Extracellular vesicle-mediated trafficking of molecular cues during human brain development.

Cellular crosstalk is an essential process influenced by numerous factors, including secreted vesicles that transfer nucleic acids, lipids, and proteins between cells. Extracellular vesicles (EVs) have been the center of many studies focusing on neurodegenerative disorders, but whether EVs display cell-type-specific features for cellular crosstalk during neurodevelopment is unknown. Here, using human-induced pluripotent stem cell-derived cerebral organoids, neural progenitors, neurons, and astrocytes, we identify heterogeneity in EV protein content and dynamics in a cell-type-specific and time-dependent manner. Our results support the trafficking of key molecules via EVs in neurodevelopment, such as the transcription factor YAP1, and their localization to differing cell compartments depending on the EV recipient cell type. This study sheds new light on the biology of EVs during human brain development.

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation.

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600-630) and two in the far red channel (633/641-680), supplemented by a single further redshifted fluorochrome (670/701-750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing.

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

HEXIM1 Diffusion in the Nucleus Is Regulated by Its Interactions with Both 7SK and P-TEFb.

How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly α coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.

[P-TEFb and Brd4: actors of the transcription pause release as therapeutical targets]. / P-TEFb et Brd4 - Acteurs de la levée de pause transcriptionnelle, possibles cibles thérapeutiques.

Most cell physiology events are dictated by the integration of perceived signals and the elaboration by cells of adapted answers via the execution of proper transcriptional programs. In order to ensure an optimal control of these answers, many regulation mechanisms have been selected throughout the evolution, thus allowing to fine-tune transcript expression. The transcriptional pause and its release by P-TEFb (Positive Transcription Elongation Factor) have been evidenced two decades ago. Since then, the importance of such mechanisms has been highlighted by the association between alterations of this machinery and the appearance of diseases. P-TEFb and Brd4 have thus recently emerged as potential therapeutical targets for cancers and AIDS notably. In this review, we present a brief case history and an up-to-date synthesis of models for transcriptional pause release. We later discuss on the pathophysiological processes associated with this mechanism and clinical trials targeting Brd4 and P-TEFb.

The extended cytoplasmic tail of the human B4GALNT2 is critical for its Golgi targeting and post-Golgi sorting.

The Sda /Cad antigen reported on glycoconjugates of human tissues has an increasingly recognized wide impact on the physio-pathology of different biological systems. The last step of its biosynthesis relies on the enzymatic activity of the ß1,4-N-acetylgalactosaminyltransferase-II (B4GALNT2), which shows the highest expression level in healthy colon. Previous studies reported the occurrence in human colonic cells of two B4GALNT2 protein isoforms that differ in the length of their cytoplasmic tail, the long isoform showing an extended 66-amino acid tail. We examined here, the subcellular distribution of the two B4GALNT2 protein isoforms in stably transfected colonic LS174T cells and in transiently transfected HeLa cells using fluorescence microscopy. While a similar subcellular distribution at the trans-Golgi cisternae level was observed for the two isoforms, our study pointed to an atypical subcellular localization of the long B4GALNT2 isoform into dynamic vesicles. We demonstrated a critical role of its extended cytoplasmic tail for its Golgi targeting and post-Golgi sorting and highlighted the existence of a newly described post-Golgi sorting signal as well as a previously undescribed fate of a Golgi glycosyltransferase. DATABASE: The proteins ß1,4GalNAcT II, ß1,4-GalT1, FucT I, FucT VI and ST3Gal IV are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4, whereas the corresponding human genes are noted B4GALNT2, B4GALT1, FUT1, FUT6 and ST3GAL4 according to the HUGO nomenclature.

Improved photodynamic effect through encapsulation of two photosensitizers in lipid nanocapsules.

Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non-toxic partners, i.e. a photosensitizer (PS), molecular oxygen (O2) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damage. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs: Protoporphyrin IX, PpIX and Hypericin, Hy) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PS-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and round internal structures. The biodistribution of LNCs was studied upon different routes of injection into Swiss nude mice; based on the obtained data, LNCs were injected intratumorally and used to slow the growth of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of conventional photodynamic therapy as well as to reduce dark toxicity.

Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance.

We have discovered and developed a series of molecules (thiazole benzenesulfonamides). HA15, the lead compound of this series, displayed anti-cancerous activity on all melanoma cells tested, including cells isolated from patients and cells that developed resistance to BRAF inhibitors. Our molecule displayed activity against other liquid and solid tumors. HA15 also exhibited strong efficacy in xenograft mouse models with melanoma cells either sensitive or resistant to BRAF inhibitors. Transcriptomic, proteomic, and biochemical studies identified the chaperone BiP/GRP78/HSPA5 as the specific target of HA15 and demonstrated that the interaction increases ER stress, leading to melanoma cell death by concomitant induction of autophagic and apoptotic mechanisms.

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms.

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.

Implementation of transportation distance for analyzing FLIM and FRET experiments.

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.