Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins.

Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation.

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes, and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well-understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

Myotubularin related protein-2 and its phospholipid substrate PIP2 control Piezo2-mediated mechanotransduction in peripheral sensory neurons.

Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.

Rhombic organization of microvilli domains found in a cell model of the human intestine.

Symmetry is rarely found on cellular surfaces. An exception is the brush border of microvilli, which are essential for the proper function of transport epithelia. In a healthy intestine, they appear densely packed as a 2D-hexagonal lattice. For in vitro testing of intestinal transport the cell line Caco-2 has been established. As reported by electron microscopy, their microvilli arrange primarily in clusters developing secondly into a 2D-hexagonal lattice. Here, atomic force microscopy (AFM) was employed under aqueous buffer conditions on Caco-2 cells, which were cultivated on permeable filter membranes for optimum differentiation. For analysis, the exact position of each microvillus was detected by computer vision; subsequent Fourier transformation yielded the type of 2D-lattice. It was confirmed, that Caco-2 cells can build a hexagonal lattice of microvilli and form clusters. Moreover, a second type of arrangement was discovered, namely a rhombic lattice, which appeared at sub-maximal densities of microvilli with (29 ± 4) microvilli / µm2. Altogether, the findings indicate the existence of a yet undescribed pattern in cellular organization.

Coordinated increase of nuclear tension and lamin-A with matrix stiffness outcompetes lamin-B receptor that favors soft tissue phenotypes.

Matrix stiffness that is sensed by a cell or measured by a purely physical probe reflects the intrinsic elasticity of the matrix and also how thick or thin the matrix is. Here, mesenchymal stem cells (MSCs) and their nuclei spread in response to thickness-corrected matrix microelasticity, with increases in nuclear tension and nuclear stiffness resulting from increases in myosin-II and lamin-A,C. Linearity between the widely varying projected area of a cell and its nucleus across many matrices, timescales, and myosin-II activity levels indicates a constant ratio of nucleus-to-cell volume, despite MSCs' lineage plasticity. Nuclear envelope fluctuations are suppressed on the stiffest matrices, and fluctuation spectra reveal a high nuclear tension that matches trends from traction force microscopy and from increased lamin-A,C. Transcriptomes of many diverse tissues and MSCs further show that lamin-A,C's increase with tissue or matrix stiffness anti-correlates with lamin-B receptor (LBR), which contributes to lipid/sterol biosynthesis. Adipogenesis (a soft lineage) indeed increases LBR:lamin-A,C protein stoichiometry in MSCs versus osteogenesis (stiff). The two factors compete for lamin-B in response to matrix elasticity, knockdown, myosin-II inhibition, and even constricted migration that disrupts and segregates lamins in situ. Matrix stiffness-driven contractility thus tenses the nucleus to favor lamin-A,C accumulation and suppress soft tissue phenotypes.

Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro.

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.

X-Ray Micro- and Nanodiffraction Imaging on Human Mesenchymal Stem Cells and Differentiated Cells.

Adult human mesenchymal stem cells show structural rearrangements of their cytoskeletal network during mechanically induced differentiation toward various cell types. In particular, the alignment of acto-myosin fibers is cell fate-dependent and can serve as an early morphological marker of differentiation. Quantification of such nanostructures on a mesoscopic scale requires high-resolution imaging techniques. Here, we use small- angle x-ray scattering with a spot size in the micro- and submicrometer range as a high-resolution and label-free imaging technique to reveal structural details of stem cells and differentiated cell types. We include principal component analysis into an automated empirical analysis scheme that allows the local characterization of oriented structures. Results on freeze-dried samples lead to quantitative structural information for all cell lines tested: differentiated cells reveal pronounced structural orientation and a relatively intense overall diffraction signal, whereas naive human mesenchymal stem cells lack these features. Our data support the hypothesis of stem cells establishing ordered structures along their differentiation process.

Mechanotransduction: use the force(s).

Mechanotransduction - how cells sense physical forces and translate them into biochemical and biological responses - is a vibrant and rapidly-progressing field, and is important for a broad range of biological phenomena. This forum explores the role of mechanotransduction in a variety of cellular activities and highlights intriguing questions that deserve further attention.

The filament sensor for near real-time detection of cytoskeletal fiber structures.

A reliable extraction of filament data from microscopic images is of high interest in the analysis of acto-myosin structures as early morphological markers in mechanically guided differentiation of human mesenchymal stem cells and the understanding of the underlying fiber arrangement processes. In this paper, we propose the filament sensor (FS), a fast and robust processing sequence which detects and records location, orientation, length, and width for each single filament of an image, and thus allows for the above described analysis. The extraction of these features has previously not been possible with existing methods. We evaluate the performance of the proposed FS in terms of accuracy and speed in comparison to three existing methods with respect to their limited output. Further, we provide a benchmark dataset of real cell images along with filaments manually marked by a human expert as well as simulated benchmark images. The FS clearly outperforms existing methods in terms of computational runtime and filament extraction accuracy. The implementation of the FS and the benchmark database are available as open source.

Force fluctuations in three-dimensional suspended fibroblasts.

Cells are sensitive to mechanical cues from their environment and at the same time generate and transmit forces to their surroundings. To test quantitatively forces generated by cells not attached to a substrate, we used a dual optical trap to suspend 3T3 fibroblasts between two fibronectin-coated beads. In this simple geometry, we measured both the cells' elastic properties and the force fluctuations they generate with high bandwidth. Cell stiffness decreased substantially with both myosin inhibition by blebbistatin and serum-starvation, but not with microtubule depolymerization by nocodazole. We show that cortical forces generated by non-muscle myosin II deform the cell from its rounded shape in the frequency regime from 0.1 to 10 Hz. The amplitudes of these forces were strongly reduced by blebbistatin and serum starvation, but were unaffected by depolymerization of microtubules. Force fluctuations show a spectrum that is characteristic for an elastic network activated by random sustained stresses with abrupt transitions.

Contractile forces sustain and polarize hematopoiesis from stem and progenitor cells.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation.

Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.

Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells.

Physical features of microenvironments such as matrix elasticity E can clearly influence cell morphology and cell phenotype, but many differences between model matrices raise questions as to whether a standard biological scale for E exists, especially in 3D as well as in 2D. An E-series of two distinct types of hydrogels are ligand-functionalized here with non-fibrous collagen and used to elucidate wide-ranging cell and cytoskeletal responses to E in both 2D and 3D matrix geometries. Cross-linked hyaluronic acid (HA) based matrices as well as standard polyacrylamide (PA) hydrogels show that, within hours of initial plating, the adhesion, asymmetric shape, and cytoskeletal order within mesenchymal stem cells generally depend on E nonmonotonically over a broad range of physiologically relevant E. In particular, with overlays of a second matrix the stiffer of the upper or lower matrix dominates key cell responses to 3D: the cell invariably takes an elongated shape that couples to E in driving cytoplasmic stress fiber assembly. In contrast, embedding cells in homogeneous HA matrices constrains cells to spherically symmetric shapes in which E drives the assembly of a predominantly cortical cytoskeleton. Non-muscle myosin II generates the forces required for key cell responses and is a target of a phospho-Tyrosine signaling pathway that likely regulates contractile assemblies and also depends nonmonotonically on E. The results can be understood in part from a theory for stress fiber polarization that couples to matrix elasticity as well as cell shape and accurately predicts cytoskeletal order in 2D and 3D, regardless of polymer system.

Preparation of collagen-coated gels that maximize in vitro myogenesis of stem cells by matching the lateral elasticity of in vivo muscle.

The physical nature of a cell's microenvironment--including the elasticity of the surrounding tissue--appears to exert a significant influence on cell morphology, cytoskeleton, and gene expression. We have previously shown that committed muscle cells will develop sarcomeric striations of skeletal muscle myosin II only when the cells are grown on a compliant gel that closely matches the passive compliance of skeletal muscle. We have more recently shown with the same types of elastic gels that mesenchymal stem cells (MSCs) maximally express myogenic genes, even in the absence of tailored soluble factors. Here, we provide detailed methods not only for how we make and nanomechanically characterize hydrogels of muscle-like elasticity, but also how we culture MSCs and characterize their myogenic induction by whole human genome transcript analysis.

Cell responses to the mechanochemical microenvironment--implications for regenerative medicine and drug delivery.

Soft-tissue cells are surprisingly sensitive to the elasticity of their microenvironment, suggesting that traditional culture plastic and glass are less relevant to tissue regeneration and chemotherapeutics than might be achieved. Cells grown on gels that mimic the elasticity of tissue reveal a significant influence of matrix elasticity on adhesion, cytoskeletal organization, and even the differentiation of human adult derived stem cells. Cellular forces and feedback are keys to how cells feel their mechanical microenvironment, but detailed molecular mechanisms are still being elucidated. This review summarizes our initial findings for multipotent stem cells and also the elasticity-coupled effects of drugs on cancer cells and smooth muscle cells. The drugs include the contractility inhibitor blebbistatin, the proliferation inhibitor mitomycin C, an apoptotis-inducing antibody against CD47, and the translation inhibitor cycloheximide. The differential effects not only lend insight into mechano-sensing of the substrate by cells, but also have important implications for regeneration and molecular therapies.

Oligomer-to-polymer transition in short ethylene glycol chains connected to mobile hydrophobic anchors.

We studied the structure of short ethylene glycol (EG) chains with N repeating units (EGN, N = 3, 6, 9, 12, and 15) connected to hydrophobic dihexadecyl chains by means of a combination of differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS/WAXS). These synthetic amphiphiles dispersed in water form planar lamellar stacks and hexagonal cylinders confining the EG chains to restricted geometries. Owing to the self-assembly of the anchoring points, the lateral density of EG chains in planar lamella can be quantitatively controlled. Furthermore, the chain-melting phase transition of the anchors enables us to "switch" the intermolecular distance reversibly. SAXS/WAXS results suggest that the shorter EG chains (N = 3, 6, and 9) assume a helical conformation in stacks of planar lamella. When the EG chains are further elongated (N = 12 and 15), the lamellar periodicities cannot be explained by a linear extrapolation of shorter oligomers, but can be interpreted well as polymer brushes following the scaling theorem. Such rich phase behaviors of EGN molecules can be used as a simple model of oligo/poly-saccharide chains on cell surfaces, which act not only as flexible repellers between neighboring cells but also as stable spacers for functional ligands.