For several decades optical tweezers have proven to be an invaluable tool in the study and analysis of a myriad biological responses and applications. However, as every tool, it can have undesirable or damaging effects upon the very sample it is help
ing to study. In this review the main negative effects of optical tweezers upon biostructures and living systems will be presented. Three are the main areas on which the review will focus: linear optical excitation within the tweezers, non-linear photonic effects, and thermal load upon the sampled volume. Additional information is provided on negative mechanical effects of optical traps on biological structures. Strategies to avoid or, in the least, minimize these negative effects will be introduced. Finally, all these effects, undesirable for the most, can have positive applications under the right conditions. Some hints in this direction will also be discussed.
Natural and synthetic phenazines are widely used in biomedical sciences. In dehydrogenase histochemistry, phenazine methosulfate (PMS) is applied as a redox reagent for coupling reduced coenzymes to the reduction of tetrazolium salts into colored for
mazans. PMS is also currently used for cytotoxicity and viability assays of cell cultures using sulfonated tetrazoliums. Under UV (340 nm) excitation, aqueous solutions of the cationic PMS show green fluorescence ({lambda}em: 526 nm), whereas the reduced hydrophobic derivative (methyl-phenazine, MPH) shows blue fluorescence ({lambda}em: 465 nm). Under UV (365 nm) excitation, cultured cells (LM2, IGROV-1, BGC-1, and 3T3-L1 adipocytes) treated with PMS (5 ug/mL, 30 min) showed cytoplasmic granules with bright blue fluorescence, which correspond to lipid droplets labeled by the lipophilic methyl-phenazine. After formaldehyde fixation blue-fluorescing droplets could be stained with oil red O. Interestingly, PMS-treated 3T3-L1 adipocytes observed under UV excitation 24 h after labeling showed large lipid droplets with a weak green emission within a diffuse pale blue-fluorescing cytoplasm, whereas a strong green emission was observed in small lipid droplets. This fluorescence change from blue to green indicates that reoxidation of methyl-phenazine to PMS can occur. Regarding cell uptake and labeling mechanisms, QSAR models predict that the hydrophilic PMS is not significantly membrane-permeant, so most PMS reduction is expected to be extracellular and associated with a plasma membrane NAD(P)H reductase. Once formed, the lipophilic and blue-fluorescing methyl-phenazine enters live cells and mainly accumulates in lipid droplets. Overall, the results reported here indicate that PMS is an excellent fluorescent probe to investigate labeling and redox dynamics of lipid droplets in cultured cells.
Cortical actin networks are highly dynamic and play critical roles in shaping the mechanical properties of cells. The actin cytoskeleton undergoes significant reorganization over the course of the cell cycle, when cortical actin transitions between o
pen patched meshworks, homogeneous distributions, and aligned bundles. Several types of myosin motor proteins, characterized by different kinetic parameters, have been involved in this reorganization of actin filaments. Given the limitations in studying the interactions of actin with myosin in vivo, we propose stochastic agent-based model simulations and develop a set of data analysis measures to assess how myosin motor proteins mediate various actin organizations. In particular, we identify individual motor parameters, such as motor binding rate and step size, that generate actin networks with different levels of contractility and different patterns of myosin motor localization. In simulations where two motor populations with distinct kinetic parameters interact with the same actin network, we find that motors may act in a complementary way, by tuning the actin network organization, or in an antagonistic way, where one motor emerges as dominant. This modeling and data analysis framework also uncovers parameter regimes where spatial segregation between motor populations is achieved. By allowing for changes in kinetic rates during the actin-myosin dynamic simulations, our work suggests that certain actin-myosin organizations may require additional regulation beyond mediation by motor proteins in order to reconfigure the cytoskeleton network on experimentally-observed timescales.
Trajectories of endosomes inside living eukaryotic cells are highly heterogeneous in space and time and diffuse anomalously due to a combination of viscoelasticity, caging, aggregation and active transport. Some of the trajectories display switching
between persistent and anti-persistent motion while others jiggle around in one position for the whole measurement time. By splitting the ensemble of endosome trajectories into slow moving sub-diffusive and fast moving super-diffusive endosomes, we analyzed them separately. The mean squared displacements and velocity auto-correlation functions confirm the effectiveness of the splitting methods. Applying the local analysis, we show that both ensembles are characterized by a spectrum of local anomalous exponents and local generalized diffusion coefficients. Slow and fast endsomes have exponential distributions of local anomalous exponents and power law distributions of generalized diffusion coefficients. This suggests that heterogeneous fractional Brownian motion is an appropriate model for both fast and slow moving endosomes. This article is part of a Special Issue entitled: Recent Advances In Single-Particle Tracking: Experiment and Analysis edited by Janusz Szwabinski and Aleksander Weron.
Maintenance of epidermal thickness is critical to the barrier function of the skin. Decreased tissue thickness, specifically in the stratum corneum (the outermost layer of the tissue), causes discomfort and inflammation, and is related to several sev
ere diseases of the tissue. In order to maintain both stratum corneum thickness and overall tissue thickness it is necessary for the system to balance cell proliferation and cell loss. Cell proliferation in the epidermis occurs in the basal layer and causes constant upwards movement in the tissue. Cell loss occurs when dead cells at the top of the tissue are lost to the environment through a process called desquamation. Desquamation is thought to occur through a gradual reduction in adhesion between cells, due to the cleaving of adhesion proteins by enzymes, in the stratum corneum. In this paper we will investigate combining a (mass action) subcellular model of desquamation with a three dimensional (cell centre based) multicellular model of the interfollicular epidermis to better understand maintenance of epidermal thickness. These investigations show that hypothesised biological models for the degradation of cell-cell adhesion from the literature are able to provide a consistent rate of cell loss in the multicellular model. This loss balances proliferation, and hence maintains a homeostatic tissue thickness. Moreover, we find that multiple proliferative cell populations in the basal layer can be represented by a single proliferative cell population, simplifying investigations with this model. The model is used to investigate a disorder (Netherton Syndrome) which disrupts desquamation. The model shows how biochemical changes can cause disruptions to the tissue, resulting in a reduced tissue thickness and consequently diminishing the protective role of the tissue. A hypothetical treatment result is also investigated. [ABR]
A major open problem in biophysics is to understand the highly heterogeneous transport of many structures inside living cells, such as endosomes. We find that mathematically it is described by spatio-temporal heterogeneous fractional Brownian motion
(hFBM) which is defined as FBM with a randomly switching anomalous exponent and random generalized diffusion coefficient. Using a comprehensive local analysis of a large ensemble of experimental endosome trajectories (> 10^5), we show that their motion is characterized by power-law probability distributions of displacements and displacement increments, exponential probability distributions of local anomalous exponents and power-law probability distributions of local generalized diffusion coefficients of endosomes which are crucial ingredients of spatio-temporal hFBM. The increased sensitivity of deep learning neural networks for FBM characterisation corroborates the development of this multi-fractal analysis. Our findings are an important step in understanding endosome transport. We also provide a powerful tool for studying other heterogeneous cellular processes.
We study the dynamics of membrane vesicle motor transport into dendritic spines, which are bulbous intracellular compartments in neurons that play a key role in transmitting signals between neurons. We consider the stochastic analog of the vesicle tr
ansport model in [Park and Fai, The Dynamics of Vesicles Driven Into Closed Constrictions by Molecular Motors. Bull. Math. Biol. 82, 141 (2020)]. The stochastic version, which may be considered as an agent-based model, relies mostly on the action of individual myosin motors to produce vesicle motion. To aid in our analysis, we coarse-grain this agent-based model using a master equation combined with a partial differential equation describing the probability of local motor positions. We confirm through convergence studies that the coarse-graining captures the essential features of bistability in velocity (observed in experiments) and waiting-time distributions to switch between steady-state velocities. Interestingly, these results allow us to reformulate the translocation problem in terms of the mean first passage time for a run-and-tumble particle moving on a finite domain with absorbing boundaries at the two ends. We conclude by presenting numerical and analytical calculations of vesicle translocation.
Axonal growth and guidance at the ventral floor plate is here followed $textit{in vivo}$ in real time at high resolution by light-sheet microscopy along several hundred micrometers of the zebrafish spinal cord. The recordings show the strikingly ster
eotyped spatio-temporal control that governs midline crossing. Commissural axons are observed crossing the ventral floor plate midline perpendicularly at about 20 microns/h, in a manner dependent on the Robo3 receptor and with a growth rate minimum around the midline, confirming previous observations. At guidance points, commissural axons are seen to decrease their growth rate and growth cones increase in size. Commissural filopodia appear to interact with the nascent neural network, and thereby trigger immediate plastic and reversible sinusoidal-shaped bending movements of neighboring commissural shafts. Ipsilateral axons extend concurrently, but straight and without bends, at three to six times higher growth rates than commissurals, indicating they project their path on a substrate-bound surface rather than relying on diffusible guidance cues. Growing axons appeared to be under stretch, an observation that is of relevance for tension-based models of cortical morphogenesis. The textit{in vivo} observations provide for a discussion of the current distinction between substrate-bound and diffusible guidance cues. The study applies the transparent zebrafish model that provides an experimental model system to explore further the cellular, molecular and physical mechanisms involved during axonal growth, guidance and midline crossing through a combination of $textit{in vitro}$ and $textit{in vivo}$ approaches.
Mechanical loading generally weakens adhesive structures and eventually leads to their rupture. However, biological systems can adapt to loads by strengthening adhesions, which is essential for maintaining the integrity of tissue and whole organisms.
Inspired by cellular focal adhesions, we suggest here a generic, molecular mechanism that allows adhesion systems to harness applied loads for self-stabilization under non-equilibrium conditions -- without any active feedback involved. The mechanism is based on conformation changes of adhesion molecules that are dynamically exchanged with a reservoir. Tangential loading drives the occupation of some stretched conformation states out of equilibrium, which, for thermodynamic reasons, leads to association of further molecules with the adhesion cluster. Self-stabilization robustly increases adhesion lifetimes in broad parameter ranges. Unlike for catch-bonds, bond dissociation rates do not decrease with force. The self-stabilization principle can be realized in many ways in complex adhesion-state networks; we show how it naturally occurs in cellular adhesions involving the adaptor proteins talin and vinculin.
A highly organized and densely packed lattice of molecular machinery within the sarcomeres of muscle cells powers contraction. Although many of the proteins that drive contraction have been studied extensively, the mechanical impact of fluid shearing
within the lattice of molecular machinery has received minimal attention. It was recently proposed that fluid flow augments substrate transport in the sarcomere, however, this analysis used analytical models of fluid flow in the molecular machinery that could not capture its full complexity. By building a finite element model of the sarcomere, we estimate the explicit flow field, and contrast it with analytical models. Our results demonstrate that viscous drag forces on sliding filaments are surprisingly small in contrast to the forces generated by single myosin molecular motors. This model also indicates that the energetic cost of fluid flow through viscous shearing with lattice proteins is likely minimal. The model also highlights a steep velocity gradient between sliding filaments and demonstrates that the maximal radial fluid velocity occurs near the tips of the filaments. To our knowledge, this is the first computational analysis of fluid flow within the highly structured sarcomere.
