Navigating across multi-dimensional space of tissue clearing parameters.

Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1914, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible.

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes.

Fluorescence protein complementation in microscopy: applications beyond detecting bi-molecular interactions.

Conventional fragments of fluorescent proteins used in bimolecular fluorescence complementation technique (BiFC), form light-emitting species only when they are kept in close proximity by interacting proteins of interest. By contrast, certain fluorescent protein fragments complement spontaneously, namely those corresponding to the 1st to 10th beta-strands (GFP1-10) and the 11th beta-strand of superfolder GFP (GFP11). They were designed as folding reporters for high throughput expression and structure biology.

SYBR Gold dye enables preferential labelling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.

Mitochondrial DNA molecules coated with proteins form compact particles called mitochondrial nucleoids. They are redistributed within mitochondrial network undergoing morphological changes. The straightforward technique to characterize nucleoids' motions is fluorescence microscopy.

Robo1 Forms a Compact Dimer-of-Dimers Assembly.

Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation.

Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 protein.

Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, we have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells (Avilov, Moisy, Munier, Schraidt, Naffakh and Cusack [12]). Here, using this virus, we studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection.

HMGB1 protein binds to influenza virus nucleoprotein and promotes viral replication.

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs).

CC2D1A is a regulator of ESCRT-III CHMP4B.

Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function.

Replication-competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle.

Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S.

Nuclear import and assembly of influenza A virus RNA polymerase studied in live cells by fluorescence cross-correlation spectroscopy.

Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential.

Site-specific characterization of HIV-1 nucleocapsid protein binding to oligonucleotides with two binding sites.

The nucleocapsid protein (NC) of HIV-1 is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. Most NC functions rely on its binding to the HIV-1 genomic RNA and its DNA copies that contain multiple and possibly interdependent binding sites. Therefore, a detailed understanding of NC binding requires a site-specific experimental approach.

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue.

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment.

Resolution of Cys and Lys labeling of alpha-crystallin with site-sensitive fluorescent 3-hydroxyflavone dye.

Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4'-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens alpha-crystallin. The labeling of SH and NH(2) groups are clearly distinguished by spectroscopic criteria.

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study.

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein.

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity.