Experimental determination of the propulsion matrix of the body of helical Magnetospirillum magneticum cells.

Helical-shaped magnetotactic bacteria provide a rare opportunity to precisely measure both the translational and rotational friction coefficients of micron-sized chiral particles. The possibility to align these cells with a uniform magnetic field allows clearly separating diffusion along and perpendicular to their longitudinal axis. Meanwhile, their corkscrew shape allows detecting rotations around their longitudinal axis, after which orientation correlation analysis can be used to retrieve rotational diffusion coefficients in the two principal directions. Using light microscopy, we measured the four principal friction coefficients of deflagellated Magnetospirillum magneticum cells, and compared our results to that expected for cylinders of comparable size. We show that for rotational motions, the overall dimensions of the cell body are what matters most, while the exact body shape has a larger influence on translational motions. To obtain a full characterization of the friction matrix of these elongated chiral particles, we also quantified the coupling between the rotation around and translation along the longitudinal axis of the cell. Our results suggest that for this bacterial species cell body rotation could significantly contribute to cellular propulsion.

Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription.

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid's partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

Inducing Microscale Structural Order in Phage Nanofilament Hydrogels with Globular Proteins.

Biological hydrogels play important physiological roles in the body. These hydrogels often contain ordered subdomains that provide mechanical toughness and other tissue-specific functionality. Filamentous bacteriophages are nanofilaments with a high aspect ratio that can self-assemble into liquid crystalline domains that could be designed to mimic ordered biological hydrogels and can thus find applications in biomedical engineering. We have previously reported hydrogels of pure cross-linked liquid crystalline filamentous phage formed at very high concentrations exhibiting a tightly packed microstructure and high stiffness. In this work, we report a method for inducing self-assembly of filamentous phage into liquid crystalline hydrogels at concentrations that are several orders of magnitude below that of lyotropic liquid crystal formation, thus creating structural order but a less densely packed microstructure. Hybrid hydrogels of M13 phage and bovine serum albumin (0.25 w/v%) were formed and shown to adsorb up to 16× their weight in water. Neither component gelled on its own at the low concentrations used, suggesting synergistic action between the two components in the formation of the hydrogel. The hybrid hydrogels exhibited repetitive self-healing under physiological conditions and at room temperature, autofluorescence in three channels, and antibacterial activity toward Escherichia coli host cells. Furthermore, the hybrid hydrogels exhibited a more than 2× higher ability to pack water compared to BSA-only hydrogels and 2× lower compression modulus compared to tightly packed M13-only hydrogels, suggesting that our method could be used to create hydrogels with tunable mechanical properties and pore structure through the addition of globular proteins, while maintaining bioactivity and microscale structural order.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane.

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6µm-2 to ≃0.1µm-2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

Using FCS to accurately measure protein concentration in the presence of noise and photobleaching.

Quantitative cell biology requires precise and accurate concentration measurements, resolved both in space and time. Fluorescence correlation spectroscopy (FCS) has been held as a promising technique to perform such measurements because the fluorescence fluctuations it relies on are directly dependent on the absolute number of fluorophores in the detection volume. However, the most interesting applications are in cells, where autofluorescence and confinement result in strong background noise and important levels of photobleaching. Both noise and photobleaching introduce systematic bias in FCS concentration measurements and need to be corrected for. Here, we propose to make use of the photobleaching inevitably occurring in confined environments to perform series of FCS measurements at different fluorophore concentration, which we show allows a precise in situ measurement of both background noise and molecular brightness. Such a measurement can then be used as a calibration to transform confocal intensity images into concentration maps. The power of this approach is first illustrated with in vitro measurements using different dye solutions, then its applicability for in vivo measurements is demonstrated in Drosophila embryos for a model nuclear protein and for two morphogens, Bicoid and Capicua.

Capicua is a fast-acting transcriptional brake.

Even though transcriptional repressors are studied with ever-increasing molecular resolution, the temporal aspects of gene repression remain poorly understood. Here, we address the dynamics of transcriptional repression by Capicua (Cic), which is essential for normal development and is commonly mutated in human cancers and neurodegenerative diseases.1,2 We report the speed limit for Cic-dependent gene repression based on live imaging and optogenetic perturbations in the early Drosophila embryo, where Cic was originally discovered.3 Our measurements of Cic concentration and intranuclear mobility, along with real-time monitoring of the activity of Cic target genes, reveal remarkably fast transcriptional repression within minutes of removing an optogenetic de-repressive signal. In parallel, quantitative analyses of transcriptional bursting of Cic target genes support a repression mechanism providing a fast-acting brake on burst generation. This work sets quantitative constraints on potential mechanisms for gene regulation by Cic.

Self-organisation and convection of confined magnetotactic bacteria.

Collective motion is found at all scales in biological and artificial systems, and extensive research is devoted to describing the interplay between interactions and external cues in collective dynamics. Magnetotactic bacteria constitute a remarkable example of living organisms for which motion can be easily controlled remotely. Here, we report a new type of collective motion where a uniform distribution of magnetotactic bacteria is rendered unstable by a magnetic field. A new state of "bacterial magneto-convection" results, wherein bacterial plumes emerge spontaneously perpendicular to an interface and develop into self-sustained flow convection cells. While there are similarities to gravity driven bioconvection and the Rayleigh-Bénard instability, these rely on a density mismatch between layers of the fluids. Remarkably, here no external forces are applied on the fluid and the magnetic field only exerts an external torque aligning magnetotactic bacteria with the field. Using a theoretical model based on hydrodynamic singularities, we capture quantitatively the instability and the observed long-time growth. Bacterial magneto-convection represents a new class of collective behaviour resulting only from the balance between hydrodynamic interactions and external alignment.

Allosteric Regulation of BH3 Proteins in Bcl-xL Complexes Enables Switch-like Activation of Bax.

Current models of apoptosis regulation by the Bcl-2 family of proteins postulate that heterodimeric interactions between family members determine whether Bax and Bak are activated to trigger cell death. Thus, the relative abundance and binding affinities between pro- and anti-apoptotic proteins determines the outcome of these interactions. Examination of these interactions using purified mitochondria and liposomes with full-length recombinant proteins revealed that Bcl-xL inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins. Allosteric regulation of this complex by the BH3 sensitizer Bad confers switch-like activity to the indirect activation of Bax. The BH3 activator cBid sequestered by Bcl-xL complexes changes from an inactive to an active form while bound to a Bcl-xL complex only when Bad is also bound. Bcl-xL complexes enable Bad to function as a non-competitive inhibitor of Bcl-xL and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad.

Misalignment between the magnetic dipole moment and the cell axis in the magnetotactic bacterium Magnetospirillum magneticum AMB-1.

While most quantitative studies of the motion of magnetotactic bacteria rely on the premise that the cells' magnetic dipole moment is aligned with their direction of motility, this assumption has so far rarely been challenged. Here we use phase contrast microscopy to detect the rotational diffusion of non-motile cells of Magnetospirillum magneticum AMB-1 around their magnetic moment, showing that in this species the magnetic dipole moment is, in fact, not exactly aligned with the cell body axis. From the cell rotational trajectories, we are able to infer the misalignment between cell magnetic moment and body axis with a precision of better than 1°, showing that it is, on average, 6°, and can be as high as 20°. We propose a method to correct for this misalignment, and perform a non-biased measurement of the magnetic moment of single cells based on the analysis of their orientation distribution. Using this correction, we show that magnetic moment strongly correlates with cell length. The existence of a range of misalignments between magnetic moment and cell axis in a population implies that the orientation and trajectories of magnetotactic bacteria placed in external magnetic fields is more complex than generally assumed, and might show some important cell-to-cell differences.

Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders.

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development.

Membrane charge and lipid packing determine polymyxin-induced membrane damage.

With the advent of polymyxin B (PmB) resistance in bacteria, the mechanisms for mcr-1 resistance are of crucial importance in the design of novel therapeutics. The mcr-1 phenotype is known to decrease membrane charge and increase membrane packing by modification of the bacterial outer membrane. We used X-ray diffraction, Molecular Dynamics simulations, electrochemistry, and leakage assays to determine the location of PmB in different membranes and assess membrane damage. By varying membrane charge and lipid tail packing independently, we show that increasing membrane surface charge promotes penetration of PmB and membrane damage, whereas increasing lipid packing decreases penetration and damage. The penetration of the PmB molecules is well described by a phenomenological model that relates an attractive electrostatic and a repulsive force opposing insertion due to increased membrane packing. The model applies well to several gram-negative bacterial strains and may be used to predict resistance strength.

Anomalous Diffusion in Inverted Variable-Lengthscale Fluorescence Correlation Spectroscopy.

Using fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1.

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Live Imaging of mRNA Transcription in Drosophila Embryos.

Live imaging has been used in recent years for the understanding of dynamic processes in biology, such as embryo development. This was made possible by a combination of advancements in microscopy, leading to improved signal-to-noise ratios and better spatial and temporal resolutions, and by the development of new fluorescence markers, allowing for the quantification of protein expression and transcriptional dynamics in vivo. Here we describe a general protocol, which can be used in standard confocal microscopes to image early Drosophila melanogaster embryos, in order to learn about the transcriptional dynamics of a fluorescently labeled RNA.

LiveFly: A Toolbox for the Analysis of Transcription Dynamics in Live Drosophila Embryos.

We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

Precision in a rush: Trade-offs between reproducibility and steepness of the hunchback expression pattern.

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene's ability to read positional information along the embryo's anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies.

3 minutes to precisely measure morphogen concentration.

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression.

On the importance of protein diffusion in biological systems: The example of the Bicoid morphogen gradient.

Morphogens are proteins that form concentration gradients in embryos and developing tissues, where they act as postal codes, providing cells with positional information and allowing them to behave accordingly. Bicoid was the first discovered morphogen, and remains one of the most studied. It regulates segmentation in flies, forming a striking exponential gradient along the anterior-posterior axis of early Drosophila embryos, and activating the transcription of multiple target genes in a concentration-dependent manner. In this review, the work done by us and by others to characterize the mobility of Bicoid in D. melanogaster embryos is presented. The central role played by the diffusion of Bicoid in both the establishment of the gradient and the activation of target genes is discussed, and placed in the context of the need for these processes to be all at once rapid, precise and robust. The Bicoid system, and morphogen gradients in general, remain amongst the most amazing examples of the coexistence, often observed in living systems, of small-scale disorder and large-scale spatial order. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

Precision of Readout at the hunchback Gene: Analyzing Short Transcription Time Traces in Living Fly Embryos.

The simultaneous expression of the hunchback gene in the numerous nuclei of the developing fly embryo gives us a unique opportunity to study how transcription is regulated in living organisms. A recently developed MS2-MCP technique for imaging nascent messenger RNA in living Drosophila embryos allows us to quantify the dynamics of the developmental transcription process. The initial measurement of the morphogens by the hunchback promoter takes place during very short cell cycles, not only giving each nucleus little time for a precise readout, but also resulting in short time traces of transcription. Additionally, the relationship between the measured signal and the promoter state depends on the molecular design of the reporting probe. We develop an analysis approach based on tailor made autocorrelation functions that overcomes the short trace problems and quantifies the dynamics of transcription initiation. Based on live imaging data, we identify signatures of bursty transcription initiation from the hunchback promoter. We show that the precision of the expression of the hunchback gene to measure its position along the anterior-posterior axis is low both at the boundary and in the anterior even at cycle 13, suggesting additional post-transcriptional averaging mechanisms to provide the precision observed in fixed embryos.