Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells
Shada Abuhattum,
Kyoohyun Kim,
Titus M. Franzmann,
Anne Esslinger,
Daniel Midtvedt,
Raimund Schluessler,
Stephanie Mollmert,
Hui-Shun Kuan,
Simon Alberti, et al.
Many organisms, including yeast cells, bacteria, nematodes, and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography (ODT) and atomic force microscopy (AFM). We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of a solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.
Comparative study of cell mechanics methods
Pei-Hsun Wu,
Dikla Raz-Ben Aroush,
Atef Asnacios,
Wei-Chiang Chen,
Maxim Dokukin,
Bryant L Doss,
Pauline Durand,
Andrew Ekpenyong,
Jochen Guck, et al.
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.
Metabolic Profiling of Human Eosinophils
Linsey Porter,
Nicole Toepfner,
Kathleen R. Bashant,
Jochen Guck,
Margaret Ashcroft,
Neda Farahi,
Edwin R. Chilvers
Immune cells face constant changes in their microenvironment, which requires rapid metabolic adaptation. In contrast to neutrophils, which are known to rely near exclusively on glycolysis, the metabolic profile of human eosinophils has not been characterized. Here, we assess the key metabolic parameters of peripheral blood-derived human eosinophils using real-time extracellular flux analysis to measure extracellular acidification rate and oxygen consumption rate, and compare these parameters to human neutrophils. Using this methodology, we demonstrate that eosinophils and neutrophils have a similar glycolytic capacity, albeit with a minimal glycolytic reserve. However, compared to neutrophils, eosinophils exhibit significantly greater basal mitochondrial respiration, ATP-linked respiration, maximum respiratory capacity, and spare respiratory capacity. Of note, the glucose oxidation pathway is also utilized by eosinophils, something not evident in neutrophils. Furthermore, using a colorimetric enzymatic assay, we show that eosinophils have much reduced glycogen stores compared to neutrophils. We also show that physiologically relevant levels of hypoxia (PO2 3 kPa), by suppressing oxygen consumption rates, have a profound effect on basal and phorbol-myristate-acetate-stimulated eosinophil and neutrophil metabolism. Finally, we compared the metabolic profile of eosinophils purified from atopic and non-atopic subjects and show that, despite a difference in the activation status of eosinophils derived from atopic subjects, these cells exhibit comparable oxygen consumption rates upon priming with IL-5 and stimulation with fMLP. In summary, our findings show that eosinophils display far greater metabolic flexibility compared to neutrophils, with the potential to use glycolysis, glucose oxidation, and oxidative phosphorylation. This flexibility may allow eosinophils to adapt better to diverse roles in host defense, homeostasis, and immunomodulation.
Accurate evaluation of size and refractive index for spherical objects
in quantitative phase imaging
Paul Mueller,
Mirjam Schuermann,
Salvatore Girardo,
Gheorghe Cojoc,
Jochen Guck
Measuring the average refractive index (RI) of spherical objects, such as suspended cells, in quantitative phase imaging (QPI) requires a decoupling of RI and size from the QPI data. This has been commonly, achieved by determining the object's radius with geometrical approaches, neglecting light-scattering. Here, we present a novel QPI Fitting algorithm that reliably uncouples the RI using Mie theory and a semi-analytical, corrected Rytov approach. We assess the range of validity of this algorithm in silico and experimentally investigate various objects (oil and protein droplets, microgel beads, cells) and noise conditions. In addition, we provide important practical cues for the analysis of spherical objects in QPI. Published by The Optical Society under the terms of the Creative Commons Attribution 4.0 License.
High-throughput single-cell mechanical phenotyping with real-time
deformability cytometry
Marta Urbanska,
Philipp Rosendahl,
Martin Kraeter,
Jochen Guck
MICROFLUIDICS IN CELL BIOLOGY, PT B: MICROFLUIDICS IN SINGLE CELLS
147
175-+
(2018)
| Journal
Mechanical properties of cells can serve as a label-free marker of cell state and function and their alterations have been implicated in processes such as cancer metastasis, leukocyte activation, or stem cell differentiation. Over recent years, new techniques for single-cell mechanical characterization at high throughput have been developed to accelerate discovery in the field of mechanical phenotyping. One such technique is real-time deformability cytometry (RT-DC), a robust technology based on microfluidics that performs continuous mechanical characterization of cells in a contactless manner at rates of up to 1000 cells per second. This tremendous throughput allows for comparison of large sample numbers and precise characterization of heterogeneous cell populations. Additionally, parameters acquired in RT-DC measurements can be used to determine the apparent Young's modulus of individual cells. In this chapter, we present practical aspects important for the implementation of the RT-DC methodology, including a description of the setup, operation principles, and experimental protocols. In the latter, we describe a variety of preparation procedures for samples originating from different sources including 2D and 3D cell cultures as well as blood and tissue-derived primary cells, and discuss obstacles that may arise during their measurements. Finally, we provide insights into standard data analysis procedures and discuss the method's performance in light of other available techniques.
Droplet-Assisted Microfluidic Fabrication and Characterization of
Multifunctional Polysaccharide Microgels Formed by Multicomponent
Reactions
Nicolas Hauck,
Nalin Seixas,
Silvia P. Centeno,
Raimund Schluelsser,
Gheorghe Cojoc,
Paul Mueller,
Jochen Guck,
Dominik Woell,
Ludger A. Wessjohann, et al.
Polysaccharide-based microgels have broad applications in multi-parametric cell cultures, cell-free biotechnology, and drug delivery. Multicomponent reactions like the Passerini three-component and the Ugi four-component reaction are shown in here to be versatile platforms for fabricating these polysaccharide microgels by droplet microfluidics with a narrow size distribution. While conventional microgel formation requires pre-modification of hydrogel building blocks to introduce certain functionality, in multicomponent reactions one building block can be simply exchanged by another to introduce and extend functionality in a library-like fashion. Beyond synthesizing a range of polysaccharide-based microgels utilizing hyaluronic acid, alginate and chitosan, exemplary in-depth analysis of hyaluronic acid-based Ugi four-component gels is conducted by colloidal probe atomic force microscopy, confocal Brillouin microscopy, quantitative phase imaging, and fluorescence correlation spectroscopy to elucidate the capability of microfluidic multicomponent reactions for forming defined polysaccharide microgel networks. Particularly, the impact of crosslinker amount and length is studied. A higher network density leads to higher Young's moduli accompanied by smaller pore sizes with lower diffusion coefficients of tracer molecules in the highly homogeneous network, and vice versa. Moreover, tailored building blocks allow for crosslinking the microgels and incorporating functional groups at the same time as demonstrated for biotin-functionalized, chitosan-based microgels formed by Ugi four-component reaction. To these microgels, streptavidin-labeled enzymes are easily conjugated as shown for horseradish peroxidase (HRP), which retains its activity inside the microgels.
Standardized microgel beads as elastic cell mechanical probes
S. Girardo,
N. Traeber,
K. Wagner,
G. Cojoc,
C. Herold,
R. Goswami,
R. Schluessler,
S. Abuhattum,
A. Taubenberger, et al.
JOURNAL OF MATERIALS CHEMISTRY B
6
(39)
6245-6261
(2018)
| Journal
Cell mechanical measurements are gaining increasing interest in biological and biomedical studies. However, there are no standardized calibration particles available that permit the cross-comparison of different measurement techniques operating at different stresses and time-scales. Here we present the rational design, production, and comprehensive characterization of poly-acrylamide (PAAm) microgel beads mimicking size and overall mechanics of biological cells. We produced mono-disperse beads at rates of 20-60 kHz by means of a microfluidic droplet generator, where the pre-gel composition was adjusted to tune the beads' elasticity in the range of cell and tissue relevant mechanical properties. We verified bead homogeneity by optical diffraction tomography and Brillouin microscopy. Consistent elastic behavior of microgel beads at different shear rates was confirmed by AFM-enabled nanoindentation and real-time deformability cytometry (RT-DC). The remaining inherent variability in elastic modulus was rationalized using polymer theory and effectively reduced by sorting based on forward-scattering using conventional flow cytometry. Our results show that PAAm microgel beads can be standardized as mechanical probes, to serve not only for validation and calibration of cell mechanical measurements, but also as cell-scale stress sensors.
Detection of human disease conditions by single-cell morpho-rheological
phenotyping of blood
Nicole Toepfner,
Christoph Herold,
Oliver Otto,
Philipp Rosendahl,
Angela Jacobi,
Martin Kraeter,
Julia Staechele,
Leonhard Menschner,
Maik Herbig, et al.
Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis.
Axonal Transport, Phase-Separated Compartments, and Neuron Mechanics - A
New Approach to Investigate Neurodegenerative Diseases
Martin Noetzel,
Gonzalo Rosso,
Stephanie Moellmert,
Anne Seifert,
Raimund Schluessler,
Kyoohyun Kim,
Andreas Hermann,
Jochen Guck
FRONTIERS IN CELLULAR NEUROSCIENCE
12
358
(2018)
| Journal
Many molecular and cellular pathogenic mechanisms of neurodegenerative diseases have been revealed. However, it is unclear what role a putatively impaired neuronal transport with respect to altered mechanical properties of neurons play in the initiation and progression of such diseases. The biochemical aspects of intracellular axonal transport, which is important for molecular movements through the cytoplasm, e.g., mitochondrial movement, has already been studied. Interestingly, transport deficiencies are associated with the emergence of the affliction and potentially linked to disease transmission. Transport along the axon depends on the normal function of the neuronal cytoskeleton, which is also a major contributor to neuronal mechanical properties. By contrast, little attention has been paid to the mechanical properties of neurons and axons impaired by neurodegeneration, and of membraneless, phase-separated organelles such as stress granules (SGs) within neurons. Mechanical changes may indicate cytoskeleton reorganization and function, and thus give information about the transport and other system impairment. Nowadays, several techniques to investigate cellular mechanical properties are available. In this review, we discuss how select biophysical methods to probe material properties could contribute to the general understanding of mechanisms underlying neurodegenerative diseases.
Real-Time Deformability Cytometry: Label-Free Functional
Characterization of Cells
Maik Herbig,
Martin Kraeter,
Katarzyna Plak,
Paul Mueller,
Jochen Guck,
Oliver Otto
Real-time deformability cytometry (RT-DC) is a microfluidic technique that allows to capture and evaluate morphology and rheology of up to 1000 cells/s in a constricted channel. The cells are deformed without mechanical contact by hydrodynamic forces and are quantified in real-time without the need of additional handling or staining procedures. Segmented pictures of the cells are stored and can be used for further analysis. RT-DC is sensitive to alterations of the cytoskeleton, which allows, e.g., to show differences in cell cycle phases, identify different subpopulations in whole blood and to study mechanical stiffening of cells entering a dormant state. The abundance of the obtainable parameters and the interpretation as mechanical readout is an analytical challenge that needs standardization. Here, we will provide guidelines for measuring and post-processing of RT-DC data.
Toll-Like Receptor-Mediated Upregulation of CXCL16 in Psoriasis
Orchestrates Neutrophil Activation
JOURNAL OF INVESTIGATIVE DERMATOLOGY
138
(2)
344-354
(2018)
| Journal
Innate immune processes are central in the development of the chronic inflammatory skin disease psoriasis. Studying stimulation of keratinocytes, monocytes, and dendritic cells by type I interferons or ligation of Tolllike receptors 1/2, 2/6, or 7, but not 7/8, resulted in enhanced surface expression and secretion of CXC chemokine ligand (CXCL) 16. The corresponding CXC chemokine receptor 6 was expressed on neutrophils whose recruitment into skin is important, especially in early psoriatic disease. Using the recently developed technique real-time deformability cytometry demonstrated that CXCL16 and IL-8 decreased the stiffness and enhanced deformation of neutrophils facilitating transmigration through vessel wall. In addition, CXCL16 potently induced migration of neutrophils and enhanced the chemotactic effect of IL-8. The positive feedback loop was supported by IL-8 enhancing CXCL16 production of neutrophils. Blocking of CXCL16 expression by effective treatment of psoriasis patients with tumor necrosis factor-alpha blockers further supported the pathogenic role of this chemokine. In summary, the data link innate immune stimulation to CXCL16 upregulation and neutrophil infiltration into skin. CXCL16 could therefore represent a potent future target for treatment of psoriasis.
Mechanical Mapping of Spinal Cord Growth and Repair in Living Zebrafish
Larvae by Brillouin Imaging
Raimund Schluessler,
Stephanie Moellmert,
Shada Abuhattum,
Gheorghe Cojoc,
Paul Mueller,
Kyoohyun Kim,
Conrad Moeckel,
Conrad Zimmermann,
Jurgen Czarske, et al.
The mechanical properties of biological tissues are increasingly recognized as important factors in developmental and pathological processes. Most existing mechanical measurement techniques either necessitate destruction of the tissue for access or provide insufficient spatial resolution. Here, we show for the first time to our knowledge a systematic application of confocal Brillouin microscopy to quantitatively map the mechanical properties of spinal cord tissues during biologically relevant processes in a contact-free and nondestructive manner. Living zebrafish larvae were mechanically imaged in all anatomical planes during development and after spinal cord injury. These experiments revealed that Brillouin microscopy is capable of detecting the mechanical properties of distinct anatomical structures without interfering with the animal's natural development. The Brillouin shift within the spinal cord remained comparable during development and transiently decreased during the repair processes after spinal cord transection. By taking into account the refractive index distribution, we explicitly determined the apparent longitudinal modulus and viscosity of different larval zebrafish tissues. Importantly, mechanical properties differed between tissues in situ and in excised slices. The presented work constitutes the first step toward an in vivo assessment of spinal cord tissue mechanics during regeneration, provides a methodical basis to identify key determinants of mechanical tissue properties, and allows us to test their relative importance in combination with biochemical and genetic factors during developmental and regenerative processes.
Real-time fluorescence and deformability cytometry
Philipp Rosendahl,
Katarzyna Plak,
Angela Jacobi,
Martin Kraeter,
Nicole Toepfner,
Oliver Otto,
Christoph Herold,
Maria Winzi,
Maik Herbig, et al.
The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.
Alterations in Cell Mechanics by Actin Cytoskeletal Changes Correlate
with Strain-Specific Rubella Virus Phenotypes for Cell Migration and
Induction of Apoptosis
The cellular cytoskeleton is central for key cellular functions, and as such is a marker for diseased and infected cell states. Here we analyzed infection with rubella virus (RV) strains with respect to phenotypes in cellular mechanical properties, cell movement, and viral cytopathogenicity. Real-time deformability cytometry (RT-DC), as a high-throughput platform for the assessment of cell mechanics, revealed a correlation of an increase in cortical filamentous-actin (F-actin) with a higher cellular stiffness. The additional reduction of stress fibers noted for only some RV strains as the most severe actin rearrangement lowered cell stiffness. Furthermore, a reduced collective and single cell migration speed in a wound healing assay was detected in addition to severe changes in cell morphology. The latter was followed by activation of caspase 3/7 as a sign for induction of apoptosis. Our study emphasizes RT-DC technology as a sensitive means to characterize viral cell populations and to implicate alterations of cell mechanical properties with cell functions. These interdependent events are not only promising options to elucidate viral spread and to understand viral pathologies within the infected host. They also contribute to any diseased cell state, as exemplified by RV as a representative agent for cytoskeletal alterations involved in a cytopathological outcome.
Contact
Cell Physics Division Prof. Vahid Sandoghdar Acting Division Head
Max Planck Institute for the Science of Light Staudtstr. 2 91058 Erlangen, Germany
