Essential magnetosome proteins MamI and MamL from magnetotactic bacteria interact in mammalian cells.

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells.

Both the transcriptional activator, Bcd, and repressor, Cic, form small mobile oligomeric clusters.

Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single-particle tracking to access a wide range of dynamical timescales.

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.

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

For over 40 years, the Bicoid- (Bcd-) 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 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.

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.

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.

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.

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. 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.

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.

Allosteric Regulation of BH3 Proteins in Bcl-x 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-x inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins.

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°.

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.

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

With the advent of polymyxin B (PmB) resistance in bacteria, the mechanisms for -1 resistance are of crucial importance in the design of novel therapeutics. The -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.

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.

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.

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.

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.

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.

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.

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.

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.

Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy.

The diffusion of macromolecules in cells and in complex fluids is often found to deviate from simple Fickian diffusion. One explanation offered for this behavior is that molecular crowding renders diffusion anomalous, where the mean-squared displacement of the particles scales as 〈r(2)〉∝t(α) with α < 1. Unfortunately, methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) probe diffusion only over a narrow range of lengthscales and cannot directly test the dependence of the mean-squared displacement (MSD) on time.

Lipid Diffusion in Supported Lipid Bilayers: A Comparison between Line-Scanning Fluorescence Correlation Spectroscopy and Single-Particle Tracking.

Diffusion in lipid membranes is an essential component of many cellular process and fluorescence a method of choice to study membrane dynamics. The goal of this work was to directly compare two common fluorescence methods, line-scanning fluorescence correlation spectroscopy and single-particle tracking, to observe the diffusion of a fluorescent lipophilic dye, DiD, in a complex five-component mitochondria-like solid-supported lipid bilayer. We measured diffusion coefficients of DFCS ~ 3 um2 * s-1 and DSPT ~ 2 um2 * s-1, respectively.

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane.

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails.