With an emphasis on the interplay between stress, strain, and the rate of flow, rheological measurements are used to investigate how cells flow rather than deform purely elastically in response to an applied force. At this point it is important to note that the simple mechanical terms elasticity and viscosity can be used as comparative quantities in cell mechanics. Many of the early cell mechanical measurements have shown links between local increases in cellular elasticity and subcellular structures such as stress fibres 25 - 27 and changes to cellular elastic and viscous properties under different treatments.
Even if thinking of a cell as an inert material, one of the main difficulties in cell mechanics is to understand the structural origins of the measured cellular mechanical properties. Indeed, cells are complex heterogeneous media containing a range of proteins, filaments, subcellular structures and organelles that can have different contributions to cell elasticity and viscosity. One particular example of this is the role of the nucleus in defining whole cell elasticity.
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The nucleus is known to be stiffer than the cytoplasmic portion of the cell. Similar nonlinear features occur during cellular rheological measurements leading to the development of scale free models see Beyond Simple Phenomenology. Nevertheless, a great deal of interesting information can be obtained via a simplistic mechanical models that enable comparative characterization of cell mechanics under genetic or pharmacological perturbations.
In the following, we place cell mechanics in the context of physiological function, describe the classical cell mechanics measurements and then move to more complex descriptions of cell mechanics and their interpretation. The interior of a single cell is a fluid, crowded with organelles, macromolecules, and structures that fulfill a variety of functions.
Networks of subcellular filaments called the cytoskeleton form higher order meshes and bundles that endow individual cells with their ability to sustain external mechanical forces. Three cytoskeletal filaments are of specific interest to cell mechanical properties; actin microfilaments, microtubules, and intermediate filaments. Actin is one of the most abundant proteins in eukaryotes that forms polarized filaments that interact with an array of ancilliary proteins.
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Despite their flexibility and high turnover rate, actin has long been known to be vital for the cell mechanically. The ability of the actin cytoskeleton to sustain mechanical stress is therefore not strongly influenced by single filament rigidity but a consequence of the higher level structures that they form and their interaction with crosslinkers and polymerizing factors. For network of filaments the entanglement length l is the confining lengthscale over which the motion of each filament is restricted via topological constraints from neighboring filaments. Chemical signaling between cells span a range of lengthscales; from hormones that travel between organs in the blood stream; to paracrine signaling between local groups of cells; small molecule signaling between contacting neighbors through gap junctions; and intracellular signaling cascades.
A wealth of recent research has shown that cells are able to sense mechanical signals and forces in their environment. Complex sensory machineries located on different cellular sites, such focal adhesion complexes 41 and more recently focal adherens junctions, 42 have been shown to make up part of the molecular machinery involved in sensing mechanical stimulation.
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The importance of sustaining, generating, and sensing mechanical forces at the cellular level is brought largely into context when examining diseases that target the cytoskeleton. Genetic disorders that disrupt the actin cytoskeleton or the binding of actin to the membrane of red blood cells, lead to abnormal cell shape and compromised function in diseases such as malaria 43 and sickle cell anemia. One recent clinical example suggests that changes in cell rheology can have consequences for the health of patients.
The alterations in cancer cells stiffness could have significant effects in their ability to squeeze through the surrounding tissue, invade and metastasize. One of the most exciting avenues of cell mechanics research is to make links between cellular level mechanosensitivity, force generation, mechanical properties and the underlying molecular mechanisms. Stretching the molecule talin using magnetic tweezers and AFM revealed binding domains for vinculin and its recruitment to focal adhesions.
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In comparison to typical materials such as metals, plastics and glass, cells are small soft objects. This has lead to the development of a vast number of cell mechanical measurement techniques, making choosing the appropriate tool puzzling. Taken broadly, mechanical measurement techniques can be put into two categories: those that can be used to apply controlled deformations and forces on part of, or on the entire cell such as magnetic bead cytometry, 16 , 18 , 51 optical tweezers, 52 - 54 cell stretchers, 55 - 58 flow rheometry 56 , 59 , 60 and AFM 61 , and those that monitor the ability of a cell to generate forces and deform its environment such as TFM 62 , 63 and micropillar arrays Local cell mechanical properties can also be extracted by tracking the motion of endogenous cellular structures, such as the movement of actin filaments, microtubules, mitochondria, or embedded particles of various sizes that are excited thermally or driven with an external force PTM 65 - In this section we outline the basic logic for choosing the appropriate experimental tool and what factors should influence this decision.
The choice of experimental tool depends on three key considerations: the compliance and the lengthscale of the cellular material under investigation, the timescale at which mechanical properties are investigated, and the required environmental and experimental conditions Figure 2. Because of the cell size and low elastic modulus, the mechanical measurement technique needs to be capable of applying and monitoring deformations and forces in the range of micro to nano meters and nano to pico newtons respectively. Typical measurement techniques that are capable of applying these deformations and forces at a high spatial accuracy include atomic force microscopy.
As the size of the cellular sample changes, for example to the multicellular aggregate and embryo level, then the experimental tool must be changed to apply larger forces over a greater area. For example mechanical shivering of multicellular aggregates has been observed using micropipette aspiration of entire aggregates rather than the more traditional single cell aspiration experiments. Second, the experimental tool can be chosen according to the frequency timescale of application and measurement of forces or deformations Figure 2 c. On the other hand PTM techniques inherently capture the frequency dependent response.
Third, a variety of experimental and environmental conditions can determine the choice of experimental tool. To date many mechanical measurements are performed in ambient conditions, far from those of the true physiological environment. Some techniques that use optical traps can cause an increase in cell temperature.
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Another goal of mechanical measurements is to observe changes in the localization of different proteins and protein activation during mechanical loading. Inverted fluorescence microscopes can be easily integrated with mechanical testing techniques 8 , 58 to image protein expression and localization.
In the next section, due to their wide range of applicability, commercial availability and ease of operation for novices, we discuss four mechanical measurement techniques in detail and summarize recent findings in our case studies. For in depth discussion of other techniques we refer readers to a recent review. The atomic force microscope is a high resolution surface characterization technique, that has become rapidly adopted for imaging and mechanical characterization of a range of biological samples 61 Figure 4 a.
One of the most widespread uses of AFM in cell mechanics is AFM force spectroscopy to measure cellular elasticity and rheology. To extract the cell elasticity, the tip of AFM cantilever is pressed against the cell while the force and the imposed cellular deformation are monitored. Considering the tip geometry and using an appropriate contact model, the elasticity of the cell can be computed from the measured force versus indentation data. Furthermore, because the levels of force and deformation can be very accurately measured over time, AFM has been applied for a variety of rheological measurements.
Using a feedback loop incorporated into most commercial systems levels of strain and stress can be controlled over time, following indentation of the cell via AFM cantilever. Another recent use of AFM involves placing a tipless cantilever on a cell entering mitosis and monitoring the temporal changes of cell rounding forces during mitosis. The use of optical methods to image cells has been well established for decades.
More recently, with the growing interest in cell mechanics there have been several methods developed that employ light trapping to manipulate part of a cell 85 - 87 or stretch the whole cell. Conservation of momentum means that there is a restoring force created by the light passing through the material that resists higher levels of refraction. Early measurements using this approach revealed the presence of a large amount of excess membrane at the cell surface that flows into the tether as it is pulled.
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Although optical tweezers provide a valuable tool for high precision measurements of small forces, there is an inherent limit on the amount of force that can be applied using these methods. In particular increasing the laser power to increase the optical forces induces local heating of the cell that might damage the cell structure and influence its mechanical properties. To increase the amount of optical forces, leaving minimal photodamage, another type of optical manipulation technique involves coupling a laser light to another optical fiber that enables trapping and stretching of the whole cell.
One shortcoming of most cell rheological techniques, including AFM, is that they require external interventions and measure the combined response of subcellular structures such as cell membrane, cytoplasm, and the nucleus by application of external forces directly on the cell surface Figure 4 a. However, in PTM Figure 4 c , localized mechanical measurements inside the cytoplasm can be achieved by tracking thermally driven motion of embedded tracer particles, without a need for a direct contact of between the cell and an external probe.
Indeed the movement of particles within the cell is perturbed by both elastic and viscous mechanical resistances. Generally PTM experiments on cells revealed a dominant elastic response at short timescales and a more viscous behavior over longer time periods. However, the magnitude and timescale of these viscoelastic responses may vary for various cell types, under different pharmacological treatments and physiological conditions.
Microscopic mechanical properties of soft material including cells can be measured by tracking the movement of embedded tracer particles. This behavior is the characteristic of normal diffusion or Brownian motion. During normal diffusion the thermal motion of a particle with size a is slowed down by only a linear viscous drag from surrounding environment.
Force sensing techniques are another type of tool that have advanced our understanding of mechanotransduction 38 and improved quantitative modeling of cellular interactions with the ECM. These techniques measure traction forces generated by the cell on the surrounding environment using different detection mechanisms such as micropillar arrays 64 or embedded beads in soft gels Figure 4 d. Traction forces drive cell spreading and migration during commonly occurring cell processes such as morphogenesis, wound healing, and tumor metastasis.
These results showed that focal adhesions exhibit either stable or oscillating force transmission to the ECM via adhesion sites and ECM stiffness modulates the dynamics of focal adhesions.
Using TFM other recent works also studied the oscillation of forces within focal adhesions and the impact of ECM compliance on force fluctuations and directed cell migration. Moreover, a QR code can be used to uniquely identify a carton of farm produce.
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