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ABSTRACT: Cells migrate through a crowded environment during processes such as metastasis or wound healing, and must generate and withstand substantial forces. The cellular motility responses to environmental forces are represented by their force-velocity relation, which has been measured for fish keratocytes but remains unexplained. Even pN opposing forces slow down lamellipodium motion by three orders of magnitude. At larger opposing forces, the retrograde flow of the actin network accelerates until it compensates for polymerization, and cell motion stalls. Subsequently, the lamellipodium adapts to the stalled state. We present a mechanism quantitatively explaining the cell's force-velocity relation and its changes upon application of drugs that hinder actin polymerization or actomyosin-based contractility. Elastic properties of filaments, close to the lamellipodium leading edge, and retrograde flow shape the force-velocity relation. To our knowledge, our results shed new light on how these migratory responses are regulated, and on the mechanics and structure of the lamellipodium.
Biophysical Journal 01/2012; 102(2):287-95. · 3.65 Impact Factor
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Biophys J. 01/2012; 102(2):287-295.
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ABSTRACT: Optical traps such as tweezers and stretchers are widely used to probe the mechanical properties of cells. Beyond their large range of applications, the use of infrared laser light in optical traps causes significant heating effects in the cell. This study investigated the effect of laser-induced heating on cell viability. Common viability assays are not very sensitive to damages caused in short periods of time or are not practicable for single cell analysis. We used cell spreading, a vital ability of cells, as a new sensitive viability marker. The optical stretcher, a two beam laser trap, was used to simulate heat shocks that cells typically experience during measurements in optical traps. The results show that about 60% of the cells survived heat shocks without vital damage at temperatures of up to 58 ± 2°C for 0.5 s. By varying the duration of the heat shocks, it was shown that 60% of the cells stayed viable when exposed to 48 ± 2°C for 5 s.
Biophysics of Structure and Mechanism 06/2011; 40(9):1109-14. · 2.44 Impact Factor
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ABSTRACT: Despite its impressive complexity the cytoskeleton succeeds to persistently organize itself and thus the cells' interior. In contrast to classical man-made machines, much of the cellular organization originates from inherent self-assembly and self-organization allowing a high degree of autonomy for various functional units. Recent experimental and theoretical studies revealed numerous examples of cytoskeleton components that arrange and organize in a self-regulative way. In the present review we want to shortly summarize some of the principle mechanisms that are able to inherently trigger and regulate the cytoskeleton organization. Although taken individually most of these regulative principles are rather simple with intuitively predictable consequences, combinations of two or more of these mechanisms can quickly give rise to very complex, unexpected behavior and might even be able to explain the formation of different functional units out of a common pool of available building blocks.
Cytoskeleton 03/2011; 68(5):259-65.
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ABSTRACT: Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reaction-diffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extension's bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility.
PLoS ONE 01/2011; 6(1):e14471. · 4.09 Impact Factor
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ABSTRACT: F-actin bundles are prominent cytoskeletal structures in eukaryotes. They provide mechanical stability in stereocilia, microvilli, filopodia, stress fibers and the sperm acrosome. Bundles are typically stabilized by a wide range of specific crosslinking proteins, most of which exhibit off-rates on the order of 1s(-1). Yet F-actin bundles exhibit structural and mechanical integrity on time scales that are orders of magnitude longer. By applying large deformations to reconstituted F-actin bundles using optical tweezers, we provide direct evidence of their differential mechanical response in vitro: bundles exhibit fully reversible, elastic response on short time scales and irreversible, elasto-plastic response on time scales that are long compared to the characteristic crosslink dissociation time. Our measurements show a broad range of characteristic relaxation times for reconstituted F-actin bundles. This can be reconciled by considering that bundle relaxation behavior is also modulated by the number of filaments, crosslinking type and occupation number as well as the consideration of defects due to filament ends.
Biophysics of Structure and Mechanism 01/2011; 40(1):93-101. · 2.44 Impact Factor
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Björn Kemper,
Patrik Langehanenberg,
Alexander Höink,
Gert von Bally,
Falk Wottowah,
Stefan Schinkinger,
Jochen Guck, Josef Käs,
Ilona Bredebusch,
Jürgen Schnekenburger,
Karin Schütze
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ABSTRACT: For a precise manipulation of particles and cells with laser light as well as for the understanding and the control of the underlying processes it is important to visualize and quantify the response of the specimens. Thus, we investigated if digital holographic microscopy (DHM) can be used in combination with microfluidics to observe optically trapped living cells in a minimally invasive fashion during laser micromanipulation. The obtained results demonstrate that DHM multi-focus phase contrast provides label-free quantitative monitoring of optical manipulation with a temporal resolution of a few milliseconds.
Journal of Biophotonics 07/2010; 3(7):425-31. · 4.34 Impact Factor
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01/2010: pages 1201-1226; , ISBN: 3642025242
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Kristian Franze,
Jens Gerdelmann,
Michael Weick,
Timo Betz,
Steve Pawlizak,
Melike Lakadamyali,
Johannes Bayer,
Katja Rillich,
Michael Gögler,
Yun-Bi Lu,
Andreas Reichenbach,
Paul Janmey, Josef Käs
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ABSTRACT: Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.
Biophysical Journal 10/2009; 97(7):1883-90. · 3.65 Impact Factor
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ABSTRACT: Rather than forming a simple and uniform nematic liquid crystal, concentrated solutions of semiflexible polymers, such as F-actin, have been observed to display a spatially periodic switching of the nematic director. When observed with polarization microscopy, these patterns appear as alternating light and dark bands, often referred to as zebra stripe patterns. Zebra stripe patterns, although not fully characterized, are due to periodic orientation distortions in the nematic order. We characterize such patterns by using a combination of two techniques. Using polarization microscopy, we quantify the periodic orientation distortions and show that the magnitude of the order parameter also varies periodically in the striped domains. When using fluorescently labeled filaments as markers, filaments spanning the striped domains are seen to undergo large angle bends. With fluorescence, clear density differences between adjacent stripes are also observed with domains of lesser density corresponding to strongly bent filaments. By directly comparing patterned areas with both polarization and fluorescence techniques, we show that periodic variation in the orientation, order parameter, filament bending, and density are correlated. We propose that these effects originate from the coupling of orientation and density that occurs for highly concentrated solutions of long semiflexible polymers subject to shear flows, as previously proposed [P. de Gennes, Mol. Cryst. Liq. Cryst. (Phila. Pa.) 34, 177 (1977)]. After cessation of shearing, strong interfilament interactions and high compressibility can lead to periodic buckling from the relaxation of filaments stretched during flows. The characterization of zebra stripe patterns presented here provides evidence that buckling in confined F-actin nematics produces strong periodic bending that is responsible for the observed features.
Physical Review E 04/2009; 79(3 Pt 1):031916. · 2.26 Impact Factor
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ABSTRACT: For a long time, neurosciences have focused on biochemical, molecular, and electrophysiological aspects of cell functioning.
However, there is an increasing awareness of the importance of biomechanics in physiology and pathology of the central nervous
system (CNS). In the first part of this review we provide physical basics necessary to understand biomechanical measurements,
we introduce the cytoskeleton as a major contributor to a cell’s passive and active mechanical behavior, and we discuss some
of the methods nowadays used to quantify mechanical properties. In the second part we present actual data on CNS mechanics,
and we discuss the impact of passive mechanical material properties and active mechanical behavior of cells on the development,
normal functioning and pathology of the CNS.
12/2008: pages 173-213;
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ABSTRACT: A flow-cytometry-based assay is presented with which the uptake of polyelectrolyte capsules can be quantified. The cavity of the capsules is loaded with the pH-sensitive dye SNARF, which emits in the red and green in alkaline and acidic environments, respectively. By recording the fluorescence intensities in the red and green channels, the localization of capsules associated with cells can be determined. Capsules adherent to the outer cell membrane fluoresce in the red due to the alkaline pH of the cell medium, whereas capsules internalized by cells fluoresce in the green due to the acidic pH in the endosomal/lysosomal/phagosomal compartments in which incorporated capsules are located. Adding the SNARF readout to the scattering signal typically derived with flow cytometry analysis allows for a more detailed quantitative analysis of particle uptake, which can also distinguish between adherent and ingested particles.
Small 10/2008; 4(10):1763-8. · 8.35 Impact Factor
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ABSTRACT: Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1-2 microm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data.
Biophysical Journal 09/2008; 95(12):5508-23. · 3.65 Impact Factor
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Alumdena Muñoz Javier,
Oliver Kreft,
Maximilian Semmling,
Susanne Kempter,
Andre G. Skirtach,
Oliver T. Bruns,
Pablo del Pino,
Mathieu F. Bedard,
Joachim Rädler, Josef Käs,
Christian Plank,
Gleb B. Sukhorukov,
Wolfgang J. Parak
Advanced Materials 08/2008; 20(22):4281 - 4287. · 13.88 Impact Factor
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ABSTRACT: We present a novel technique to noninvasively control the growth and turning behavior of an extending neurite. A highly focused infrared laser, positioned at the leading edge of a neurite, has been found to induce extension/turning toward the beam's center. This technique has been used successfully to guide NG108-15 and PC12 cell lines [Ehrlicher, A., Betz, T., Stuhrmann, B., Koch, D. Milner, V. Raizen, M. G., and Kas, J. (2002). Guiding neuronal growth with light. Proc. Natl. Acad. Sci. USA 99, 16024-16028], as well as primary rat and mouse cortical neurons [Stuhrmann, B., Goegler, M., Betz, T., Ehrlicher, A., Koch, D., and Kas, J. (2005). Automated tracking and laser micromanipulation of cells. Rev. Sci. Instr. 76, 035105]. Optical guidance may eventually be used alone or with other methods for controlling neurite extension in both research and clinical applications.
Methods in cell biology 02/2007; 83:495-520. · 2.05 Impact Factor
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Yun-Bi Lu,
Kristian Franze,
Gerald Seifert,
Christian Steinhäuser,
Frank Kirchhoff,
Hartwig Wolburg,
Jochen Guck,
Paul Janmey,
Er-Qing Wei, Josef Käs,
Andreas Reichenbach
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ABSTRACT: One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word "gammalambdaiotaalpha" means "glue") or provide a robust scaffold for them ("support cells"). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft ("rubber elastic"), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.
Proceedings of the National Academy of Sciences 12/2006; 103(47):17759-64. · 9.68 Impact Factor
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ABSTRACT: The structural models created to understand the cytoskeletal mechanics of cells in suspension are described here. Suspended cells can be deformed by well-defined surface stresses in an Optical Stretcher [Guck, J., Ananthakrishnan, R., Mahmood, H., Moon, T.J., Cunningham, C.C., Käs, J., 2001. The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81(2), 767-784], a two-beam optical trap designed for the contact-free deformation of cells. Suspended cells have a well-defined cytoskeleton, displaying a radially symmetric actin cortical network underlying the cell membrane with no actin stress fibers, and microtubules and intermediate filaments in the interior. Based on experimental data using suspended fibroblasts, we create two structural models: a thick shell actin cortex model that describes cell deformation for a localized stress distribution on these cells and a three-layered model that considers the entire cytoskeleton when a broad stress distribution is applied. Applying the models to data, we obtain a (actin) cortical shear moduli G of approximately 220 Pa for normal fibroblasts and approximately 185 Pa for malignantly transformed fibroblasts. Additionally, modeling the cortex as a transiently crosslinked isotropic actin network, we show that actin and its crosslinkers must be co-localized into a tight shell to achieve these cortical strengths. The similar moduli values and cortical actin and crosslinker densities but different deformabilities of the normal and cancerous cells suggest that a cell's structural strength is not solely determined by cytoskeletal composition but equally importantly by (actin) cytoskeletal architecture via differing cortical thicknesses. We also find that although the interior structural elements (microtubules, nucleus) contribute to the deformed cell's exact shape via their loose coupling to the cortex, it is the outer actin cortical shell (and its thickness) that mainly determines the cell's structural response.
Journal of Theoretical Biology 10/2006; 242(2):502-16. · 2.21 Impact Factor
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Jochen Guck,
Stefan Schinkinger,
Bryan Lincoln,
Falk Wottawah,
Susanne Ebert,
Maren Romeyke,
Dominik Lenz,
Harold M Erickson,
Revathi Ananthakrishnan,
Daniel Mitchell, Josef Käs,
Sydney Ulvick,
Curt Bilby
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ABSTRACT: The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.
Biophysical Journal 06/2005; 88(5):3689-98. · 3.65 Impact Factor
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ABSTRACT: A step stress deforming suspended cells causes a passive relaxation, due to a transiently cross-linked isotropic actin cortex underlying the cellular membrane. The fluid-to-solid transition occurs at a relaxation time coinciding with unbinding times of actin cross-linking proteins. Elastic contributions from slowly relaxing entangled filaments are negligible. The symmetric geometry of suspended cells ensures minimal statistical variability in their viscoelastic properties in contrast with adherent cells and thus is defining for different cell types. Mechanical stimuli on time scales of minutes trigger active structural responses.
Physical Review Letters 04/2005; 94(9):098103. · 7.37 Impact Factor
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ABSTRACT: The transition from localized to systemic spreading of bacteria, viruses, and other agents is a fundamental problem that spans medicine, ecology, biology, and agriculture science. We have conducted experiments and simulations in a simple one-dimensional system to determine the spreading of bacterial populations that occurs for an inhomogeneous environment under the influence of external convection. Our system consists of a long channel with growth inhibited by uniform ultraviolet (UV) illumination except in a small "oasis", which is shielded from the UV light. To mimic blood flow or other flow past a localized infection, the oasis is moved with a constant velocity through the UV-illuminated "desert". The experiments are modeled with a convective reaction-diffusion equation. In both the experiment and model, localized or extinct populations are found to develop, depending on conditions, from an initially localized population. The model also yields states where the population grows everywhere. Further, the model reveals that the transitions between localized, extended, and extinct states are continuous and nonhysteretic. However, it does not capture the oscillations of the localized population that are observed in the experiment.
Biophysical Journal 08/2004; 87(1):75-80. · 3.65 Impact Factor
