Insights into the micromechanical properties of the metaphase spindle.

The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.

Membrane molecular crowding enhances MreB polymerization to shape synthetic cells from spheres to rods.

Executing gene circuits by cell-free transcription-translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.

Green function of correlated genes in a minimal mechanical model of protein evolution.

The function of proteins arises from cooperative interactions and rearrangements of their amino acids, which exhibit large-scale dynamical modes. Long-range correlations have also been revealed in protein sequences, and this has motivated the search for physical links between the observed genetic and dynamic cooperativity. We outline here a simplified theory of protein, which relates sequence correlations to physical interactions and to the emergence of mechanical function. Our protein is modeled as a strongly coupled amino acid network with interactions and motions that are captured by the mechanical propagator, the Green function. The propagator describes how the gene determines the connectivity of the amino acids and thereby, the transmission of forces. Mutations introduce localized perturbations to the propagator that scatter the force field. The emergence of function is manifested by a topological transition when a band of such perturbations divides the protein into subdomains. We find that epistasis-the interaction among mutations in the gene-is related to the nonlinearity of the Green function, which can be interpreted as a sum over multiple scattering paths. We apply this mechanical framework to simulations of protein evolution and observe long-range epistasis, which facilitates collective functional modes.

Hydrodynamics and collective behavior of the tethered bacterium Thiovulum majus.

The ecology and dynamics of many microbial systems, particularly in mats and soils, are shaped by how bacteria respond to evolving nutrient gradients and microenvironments. Here we show how the response of the sulfur-oxidizing bacterium Thiovulum majus to changing oxygen gradients causes cells to organize into large-scale fronts. To study this phenomenon, we develop a technique to isolate and enrich these bacteria from the environment. Using this enrichment culture, we observe the formation and dynamics of T. majus fronts in oxygen gradients. We show that these dynamics can be understood as occurring in two steps. First, chemotactic cells moving up the oxygen gradient form a front that propagates with constant velocity. We then show, through observation and mathematical analysis, that this front becomes unstable to changes in cell density. Random perturbations in cell density create oxygen gradients. The response of cells magnifies these gradients and leads to the formation of millimeter-scale fluid flows that actively pull oxygenated water through the front. We argue that this flow results from a nonlinear instability excited by stochastic fluctuations in the density of cells. Finally, we show that the dynamics by which these modes interact can be understood from the chemotactic response of cells. These results provide a mathematically tractable example of how collective phenomena in ecological systems can arise from the individual response of cells to a shared resource.

Fundamental amino acid mass distributions and entropy costs in proteomes.

We examine whether the frequency of amino acids across an organism's proteome is primarily determined by optimization to function or other factors, such as the structure of the genetic code. Considering all available proteins together, we first point out that the frequency of an amino acid in a proteome negatively correlates with its mass, suggesting that the genome preserves a fundamental distribution ruled by simple energetics. Given the universality of such distributions, one can use outliers, cysteine and leucine, to identify amino acids that deviate from this simple rule for functional purposes and examine those functions. We quantify the strength of such selection as the entropic cost outliers pay to defy the mass-frequency relation. Codon degeneracy of an amino acid partially explains the correlation between mass and frequency: light amino acids being typically encoded by highly degenerate codon families, with the exception of arginine. While degeneracy may be a factor in hard wiring the relationship between mass and frequency in proteomes, it does not provide a complete explanation. By examining extremophiles, we are able to show that this law weakens with temperature, likely due to protein stability considerations, thus the environment is essential.

Fast-moving bacteria self-organize into active two-dimensional crystals of rotating cells.

We investigate a new form of collective dynamics displayed by Thiovulum majus, one of the fastest-swimming bacteria known. Cells spontaneously organize on a surface into a visually striking two-dimensional hexagonal lattice of rotating cells. As each constituent cell rotates its flagella, it creates a tornadolike flow that pulls neighboring cells towards and around it. As cells rotate against their neighbors, they exert forces on one another, causing the crystal to rotate and cells to reorganize. We show how these dynamics arise from hydrodynamic and steric interactions between cells. We derive the equations of motion for a crystal, show that this model explains several aspects of the observed dynamics, and discuss the stability of these active crystals.

Effects of long DNA folding and small RNA stem-loop in thermophoresis.

In thermophoresis, with the fluid at rest, suspensions move along a gradient of temperature. In an aqueous solution, a PEG polymer suspension is depleted from the hot region and builds a concentration gradient. In this gradient, DNA polymers of different sizes can be separated. In this work the effect of the polymer structure for genomic DNA and small RNA is studied. For genome-size DNA, individual single T4 DNA is visualized and tracked in a PEG solution under a temperature gradient built by infrared laser focusing. We find that T4 DNA follows steps of depletion, ring-like localization, and accumulation patterns as the PEG volume fraction is increased. Furthermore, a coil-globule transition for DNA is observed for a large enough PEG volume fraction. This drastically affects the localization position of T4 DNA. In a similar experiment, with small RNA such as ribozymes we find that the stem-loop folding of such polymers has important consequences. The RNA polymers having a long and rigid stem accumulate, whereas a polymer with stem length less than 4 base pairs shows depletion. Such measurements emphasize the crucial contribution of the double-stranded parts of RNA for thermal separation and selection under a temperature gradient. Because huge temperature gradients are present around hydrothermal vents in the deep ocean seafloor, this process might be relevant, at the origin of life, in an RNA world hypothesis. Ribozymes could be selected from a pool of random sequences depending on the length of their stems.

A concentration-dependent switch in the bacterial response to temperature.

We observed that bacteria grown below a critical concentration, in batch-mode cultures, swim towards warm regions when subjected to a temperature gradient. Above that concentration, they swim towards colder regions. Our findings indicate that the secreted intercellular signal, glycine, mediates this switch through methylation of Tsr receptors. At high bacterial concentration, the switch is reinforced by an inversion of the Tar/Tsr expression ratio.

Effects of pressure and temperature on the binding of RecA protein to single-stranded DNA.

The binding and polymerization of RecA protein to DNA is required for recombination, which is an essential function of life. We study the pressure and temperature dependence of RecA binding to single-stranded DNA in the presence of adenosine 5'-[γ-thio]triphosphate (ATP[γ-S]), in a temperature regulated high pressure cell using fluorescence anisotropy. Measurements were possible at temperatures between 5-60 °C and pressures up to 300 MPa. Experiments were performed on Escherichia coli RecA and RecA from a thermophilic bacteria, Thermus thermophilus. For E. coli RecA at a given temperature, binding is a monotonically decreasing and reversible function of pressure. At atmospheric pressure, E. coli RecA binding decreases monotonically up to 42 °C, where a sharp transition to the unbound state indicates irreversible heat inactivation. T. thermophilus showed no such transition within the temperature range of our apparatus. Furthermore, we find that binding occurs for a wider range of pressure and temperature for T. thermophilus compared to E. coli RecA, suggesting a correlation between thermophilicity and barophilicity. We use a two-state model of RecA binding/unbinding to extract the associated thermodynamic parameters. For E. coli, we find that the binding/unbinding phase boundary is hyperbolic. Our results of the binding of RecA from E. coli and T. thermophilus show adaptation to pressure and temperature at the single protein level.

Development of an artificial cell, from self-organization to computation and self-reproduction.

This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.

Pressure and temperature dependence of growth and morphology of Escherichia coli: experiments and stochastic model.

We have investigated the growth of Escherichia coli, a mesophilic bacterium, as a function of pressure (P) and temperature (T). Escherichia coli can grow and divide in a wide range of pressure (1-400 atm) and temperature (23-40°C). For T > 30°C, the doubling time of E. coli increases exponentially with pressure and exhibits a departure from exponential behavior at pressures between 250 and 400 atm for all the temperatures studied in our experiments. The sharp change in doubling time is followed by a sharp change in phenotypic transition of E. coli at high pressures where bacterial cells switch to an elongating cell type. We propose a model that this phenotypic change in bacteria at high pressures is an irreversible stochastic process, whereas the switching probability to elongating cell type increases with increasing pressure. The model fits well the experimental data. We discuss our experimental results in the light of structural and thus functional changes in proteins and membranes.

Examining how the spatial organization of chromatin signals influences metaphase spindle assembly.

During cell division, the proper assembly of a microtubule-based bipolar spindle depends on signals from chromatin. However, it is unknown how the spatial organization of chromatin signals affects spindle morphology. Here, we use paramagnetic chromatin beads, and magnetic fields for their alignment in cell-free extracts, to examine the spatial components of signals that regulate spindle assembly. We find that for linear chromatin-bead arrays that vary by eightfold in length, metaphase spindle size and shape are constant. Our findings indicate that, although chromatin provides cues for microtubule formation, metaphase spindle organization, which is controlled by microtubule-based motors, is robust to changes in the shape of chromatin signals.

Universal protein fluctuations in populations of microorganisms.

The copy number of any protein fluctuates among cells in a population; characterizing and understanding these fluctuations is a fundamental problem in biophysics. We show here that protein distributions measured under a broad range of biological realizations collapse to a single non-gaussian curve under scaling by the first two moments. Moreover, in all experiments the variance is found to depend quadratically on the mean, showing that a single degree of freedom determines the entire distribution. Our results imply that protein fluctuations do not reflect any specific molecular or cellular mechanism, and suggest that some buffering process masks these details and induces universality.

Effects of population density and chemical environment on the behavior of Escherichia coli in shallow temperature gradients.

In shallow temperature gradients, changes in temperature that bacteria experience occur over long time scales. Therefore, slow processes such as adaptation, metabolism, chemical secretion and even gene expression become important. Since these are cellular processes, the cell density is an important parameter that affects the bacteria's response. We find that there are four density regimes with distinct behaviors. At low cell density, bacteria do not cause changes in their chemical environment; however, their response to the temperature gradient is strongly influenced by it. In the intermediate cell-density regime, the consumption of nutrients becomes significant and induces a gradient of nutrients opposing the temperature gradient due to higher consumption rate at the high temperature. This causes the bacteria to drift toward low temperature. In the high cell-density regime, interactions among bacteria due to secretion of an attractant lead to a strong local accumulation of bacteria. This together with the gradient of nutrients, resulted from the differential consumption rate, creates a fast propagating pulse of bacterial density. These observations are a result of classical nonlinear population dynamics. At extremely high cell density, a change in the physiological state of the bacteria is observed. The bacteria, at the individual level, become cold seeking. This appears initially as a result of a change in the methylation level of the two most abundant sensing receptors, Tsr and Tar. It is further enforced at an even higher cell density by a change in the expression level of these receptors.

Thermal separation: interplay between the Soret effect and entropic force gradient.

Thermophoresis, the Soret effect, depletes a high concentration of a polyethylene glycol polymer solution from the hot region and builds a concentration gradient. In such a solution, solutes of small concentration experience thermophoresis and polyethylene glycol concentration-dependent restoring forces. We report that by using focused laser heating and varying the polyethylene glycol concentration one observes geometrical localizations of solutes like DNA and RNA into patterns such as a ring. For DNA up to 5.6 kbp, the ring size decreases following a behavior analogous to a gel electrophoresis separation. Above 5.6 kbp, the ring diameter increases with the DNA length. Mixtures of DNA and RNA can be separated as well as different RNA lengths. Separation of colloids is also observed. The experiments might be relevant for the separation of small RNA ribozymes in an early stage of life.

Degeneracy of the genetic code and stability of the base pair at the second position of the anticodon.

With an analysis of the structural constraints of the anticodon-codon interaction within the decoding center of the ribosome, we show that the extent of degeneracy at the third position of the anticodon is determined by the level of stability of the base pair at the second position.

High-Q microsphere biosensor - analysis for adsorption of rodlike bacteria.

Theory is developed for frequency shift and linewidth-broadening induced by rodlike bacteria bound to micro-optical resonators. Optical shift of whispering gallery modes (WGMs) is modeled by introducing a form factor that accounts for random horizontal orientation of cylindrical bacteria bound by their high refractive index cell walls. Linewidth-broadening is estimated from scattering losses. Analytic results are confirmed by measurement using E.Coli as model system (~10(2) bacteria/mm(2) sensitivity), establishing the WGM biosensor as sensitive technique for detection and analysis of micro-organisms.

Toward an artificial cell based on gene expression in vesicles.

We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.

Biophysical basis for convergent evolution of two veil-forming microbes.

Microbes living in stagnant water typically rely on chemical diffusion to draw nutrients from their environment. The sulfur-oxidizing bacterium Thiovulum majus and the ciliate Uronemella have independently evolved the ability to form a 'veil', a centimetre-scale mucous sheet on which cells organize to produce a macroscopic flow. This flow pulls nutrients through the community an order of magnitude faster than diffusion. To understand how natural selection led these microbes to evolve this collective behaviour, we connect the physical limitations acting on individual cells to the cell traits. We show how diffusion limitation and viscous dissipation have led individual T. majus and Uronemella cells to display two similar characteristics. Both of these cells exert a force of approximately 40 pN on the water and attach to boundaries by means of a mucous stalk. We show how the diffusion coefficient of oxygen in water and the viscosity of water define the force the cells must exert. We then show how the hydrodynamics of filter-feeding orient a microbe normal to the surface to which it attaches. Finally, we combine these results with new observations of veil formation and a review of veil dynamics to compare the collective dynamics of these microbes. We conclude that this convergent evolution is a reflection of similar physical limitations imposed by diffusion and viscosity acting on individual cells.

Signal and noise in bridging PCR.

BACKGROUND: In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR) can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA. RESULTS: In this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N) of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA. CONCLUSIONS: In the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.