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Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

ABSTRACT: A single-cell assay of active and passive intracellular mechanical properties of mammalian cells could give significant insight into cellular processes. Force spectrum microscopy (FSM) is one such technique, which combines the spontaneous motion of probe particles and the mechanical properties of the cytoskeleton measured by active microrheology using optical tweezers to determine the force spectrum of the cytoskeleton. A simpler and noninvasive method to perform FSM would be very useful, enabling its widespread adoption. Here, we develop an alternative method of FSM using measurement of the fluctuating motion of mitochondria. Mitochondria of the C3H-10T1/2 cell line were labeled and tracked using confocal microscopy. Mitochondrial probes were selected based on morphological characteristics, and their mean-square displacement, creep compliance, and distributions of directional change were measured. We found that the creep compliance of mitochondria resembles that of particles in viscoelastic media. However, comparisons of creep compliance between controls and cells treated with pharmacological agents showed that perturbations to the actomysoin network had surprisingly small effects on mitochondrial fluctuations, whereas microtubule disruption and ATP depletion led to a significantly decreased creep compliance. We used properties of the distribution of directional change to identify a regime of thermally dominated fluctuations in ATP-depleted cells, allowing us to estimate the viscoelastic parameters for a range of timescales. We then determined the force spectrum by combining these viscoelastic properties with measurements of spontaneous fluctuations tracked in control cells. Comparisons with previous measurements made using FSM revealed an excellent match.

SUBMITTER: Xu W

PROVIDER: S-EPMC6026348 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

Xu Wenlong W Alizadeh Elaheh E Prasad Ashok A

Biophysical journal 20180601 12

A single-cell assay of active and passive intracellular mechanical properties of mammalian cells could give significant insight into cellular processes. Force spectrum microscopy (FSM) is one such technique, which combines the spontaneous motion of probe particles and the mechanical properties of the cytoskeleton measured by active microrheology using optical tweezers to determine the force spectrum of the cytoskeleton. A simpler and noninvasive method to perform FSM would be very useful, enabli ...[more]

PMID: 29925029

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Project description:We present a magnetic force-based direct drive modulation method to measure local nano-rheological properties of soft materials across a broad frequency range (10 Hz to 2 kHz) using colloid-attached atomic force microscope (AFM) probes in liquid. The direct drive method enables artefact-free measurements over several decades of excitation frequency, and avoids the need to evaluate medium-induced hydrodynamic drag effects. The method was applied to measure the local mechanical properties of polyacrylamide hydrogels. The frequency-dependent storage stiffness, loss stiffness, and loss tangent (tan??) were quantified for hydrogels having high and low crosslinking densities by measuring the amplitude and the phase response of the cantilever while the colloid was in contact with the hydrogel. The frequency bandwidth was further expanded to lower effective frequencies (0.1 Hz to 10 Hz) by obtaining force-displacement (FD) curves. Slow FD measurements showed a recoverable but highly hysteretic response, with the contact mechanical behaviour dependent on the loading direction: approach curves showed Hertzian behaviour while retraction curves fit the JKR contact mechanics model well into the adhesive regime, after which multiple detachment instabilities occurred. Using small amplitude dynamic modulation to explore faster rates, the load dependence of the storage stiffness transitioned from Hertzian to a dynamic punch-type (constant contact area) model, indicating significant influence of material dissipation coupled with adhesion. Using the appropriate contact model across the full frequency range measured, the storage moduli were found to remain nearly constant until an increase began near ?100 Hz. The softer gels' storage modulus increased from 7.9 ± 0.4 to 14.5 ± 2.1 kPa (?85%), and the stiffer gels' storage modulus increased from 16.3 ± 1.1 to 31.7 ± 5.0 kPa (?95%). This increase at high frequencies may be attributed to a contribution from solvent confinement in the hydrogel (poroelasticity). The storage moduli measured by both macro-rheometry and AFM FD curves were comparable to those measured using the modulation method at their overlapping frequencies (10-25 Hz). In all cases, care was taken to ensure the contact mechanics models were applied within the important limit of small relative deformations. This study thus highlights possible transitions in the probe-material contact mechanical behaviour for soft matter, especially when the applied strain rates and the material relaxation rates become comparable. In particular, at low frequencies, the modulus follows Hertzian contact mechanics, while at high frequencies adhesive contact is well represented by punch-like behaviour. More generally, use of the Hertz model on hydrogels at high loading rates, at high strains, or during the retraction portion of FD curves, leads to significant errors in the calculated moduli.

Dataset Information

Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

Publications

Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

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