Cell mechanics controls important cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization, and trafficking inside the cytoplasm. Yet, reported values of cell stiffness and viscosity vary strongly, suggesting disagreements. To address this issue and illustrate the complementarity of different instruments, we present, analyze, and critically compare measurements conducted by some of the most widelyused methods of cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-platesrheometry, cell monolayer rheology, and optical stretcher.These measurements highlight that elastic and viscous moduliofbreast cancer cell MCF-7 can vary 1,000 fold and 100fold,respectively. We discussthe sources ofthese variations,including the level of applied mechanical stress and rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.
Initiation of acute graft-versus-host disease by angiogenesis
Katarina Riesner,
Yu Shi,
Angela Jacobi,
Martin Kraeter,
Martina Kalupa,
Aleixandria McGearey,
Sarah Mertlitz,
Steffen Cordes,
Jens-Florian Schrezenmeier, et al.
The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue-infiltrating leukocytes. However, it is not known if angiogenesis has an active role during the initiation of inflammation or if it is merely a secondary effect occurring in response to stimuli by tissue-infiltrating leukocytes. Here, we show that angiogenesis precedes leukocyte infiltration in experimental models of inflammatory bowel disease and acute graft-versus-host disease (GVHD). We found that angiogenesis occurred as early as day12 after allogeneic transplantation mainly in GVHD typical target organs skin, liver, and intestines, whereas no angiogenic changes appeared due to conditioning or syngeneic transplantation. The initiation phase of angiogenesis was not associated with classical endothelial cell (EC) activation signs, such as Vegfa/VEGFR112 upregulation or increased adhesion molecule expression. During early GVHD at day12, we found significant metabolic and cytoskeleton changes in target organ ECs in gene array and proteomic analyses. These modifications have significant functional consequences as indicated by profoundly higher deformation in real-time deformability cytometry. Our results demonstrate that metabolic changes trigger alterations in cell mechanics, leading to enhanced migratory and proliferative potential of ECs during the initiation of inflammation. Our study adds evidence to the hypothesis that angiogenesis is involved in the initiation of tissue inflammation during GVHD.
V-ATPase inhibition increases cancer cell stiffness and blocks membrane
related Ras signaling - a new option for HCC therapy
Karin Bartel,
Maria Winzi,
Melanie Ulrich,
Andreas Koeberle,
Dirk Menche,
Oliver Werz,
Rolf Mueller,
Jochen Guck,
Angelika M. Vollmar, et al.
Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide and the third leading cause of cancer-related death. However, therapy options are limited leaving an urgent need to develop new strategies. Currently, targeting cancer cell lipid and cholesterol metabolism is gaining interest especially regarding HCC. High cholesterol levels support proliferation, membrane-related mitogenic signaling and increase cell softness, leading to tumor progression, malignancy and invasive potential. However, effective ways to target cholesterol metabolism for cancer therapy are still missing. The V-ATPase inhibitor archazolid was recently shown to interfere with cholesterol metabolism. In our study, we report a novel therapeutic potential of V-ATPase inhibition in HCC by altering the mechanical phenotype of cancer cells leading to reduced proliferative signaling. Archazolid causes cellular depletion of free cholesterol leading to an increase in cell stiffness and membrane polarity of cancer cells, while hepatocytes remain unaffected. The altered membrane composition decreases membrane fluidity and leads to an inhibition of membrane-related Ras signaling resulting decreased proliferation in vitro and in vivo. V-ATPase inhibition represents a novel link between cell biophysical properties and proliferative signaling selectively in malignant HCC cells, providing the basis for an attractive and innovative strategy against HCC.
Single-cell mechanical phenotype is an intrinsic marker of reprogramming
and differentiation along the mouse neural lineage
Marta Urbanska,
Maria Winzi,
Katrin Neumann,
Shada Abuhattum,
Philipp Rosendahl,
Paul Mueller,
Anna Taubenberger,
Konstantinos Anastassiadis,
Jochen Guck
Development
144
(23 SI)
4313-4321
(2017)
| Journal
Cellular reprogramming is a dedifferentiation process during which cells continuously undergo phenotypical remodeling. Although the genetic and biochemical details of this remodeling are fairly well understood, little is known about the change in cell mechanical properties during the process. In this study, we investigated changes in the mechanical phenotype of murine fetal neural progenitor cells (fNPCs) during reprogramming to induced pluripotent stem cells (iPSCs). We find that fNPCs become progressively stiffer en route to pluripotency, and that this stiffening is mirrored by iPSCs becoming more compliant during differentiation towards the neural lineage. Furthermore, we show that the mechanical phenotype of iPSCs is comparable with that of embryonic stem cells. These results suggest that mechanical properties of cells are inherent to their developmental stage. They also reveal that pluripotent cells can differentiate towards a more compliant phenotype, which challenges the view that pluripotent stem cells are less stiff than any cells more advanced developmentally. Finally, our study indicates that the cell mechanical phenotype might be utilized as an inherent biophysical marker of pluripotent stem cells.
Numerical Simulation of Real-Time Deformability Cytometry To Extract
Cell Mechanical Properties
M. Mokbel,
D. Mokbel,
A. Mietke,
N. Traeber,
S. Girardo,
O. Otto,
Jochen Guck,
S. Aland
The measurement of cell stiffness is an important part of biological research with diverse applications in biology, biotechnology and medicine. Real-time deformability cytometry (RT-DC) is a new method to probe cell stiffness at high throughput by flushing cells through a microfluidic channel where cell-deformation provides an indicator for cell stiffness (Otto et al. Real-time deformability cytometry: on-the-fly cell 725 mechanical phenotyping. Nat. Methods 2015, 12, 199-202). Here, we propose a full numerical model for single cells in a flow channel to quantitatively relate cell deformation to mechanical parameters. Thereby the cell is modeled as a viscoelastic material surrounded by a thin shell cortex, subject to bending stiffness and cortical surface tension. For small deformations our results show good agreement with a previously developed analytical model that neglects the influence of cell deformation on the fluid flow (Mietke et al. Extracting Cell Stiffness from Real Time Deformability Cytometry: 728 Theory and Experiment. Biophys. J. 2015, 109, 2023-2036). Including linear elasticity as well as neo-Hookean hyperelasticity, our model is valid in a wide range of cell deformations and allows to extract cell stiffness for largely deformed cells. We introduce a new measure for cell deformation that is capable to distinguish between deformation effects stemming from cell cortex and cell bulk elasticity. Finally, we demonstrate the potential of the method to simultaneously quantify multiple mechanical cell parameters by RT-DC.
Mechanical mismatch between Ras transformed and untransformed epithelial
cells
Corinne Gullekson,
Gheorghe Cojoc,
Mirjam Schuermann,
Jochen Guck,
Andrew Pelling
The organization of the actin cytoskeleton plays a key role in regulating cell mechanics. It is fundamentally altered during transformation, affecting how cells interact with their environment. We investigated mechanical properties of cells expressing constitutively active, oncogenic Ras (Ras(V12)) in adherent and suspended states. To do this, we utilized atomic force microscopy and a microfluidic optical stretcher. We found that adherent cells stiffen and suspended cells soften with the expression of constitutively active Ras. The effect on adherent cells was reversed when contractility was inhibited with the ROCK inhibitor Y-27632, resulting in softer Ras(V12) cells. Our findings suggest that increased ROCK activity as a result of Ras has opposite effects on suspended and adhered cells. Our results also establish the importance of the activation of ROCK by Ras and its effect on cell mechanics.
Plasmodium falciparum erythrocyte-binding antigen 175 triggers a
biophysical change in the red blood cell that facilitates invasion
Marion Koch,
Katherine E. Wright,
Oliver Otto,
Maik Herbig,
Nichole D. Salinas,
Niraj H. Tolia,
Timothy J. Satchwell,
Jochen Guck,
Nicholas J. Brooks, et al.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
114
(16)
4225-4230
(2017)
| Journal
Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.
Mechanical deformation induces depolarization of neutrophils
Andrew E. Ekpenyong,
Nicole Toepfner,
Christine Fiddler,
Maik Herbig,
Wenhong Li,
Gheorghe Cojoc,
Charlotte Summers,
Jochen Guck,
Edwin R. Chilvers
The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.
Actin stress fiber organization promotes cell stiffening and
proliferation of pre-invasive breast cancer cells
Sandra Tavares,
Andre Filipe Vieira,
Anna Verena Taubenberger,
Margarida Araujo,
Nuno Pimpao Martins,
Catarina Bras-Pereira,
Antonio Polonia,
Maik Herbig,
Clara Barreto, et al.
Studies of the role of actin in tumour progression have highlighted its key contribution in cell softening associated with cell invasion. Here, using a human breast cell line with conditional Src induction, we demonstrate that cells undergo a stiffening state prior to acquiring malignant features. This state is characterized by the transient accumulation of stress fibres and upregulation of Ena/VASP-like (EVL). EVL, in turn, organizes stress fibres leading to transient cell stiffening, ERK-dependent cell proliferation, as well as enhancement of Src activation and progression towards a fully transformed state. Accordingly, EVL accumulates predominantly in premalignant breast lesions and is required for Src-induced epithelial overgrowth in Drosophila. While cell softening allows for cancer cell invasion, our work reveals that stress fibre-mediated cell stiffening could drive tumour growth during premalignant stages. A careful consideration of the mechanical properties of tumour cells could therefore offer new avenues of exploration when designing cancer-targeting therapies.
Roadmap for optofluidics
Paolo Minzioni,
Roberto Osellame,
Cinzia Sada,
S. Zhao,
F. G. Omenetto,
Kristinn B. Gylfason,
Tommy Haraldsson,
Yibo Zhang,
Aydogan Ozcan, et al.
Optofluidics, nominally the research area where optics and fluidics merge, is a relatively new research field and it is only in the last decade that there has been a large increase in the number of optofluidic. applications, as well as in the number of research groups, devoted to the topic. Nowadays optofluidics applications include, without being limited to, lab-on-a-chip devices, fluid-based and controlled lenses, optical sensors for fluids and for suspended particles, biosensors, imaging tools, etc. The long list of potential optofluidics applications, which have been recently demonstrated, suggests that optofluidic technologies will become more and more common in everyday life in the future, causing a significant impact on many aspects of our society. A characteristic of this research field, deriving from both its interdisciplinary origin and applications, is that in order to develop suitable solutions a. combination of a deep knowledge in different fields, ranging from materials science to photonics, from microfluidics to molecular biology and biophysics,. is often required. As a direct consequence, also being able to understand the long-term evolution of optofluidics research is not. easy. In this article, we report several expert contributions on different topics. so as to provide guidance for young scientists. At the same time, we hope that this document will also prove useful for funding institutions and stakeholders. to better understand the perspectives and opportunities offered by this research field.
Bone marrow niche-mimetics modulate HSPC function via integrin signaling
Martin Kraeter,
Angela Jacobi,
Oliver Otto,
Stefanie Tietze,
Katrin Mueller,
David M. Poitz,
Sandra Palm,
Valentina M. Zinna,
Ulrike Biehain, et al.
The bone marrow (BM) microenvironment provides critical physical cues for hematopoietic stem and progenitor cell (HSPC) maintenance and fate decision mediated by cell-matrix interactions. However, the mechanisms underlying matrix communication and signal transduction are less well understood. Contrary, stem cell culture is mainly facilitated in suspension cultures. Here, we used bone marrow-mimetic decellularized extracellular matrix (ECM) scaffolds derived from mesenchymal stromal cells (MSCs) to study HSPC-ECM interaction. Seeding freshly isolated HSPCs adherent (AT) and non-adherent (SN) cells were found. We detected enhanced expansion and active migration of AT-cells mediated by ECM incorporated stromal derived factor one. Probing cell mechanics, AT-cells displayed naive cell deformation compared to SN-cells indicating physical recognition of ECM material properties by focal adhesion. Integrin alpha IIb (CD41), alpha V (CD51) and beta 3 (CD61) were found to be induced. Signaling focal contacts via ITG beta 3 were identified to facilitate cell adhesion, migration and mediate ECM-physical cues to modulate HSPC function.
Three-dimensional correlative single-cell imaging utilizing fluorescence and refractive index tomography
Mirjam Schuermann,
Gheorghe Cojoc,
Salvatore Girardo,
Elke Ulbricht,
Jochen Guck,
Paul Mueller
Journal of Biophotonics
11
(3)
UNSP e201700145
(2017)
| Journal
Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.
Enlightening discriminative network functional modules behind Principal
Component Analysis separation in differential-omic science studies
Sara Ciucci,
Yan Ge,
Claudio Duran,
Alessandra Palladini,
Victor Jimenez-Jimenez,
Luisa Maria Martinez-Sanchez,
Yuting Wang,
Susanne Sales,
Andrej Shevchenko, et al.
Omic science is rapidly growing and one of the most employed techniques to explore differential patterns in omic datasets is principal component analysis (PCA). However, a method to enlighten the network of omic features that mostly contribute to the sample separation obtained by PCA is missing. An alternative is to build correlation networks between univariately-selected significant omic features, but this neglects the multivariate unsupervised feature compression responsible for the PCA sample segregation. Biologists and medical researchers often prefer effective methods that offer an immediate interpretation to complicated algorithms that in principle promise an improvement but in practice are difficult to be applied and interpreted. Here we present PC-corr: a simple algorithm that associates to any PCA segregation a discriminative network of features. Such network can be inspected in search of functional modules useful in the definition of combinatorial and multiscale biomarkers from multifaceted omic data in systems and precision biomedicine. We offer proofs of PC-corr efficacy on lipidomic, metagenomic, developmental genomic, population genetic, cancer promoteromic and cancer stem-cell mechanomic data. Finally, PC-corr is a general functional network inference approach that can be easily adopted for big data exploration in computer science and analysis of complex systems in physics.
Niche WNT5A regulates the actin cytoskeleton during regeneration of
hematopoietic stem cells
Christina Schreck,
Rouzanna Istvanffy,
Christoph Ziegenhain,
Theresa Sippenauer,
Franziska Ruf,
Lynette Henkel,
Florian Gaertner,
Beate Vieth,
M. Carolina Florian, et al.
JOURNAL OF EXPERIMENTAL MEDICINE
214
(1)
165-181
(2017)
| Journal
Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin(-)SCA-1(+) KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp(185) leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.
Mechanical Strain Promotes Oligodendrocyte Differentiation by Global Changes of Gene Expression
Anna Jagielska,
Alexis L. Lowe,
Ekta Makhija,
Liliana Wroblewska,
Jochen Guck,
Robin J. M. Franklin,
G. V. Shivashankar,
Krystyn J. Van Vliet
FRONTIERS IN CELLULAR NEUROSCIENCE
11
93
(2017)
| Journal
Differentiation of oligodendrocyte progenitor cells (OPC) to oligodendrocytes and subsequent axon myelination are critical steps in vertebrate central nervous system (CNS) development and regeneration. Growing evidence supports the significance of mechanical factors in oligodendrocyte biology. Here, we explore the effect of mechanical strains within physiological range on OPC proliferation and differentiation, and strain-associated changes in chromatin structure, epigenetics, and gene expression. Sustained tensile strain of 10-15% inhibited OPC proliferation and promoted differentiation into oligodendrocytes. This response to strain required specific interactions of OPCs with extracellular matrix ligands. Applied strain induced changes in nuclear shape, chromatin organization, and resulted in enhanced histone deacetylation, consistent with increased oligodendrocyte differentiation. This response was concurrent with increased mRNA levels of the epigenetic modifier histone deacetylase Hdac11. Inhibition of HDAC proteins eliminated the strain-mediated increase of OPC differentiation, demonstrating a role of HDACs in mechanotransduction of strain to chromatin. RNA sequencing revealed global changes in gene expression associated with strain. Specifically, expression of multiple genes associated with oligodendrocyte differentiation and axon-oligodendrocyte interactions was increased, including cell surface ligands (Ncam, ephrins), cyto-and nucleo-skeleton genes (Fyn, actinins, myosin, nesprin, Sun1), transcription factors (Sox10, Zfp191, Nkx2.2), and myelin genes (Cnp, Plp, Mag). These findings show how mechanical strain can be transmitted to the nucleus to promote oligodendrocyte differentiation, and identify the global landscape of signaling pathways involved in mechanotransduction. These data provide a source of potential new therapeutic avenues to enhance OPC differentiation in vivo.
High-throughput cell mechanical phenotyping for label-free titration
assays of cytoskeletal modifications
Stefan Golfier,
Philipp Rosendahl,
Alexander Mietke,
Maik Herbig,
Jochen Guck,
Oliver Otto
The mechanical fingerprint of cells is inherently linked to the structure of the cytoskeleton and can serve as a label-free marker for cell homeostasis or pathologic states. How cytoskeletal composition affects the physical response of cells to external loads has been intensively studied with a spectrum of techniques, yet quantitative and statistically powerful investigations in the form of titration assays are hampered by the low throughput of most available methods. In this study, we employ real-time deformability cytometry (RT-DC), a novel microfluidic tool to examine the effects of biochemically modified F-actin and microtubule stability and nuclear chromatin structure on cell deformation in a human leukemia cell line (HL60). The high throughput of our method facilitates extensive titration assays that allow for significance assessment of the observed effects and extraction of half-maximal concentrations for most of the applied reagents. We quantitatively show that integrity of the F-actin cortex and microtubule network dominate cell deformation on millisecond timescales probed with RT-DC. Drug-induced alterations in the nuclear chromatin structure were not found to consistently affect cell deformation. The sensitivity of the high-throughput cell mechanical measurements to the cytoskeletal modifications we present in this study opens up new possibilities for label-free dose-response assays of cytoskeletal modifications.
Volume Transitions of Isolated Cell Nuclei Induced by Rapid Temperature
Increase
Chii J. Chan,
Wenhong Li,
Gheorghe Cojoc,
Jochen Guck
Understanding the physical mechanisms governing nuclear mechanics is important as it can impact gene expression and development. However, how cell nuclei respond to external cues such as heat is not well understood. Here, we studied the material properties of isolated nuclei in suspension using an optical stretcher. We demonstrate that isolated nuclei regulate their volume in a highly temperature-sensitive manner. At constant temperature, isolated nuclei behaved like passive, elastic and incompressible objects, whose volume depended on the pH and ionic conditions. When the temperature was increased suddenly by even a few degrees Kelvin, nuclei displayed a repeatable and reversible temperature-induced volume transition, whose sign depended on the valency of the solvent. Such phenomenon is not observed for nuclei subjected to slow heating. The transition temperature could be shifted by adiabatic changes of the ambient temperature, and the magnitude of temperature-induced volume transition could be modulated by modifying the chromatin compaction state and remodeling processes. Our findings reveal that the cell nucleus can be viewed as a highly charged polymer gel with intriguing thermoresponsive properties, which might play a role in nuclear volume regulation and thermosensing in living cells.
Contact
Cell Physics Division Prof. Vahid Sandoghdar Acting Division Head
Max Planck Institute for the Science of Light Staudtstr. 2 91058 Erlangen, Germany
