Publications

Biological patterns that emerge during the morphogenesis of multicellular organisms can display high precision at large scales, while at cellular scales, cells exhibit large fluctuations stemming from cell-cell differences in molecular copy numbers also called demographic noise. We study the conflicting interplay between high precision and demographic noise in trichome patterns on the epidermis of wild-type Arabidopsis thaliana leaves, as a two-dimensional model system. We carry out a statistical characterization of these patterns and show that their power spectra display fat tails - a signature compatible with noise-driven stochastic Turing patterns - which are absent in power spectra of patterns driven by deterministic instabilities. We then present a theoretical model that includes demographic noise stemming from birth- death processes of genetic regulators which we study analytically and by stochastic simulations. The model captures the observed experimental features of trichome patterns.

Circadian clocks display remarkable reliability despite significant stochasticity in biomolecular reactions. We study the dynamics of a circadian clock-controlled gene at the individual cell level in Anabaena sp. PCC 7120, a multicellular filamentous cyanobacterium. We found significant synchronization and spatial coherence along filaments, clock coupling due to cell-cell communication, and gating of the cell cycle. Furthermore, we observed low-amplitude circadian oscillatory transcription of kai genes comprising the post-transcriptional core oscillatory circuit, and high-amplitude oscillations of rpaA coding for the master regulator transducing the core clock output. Transcriptional oscillations of rpaA suggest an additional level of regulation. A stochastic, one-dimensional toy model of coupled clock cores and their phosphorylation states shows that demographic noise can seed stochastic oscillations outside the region where deterministic limit cycles with circadian periods occur. The model reproduces the observed spatio-temporal coherence along filaments, and provides a robust description of coupled circadian clocks in a multicellular organism.

Under nitrogen-poor conditions, multicellular cyanobacteria such as Anabaena sp. PCC 7120 undergo a process of differentiation, forming nearly regular, developmental patterns of individual nitrogen-fixing cells, called heterocysts, interspersed between intervals of vegetative cells that carry out photosynthesis. Developmental pattern formation is mediated by morphogen species that can act as activators and inhibitors, some of which can diffuse along filaments. We survey the limitations of the classical, deterministic Turing mechanism that has been often invoked to explain pattern formation in these systems, and then, focusing on a simpler system governed by birth-death processes, we illustrate pedagogically a recently proposed paradigm that provides a much more robust description of pattern formation: stochastic Turing patterns. We emphasize the essential role that cell-to-cell differences in molecular numberscaused by inevitable fluctuations in gene expressionplay, the so called demographic noise, in seeding the formation of stochastic Turing patterns over a much larger region of parameter space, compared to their deterministic counterparts.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Cyanobacteria carry out oxygenic photosynthesis, play a key role in the cycling of carbon and nitrogen in the biosphere, and have had a large impact on the evolution of life and the Earth itself. Many cyanobacterial strains exhibit a multicellular lifestyle, growing as filaments that can be hundreds of cells long and endowed with intercellular communication. Furthermore, under depletion of combined nitrogen, filament growth requires the activity of two interdependent cell types: vegetative cells that fix CO2 and heterocysts that fix N2. Intercellular molecular transfer is essential for signaling involved in the regulation of heterocyst differentiation and for reciprocal nutrition of heterocysts and vegetative cells. Here we review various aspects of multicellularity in cyanobacterial filaments and their differentiation, including filament architecture with emphasis on the structures used for intercellular communication; we survey theoretical models that have been put forward to understand heterocyst patterning and discuss the factors that need to be considered for these models to reflect the biological entity; and finally, since cell division in filamentous cyanobacteria has the peculiarity of producing linked instead of independent cells, we review distinct aspects of cell division in these organisms.

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell's biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ~10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′-or5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

The SOS genetic network is responsible for the repair/bypass of DNA damage in bacterial cells. While the initial stages of the response have been well characterized, less is known about the dynamics of the response after induction and its shutoff. To address this, we followed the response of the SOS network in living individual Escherichia coli cells. The promoter activity (PA) of SOS genes was monitored using fluorescent protein-promoter fusions, with high temporal resolution, after ultraviolet irradiation activation. We find a temporal pattern of discrete activity peaks masked in studies of cell populations. The number of peaks increases, while their amplitude reaches saturation, as the damage level is increased. Peak timing is highly precise from cell to cell and is independent of the stage in the cell cycle at the time of damage. Evidence is presented for the involvement of the umuDC operon in maintaining the pattern of PA and its temporal precision, providing further evidence for the role UmuD cleavage plays in effecting a timed pause during the SOS response, as previously proposed. The modulations in PA we observe share many features in common with the oscillatory behavior recently observed in a mammalian DNA damage response. Our results, which reveal a hitherto unknown modulation of the SOS response, underscore the importance of carrying out dynamic measurements at the level of individual living cells in order to unravel how a natural genetic network operates at the systems level.

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed λ-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32°C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-tonoise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the method’s use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.

Using rheometry and light scattering, we have studied the viscoelasticity of gels formed by the kinetically arrested phase separation in an emulsion-polymer mixture; to prevent the gels from collapsing under their own weight, we have used an isopycnic solvent. At constant osmotic pressure (set by the polymer concentration) and droplet volume fractions φ well above the gelation transition, we find the elastic modulus to increase roughly linearly with φ, indicating an entropic elasticity based on the cluster packing.

Powder mixtures partially filling horizontal, slowly rotating tubes segregate under certain conditions into bands of different composition along the tube axis. The one-dimensional patterns of bands evolve in time through coarsening. Recent work on the origin and dynamics of axial segregation of powder mixtures is reviewed.

A binary mixture of granular materials in a horizontal, rotating tube segregates under certain circumstances into an alternating pattern of bands of two types along the tube axis, each type being rich in one idf the components. The pattern coarsens with time through band shrinking and merging, until a steady state characterized by some average bandwidth is reached. We have studied experimentally the long-term evolution of segregation patterns in sand-glass bead mixtures. We observe that during coarsening, the two types of band evolve differently, indicating that another transport mechanism is at work besides diffusion. We propose that glass beads are transported axially due to "avalanche waves," a hallmark of the discontinuous flow characterizing sand-rich bands.

We have studied experimentally phase separation in binary mixtures comprised of nearly monodisperse emulsion droplets of two different sizes. For three values of ξ, where ξ

We have studied segregation in binary mixtures of different granular media subjected to rotation in horizontal tubes. Mixtures separate into bands of different relative concentrations arranged along the axis of the tubes. Axial modulations of the tube radius lock bands in space and induce segregation in otherwise nonsegregating mixtures of glass beads. The segregation results from an instability analogous to spinodal decomposition, and we present a model which describes this instability.

We present a preliminary account of an experimental study of the influence of nonionic polymers on the draining process and thickness of freely suspended vertical soap films. We studied films of both cationic and anionic surfactants. While the presence of a polymer leads to a monotonous reduction in thickness with concentration in the former case, the behavior in the latter is very different: an increase in polymer concentration leads to an increase in thickness of black films and to their eventual loss of stability through disruption of micellar stratification.

We have studied experimentally the coarsening of two-dimensional soap froths in the presence of pinning centers. When the average bubble size is smaller than the average interpin distance, the growth is unaffected. When both dimensions become comparable the froth enters a crossover regime followed by a pinned state where growth stops. The nature of the long-time configurations depends strongly on the size of the pins relative to the size of the plateau borders. For thick pins, the final configurations consist of combinations of Steiner trees of small numbers of points with pins located at vertices of the film network. For thin pins the final average bubble size is larger than in the case of thick pins and the final configurations are much more varied. Qualitatively similar behavior is observed in grain growth in metals with impurities.

The mixing of identical grains in a granular flow was experimentally studied and found to be a self-diffusive process. The flow, produced by vertically vibrating a horizontal layer of grains, is two-dimensional allowing us to study self-diffusion in the perpendicular direction to the flow plane. The self-diffusion coefficient depends only on the flow velocity and not on the independent values of frequency and amplitude of vibration. Its growth with the flow velocity is consistent with a linear dependence. Our findings support the picture of the velocity field as consisting of convective and fluctuating parts and agree qualitatively with recent hydrodynamical models of granular flows.