Living cells exhibit an important out-of-equilibrium mechanical activity, mainly due to the forces generated by molecular motors. These motor proteins, acting individually or collectively on the cytoskeleton, contribute to the violation of the fluctuation-dissipation theorem in living systems. In this work we probe the cytoskeletal out-of-equilibrium dynamics by performing simultaneous active and passive microrheology experiments, using the same micron-sized probe specifically bound to the actin cortex. The free motion of the probe exhibits a constrained, subdiffusive behavior at short time scales (t < 2s), and a directed, superdiffusive behavior at larger time scales, while, in response to a step force, its creep function presents the usual weak power law dependence with time. Combining the results of both experiments, we precisely measure for the first time the power spectrum of the force fluctuations exerted on this probe, which lies more than one order of magnitude above the spectrum expected at equilibrium, and greatly depends on frequency. We retrieve an effective temperature Teff of the system, as an estimate of the departure from thermal equilibrium. This departure is especially pronounced on long time scales, where Teff bears the footprint of the cooperative activity of motors pulling on the actin network. ATP depletion reduces the fluctuating force amplitude and results in a sharp decrease of Teff towards equilibrium.
Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g., phagocytosis) and pathological conditions (e.g., cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in localized measurements of ROS/RNS insid…
Sulfobetaine-Phosphonate Block Copolymer Coated Iron Oxide Nanoparticles for Genomic Locus Targeting and Magnetic Micromanipulation in the Nucleus of Living Cells
In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss sol…
Recently, the implementation of plasmonic nanoantennas has opened new possibilities to investigate the nanoscale dynamics of individual biomolecules in living cell. However, studies have yet been restricted to single molecular species as the narrow wavelength resonance of gold-based nanostructures precludes the simultaneous interrogation of different fluorescently labeled molecules. Here we exploited broadband aluminum-based nanoantennas carved at the apex of near-field probes to resolve nanoscale-dynamic molecular interactions on intact living cell membranes. Through multicolor excitation, we…
Microrheology, the study of fluids on micron length-scales, promises to reveal insights into cellular biology, including mechanical biomarkers of disease and the interplay between biomechanics and cellular function. Here a minimally-invasive passive microrheology technique is applied to individual living cells by chemically binding a bead to the surface of a cell, and observing the mean squared displacement of the bead at timescales ranging from milliseconds to 100s of seconds. Measurements are repeated over the course of hours, and presented alongside novel analysis to quantify changes in the…
The infection pathway of virus in living cell is of interest from the viewpoint of the physics of diffusion. Here, recent developments about a diffusion theory for the infection pathway of an adeno-associated virus in cytoplasm of a living HeLa cell are reported. Generalizing fractional kinetics successfully modeling anomalous diffusion, a theory for describing the infection pathway of the virus over the cytoplasm is presented. The statistical property of the fluctuations of the anomalous-diffusion exponent is also discussed based on a maximum-entropy-principle approach. In addition, an issue …
Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living…
The life of a cell is governed by highly dynamical microscopic processes. Two notable examples are the diffusion of membrane receptors and the kinetics of transcription factors governing the rates of gene expression. Different fluorescence imaging techniques have emerged to study molecular dynamics. Among them, fluorescence correlation spectroscopy (FCS) and single-particle tracking (SPT) have proven to be instrumental to our understanding of cell dynamics and function. The analysis of SPT and FCS is an ongoing effort, and despite decades of work, much progress remains to be done. In this pape…
Proteins control many vital functions in living cells, such as cell growth and cell division. Reliable coordination of these functions requires the spatial and temporal organizaton of proteins inside cells, which encodes information about the cell's geometry and the cell-cycle stage. Such protein patterns arise from protein transport and reaction kinetics, and they can be controlled by various guiding cues within the cell. Here, we review how protein patterns are guided by cell size and shape, by other protein patterns that act as templates, and by the mechanical properties of the cell. The ba…
Trying to identify the entropy production within a cell has been part of debates and studies in the last century. First the idea was to make a resemblance of a cell with a Carnot engine, which is the most thermodynamically perfect machine. This approach was clearly not the best, since the yield achieved within a cell cannot be ideal, but can we even measure it? Several models approach the living cell, since the very simple one (e.g. Prigogine model) to more elaborated proposals. The concept of entropy has been the centre of discussions within several scientific fields. To interpret how entropy…
In video-records, objects moving in intracellular regions are often hardly detectable and identifiable. In order to squeeze the information on the intracellular flows, we propose an automatic method of reconstruction of intracellular flow velocity fields based only on a recorded video of an unstained cell. The basis of the method is detection of speeded-up robust features (SURF) and assembling them into trajectories. Two components of motion -- direct and Brownian -- are separated by an original method based on minimum covariance estimation. The Brownian component gives a spatially resolved di…
In this paper, we bring together our efforts in identifying and understanding nonequilibrium phase segregation driven by active processes in the living cell, with special focus on the segregation of cell membrane components driven by active contractile stresses arising from cortical actomyosin. This also has implications for active segregation dynamics in membraneless regions within the cytoplasm and nucleus (3d). We formulate an active version of the Flory-Huggins theory that incorporates a contribution from fluctuating active stresses. Apart from knitting together some of our past theoretica…
We report a new approach to controllable thermal stimulation of a single living cell and its compartments. The technique is based on the use of a single polycrystalline diamond particle containing silicon-vacancy (SiV) color centers. Due to the presence of amorphous carbon at its intercrystalline boundaries, such a particle is an efficient light absorber and becomes a local heat source when illuminated by a laser. Furthermore, the temperature of such a local heater is tracked by the spectral shift of the zero-phonon line of SiV centers [2]. Thus, the diamond particle acts simultaneously as a h…
A statistical analysis of particle trajectories in living cells
分子生物学和荧光显微镜成像的最新进展使活细胞中单分子动力学的推断成为可能。这种推断允许确定细胞的组织和功能。用跟踪算法计算的细胞中颗粒的轨迹可以用扩散过程建模。三种类型的扩散被认为是：（i）自由扩散;（ii）subdiffusion或（iii）superdiffusion。均方位移（MSD）通常用于确定活细胞中颗粒的不同类型的动力学（Qian，Sheetz和Elson 1991）。在这里，我们提出了一个非参数的三决策测试作为替代的MSD方法。对原假设--自由扩散--的拒绝伴随着对替代方向（亚扩散或超扩散）的主张。例如，我们研究了在原假设下以及在生物物理学文献中目前考虑的参数替代方案下（Monnier et al，2012）检验统计量的渐近行为。此外，我们调整Benjamini和Hochberg（2000）的程序，以适应三决策测试设置，以便将测试程序应用于独立轨迹的集合。通过蒙特卡罗实验证实了该方法的性能优于MSD方法。该方法被证明对应于在荧光显微镜观察到的蛋白质动力学的真实数据集。
Engineered living materials (ELMs) are the most relevant contemporary revolution in materials science and engineering. These ELMs aim to outperform current examples of "smart", active or multifunctional materials, enabling countless industrial and societal applications. The "living" materials facilitate unique properties, including autonomy, intelligent responses, self-repair, and even self-replication. Within this dawning field, most reviews and documents have divided ELMs into biological ELMs, which are solely made of cells, and hybrid living materials, which consist of abiotic chassis and l…
In their work [Proc. Natl. Acad. Sci. USA 112 (2015) E5725], Bosse et al. experimentally showed that virus capsid exhibits not only normal diffusion but also anomalous diffusion in nucleus of a living cell. There, it was found that the distribution of fluctuations of the diffusion exponent characterizing them takes the Gaussian form, which is, quite remarkably, the same form for two different types of the virus. This suggests high robustness of such fluctuations. Here, the statistical property of local fluctuations of the diffusion exponent of the virus capsid in the nucleus is studied. A maxi…
The spatiotemporal organisation of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting and endocytosis. Earlier studies have shown that multiple types of membrane proteins including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered GPI-anchored proteins (GPI-APs) form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nan…
