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Life at the Nanoscale: Atomic Force Microscopy of Live Cells David Alsteens, Vincent Dupres, Claire Verbelen, Guillaume Andre, and Yves F. Dufrêne Copyright © 2009 by Pan Stanford Publishing Pte Ltd www.panstanford.com
Investigating Mammalian Cell Nanomechanics with Simultaneous Optical and Atomic Force Microscopy
Yaron R. Silberberg1, Louise Guolla2 and Andrew E. Pelling2
1Laboratory of Plasma Membrane and Nuclear Signalling, Graduate School of Biostudies, Kyoto University 1-1, Yoshida-Konoecho, Sakyo-ku, Kyoto, 606-8501, Japan. 2Department of Physics, University of Ottawa, MacDonald Hall, 150 Louis Pasteur, Ottawa, ON K1N 6N5, Canada. email@example.com
1.1 CELLULAR STRUCURE AND NANOMECHANICS
The living cell is embedded in a complex mechanical environment, in which its behaviour is constantly influenced by mechanical cues arriving from the extracellular matrix and from neighbouring cells. These signals regulate various cellular processes including differentiation, gene expression, mitosis, development, gastrulations and apoptosis.1-15 Hence, understanding the mechanisms that are involved in cellular transduction of forces is crucial for understanding how those forces affect the living cell. Advances in live cell staining and imaging techniques allow the observation of intracellular structures with high temporal and spatial resolution. In addition, tools such as atomic force microscopy (AFM)16 allow for the high-precision measurement and application of forces in the nano- and pico-Newton scale.17 The ability to visualize changes in the intracellular architecture of the living cell in real time, in response to locally applied extracellular perturbations, together with quantified measurements of changes in cell elasticity, can provide insights into the immediate effect of stress on the behaviour of the cell and on the mechanism in which forces are transmitted through the cell.11,18-20
The cellular cytoskeleton and organelles are some of the major elements responsible for modulating and controlling the mechanical properties of the cell. Moreover, internal remodeling and deformation of this complex network is highly dependent of the mechanics, topography and biochemistry of the
2 Investigating Mammalian Cell Nanomechanics with Simultaneous Optical and Atomic Force Microscopy microenvironment.1-13 The cytoskeleton is an elaborated network of filamentous protein fibres spread throughout the cytoplasm. The cytoskeleton provides mechanical stability and often regulates controlled and dynamic mechanical processes such as migration, chromosome separation during mitosis and muscle contractions. The cytoskeleton also forms an elaborated network of tracks on which cargos, both membrane-bound such as the Golgi and mitochondria and non membrane-bound such as mRNA and protein, can be transported.21,22 Three major types of filaments that make up the cytoskeleton which include the actin filaments, intermediate filaments and microtubules.23
Actin filaments are typically located below the plasma membrane and are cross-linked by a variety of proteins, including motor proteins such as myosin, which can generate forces and perform mechanical work. They are assembled from subunits called G-actin and are roughly 8 nm thick in diameter. The filaments are also linked to the plasma membrane through the Ezrin-Radixin- Moesin (ERM) proteins and membrane-spanning integrins, allowing signals from the extracellular matrix to be transmitted to the cytoskeleton, and vice versa.24-27 Microtubules (Figure 1.1b) are hollow, cylindrical filaments of approximately 25 nm in diameter, which are formed by the assembly of tubulin monomers. Individual microtubules originate from a centrosome near the nucleus, and can span the entire cell. They play an important role in organelle transport and organization, in cell division and chromosome distribution, and in mechanical stabilisation of the cell.28 Intermediate filaments (Figure 1.1c), unlike actin filaments and microtubules, are not polarised and are made of elongated polypeptide rods that are arranged in a coiled-coil structure of about 8-10 nm in diameter. They are located in two separate systems, one in the nucleus and one in the cytoplasm. Their main role is believed to be that of a passive mechanical absorber to provide structural reinforcement, particularly in cells that need to withstand strong mechanical stress such as epithelial cells.29,30 Apart from the structural contribution, intermediate filaments also have cell-type specific physiological roles and contribute to some gene-expression programmes.29
Figure 1.1 The cytoskeleton of mouse fibroblasts consists of actin (a), microtubules (b) and intermediate filaments (c). Scale bars = 10 um.
1.2 APPROACHES TO STUDYING FORCE TRANSMISSION IN CELLS 3
1.2 APPROACHES TO STUDYING FORCE TRANSMISSION IN CELLS
Historically, interest in the mechanical properties of cells and tissues stems almost from the moment of their discovery. Using some of the first microscopes in the seventeenth century, motion of particles in and around cells was observed. From these microscopic movements, early scientists postulated that measurements could be taken that would allow for estimates of viscosity and other physical properties.31 Technology at the time did not allow for quantitative measurements and it was not until the early twentieth century that many physical properties began to be determined.31 Many research groups around the world are investigating the phenomena of mechanotransduction and force transmission through cells, using a variety of techniques, and several different models now exist to explain the observed effects. Though the exact process of mechanotransduction and force transmission and their pathways have yet to be elucidated, there is consensus in which cellular structures appear to play an important part. Foremost among these are the cytoskeleton and its connections to the extracellular environment through the ERMs, focal adhesion complexes and mechanosensitive ion channels.
In the late 1980s, a variety of approaches were being employed to determine the mechanical properties of living cells and intracellular structures.32- 35 The most commonly used techniques at the time were micropipette aspiration,34 a rudimentary cell poker,36,37 and application of a shear, twisting force using magnetic fields and ferromagnetic beads.32,33,38,39 Micropipette aspiration involves suction of a portion of the cell into a tube with a diameter of a few micrometres (usually between 1-8 µm), using a known suction pressure (typically between 0.1-105 Pa). The geometry and known pressure are then used to determine the mechanical properties of the cell.40 Early work investigated the viscoelasticity and cortical tension of red blood cells.34
Magnetic tweezers were later developed to utilize magnetic fields to generate forces on small paramagnetic beads with a typical size of 0.1-5 µm. Resulting displacements of the beads can then be used to deduce rheological properties of living cell. Beads were functionalized and bound to integrin receptors on the cell membrane to measure viscoelastic properties of fibroblast cells41 and their response to deformation.42 A series of experiments38 using magnetic twisting cytometry clarified that applied force was transmitted through integrin receptors found at focal adhesions, which are directly connected to the cytoskeleton. Cells with RGD-coated ferromagnetic beads attached to integrin receptors experienced a force-dependant increase in stiffness, while beads attached to other receptors did not experience the same effect. It was also found that this effect was proportional to an increased number of connections to the extracellular matrix (ECM). Together, this indicates that integrins act as mechanoreceptors which transmit signals to the cytoskeleton from the extracellular matrix and directly modulates cell rigidity. Published evidence supports the transmission of force through focal adhesions using a combination
4 Investigating Mammalian Cell Nanomechanics with Simultaneous Optical and Atomic Force Microscopy of micromanipulation with glass needles and cells expressing green fluorescent protein (GFP) conjugated to actin.43
Advances in optical technology have also led to several interesting approaches to studying cell nanomechanics. Optical tweezers (laser traps) are a highly sensitive technique in which dielectric spherical beads are trapped at the focus of a laser beam.44 The surface of the bead is functionalized and can be attached to a cell membrane or other molecules. The laser beam creates a field that ‘traps’ the bead at the focal point, allowing measurement of forces acting on the bead. Using this method, forces such as those generated by single molecules such as kinesin motors45 and cytoskeleton-integrin linkage46 were successfully measured. The ability to apply a controlled and localized force to a cell demonstrated that increased force on focal adhesion complexes and stress fibres leads to an increased calcium ion influx near those focal adhesion complexes. This supports the theory that mechanosensitive ion channels can be activated by increased tension in the cytoskeleton.47 It is also possible to use a focussed laser with enough precision to sever a single cytoskeleton filament, known as laser ablation.48 A series of laser ablation experiments20 demonstrated stress fibres reveals that they mechanically retract following severing (as opposed to depolymerization), and form pseudo focal adhesion sites along the basal membrane as they slide