Tracing endogenous proteins in living cells through electrotransfer of mRNA encoding chromobodies.

Chromobodies made of nanobodies fused to fluorescent proteins are powerful tools for targeting and tracing intracellular proteins in living cells. Typically, this is achieved by transfecting plasmids encoding the chromobodies. However, an excess of unbound chromobody relative to the endogenous antigen can result in high background fluorescence in live cell imaging. Here, we overcome this problem by using mRNA encoding chromobodies. Our approach allows one to precisely control the amount of chromobody expressed inside the cell by adjusting the amount of transfected mRNA. To challenge our method, we evaluate three chromobodies targeting intracellular proteins of different abundance and cellular localization, namely lamin A/C, Dnmt1 and actin. We demonstrate that the expression of chromobodies in living cells by transfection of tuned amounts of the corresponding mRNAs allows the accurate tracking of their cellular targets by time-lapse fluorescence microscopy.

Quantitative live imaging reveals a direct interaction between CD44v6 and MET in membrane domains upon activation with both MET ligands, HGF and internalin B.

Deregulation of the receptor tyrosine kinase MET/hepatocyte growth factor (HGF) pathway results in several pathological processes involved in tumor progression and metastasis. In a different context, MET can serve as an entry point for the bacterium Listeria monocytogenes, when activated by the internalin B (InlB) protein during infection of non-phagocytic cells. We have previously demonstrated that MET requires CD44v6 for its ligand-induced activation. However, the stoichiometry and the steps required for the formation of this complex, are still unknown. In this work, we studied the dynamics of the ligand-induced interaction of CD44v6 with MET at the plasma membrane. Using Förster resonance energy transfer-based fluorescence lifetime imaging microscopy in T-47D cells, we evidenced a direct interaction between MET and CD44v6 promoted by HGF and InlB in live cells. In the absence of MET, fluorescence correlation spectroscopy experiments further showed the dimerization of CD44v6 and the increase of its diffusion induced by HGF and InlB. In the presence of MET, stimulation of the cells by HGF or InlB significantly decreased the diffusion of CD44v6, in line with the formation of a ternary complex of MET with CD44v6 and HGF/InlB. Finally, similarly to HGF/InlB, disruption of liquid-ordered domains (Lo) by methyl-ß-cyclodextrin increased CD44v6 mobility suggesting that these factors induce the exit of CD44v6 from the Lo domains. Our data led us to propose a model for MET activation, where CD44v6 dimerizes and diffuses rapidly out of Lo domains to form an oligomeric MET/ligand/CD44v6 complex that is instrumental for MET activation.

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains.

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton.

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end-directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. In vivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of newborn neurons independently of its motility on microtubules. We show that Kif21b directly binds and regulates the actin cytoskeleton both in vitro and in vivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

Assembly dynamics and structure of an aegerolysin, ostreolysin A6.

Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

V-ATPase modulates exocytosis in neuroendocrine cells through the activation of the ARNO-Arf6-PLD pathway and the synthesis of phosphatidic acid.

Although there is mounting evidence indicating that lipids serve crucial functions in cells and are implicated in a growing number of human diseases, their precise roles remain largely unknown. This is particularly true in the case of neurosecretion, where fusion with the plasma membrane of specific membrane organelles is essential. Yet, little attention has been given to the role of lipids. Recent groundbreaking research has emphasized the critical role of lipid localization at exocytotic sites and validated the essentiality of fusogenic lipids, such as phospholipase D (PLD)-generated phosphatidic acid (PA), during membrane fusion. Nevertheless, the regulatory mechanisms synchronizing the synthesis of these key lipids and neurosecretion remain poorly understood. The vacuolar ATPase (V-ATPase) has been involved both in vesicle neurotransmitter loading and in vesicle fusion. Thus, it represents an ideal candidate to regulate the fusogenic status of secretory vesicles according to their replenishment state. Indeed, the cytosolic V1 and vesicular membrane-associated V0 subdomains of V-ATPase were shown to dissociate during the stimulation of neurosecretory cells. This allows the subunits of the vesicular V0 to interact with different proteins of the secretory machinery. Here, we show that V0a1 interacts with the Arf nucleotide-binding site opener (ARNO) and promotes the activation of the Arf6 GTPase during the exocytosis in neuroendocrine cells. When the interaction between V0a1 and ARNO was disrupted, it resulted in the inhibition of PLD activation, synthesis of phosphatidic acid during exocytosis, and changes in the timing of fusion events. These findings indicate that the separation of V1 from V0 could function as a signal to initiate the ARNO-Arf6-PLD1 pathway and facilitate the production of phosphatidic acid, which is essential for effective exocytosis in neuroendocrine cells.

Thienoguanosine, a unique non-perturbing reporter for investigating rotational dynamics of DNA duplexes and their complexes with proteins.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine (thG), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of thG-labelled dsDNA of different lengths. thG behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe thG-labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, thG was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, thG thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes.

Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop.

Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44fl/fl;VillinCreERT2 mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration.

Kinetics of protein-assisted nucleic acid interconversion monitored by transient time resolved fluorescence in microfluidic droplets.

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.

Intermolecular dark resonance energy transfer (DRET): upgrading fluorogenic DNA sensing.

The sensitivity of FRET-based sensing is usually limited by the spectral overlaps of the FRET donor and acceptor, which generate a poor signal-to-noise ratio. To overcome this limitation, a quenched donor presenting a large Stokes shift can be combined with a bright acceptor to perform Dark Resonance Energy Transfer (DRET). The consequent fluorogenic response from the acceptor considerably improves the signal-to-noise ratio. To date, DRET has mainly relied on a donor that is covalently bound to the acceptor. In this context, our aim was to develop the first intermolecular DRET pair for specific sensing of nucleic acid sequences. To this end, we designed DFK, a push-pull probe based on a fluorenyl π-platform that is strongly quenched in water. DFK was incorporated into a series of oligonucleotides and used as a DRET donor with Cy5-labeled complementary sequences. In line with our expectations, excitation of the dark donor in the double-labeled duplex switched on the far-red Cy5 emission and remained free of cross-excitation. The DRET mechanism was supported by time-resolved fluorescence measurements. This concept was then applied with binary probes, which confirmed the distance dependence of DRET as well as its potency in detecting sequences of interest with low background noise.

The use of pore-forming toxins to image lipids and lipid domains.

Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.

What Makes Thienoguanosine an Outstanding Fluorescent DNA Probe?

Thienoguanosine (thG) is an isomorphic guanosine (G) surrogate that almost perfectly mimics G in nucleic acids. To exploit its full potential and lay the foundation for future applications, 20 DNA duplexes, where the bases facing and neighboring thG were systematically varied, were thoroughly studied using fluorescence spectroscopy, molecular dynamics simulations, and mixed quantum mechanical/molecular mechanics calculations, yielding a comprehensive understanding of its photophysics in DNA. In matched duplexes, thG's hypochromism was larger for flanking G/C residues but its fluorescence quantum yield (QY) and lifetime values were almost independent of the flanking bases. This was attributed to high duplex stability, which maintains a steady orientation and distance between nucleobases, so that a similar charge transfer (CT) mechanism governs the photophysics of thG independently of its flanking nucleobases. thG can therefore replace any G residue in matched duplexes, while always maintaining similar photophysical features. In contrast, the local destabilization induced by a mismatch or an abasic site restores a strong dependence of thG's QY and lifetime values on its environmental context, depending on the CT route efficiency and solvent exposure of thG. Due to this exquisite sensitivity, thG appears ideal for monitoring local structural changes and single nucleotide polymorphism. Moreover, thG's dominant fluorescence lifetime in DNA is unusually long (9-29 ns), facilitating its selective measurement in complex media using a lifetime-based or a time-gated detection scheme. Taken together, our data highlight thG as an outstanding emissive substitute for G with good QY, long fluorescence lifetimes, and exquisite sensitivity to local structural changes.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Excited-State Dynamics of Thienoguanosine, an Isomorphic Highly Fluorescent Analogue of Guanosine.

Thienoguanosine (th G) is an isomorphic analogue of guanosine with promising potentialities as fluorescent DNA label. As a free probe in protic solvents, th G exists in two tautomeric forms, identified as the H1, being the only one observed in nonprotic solvents, and H3 keto-amino tautomers. We herein investigate the photophysics of th G in solvents of different polarity, from water to dioxane, by combining time-resolved fluorescence with PCM/TD-DFT and CASSCF calculations. Fluorescence lifetimes of 14.5-20.5 and 7-13 ns were observed for the H1 and H3 tautomers, respectively, in the tested solvents. In methanol and ethanol, an additional fluorescent decay lifetime (≈3 ns) at the blue emission side (λ≈430 nm) as well as a 0.5 ns component with negative amplitude at the red edge of the spectrum, typical of an excited-state reaction, were observed. Our computational analysis explains the solvent effects observed on the tautomeric equilibrium. The main radiative and nonradiative deactivation routes have been mapped by PCM/TD-DFT calculations in solution and CASSCF in the gas phase. The most easily accessible conical intersection, involving an out-of plane motion of the sulfur atom in the five-membered ring of th G, is separated by a sizeable energy barrier (≥0.4 eV) from the minimum of the spectroscopic state, which explains the large experimental fluorescence quantum yield.

GUV-AP: multifunctional FIJI-based tool for quantitative image analysis of Giant Unilamellar Vesicles.

MOTIVATION: Giant Unilamellar Vesicles (GUVs) are widely used synthetic membrane systems that mimic native membranes and cellular processes. Various fluorescence imaging techniques can be employed for their characterization. In order to guarantee a fast and unbiased analysis of imaging data, the development of automated recognition and processing steps is required. RESULTS: We developed a fast and versatile Fiji-based macro for the analysis of digital microscopy images of GUVs. This macro was designed to investigate membrane dye incorporation and protein binding to membranes. Moreover, we propose a fluorescence intensity-based method to quantitatively assess protein binding. AVAILABILITY AND IMPLEMENTATION: The ImageJ distribution package FIJI is freely available online: https://imagej.net/Fiji. The macro file GUV-AP.ijm is available at https://github.com/AG-Roemer/GUV-AP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Probing Polarity and Heterogeneity of Lipid Droplets in Live Cells Using a Push-Pull Fluorophore.

Lipid droplets (LDs) are organelles composed of a lipid core surrounded by a phospholipid monolayer. Lately, LDs have attracted considerable attention due to recent studies demonstrating their role in a variety of physiological processes as well as diseases. Herein we synthesized a push-pull molecule named DAF (Dimethyl Aniline Furaldehyde) that possesses a strong positive solvatochromism in emission of 119 nm from toluene to methanol. Its impressive fluorogenic properties from water to oil (2000-fold) as well as its high quantum yields (up to 0.97) led us to investigate its ability to sense the distribution of polarity in live cells by fluorescence ratiometric imaging. When added to live cells and excited at 405 nm, DAF immediately and brightly stain lipid droplets using a blue channel (410-500 nm) and cytoplasm in a red channel (500-600 nm). DAF also proved to be compatible with fixation thus allowing 3D imaging of LDs in their cytoplasm environment. Taking advantage of DAF emission in two distinct channels, ratiometric imaging was successfully performed and led to the polarity mapping of the cell unraveling some heterogeneity in polarity within LDs of the same cell.