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Recently, it has been shown that the long coiled-coil membrane tether protein early endosome antigen 1 (EEA1) switches from a rigid to a flexible conformation upon binding of a signaling protein to its free end. This flexibility switch represents a motorlike activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. It was hypothesized that the binding-induced signal could propagate along the coiled coil and lead to conformational changes through the localized domains of the protein chain that deviate from a perfect coiled-coil structure. To elucidate, if upon binding of a single protein the corresponding mechanical signal could propagate through the whole 200-nm-long chain, we propose a simplified description of the coiled coil as a one-dimensional Frenkel-Kontorova chain. Using numerical simulations, we find that an initial perturbation of the chain can propagate along its whole length in the presence of thermal fluctuations. This may enable the change of the configuration of the entire molecule and thereby affect its stiffness. Our work sheds light on intramolecular communication and force generation in long coiled-coil proteins.

We present a minimal model to study the effects of pH on liquid phase separation of macromolecules. Our model describes a mixture composed of water and macromolecules that exist in three different charge states and have a tendency to phase separate. This phase separation is affected by pH via a set of chemical reactions describing protonation and deprotonation of macromolecules, as well as self-ionization of water. We consider the simple case in which interactions are captured by Flory-Huggins interaction parameters corresponding to Debye screening lengths shorter than a nanometer, which is relevant to proteins inside biological cells under physiological conditions. We identify the conjugate thermodynamic variables at chemical equilibrium and discuss the effective free energy at fixed pH. First, we study phase diagrams as a function of macromolecule concentration and temperature at the isoelectric point of the macromolecules. We find a rich variety of phase diagram topologies, including multiple critical points, triple points, and first-order transition points. Second, we change the pH relative to the isoelectric point of the macromolecules and study how phase diagrams depend on pH. We find that these phase diagrams as a function of pH strongly depend on whether oppositely charged macromolecules or neutral macromolecules have a stronger tendency to phase separate. One key finding is that we predict the existence of a reentrant behavior as a function of pH. In addition, our model predicts that the region of phase separation is typically broader at the isoelectric point. This model could account for both in vitro phase separation of proteins as a function of pH and protein phase separation in yeast cells for pH values close to the isoelectric point of many cytosolic proteins.

Many organisms, including yeast cells, bacteria, nematodes and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography and atomic force microscopy. We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

Microcolonies are aggregates of a few dozen to a few thousand cells exhibited by many bacteria. The formation of microcolonies is a crucial step towards the formation of more mature bacterial communities known as biofilms, but also marks a significant change in bacterial physiology. Within a microcolony, bacteria forgo a single cell lifestyle for a communal lifestyle hallmarked by high cell density and physical interactions between cells potentially altering their behaviour. It is thus crucial to understand how initially identical single cells start to behave differently while assembling in these tight communities. Here we show that cells in the microcolonies formed by the human pathogen Neisseria gonorrhoeae (Ng) present differential motility behaviors within an hour upon colony formation. Observation of merging microcolonies and tracking of single cells within microcolonies reveal a heterogeneous motility behavior: cells close to the surface of the microcolony exhibit a much higher motility compared to cells towards the center. Numerical simulations of a biophysical model for the microcolonies at the single cell level suggest that the emergence of differential behavior within a multicellular microcolony of otherwise identical cells is of mechanical origin. It could suggest a route toward further bacterial differentiation and ultimately mature biofilms., Comment: 29 pages, 5 figures, supplementary information attached

Many bacteria rely on active cell appendages, such as type IV pili, to move over substrates and interact with neighboring cells. Here, we study the motion of individual cells and bacterial colonies, mediated by the collective interactions of multiple pili. It was shown experimentally that the substrate motility of Neisseria gonorrhoeae cells can be described as a persistent random walk with a persistence length that exceeds the mean pili length. Moreover, the persistence length increases for a higher number of pili per cell. With the help of a simple, tractable stochastic model, we test whether a tug-of-war without directional memory can explain the persistent motion of single Neisseria gonorrhoeae cells. While the persistent motion of single cells indeed emerges naturally in the model, a tug-of-war alone is not capable of explaining the motility of microcolonies, which becomes weaker with increasing colony size. We suggest sliding friction between the microcolonies and the substrate as the missing ingredient. While such friction almost does not affect the general mechanism of single cell motility, it has a strong effect on colony motility. We validate the theoretical predictions by using a three-dimensional computational model that includes explicit details of the pili dynamics, force generation and geometry of cells., Comment: 25 pages, 17 figures

Bacterial chemotaxis is one of the most extensively studied adaptive responses in cells. Many bacteria are able to bias their apparently random motion to produce a drift in the direction of the increasing chemoattractant concentration. It has been recognized that the particular motility pattern employed by moving bacteria has a direct impact on the efficiency of chemotaxis. The linear theory of chemotaxis pioneered by de Gennes allows for calculation of the drift velocity in small gradients for bacteria with basic motility patterns. However, recent experimental data on several bacterial species highlighted the motility pattern where the almost straight runs of cells are interspersed with turning events leading to the reorientation of the cell swimming directions with two distinct angles following in strictly alternating order. In this manuscript we generalize the linear theory of chemotaxis to calculate the chemotactic drift speed for the motility pattern of bacteria with two turning angles. By using the experimental data on motility parameters of {\em V. alginolyticus } bacteria we can use our theory to relate the efficiency of chemotaxis and the size of bacterial cell body. The results of this work can have a straightforward extension to address most general motility patterns with alternating angles, speeds and durations of runs.

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001, eLife digest The DNA in a fertilized egg contains all the information required to form an animal’s body. In order for the animal to develop properly, particular genes encoded in the DNA are only active at specific times. The DNA is wrapped around proteins called histones, which allows the DNA to be tightly packed inside the cell. However, histones can block other proteins called transcription factors from binding to the DNA to activate the genes. Young embryos initially develop with all of their genes switched off, relying on the nutrients and other molecules provided by their mother. After some time, the embryo starts to switch on its own genes to take control of its own development, but it was not clear how this happens. Joseph et al. investigated how genes are activated in zebrafish embryos, which are often used as models to study how animals develop. The experiments show that competition between histones and transcription factors for binding to DNA controls when genes are switched on. In young fish embryos, there are so many histones present that transcription factors have no opportunity to bind to DNA. Over time, however, the numbers of histones decrease, allowing transcription factors to bind to DNA and switch on genes. Histones and transcription factors regulate the activity of genes throughout the life of the animal. Therefore, competition between these two types of protein may also control gene activity in other situations. A better understanding of how gene activity is controlled could allow researchers to more easily grow different types of cell in the laboratory or to reprogram specific cells in the body. As such, these new findings may aid the development of therapies to regenerate organs or tissues that have been damaged by injury or disease. DOI: http://dx.doi.org/10.7554/eLife.23326.002

In eukaryotes, gene expression depends on chromatin organization. However, how chromatin affects the transcription dynamics of individual RNA polymerases has remained elusive. Here, we use dual trap optical tweezers to study single yeast RNA polymerase II (Pol II) molecules transcribing along a DNA template with two nucleosomes. The slowdown and the changes in pausing behavior within the nucleosomal region allow us to determine a drift coefficient,., which characterizes the ability of the enzyme to recover from a nucleosomal backtrack. Notably, chi can be used to predict the probability to pass the first nucleosome. Importantly, the presence of a second nucleosome changes chi in a manner that depends on the spacing between the two nucleosomes, as well as on their rotational arrangement on the helical DNA molecule. Our results indicate that the ability of Pol II to pass the first nucleosome is increased when the next nucleosome is turned away from the first one to face the opposite side of the DNA template. These findings help to rationalize how chromatin arrangement affects Pol II transcription dynamics.

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state. DOI: http://dx.doi.org/10.7554/eLife.09347.001, eLife digest Most organisms live in unpredictable environments, which can often lead to nutrient shortages and other conditions that limit their ability to grow. To survive in these harsh conditions, many organisms adopt a dormant state in which their metabolism slows down to conserve vital energy. When the environmental conditions improve, the organisms can return to their normal state and continue to grow. The interior of cells is known as the cytoplasm. It is very crowded and contains many molecules and compartments called organelles that carry out a variety of vital processes. The cytoplasm has long been considered to be fluid-like in nature, but recent evidence suggests that in bacterial cells it can solidify to resemble a soft glass-type material under certain conditions. When cells become dormant they stop dividing and reorganise their cytoplasm in several ways; for example, the water content drops and many essential proteins form storage compartments. However, it was not clear how cells regulate the structure of the cytoplasm to enter into or exit from dormancy. Now, Munder et al. analyse the changes that occur in the cytoplasm when baker’s yeast cells enter a dormant state. The experiments show that when yeast cells are deprived of energy – as happens during dormancy – the cytoplasm becomes more acidic than normal. This limits the ability of molecules and organelles to move around the cytoplasm. Similar results were also seen in other types of fungi and an amoeba. Munder et al. found that this increase in acidity during dormancy causes many proteins to interact with each other and form large clumps or filament structures that result in the cytoplasm becoming stiffer. A separate study by Joyner et al. found that when yeast cells are starved of sugar, two large molecules are less able to move around the cell interior. Together, the findings of the studies suggest that the interior of cells can undergo a transition from a fluid-like to a more solid-like state to protect the cells from damage when energy is in short supply. The next challenge is to understand the molecular mechanisms that cause the physical properties of the cytoplasm to change under different conditions. DOI: http://dx.doi.org/10.7554/eLife.09347.002

Here, we show that a problem of forced polymer loops can be mapped to an asymmetric simple exclusion process with reflecting boundary conditions. The dynamics of the particle system can be solved exactly using the Bethe ansatz. We thus can fully describe the relaxation dynamics of forced polymer loops. In the steady state, the conformation of the loop can be approximated by a combination of Fermi–Dirac and Brownian bridge statistics, while the exact solution is found by using the fermion integer partition theory. With the theoretical framework presented here we establish a link between the physics of polymers and statistics of many-particle systems opening new paths of exploration in both research fields. Our result can be applied to the dynamics of the biopolymers which form closed loops. One such example is the active pulling of chromosomal loops during meiosis in yeast cells which helps to align chromosomes for recombination in the viscous environment of the cell nucleus.

During recombination, the DNA of parents exchange their genetic information to give rise to a genetically unique offspring. For recombination to occur, homologous chromosomes need to find each other and align with high precision. Fission yeast solves this problem by folding chromosomes in loops and pulling them through the viscous nucleoplasm. We propose a theory of pulled polymer loops to quantify the effect of drag forces on the alignment of chromosomes. We introduce an external force field to the concept of a Brownian bridge and thus solve for the statistics of loop configurations in space.

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.

Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface. We use it to describe formation of multiple colonies with sizes consistent with experimental data and to discuss their dissolution., Comment: 3 figures, 1 Supplementary Material

We used microfluidic tools and high-speed time-lapse microscopy to record trajectories of the soil bacterium Pseudomonas putida in a confined environment with cells swimming in close proximity to a glass-liquid interface. While the general swimming pattern is preserved, when compared to swimming in the bulk fluid, our results show that cells in the presence of two solid boundaries display more frequent reversals in swimming direction and swim faster. Additionally, we observe that run segments are no longer straight and that cells swim on circular trajectories, which can be attributed to the hydrodynamic wall effect. Using the experimentally observed parameters together with a recently presented analytic model for a run-reverse random walker, we obtained additional insight on how the spreading behavior of a cell population is affected under confinement. While on short time scales, the mean square displacement of confined swimmers grows faster as compared to the bulk fluid case, our model predicts that for large times the situation reverses due to the strong increase in effective rotational diffusion.

Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting.