Targeted Single Particle Tracking with Upconverting Nanoparticles.

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them water-dispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin-streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptor-antibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

Thienoguanosine brightness in DNA duplexes is governed by the localization of itsππ* excitation in the lowest energy absorption band.

Thienoguanosine (thG) is an isomorphic fluorescent guanosine (G) surrogate, which almost perfectly mimics the natural G in DNA duplexes and may therefore be used to sensitively investigate for example protein-induced local conformational changes. To fully exploit the information given by the probe, we carefully re-investigated the thG spectroscopic properties in 12-bp duplexes, when the Set and Ring Associated (SRA) domain of UHRF1 flips its 5' flanking methylcytosine (mC). The SRA-induced flipping of mC was found to strongly increase the fluorescence intensity of thG, but this increase was much larger when thG was flanked in 3' by a C residue as compared to an A residue. Surprisingly, the quantum yield and fluorescence lifetime values of thG were nearly constant, regardless of the presence of SRA and the nature of the 3' flanking residue, suggesting that the differences in fluorescence intensities might be related to changes in absorption properties. We evidenced that thG lowest energy absorption band in the duplexes can be deconvoluted into two bands peaking at ∼350 nm and ∼310 nm, respectively red-shifted and blue-shifted, compared to the spectrum of thG monomer. Using quantum mechanical calculations, we attributed the former to a nearly pureππ* excitation localized on thG and the latter to excited states with charge transfer character. The amplitude of thG red-shifted band strongly increased when its 3' flanking C residue was replaced by an A residue in the free duplex, or when its 5' flanking mC residue was flipped by SRA. As only the species associated with the red-shifted band were found to be emissive, the highly unusual finding of this work is that the brightness of thG in free duplexes as well as its changes on SRA-induced mC flipping almost entirely depend on the relative population and/or absorption coefficient of the red-shifted absorbing species.

CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling.

Adipose tissues dynamically remodel their cellular composition in response to external cues by stimulating beige adipocyte biogenesis; however, the developmental origin and pathways regulating this process remain insufficiently understood owing to adipose tissue heterogeneity. Here, we employed single-cell RNA-seq and identified a unique subset of adipocyte progenitor cells (APCs) that possessed the cell-intrinsic plasticity to give rise to beige fat. This beige APC population is proliferative and marked by cell-surface proteins, including PDGFRα, Sca1, and CD81. Notably, CD81 is not only a beige APC marker but also required for de novo beige fat biogenesis following cold exposure. CD81 forms a complex with αV/ß1 and αV/ß5 integrins and mediates the activation of integrin-FAK signaling in response to irisin. Importantly, CD81 loss causes diet-induced obesity, insulin resistance, and adipose tissue inflammation. These results suggest that CD81 functions as a key sensor of external inputs and controls beige APC proliferation and whole-body energy homeostasis.

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.

BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.

Thermoregulation via Temperature-Dependent PGD2 Production in Mouse Preoptic Area.

Body temperature control is essential for survival. In mammals, thermoregulation is mediated by the preoptic area of anterior hypothalamus (POA), with ∼30% of its neurons sensitive to brain temperature change. It is still unknown whether and how these temperature-sensitive neurons are involved in thermoregulation, because for eight decades they have only been identified via electrophysiological recording. By combining single-cell RNA-seq with whole-cell patch-clamp recordings, we identified Ptgds as a genetic marker for temperature-sensitive POA neurons. Then, we demonstrated these neurons' role in thermoregulation via chemogenetics. Given that Ptgds encodes the enzyme that synthesizes prostaglandin D2 (PGD2), we further explored its role in thermoregulation. Our study revealed that rising temperature of POA alters the activity of Ptgds-expressing neurons so as to increase PGD2 production. PGD2 activates its receptor DP1 and excites downstream neurons in the ventral medial preoptic area (vMPO) that mediates body temperature decrease, a negative feedback loop for thermoregulation.

Biogenesis, delivery, and function of extracellular RNA.

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

Site-Selective Monitoring of the Interaction of the SRA Domain of UHRF1 with Target DNA Sequences Labeled with 2-Aminopurine.

UHRF1 plays a central role in the maintenance and transmission of epigenetic modifications by recruiting DNMT1 to hemimethylated CpG sites via its SET and RING-associated (SRA) domain, ensuring error-free duplication of methylation profiles. To characterize SRA-induced changes in the conformation and dynamics of a target 12 bp DNA duplex as a function of the methylation status, we labeled duplexes by the environment-sensitive probe 2-aminopurine (2-Ap) at various positions near or far from the central CpG recognition site containing either a nonmodified cytosine (NM duplex), a methylated cytosine (HM duplex), or methylated cytosines on both strands (BM duplex). Steady-state and time-resolved fluorescence indicated that binding of SRA induced modest conformational and dynamical changes in NM, HM, and BM duplexes, with only slight destabilization of base pairs, restriction of global duplex flexibility, and diminution of local nucleobase mobility. Moreover, significant restriction of the local motion of residues flanking the methylcytosine in the HM duplex suggested that these residues are more rigidly bound to SRA, in line with a slightly higher affinity of the HM duplex as compared to that of the NM or BM duplex. Our results are consistent with a "reader" role, in which the SRA domain scans DNA sequences for hemimethylated CpG sites without perturbation of the structure of contacted nucleotides.

Characterization of the structural modifications accompanying the loss of HBsAg particle immunogenicity.

The aim of this work was to further understand the relationship between the immunogenicity and the structure of Hepatitis B surface antigen (HBsAg) particles used in Hepatitis B vaccines. To reach this aim, we compared by using a large range of techniques, the structure and properties of untreated particles with those of particles stored for 3 weeks at +60°C, a treatment which resulted in a loss of HBsAg antigenicity (toward RF-1 mAb) and immunogenicity (in mice). While untreated particles imaged by electron microscopy and atomic force microscopy appeared as isolated nanoparticles of ∼ 20nm, heated particles appeared as long chains of particle aggregates with a partial loss of their protein protrusions. Moreover, infrared spectroscopy and circular dichroism revealed that the secondary structure of the S proteins was significantly affected, with a loss of 10% of their α-helix content. Steady-state and time-resolved fluorescence data further revealed strong modifications of the most emitting Trp residues at the particle surface, confirming significant changes in the conformation of the S proteins. Moreover, modifications in the organization of both the lipid core and lipid membrane surface of the heated particles were evidenced by environment-sensitive 3-hydroxyflavone probes. Taken together, our data evidenced a clear relationship between the bona fide S protein structure and lipid organization notably at the particle surface and the particle immunogenicity.

The structure of HBsAg particles is not modified upon their adsorption on aluminium hydroxide gel.

Current Hepatitis B vaccines are based on recombinant Hepatitis B surface antigen (HBsAg) virus-like particles adsorbed on aluminium (Al) gel. These particles exhibit a lipoprotein-like structure with about 70 protein S molecules in association with various types of lipids. To determine whether the adsorption on Al gel affects HBsAg structure, we investigated the effect of adsorption and mild desorption processes on the protein and lipid parts of the particles, using various techniques. Electron microscopy showed that the size and morphology of native and desorbed HBsAg particles were comparable. Moreover, infrared and Raman spectroscopy revealed that the secondary structure of the S proteins was not affected by the adsorption/desorption process. Affinity measurements with Surface Plasmon Resonance showed no difference between native and desorbed HBsAg for HBsAg-specific RF-1 monoclonal antibody. Steady-state and time-resolved fluorescence data of the intrinsic fluorescence of the S proteins further indicated that the adsorption/desorption of HBsAg particles on Al gel did not modify the environment of the most emitting Trp residues, confirming that the conformation of the S proteins remains intact. Moreover, using environment-sensitive 3-hydroxyflavone probes, no significant changes of the lipid core and lipid membrane surface of the HBsAg particles were observed during the adsorption/desorption process. Finally, the ratio between lipids and proteins in the particles was found to be similar before and after the adsorption/desorption process. Taken together, our data show that adsorption on Al gel does not affect the structure of the HBsAg particles.

Characterization of the mechanisms of HIV-1 Vpr(52-96) internalization in cells.

Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52-96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52-96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52-96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-ß-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52-96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.

Characterization of the lipid and protein organization in HBsAg viral particles by steady-state and time-resolved fluorescence spectroscopy.

Hepatitis B surface antigen (HBsAg) particles, produced in the yeast Hansenula polymorpha, are 20 nm particles, composed of S surface viral proteins and host-derived lipids. Since the detailed structure of these particles is still missing, we further characterized them by fluorescence techniques. Fluorescence correlation spectroscopy indicated that the particles are mainly monomeric, with about 70 S proteins per particle. The S proteins were characterized through the intrinsic fluorescence of their thirteen Trp residues. Fluorescence quenching and time-resolved fluorescence experiments suggest the presence of both low emissive embedded Trp residues and more emissive Trp residues at the surface of the HBsAg particles. The low emission of the embedded Trp residues is consistent with their close proximity in alpha-helices. Furthermore, S proteins exhibit restricted movement, as expected from their tight association with lipids. The lipid organization of the particles was studied using viscosity-sensitive DPH-based probes and environment sensitive 3-hydroxyflavone probes, and compared to lipid vesicles and low density lipoproteins (LDLs), taken as models. Like LDLs, the HBsAg particles were found to be composed of an ordered rigid lipid interface, probably organized as a phospholipid monolayer, and a more hydrophobic and fluid inner core, likely composed of triglycerides and free fatty acids. However, the lipid core of HBsAg particles was substantially more polar than the LDL one, probably due to its larger content in proteins and its lower content in sterols. Based on our data, we propose a structural model for HBsAg particles where the S proteins deeply penetrate into the lipid core.