

bioRxiv2023

Reliable, standardized measurements for cell mechanical properties

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 (Youngs 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.

biophysics↗

bioRxiv2021

A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

plant biology↗

bioRxiv2020

Homophilic and heterophilic cadherin bond rupture forces in homo- or hetero-cellular systems measured by AFM based SCFS

Cadherins enable intercellular adherens junctions to withstand tensile forces in tissues, e.g. generated by intracellular actomyosin contraction. Single molecule force spectroscopy experiments in in-vitro experiments can reveal the cadherin-cadherin extracellular region binding dynamics such as bond formation and strength. However, characterization of cadherin homophilic and heterophilic binding in their native conformational and functional state in living cells has rarely been done. Here, we used Atomic Force Microscopy (AFM) based Single cell force Spectroscopy (SCFS) to measure rupture forces of homophilic and heterophilic bond formation of N-, OB- and E-cadherins in living fibroblast and epithelial cells in homo- and hetero-cellular arrangements, i.e. between same type of cells and between cells of different type. In addition, we used indirect immunofluorescence labelling to study and correlate the expression of these cadherins in intercellular adherens junctions. We showed that N/N and E/E cadherin homophilic bindings are stronger than N/OB, E/N and E/OB heterophilic bindings. Disassembly of intracellular actin filaments reduces the cadherin bond rupture forces suggesting a contribution of actin filaments in cadherin extracellular binding. Inactivation of myosin did not affect the cadherin rupture force in both homo- and hetero-cellular arrangements. Whereas, myosin inactivation particularly strengthened the N/OB heterophilic bond and reinforced the other cadherins homophilic bonds.

biophysics↗