Bacterial variability in the mammalian gut captured by a single-cell synthetic oscillator.

Synthetic gene oscillators have the potential to control timed functions and periodic gene expression in engineered cells. Such oscillators have been refined in bacteria in vitro, however, these systems have lacked the robustness and precision necessary for applications in complex in vivo environments, such as the mammalian gut. Here, we demonstrate the implementation of a synthetic oscillator capable of keeping robust time in the mouse gut over periods of days. The oscillations provide a marker of bacterial growth at a single-cell level enabling quantification of bacterial dynamics in response to inflammation and underlying variations in the gut microbiota. Our work directly detects increased bacterial growth heterogeneity during disease and differences between spatial niches in the gut, demonstrating the deployment of a precise engineered genetic oscillator in real-life settings.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

Development of MAST: A Microscopy-Based Antimicrobial Susceptibility Testing Platform.

Antibiotic resistance is compromising our ability to treat bacterial infections. Clinical microbiology laboratories guide appropriate treatment through antimicrobial susceptibility testing (AST) of patient bacterial isolates. However, increasingly, pathogens are developing resistance to a broad range of antimicrobials, requiring AST of alternative agents for which no commercially available testing methods are available. Therefore, there exists a significant AST testing gap in which current methodologies cannot adequately address the need for rapid results in the face of unpredictable susceptibility profiles. To address this gap, we developed a multicomponent, microscopy-based AST (MAST) platform capable of AST determinations after only a 2 h incubation. MAST consists of a solid-phase microwell growth surface in a 384-well plate format, inkjet printing-based application of both antimicrobials and bacteria at any desired concentrations, automated microscopic imaging of bacterial replication, and a deep learning approach for automated image classification and determination of antimicrobial minimal inhibitory concentrations (MICs). In evaluating a susceptible strain set, 95.8% were within ±1 and 99.4% were within ±2, twofold dilutions, respectively, of reference broth microdilution MIC values. Most (98.3%) of the results were in categorical agreement. We conclude that MAST offers promise for rapid, accurate, and flexible AST to help address the antimicrobial testing gap.

Reconstitution of proteins on electroformed giant unilamellar vesicles.

In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on identifying and isolating an essential set of soluble biomolecules, such as proteins, DNA, and RNA, reconstitution of membrane-based processes involves the additional challenge of forming and working with lipid bilayer membranes with composition, fluidity, and mechanical properties appropriate for the process at hand. Here we discuss a selection of methods for forming synthetic lipid bilayer membranes and present a versatile electroformation protocol that our lab uses for reconstituting proteins on giant unilamellar vesicles. This synthetic membrane-based approach to reconstitution offers the ability to study protein organization and activity at membranes under more cell-like conditions, addressing a central challenge to accomplishing the grand goal of "building the cell."

Genetic oscillations. A Doppler effect in embryonic pattern formation.

During embryonic development, temporal and spatial cues are coordinated to generate a segmented body axis. In sequentially segmenting animals, the rhythm of segmentation is reported to be controlled by the time scale of genetic oscillations that periodically trigger new segment formation. However, we present real-time measurements of genetic oscillations in zebrafish embryos showing that their time scale is not sufficient to explain the temporal period of segmentation. A second time scale, the rate of tissue shortening, contributes to the period of segmentation through a Doppler effect. This contribution is modulated by a gradual change in the oscillation profile across the tissue. We conclude that the rhythm of segmentation is an emergent property controlled by the time scale of genetic oscillations, the change of oscillation profile, and tissue shortening.

The segmentation clock: inherited trait or universal design principle?

Metamerism is a widespread feature of multicellular body plans; however, our understanding of the underlying mechanisms that generate these patterns is currently based on only a few model organisms. In particular, vertebrate embryos use a segmentation clock to rhythmically and sequentially add segments in concert with posterior elongation of their body. Recent evidence of a segmentation clock acting in arthropods indicates that this mechanism may be a widely used strategy for generating serial anatomy in animals. Whether this is due to homology or convergence is not yet known, but the recent discovery of an oscillatory process associated with the production of sequential root primordia in plants suggests that a segmentation clock is a fundamental patterning principle in growing tissues, independent of ancestry. In this review, we consider the principles of the segmentation clock that may be conserved across the animal and plant kingdoms, and discuss opportunities for cross-fertilization between these active fields of research.

Death receptors DR6 and TROY regulate brain vascular development.

Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development.

Forming giant vesicles with controlled membrane composition, asymmetry, and contents.

Growing knowledge of the key molecular components involved in biological processes such as endocytosis, exocytosis, and motility has enabled direct testing of proposed mechanistic models by reconstitution. However, current techniques for building increasingly complex cellular structures and functions from purified components are limited in their ability to create conditions that emulate the physical and biochemical constraints of real cells. Here we present an integrated method for forming giant unilamellar vesicles with simultaneous control over (i) lipid composition and asymmetry, (ii) oriented membrane protein incorporation, and (iii) internal contents. As an application of this method, we constructed a synthetic system in which membrane proteins were delivered to the outside of giant vesicles, mimicking aspects of exocytosis. Using confocal fluorescence microscopy, we visualized small encapsulated vesicles docking and mixing membrane components with the giant vesicle membrane, resulting in exposure of previously encapsulated membrane proteins to the external environment. This method for creating giant vesicles can be used to test models of biological processes that depend on confined volume and complex membrane composition, and it may be useful in constructing functional systems for therapeutic and biomaterials applications.

Inkjet formation of unilamellar lipid vesicles for cell-like encapsulation.

Encapsulation of macromolecules within lipid vesicles has the potential to drive biological discovery and enable development of novel, cell-like therapeutics and sensors. However, rapid and reliable production of large numbers of unilamellar vesicles loaded with unrestricted and precisely-controlled contents requires new technologies that overcome size, uniformity, and throughput limitations of existing approaches. Here we present a high-throughput microfluidic method for vesicle formation and encapsulation using an inkjet printer at rates up to 200 Hz. We show how multiple high-frequency pulses of the inkjet's piezoelectric actuator create a microfluidic jet that deforms a bilayer lipid membrane, controlling formation of individual vesicles. Variations in pulse number, pulse voltage, and solution viscosity are used to control the vesicle size. As a first step toward cell-like reconstitution using this method, we encapsulate the cytoskeletal protein actin and use co-encapsulated microspheres to track its polymerization into a densely entangled cytoskeletal network upon vesicle formation.

Unilamellar vesicle formation and encapsulation by microfluidic jetting.

Compartmentalization of biomolecules within lipid membranes is a fundamental requirement of living systems and an essential feature of many pharmaceutical therapies. However, applications of membrane-enclosed solutions of proteins, DNA, and other biologically active compounds have been limited by the difficulty of forming unilamellar vesicles with controlled contents in a repeatable manner. Here, we demonstrate a method for simultaneously creating and loading giant unilamellar vesicles (GUVs) using a pulsed microfluidic jet. Akin to blowing a bubble, the microfluidic jet deforms a planar lipid bilayer into a vesicle that is filled with solution from the jet and separates from the planar bilayer. In contrast with existing techniques, our method rapidly generates multiple monodisperse, unilamellar vesicles containing solutions of unrestricted composition and molecular weight. Using the microfluidic jetting technique, we demonstrate repeatable encapsulation of 500-nm particles into GUVs and show that functional pore proteins can be incorporated into the vesicle membrane to mediate transport. The ability of microfluidic jetting to controllably encapsulate solutions inside of GUVs creates new opportunities for the study and use of compartmentalized biomolecular systems in science, industry, and medicine.

Membrane-induced bundling of actin filaments.

Dynamic interplay between the plasma membrane and underlying cytoskeleton is essential for cellular shape change. Spatial organization of actin filaments, whose growth generates membrane deformations during motility 1, phagocytosis 2, endocytosis 3, and cytokinesis 4, is mediated by specific protein-protein interactions that branch, crosslink, and bundle filaments into networks that interact with the membrane. Although membrane curvature has been found to influence binding of proteins with curvature-sensitive domains 5, the direct effect of membrane elasticity on cytoskeletal network organization is not clear. Here we show through in vitro reconstitution and elastic modeling that a lipid bilayer can drive the emergence of bundled actin filament protrusions from branched actin filament networks, thus playing a role normally attributed to actin-binding proteins. Formation of these filopodium-like protrusions with only a minimal set of purified proteins points to an active participation of the membrane in organizing actin filaments at the plasma membrane. In this way, elastic interactions between the membrane and cytoskeleton can cooperate with accessory proteins to drive cellular shape change.