Activity driven fluctuations in living cells

We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.Comment: 6 pages, 4 figure

Energetics of active fluctuations in living cells

The nonequilibrium activity taking place in a living cell can be monitored with a tracer embedded in the medium. While microrheology experiments based on optical manipulation of such probes have become increasingly standard, we put forward a number of experiments with alternative protocols that, we claim, will provide new insight into the energetics of active fluctuations. These are based on either performing thermodynamic--like cycles in control-parameter space, or on determining response to external perturbations of the confining trap beyond simple translation. We illustrate our proposals on an active itinerant Brownian oscillator modeling the dynamics of a probe embedded in a living medium

Force measurements with optical tweezers inside living cells

The force exerted by optical tweezers can be measured by tracking the momentum changes of the trapping beam, a method which is more general and powerful than traditional calibration techniques as it is based on first principles, but which has not been brought to its full potential yet, probably due to practical difficulties when combined with high-NA optical traps, such as the necessity to capture a large fraction of the scattered light. We show that it is possible to measure forces on arbitrary biological objects inside cells without an in situ calibration, using this approach. The instrument can be calibrated by measuring three scaling parameters that are exclusively determined by the design of the system, thus obtaining a conversion factor from volts to piconewtons that is theoretically independent of the physical properties of the sample and its environment. We prove that this factor keeps valid inside cells as it shows good agreement with other calibration methods developed in recent years for viscoelastic media. Finally, we apply the method to measuring the stall forces of kinesin and dynein in living A549 cells.Publisher PD

Johnson-Kendall-Roberts theory applied to living cells

Johnson-Kendall-Roberts (JKR) theory is an accurate model for strong adhesion energies of soft slightly deformable material. Little is known about the validity of this theory on complex systems such as living cells. We have addressed this problem using a depletion controlled cell adhesion and measured the force necessary to separate the cells with a micropipette technique. We show that the cytoskeleton can provide the cells with a 3D structure that is sufficiently elastic and has a sufficiently low deformability for JKR theory to be valid. When the cytoskeleton is disrupted, JKR theory is no longer applicable

Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells

Identification of novel protein interactions and their mediators is fundamental in understanding cellular processes and is necessary for protein-targeted therapy. Evidently high throughput formatting of these applications in living cells would be beneficial, however no adequate system exists. We present a novel platform technology for the high throughput screening and verification of protein interactions in living cells. The platform's series of Dual Expression Recombinase Based (DERB) destiny vectors individually encode two sets of recombinase recognizable sequences for inserting the protein open reading frame (ORF) of interest, two sets of promoters and reporter tags in frame with the ORFs for detecting interactions. Introduction into living cells (prokaryotic and eukaryotic) enables the detection of protein interactions by fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC). The DERB platform shows advantages over current commercialized systems by DERB vectors validated through proof-of-principle experiments and the identification of an unknown interaction