Elsevier

Acta Biomaterialia

Volume 1, Issue 3, May 2005, Pages 295-303
Acta Biomaterialia

Novel dynamic rheological behavior of individual focal adhesions measured within single cells using electromagnetic pulling cytometry

https://doi.org/10.1016/j.actbio.2005.02.003Get rights and content

Abstract

The rheology of cells and sub-cellular structures, such as focal adhesions, are important for cell form and function. Here we describe electromagnetic pulling cytometry (EPC), a technique to analyze cell rheology by applying dynamic tensional forces to ligand-coated magnetic microbeads bound to cell surface integrin receptors. EPC utilizes an electromagnetic microneedle that is integrated with a computerized control and image acquisition system and an inverted microscope and CCD camera to monitor bead displacement. Arbitrary force regimens may be defined over a wide range of frequency (DC to 10 Hz) and force (100 pN to 10 nN). With EPC, the viscoelastic creep response of individual focal adhesions was measured over three decades in time using RGD-coated magnetic microbeads bound to integrins that induce local focal adhesion assembly and coupling to the internal cytoskeleton. These data were compared to the power-law-like predictions from the soft glassy model of cell rheology proposed by Fabry et al. [14]. Although power-law-like behavior was observed in some focal adhesions, 52% of these structures did not exhibit power-law-like behavior, but instead exhibited either a multi-phase response characterized by abrupt changes in slope or experienced a retraction in the opposite direction to the applied force, especially in response to prolonged force application. These data suggest that while the soft glassy model may provide reasonable estimates for aggregate mechanical behavior of living cells, the rheological behavior of individual focal adhesions may be more heterogeneous and complex than suggested by the soft glassy model. These results are considered in context with the hierarchical nature of cytoskeletal architecture.

Introduction

The mechanical properties of living cells govern how they sense and respond to mechanical stress because they determine how cells deform in response to force, as well as how stresses are transmitted to molecular support elements inside the cell. Cells sense mechanical stress through transmembrane integrin receptors that link extracellular matrix (ECM) proteins to intracellular focal adhesion components which, in turn, transfer stress to and from the internal cytoskeleton [1]. The structural backbone of the focal adhesion also orients much of the cell’s signal transduction machinery [2], [3]. This architectural arrangement facilitates mechano-chemical conversion when mechanical signals are applied to integrins; these signals influence virtually all cell behaviors, including growth, differentiation, contraction, motility and apoptosis [4], [5], [6], [7], [8].

Early studies of cell mechanics typically relied on measurements of cell deformation in response to static loads applied to the whole cell membrane, and early investigators routinely proposed simple viscoelastic models to describe the cell mechanical response [9], [10]. Later studies using more sensitive techniques, such as optical tweezers and magnetic cytometry, permitted mechanical force application to specific cell surface receptors, and revealed that the mechanical properties of the cell differ greatly depending on the receptor probed for analysis. For example, cells were found to be relatively stiff when probed through transmembrane adhesion receptors, such as integrins, that link ECM to the internal cytoskeletal lattice composed of linked microfilaments, microtubules and intermediate filaments [1], [11]. In contrast, when the same stresses were applied to transmembrane metabolic receptors, growth factor receptors, or histocompatibility antigens that only link to the submembranous cortical cytoskeleton (actin–spectrin–ankryin lattice) the same cells were found to be highly flexible. Thus, cells appear to exhibit complex mechanical behavior due to the load-bearing functions of discrete molecular networks inside the cell and their hierarchical structure [12].

On the other hand, recent measurements with oscillatory magnetic twisting cytometry have led to a more complex, viscoelastic structural dampening model of the cell that describes the generalized mechanical behavior of cells as a soft glassy material [13], [14]. Specifically, the viscoelastic storage and loss moduli (G′ and G″, respectively) of cells were found to exhibit power-law-like behavior when analyzed in the frequency domain, and the slopes of these relationships are well-described by structural damping theory. However, it is not clear how this generalized model relates to the structural complexity of living cells at the sub-cellular scale. For example, it is not clear whether the structural damping theory applies to the behavior of the internal cytoskeleton, the cortical shell, or both. This is a particularly pertinent question because the analysis of data from the magnetic twisting cytometry studies used to develop the soft glassy model of cell mechanics excluded results from magnetic beads that were most tightly bound to the cytoskeleton and moved little in response to magnetic stress [14], [15]. In these experiments, the viscoelastic response also was averaged over many beads, potentially masking, or altogether excluding, those beads that exhibited markedly smaller displacements. Additional support for the structural dampening law has been provided by atomic force microscopy, however, the probes used in this study were not directly coupled to the internal cytoskeleton via integrin linkages and therefore likely probed the cortical membrane rather than the internal cytoskeleton [16]. The question of whether structural damping theory applies to the whole cell or to particular sub-cellular domains remains unanswered.

The past studies that generated data consistent with the structural dampening theory were analyzed in the frequency domain [13], [14], [17]. However, a direct consequence of this theory is that the viscoelastic creep in the time domain also will follow a power-law relationship [13], [18]. We therefore set out to evaluate whether the viscoelastic creep response of individual integrin-dependent focal adhesions exhibits a temporal power-law-like relationship in living endothelial cells, as predicted by the structural dampening model. To accomplish this goal, we developed a new computerized system for electromagnetic pulling cytometry (EPC) that is capable of applying dynamic tensional forces to ligand-coated magnetic beads bound to receptors on the cell membrane, while measuring lateral nanometer scale bead displacements with high temporal resolution.

Section snippets

Experimental procedure

Bovine capillary endothelial cells were cultured in 10% CO2 at 37 °C on gelatin-coated tissue culture dishes in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL) supplemented with 10% calf serum (CS; Hyclone), 10 mM Hepes (JRH-Biosciences), 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (GPS, Gibco-BRL), as previously described [19]. Cells were serum-deprived for 12–24 h in defined medium containing DMEM containing 1% BSA, GPS, 10 μg/mL human high density lipoprotein (Intracell),

Results

We have previously developed and used magnetic twisting cytometry [1], a magnetic tweezer [23] and a permanent magnetic needle [24] to measure the static mechanical properties of living cultured cells. In all of these methods, controlled mechanical stresses were applied to specific cell surface receptors by exposing cells bound to ligand-coated magnetic beads to oriented magnetic fields. To more effectively probe the dynamic viscoelastic behavior of living cells, we developed a new form of

Discussion

To probe the rheological behavior of living cells and individual focal adhesions, we developed a form of magnetic cytometry—EPC—that incorporates a computer-controlled electromagnetic microneedle [22] to apply a wide range of static or dynamic tensional forces (up to 10 nN) via ligand-coated magnetic microbeads bound to membrane surface receptors. These studies show that when combined with high-speed image and data acquisition, EPC is capable of measuring the dynamic rheological behavior of

Acknowledgement

This work was supported by grants from DoD/DURINT (#N00014-01-1-0782) and NIH CA-45548. We thank Dimitrijie Stamenovic for his insightful comments.

References (35)

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1

Currently located within the Department of Biomedical Engineering, Tulane University, New Orleans, LA 70118, United States.

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