Mapping intracellular mechanics on micropatterned substrates

Abstract

The mechanical properties of cells impact on their architecture, their migration, intracellular trafficking, and many other cellular functions and have been shown to be modified during cancer progression. We have developed an approach to map the intracellular mechanical properties of living cells by combining micropatterning and optical tweezers-based active microrheology. We optically trap micrometersized beads internalized in cells plated on crossbow-shaped adhesive micropatterns and track their displacement following a step displacement of the cell. The local intracellular complex shear modulus is measured from the relaxation of the bead position assuming that the intracellular microenvironment of the bead obeys power-law rheology. We also analyze the data with a standard viscoelastic model and compare with the power-law approach. We show that the shear modulus decreases from the cell center to the periphery and from the cell rear to the front along the polarity axis of the micropattern. We use a variety of inhibitors to quantify the spatial contribution of the cytoskeleton, intracellular membranes, and ATPdependent active forces to intracellular mechanics and apply our technique to differentiate normal and cancer cells.ell mechanics play a crucial role in many cellular functions that are critical during development or are altered during pathologies such as cancers. For instance, cell migration, cell adhesion, and cell division have all been shown to depend on cell mechanics (1-5). Most studies have focused on the mechanical cross talk between the cell and its microenvironment at the whole-cell or the tissue level. It has been shown recently that intracellular mechanics could also be used to differentiate cancer cells from normal cells (6-9). Several intracellular elements participate in the mechanical response of the cytoplasm, and most of them may be modified during cancer progression. The three types of fibers constituting the cytoskeleton, actin, microtubules, and intermediate filaments, clearly contribute to cell mechanics (10-13). The role of internal membranes, molecular crowding, or active force generators in the cytoplasm is less documented but could also be significant. During cancer development, signaling associated to the cytoskeleton as well as membrane trafficking, cell polarity, and intracellular organization are deregulated (14-16), supporting the idea that the mechanics of the interior of cancer cells could differ from that of normal cells.Passive or active microrheology techniques have been developed to measure intracellular mechanical properties, mostly based on the tracking of endogenous granules or internalized particles (17)(18)(19)(20)(21)(22). The experimental results are generally analyzed using two main classes of models, viscoelastic models with a finite number of viscous (dashpots) and elastic (springs) elements and power-law (PL) models (see refs. 23-26 for reviews). An increasing amount of experimental evidence points to weak PL rheology as a common feature of cell mechanical res...

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts

“…The order of magnitude of E cortex as characterised with the OT is consistent with previous measurements [Robert et al., ; Salbreux et al., ; Guo et al., ]. On the other hand, we measure E interior of the order of 100 Pa, while the expected order of magnitude for the stiffness of the cytoplasm is a few to a few tens of Pa [Wirtz, ; Robert et al., ; Guo et al., ; Mandal et al., ]. We thus conclude that the nucleus probably plays a role in the measured stiffness of the interior of the cell, and that the over‐expression of functional desmin could increase the value of E interior both by increasing the rigidity of the cytoplasm and by reinforcing the linkage between the nucleus and the other filaments of the cytoskeleton [Huber et al., ; Leduc and Etienne‐Manneville, ].…”

Are cancer cells really softer than normal cells?

“…In breast and ovarian cancer cells and in thyroid cells, the actin network is less dense and contains less stress fibres (Gal & Weihs, ; Ketene et al., ; Prabhune et al., ; Calzado‐Martin et al., ). Similar observations were made in metastatic MDA‐MB‐231 breast cancer cells plated on adhesive micropatterns to standardise their intracellular organisation when compared with non‐tumorigenic MCF‐10A cells, with changes in actin organisation correlating with a strong decrease in cell stiffness in the metastatic cell line (Mandal et al ., ). Interestingly, disrupting actin filament polymerisation in normal cells induces a decrease in cell stiffness but also reduces the variability of the mechanical measurements (Cai et al ., ; Grady et al ., ) as observed in cancer cells (Cross et al ., ; Xu et al ., ; Grady et al ., ).…”

“…The organisation of the microtubule network in three breast cancer cell lines with different metastatic potential did not show any significant variations (Calzado‐Martín et al ., ). However, when non‐tumorigenic MCF‐10A and metastatic MDA‐MB‐231 breast cancer cells were plated on adhesive micropatterns, subtle differences in microtubule distribution could be revealed with a more uniform and less intricate network in the metastatic cells (Mandal et al ., ).…”

“…It was shown for instance that the local intracellular stiffness increases in the proximity of the Golgi apparatus (Guet et al ., ). Moreover, dispersion of Golgi membranes by brefeldin‐A has recently been reported to slightly increase intracellular rigidity (Mandal et al ., ). Interestingly, similarly to experiments performed at the scale of the whole cell (Bonakdar et al ., ), when a stress is applied on the Golgi apparatus, this organelle does not relax back to its original shape and actin contributes to its rheology (Guet et al ., ).…”

“…However, the link between ATP‐dependent forces and cell rigidity is not clear and conflicting results have been reported. Optical tweezers active microrheology using 0.5 μm diameter beads oscillating in the 0.1–100 Hz frequency range has shown that ATP depletion softens the cell interior about twofold (Guo et al ., ), while an opposite effect was measured with a protocol based on the relaxation of 2 μm diameter beads within an optical trap (Mandal et al ., ). The last result is consistent with the lower tracer particle displacement and lower intracellular forces measured in the absence of ATP (Guo et al ., ).…”