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The pathogenic bacteriaBacillus cereus,Bacillus anthracisandBacillus thuringiensisform spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in liveB. cereuscells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes duringB. cereussporulation. We used these markers to characterize a subpopulation of engulfment-defectiveB. cereuscells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organismBacillus subtilis, the activity of σKis not strictly linked to the state of forespore development inB. cereus.

Most bacteria are encased into a load-bearing rigid framework, the cell wall (CW). The peptidoglycan (PG) layer, a network composed of glycan strands cross-linked by stem peptides, is the main component of the CW. During PG synthesis, precursors are first synthetized intracellularly, before being incorporated into the existing PG meshwork by transglycosylation (TG) and transpeptidation (TP) reactions. Covalent modifications of the PG meshwork such as amidation and acetylation participate in PG homeostasis by regulating PG-associated enzymes like PG hydrolases.Because of its essential role, PG synthesis represents a primary target for antibiotic action. Here, we investigated the effect on PG composition of antibiotics targeting intracellular and extracellular steps of PG synthesis: inhibitors of PG precursors synthesis (fosfomycin, D-cycloserine, bacitracin and tunicamycin) and TG/TP inhibitors (vancomycin and penicillin G), respectively. Our study revealed interesting correlations between crosslinking and both de-N-acetylation and amidation of the sacculus. A thorough analysis of muropeptides composition put into light an unexpected anti-correlation between the degree of PG crosslinking and accumulation of de-amidated disaccharide-tripeptide monomer subunit (M3) in the presence of TP inhibitors. We confirmed these observations by analyzing mutants of the PG synthesis pathway.

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for Gram-positive bacteriaBacillus subtilis, the average cell length varies as a function of the growth rate but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width inB. subtilis. Using high-content screening (HCS) fluorescence microscopy and semi-automated measurement of single-cell dimensions, we screened a library of ~ 4000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control inB. subtilis.

International audience; Virus infection causes major rearrangements in the subcellular architecture of eukaryotes, but its impact in prokaryotic cells was much less characterized. Here, we show that infection of the bacterium Bacillus subtilis by bacteriophage SPP1 leads to a hijacking of host replication proteins to assemble hybrid viral–bacterial replisomes for SPP1 genome replication. Their biosynthetic activity doubles the cell total DNA content within 15 min. Replisomes operate at several independent locations within a single viral DNA focus positioned asymmetrically in the cell. This large nucleoprotein complex is a self-contained compartment whose boundaries are delimited neither by a membrane nor by a protein cage. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging. The resulting DNA-filled capsids do not remain associated to the DNA transactions compartment. They bind to phage tails to build infectious particles that are stored in warehouse compartments spatially independent from the viral DNA. Free SPP1 structural proteins are recruited to the dynamic phage-induced compartments following an order that recapitulates the viral particle assembly pathway. These findings show that bacteriophages restructure the crowded host cytoplasm to confine at different cellular locations the sequential processes that are essential for their multiplication.

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Total internal reflection fluorescence (TIRF) microscopy allows the visualization of the dynamic membrane-associated actin-like MreB filaments in live bacterial cells with high temporal resolution. This chapter describes computerized analysis methods to quantitatively characterize the dynamics and morphological properties of MreB assemblies. These include how to (1) segment bacterial cells, (2) perform single-particle tracking (SPT) of MreB filamentous structures, (3) classify their dynamic modes using mean squared displacement (MSD) analysis, and (4) measure their dimensions and orientation.

Total internal reflection fluorescence (TIRF) microscopy allows the visualization of the dynamic membrane-associated actin-like MreB filaments in live bacterial cells with high temporal resolution. This chapter describes computerized analysis methods to quantitatively characterize the dynamics and morphological properties of MreB assemblies. These include how to (1) segment bacterial cells, (2) perform single-particle tracking (SPT) of MreB filamentous structures, (3) classify their dynamic modes using mean squared displacement (MSD) analysis, and (4) measure their dimensions and orientation.

The construction of the bacterial cell envelope is a fundamental topic, as it confers its integrity to bacteria and is consequently the target of numerous antibiotics. MreB is an essential protein suspected to regulate the cell wall synthetic machineries. Despite two decades of study, its localization remains the subject of controversies, its description ranging from helical filaments spanning the entire cell to small discrete entities. The true structure of these filaments is important because it impacts the model describing how the machineries building the cell wall are associated, how they are coordinated at the scale of the entire cell, and how MreB mediates this regulation. Our results shed light on this debate, revealing the size of native filaments in B. subtilis during growth. They argue against models where MreB filament size directly affects the speed of synthesis of the cell wall and where MreB would coordinate distant machineries along the side wall., The actin-like MreB protein is a key player of the machinery controlling the elongation and maintenance of the cell shape of most rod-shaped bacteria. This protein is known to be highly dynamic, moving along the short axis of cells, presumably reflecting the movement of cell wall synthetic machineries during the enzymatic assembly of the peptidoglycan mesh. The ability of MreB proteins to form polymers is not debated, but their structure, length, and conditions of establishment have remained unclear and the subject of conflicting reports. Here we analyze various strains of Bacillus subtilis, the model for Gram-positive bacteria, and we show that MreB forms subdiffraction-limited, less than 200 nm-long nanofilaments on average during active growth, while micron-long filaments are a consequence of artificial overaccumulation of the protein. Our results also show the absence of impact of the size of the filaments on their speed, orientation, and other dynamic properties conferring a large tolerance to B. subtilis toward the levels and consequently the lengths of MreB polymers. Our data indicate that the density of mobile filaments remains constant in various strains regardless of their MreB levels, suggesting that another factor determines this constant.

International audience; Cancer-germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer-germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T-cell receptor specific for the P1A(35-43) peptide associated with H-2L(d), although able to induce regression of P1A-expressing P815 mastocytoma cells, were much less effective against P1A-expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A-specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H-2L(d). The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video-microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A-expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB-Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB-Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour-CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule-1, the ligand for LFA-1, which was not detected on the melanoma cells.

Cancer‐germline genes in both humans and mice have been shown to encode antigens susceptible to targeting by cytotoxic CD8 T effector cells (CTL). We analysed the ability of CTL to kill different tumour cell lines expressing the same cancer‐germline gene P1A (Trap1a). We previously demonstrated that CTL expressing a T‐cell receptor specific for the P1A35–43 peptide associated with H‐2Ld, although able to induce regression of P1A‐expressing P815 mastocytoma cells, were much less effective against P1A‐expressing melanoma cells. Here, we analysed parameters of the in vitro interaction between P1A‐specific CTL and mastocytoma or melanoma cells expressing similar levels of the P1A gene and of surface H‐2Ld. The mastocytoma cells were more sensitive to cytolysis than the melanoma cells in vitro. Analysis by video‐microscopy of early events required for target cell killing showed that similar patterns of increase in cytoplasmic Ca2+ concentration ([Ca2+]i) were induced by both types of P1A‐expressing tumour cells. However, the use of CTL expressing a fluorescent granzyme B (GZMB‐Tom) showed a delay in the migration of cytotoxic granules to the tumour interaction site, as well as a partially deficient GZMB‐Tom exocytosis in response to the melanoma cells. Among surface molecules possibly affecting tumour–CTL interactions, the mastocytoma cells were found to express intercellular adhesion molecule‐1, the ligand for LFA‐1, which was not detected on the melanoma cells.

Background The spatiotemporal behavior of chromatin is an important control mechanism of genomic function. Studies in Saccharomyces cerevisiae have broadly contributed to demonstrate the functional importance of nuclear organization. Although in the wild yeast survival depends on their ability to withstand adverse conditions, most of these studies were conducted on cells undergoing exponential growth. In these conditions, as in most eukaryotic cells, silent chromatin that is mainly found at the 32 telomeres accumulates at the nuclear envelope, forming three to five foci. Results Here, combining live microscopy, DNA FISH and chromosome conformation capture (HiC) techniques, we report that chromosomes adopt distinct organizations according to the metabolic status of the cell. In particular, following carbon source exhaustion the genome of long-lived quiescent cells undergoes a major spatial re-organization driven by the grouping of telomeres into a unique focus or hypercluster localized in the center of the nucleus. This change in genome conformation is specific to quiescent cells able to sustain long-term viability. We further show that reactive oxygen species produced by mitochondrial activity during respiration commit the cell to form a hypercluster upon starvation. Importantly, deleting the gene encoding telomere associated silencing factor SIR3 abolishes telomere grouping and decreases longevity, a defect that is rescued by expressing a silencing defective SIR3 allele competent for hypercluster formation. Conclusions Our data show that mitochondrial activity primes cells to group their telomeres into a hypercluster upon starvation, reshaping the genome architecture into a conformation that may contribute to maintain longevity of quiescent cells. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0766-2) contains supplementary material, which is available to authorized users.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τd and the squared waist of observation w2. Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

International audience; Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)(1). Single-molecule videomicroscopy, offering millisecond and nanometric resolution(1-11), allows a detailed representation of membrane organization(12-14) by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis(15,16). Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.The nanometric accuracy(17) obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods(17) such as STORM18, PALM(19,20), RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

The plasma membrane delineating the cell is a complex multicomponent assembly of lipids and proteins. Deciphering the lateral organization of this supramolecular complex on appropriate length and temporal scales is necessary to unravel its implication in biological function. Here, we show how measurements of diffusion may be taken to shed light on membrane function. We first describe the current methods used to report lateral diffusion of membrane components. We then focus on one fluorescent correlation spectroscopy (FCS)-based method, namely, the spot variation FCS (svFCS), which allows the characterization of the modes of molecular confinement within the plasma membrane of living cells. We next illustrate with different biological systems the progress made toward improving our understanding of cell membrane function. We also discuss the findings with regard to the current view of nanoscale domains/assemblies as a general membrane-organizing principle.

International audience; The dispersion properties of surface modes supported by metal/dielectric interfaces perforated by twodimensional arrays of subwavelength apertures are studied. An analytical surface-impedance model is derived. It provides closed-form expressions for the dispersion relation of surface modes and allows to take into account the aperture geometry and the dispersive and dissipative properties of the metal. The model predicts the dispersion relation and the mode lifetime, in quantitative agreement with fully vectorial computational results and with experimental data obtained at optical frequencies. The analysis reveals a double plasmonic band gap in the center of the Brillouin zone for a square array. It additionally clarifies the effects of various different geometrical and material parameters on the surface properties.

We report on high-accuracy angle-resolved optical transmission measurements through anisotropic 2D plasmonic crystals made of gold films with large-area rectangular arrays of nanoscale square holes, deposited on GaAs substrates. The measurements reveal the dispersion relations of air-gold and gold-GaAs surface plasmon polaritons. The crystal anisotropy induces a separation between plasmonic modes propagating in different directions. Their symmetry and dispersion properties are discussed.

International audience; The impact of the periodicity on the dispersion relation of surface modes supported by two dimensional (2D) structured metallic films is experimentally and numerically investigated between 1 and 5 μm. A coupling between modes propagating in two nearly orthogonal directions forming an angle of 77° is evidenced for a rectangular array of holes perforated in a gold film. The coupling results in a narrow band gap for nonzero wavevectors and in a coupled mode with a large density of states and a strong radiative damping. We show that the gap location in the (ω,k) space can be precisely tuned by changing the 2D period.

Subwavelength dielectric and metallic gratings embedded in vacuum can act as highly-resonant spectral filters. We review the theoretical principles for the design of symmetric dielectric and metal gratings to conceive efficient optical filters in the mid and far infrared range, and we show how both resonance width and resonance wavelength can be tuned. We describe an original process for the fabrication of free-standing SiC gratings, and we present the first samples obtained with 10 mm period. Experimental angularly resolved transmission spectra show evidences of their filtering properties.

Thin nanostructured metal films allow to control radiative and non-radiative losses of surface plasmon polariton modes without changing their group velocities. This effect is studied in plasmonic waveguides made of thin gold films drilled with very narrow slits and deposited on a GaAs substrate. The analysis is supported by high-resolution angle-resolved transmission measurements and rigorous electromagnetic calculations. We show that the excitation of air/gold and gold/GaAs surface waves leads to Fano-type resonances with specific light localization into the slits. As a result, gold/GaAs surface waves induce a modulation of radiative and non-radiative losses of air/gold surface waves. The minimum and maximum of the Fano-type resonance introduce two propagation regimes. In the radiative propagation regime, the losses due to the absorption are negligible, whereas an efficient inhibition of free-space coupling is demonstrated in low-loss propagation regime.

High-resolution angle-resolved transmission measurements are carried out through nanostructured plasmonic waveguides made of thin gold films with very narrow slits deposited on GaAs substrates. They reveal transmission intensity modulations along the air/metal surface plasmon band, due to the presence of metal/substrate surface modes. Two propagation regimes have been identified. In this plasmonic waveguide, the radiative emission can be enhanced or inhibited by a slight modification of the refractive index of the substrate (δn∕n≃1%), allowing to control the propagation regime of surface plasmon waves along the air/metal interface.

International audience; Performing label free coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) in endoscope imaging is a challenge, with huge potential clinical benefit. To date, this goal has remained inaccessible because of the inherent coherent Raman noise that is generated in the fiber itself. By developing double-clad hollow core photonic crystal fiber, we demonstrate coherent anti-Stokes Raman scattering and stimulated Raman scattering in an "endoscope-like‟ scheme. Both the excitation beams and the collected CARS and SRS signals travel through the same fiber. No CARS and SRS signals are generated within the hollow core fiber even for temporally overlapping pump and Stokes beams, leading to excellent image quality. The CARS and SRS signals generated in the sample are coupled back into a high numerical aperture multimode cladding surrounding the central photonic crystal cladding. We demonstrate this scheme by imaging molecular vibrational bonds of organic crystal deposited on a glass surface.

MreB proteins are actin homologs present in nonspherical bacteria. They assemble into membrane-associated discrete filamentous structures that exhibit different dynamic behaviors along the bacterial sidewalls. Total internal reflection fluorescence (TIRF) microscopy, a sensitive method for studying molecular events at cell surfaces with high contrast and temporal resolution, is a method of choice to characterize the localization and dynamics of cortical MreB assemblies in vivo. This chapter describes the methods for visualizing fluorescently tagged MreB proteins in live Bacillus subtilis cells. We detail how to (1) grow B. subtilis strains for reproducible TIRF observations, (2) immobilize cells on agarose pads and (3) in CellASIC ® microfluidic plates, and (4) acquire TIRF images and time lapses.