OBJECTIVES: Reports on gadolinium (Gd) retention in soft tissues after administration of Gd-based contrast agents (GBCAs) raise concerns about Gd-induced changes in the biophysical properties of cells and tissues. Here, we investigate if clinical GBCAs of both classes of linear and macrocyclic structure cause changes in the mechanical properties of leukocytes in human blood samples. MATERIAL AND METHODS: Real-time deformability cytometry was applied to human blood samples from 6 donors. The samples were treated with 1 mM gadoteric acid (Dotarem), gadopentetic acid (Magnevist), gadobutrol (Gadovist), or Gd trichloride at 37°C for 1 hour to mimic clinical doses of GBCAs and exposure times. Leukocyte subtypes—lymphocytes, monocytes, and neutrophils—were identified based on their size and brightness and analyzed for deformability, which is inversely correlated with cellular stiffness. RESULTS: We observed significant stiffening (3%–13%, P < 0.01) of all investigated leukocyte subtypes, which was most pronounced for lymphocytes, followed by neutrophils and monocytes, and the effects were independent of the charge and steric structure of the GBCA applied. In contrast, no changes in cell size and brightness were observed, suggesting that deformability and cell stiffness measured by real-time deformability cytometry are sensitive to changes in the physical phenotypes of leukocytes after GBCA exposure. CONCLUSIONS: Real-time deformability cytometry might provide a quantitative blood marker for critical changes in the physical properties of blood cells in patients undergoing GBCA-enhanced magnetic resonance imaging.
Passive coupling of membrane tension and cell volume during active response of cells to osmosis
Chloé Roffay,
Guillaume Molinard,
Kyoohyun Kim,
Marta Urbanska,
Virginia Andrade,
Victoria Barbarasa,
Paulina Nowak,
Vincent Mercier,
José García-Calvo, et al.
Proceedings of the National Academy of Sciences of the United States of America
118
(47)
e2103228118
(2021)
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During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.
Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy
Vaibhav Mahajan,
Timon Beck,
Paulina Gregorczyk,
André Ruland,
Simon Alberti,
Jochen Guck,
Carsten Werner,
Raimund Schlüßler,
Anna V. Taubenberger
Cancers / Molecular Diversity Preservation International (MDPI)
13
(21)
5549
(2021)
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Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.
Matrix stiffness mechanosensing modulates the expression and distribution of transcription factors in Schwann cells
Gonzalo Rosso,
Daniel Wehner,
Christine Schweitzer,
Stephanie Möllmert,
Elisabeth Sock,
Jochen Guck,
Victor Shahin
Bioengineering & Translational Medicine
e10257
(2021)
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After peripheral nerve injury, mature Schwann cells (SCs) de-differentiate and undergo cell reprogramming to convert into a specialized cell repair phenotype that promotes nerve regeneration. Reprogramming of SCs into the repair phenotype is tightly controlled at the genome level and includes downregulation of pro-myelinating genes and activation of nerve repair-associated genes. Nerve injuries induce not only biochemical but also mechanical changes in the tissue architecture which impact SCs. Recently, we showed that SCs mechanically sense the stiffness of the extracellular matrix and that SC mechanosensitivity modulates their morphology and migratory behavior. Here, we explore the expression levels of key transcription factors and myelin-associated genes in SCs, and the outgrowth of primary dorsal root ganglion (DRG) neurites, in response to changes in the stiffness of generated matrices. The selected stiffness range matches the physiological conditions of both utilized cell types as determined in our previous investigations. We find that stiffer matrices induce upregulation of the expression of transcription factors Sox2, Oct6, and Krox20, and concomitantly reduce the expression of the repair-associated transcription factor c-Jun, suggesting a link between SC substrate mechanosensing and gene expression regulation. Likewise, DRG neurite outgrowth correlates with substrate stiffness. The remarkable intrinsic physiological plasticity of SCs, and the mechanosensitivity of SCs and neurites, may be exploited in the design of bioengineered scaffolds that promote nerve regeneration upon injury.
Physical phenotype of blood cells is altered in COVID-19
Markéta Kubánková,
Bettina Hohberger,
Jakob Hoffmanns,
Julia Fürst,
Martin Herrmann,
Jochen Guck,
Martin Kräter
Biophysical Journal
120
(14)
2838-2847
(2021)
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Clinical syndrome coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 is characterized by rapid spreading and high mortality worldwide. Although the pathology is not yet fully understood, hyperinflammatory response and coagulation disorders leading to congestions of microvessels are considered to be key drivers of the still-increasing death toll. Until now, physical changes of blood cells have not been considered to play a role in COVID-19 related vascular occlusion and organ damage. Here, we report an evaluation of multiple physical parameters including the mechanical features of five frequent blood cell types, namely erythrocytes, lymphocytes, monocytes, neutrophils, and eosinophils. More than four million blood cells of 17 COVID-19 patients at different levels of severity, 24 volunteers free from infectious or inflammatory diseases, and 14 recovered COVID-19 patients were analyzed. We found significant changes in lymphocyte stiffness, monocyte size, neutrophil size and deformability, and heterogeneity of erythrocyte deformation and size. Although some of these changes recovered to normal values after hospitalization, others persisted for months after hospital discharge, evidencing the long-term imprint of COVID-19 on the body.
HIF2α is a Direct Regulator of Neutrophil Motility
Sundary Sormendi,
Mathieu Deygas,
Anupam Sinha,
Anja Krüger,
Ioannis Kourtzelis,
Gregoire Le Lay,
Mathilde Bernard,
Pablo J. Sáez,
Michael Gerlach, et al.
Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2α) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.
A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord
Leonardo Cavone,
Tess McCann,
Louisa K. Drake,
Erika A. Aguzzi,
Ana-Maria Oprisoreanu,
Elisa Pedersen,
Soe Sandi,
Jathurshan Selvarajah,
Themistoklis M. Tsarouchas, et al.
Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.
Know How to Regrow-Axon Regeneration in the Zebrafish Spinal Cord
The capacity for long-distance axon regeneration and functional recovery after spinal cord injury is poor in mammals but remarkable in some vertebrates, including fish and salamanders. The cellular and molecular basis of this interspecies difference is beginning to emerge. This includes the identification of target cells that react to the injury and the cues directing their pro-regenerative responses. Among existing models of successful spinal cord regeneration, the zebrafish is arguably the most understood at a mechanistic level to date. Here, we review the spinal cord injury paradigms used in zebrafish, and summarize the breadth of neuron-intrinsic and -extrinsic factors that have been identified to play pivotal roles in the ability of zebrafish to regenerate central nervous system axons and recover function.
Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation
Alena Uvizl,
Ruchi Goswami,
Shanil Durgeshkumar Gandhi,
Martina Augsburg,
Frank Buchholz,
Jochen Guck,
Jörg Mansfeld,
Salvatore Girardo
Optical trapping is a vital tool in biology, allowing precise optical manipulation of nanoparticles, micro-robots, and cells. Due to the low risk of photodamage and high trap stiffness, fiber-based dual-beam traps are widely used for optical manipulation of large cells. Besides trapping, advanced applications like 3D refractive index tomography need a rotation of cells, which requires precise control of the forces, for example, the acting-point of the forces and the intensities in the region of interest (ROI). A precise rotation of large cells in 3D about arbitrary axes has not been reported yet in dual-beam traps. We introduce a novel dual-beam optical trap in which a multi-core fiber (MCF) is transformed to a phased array, using wavefront shaping and computationally programmable light. The light-field distribution in the trapping region is holographically controlled within 0.1 s, which determines the orientation and the rotation axis of the cell with small retardation. We demonstrate real-time controlled rotation of HL60 cells about all 3D axes with a very high degree of freedom by holographic controlled light through an MCF with a resolution close to the diffraction limit. For the first time, the orientation of the cell can be precisely controlled about all 3D axes in a dual-beam trap. MCFs provide much higher flexibility beyond the bulky optics, enabling lab-on-a-chip applications and can be easily integrated for applications like contactless cell surgery, refractive index tomography, cell-elasticity measurement, which require precise 3D manipulation of cells.
Toward deep biophysical cytometry: prospects and challenges
Kelvin C.M. Lee,
Jochen Guck,
Keisuke Goda,
Kevin K. Tsia
Trends in Biotechnology
39
(12)
1249-1262
(2021)
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Recent advances in biophysical cytometry now make it possible to recapitulate cellular heterogeneity at the levels of throughput, precision, specificity, and sensitivity that were once inconceivable. Technological developments in state-of-the-art biophysical cytometry include single-cell mass assays, cell traction force assays, deformability and impedance cytometry, and label-free imaging cytometry. Next-generation biophysical cytometry could be more comprehensive and information-rich by exploring multimodal integration, such as simultaneous read-out of cell mass, stiffness, and morphology. How molecular signatures translate into cellular biophysical properties is not fully understood. Advanced techniques involving microfluidics, imaging, and deep learning could investigate this link. Standardizing the protocols and datasets of biophysical cytometry will be crucial to ensure wide dissemination.
The Xenopus spindle is as dense as the surrounding cytoplasm
Abin Biswas,
Kyoohyun Kim,
Gheorghe Cojoc,
Jochen Guck,
Simone Reber
The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle’s material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle’s mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle’s emergent physical properties—essential to advance predictive frameworks of spindle assembly and function.
Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response
Joan-Carles Escolano,
Anna V. Taubenberger,
Shada Abuhattum,
Christine Schweitzer,
Aleeza Farrukh,
Aránzazu del Campo,
Clare E. Bryant,
Jochen Guck
Frontiers in Cell and Developmental Biology
9
639815
(2021)
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Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.
AIDeveloper: deep learning image classification in life science and beyond
Martin Kräter,
Shada Abuhattum Hofemeier,
Despina Soteriou,
Angela Jacobi,
Thomas Krüger,
Jochen Guck,
Maik Herbig
Artificial intelligence (AI)‐based image analysis has increased drastically in recent years. However, all applications use individual solutions, highly specialized for a particular task. Here, an easy‐to‐use, adaptable, and open source software, called AIDeveloper (AID) to train neural nets (NN) for image classification without the need for programming is presented. AID provides a variety of NN‐architectures, allowing to apply trained models on new data, obtain performance metrics, and export final models to different formats. AID is benchmarked on large image datasets (CIFAR‐10 and Fashion‐MNIST). Furthermore, models are trained to distinguish areas of differentiated stem cells in images of cell culture. A conventional blood cell count and a blood count obtained using an NN are compared, trained on >1.2 million images, and demonstrated how AID can be used for label‐free classification of B‐ and T‐cells. All models are generated by non‐programmers on generic computers, allowing for an interdisciplinary use.
Mechanical properties of cell- and microgel
bead-laden oxidized alginate-gelatin hydrogels
Thomas Distler,
Lena Kretzschmar,
Dominik Schneidereit,
Salvatore Girardo,
Ruchi Goswami,
Oliver Friedrich,
Rainer Detsch,
Jochen Guck,
Aldo R. Boccaccini, et al.
Biomaterials Science
(9)
3051-3068
(2021)
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3D-printing technologies, such as biofabrication, capitalize on the homogeneous distribution and growth of cells inside biomaterial hydrogels, ultimately aiming to allow for cell differentiation, matrix remodeling, and functional tissue analogues. However, commonly, only the mechanical properties of the bioinks or matrix materials are assessed, while the detailed influence of cells on the resulting mechanical properties of hydrogels remains insufficiently understood. Here, we investigate the properties of hydrogels containing cells and spherical PAAm microgel beads through multi-modal complex mechanical analyses in the small- and large-strain regimes. We evaluate the individual contributions of different filler concentrations and a non-fibrous oxidized alginate-gelatin hydrogel matrix on the overall mechanical behavior in compression, tension, and shear. Through material modeling, we quantify parameters that describe the highly nonlinear mechanical response of soft composite materials. Our results show that the stiffness significantly drops for cell- and bead concentrations exceeding four million per milliliter hydrogel. In addition, hydrogels with high cell concentrations (≥6 mio ml−1) show more pronounced material nonlinearity for larger strains and faster stress relaxation. Our findings highlight cell concentration as a crucial parameter influencing the final hydrogel mechanics, with implications for microgel bead drug carrier-laden hydrogels, biofabrication, and tissue engineering.
A switch in pdgfrb+ cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord
Vasiliki Tsata,
Stephanie Möllmert,
Christine Schweitzer,
Julia Kolb,
Conrad Möckel,
Benjamin Böhm,
Gonzalo Rosso,
Christian Lange,
Mathias Lesche, et al.
In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM.
Contact
Cell Physics Division Prof. Vahid Sandoghdar Acting Division Head
Max Planck Institute for the Science of Light Staudtstr. 2 91058 Erlangen, Germany
