4TM-TRPM8 channels are new gatekeepers of the ER-mitochondria Ca2+ transfer.

Calcium (Ca2+) release from the endoplasmic reticulum plays an important role in many cell-fate defining cellular processes. Traditionally, this Ca2+ release was associated with the ER Ca2+ release channels, inositol 1,4,5­triphosphate receptor (IP3R) and ryanodine receptor (RyR). Lately, however, other calcium conductances have been found to be intracellularly localized and to participate in cell fate regulation. Nonetheless, molecular identity and functional properties of the ER Ca2+ release mechanisms associated with multiple diseases, e.g. prostate cancer, remain unknown. Here we identify a new family of transient receptor potential melastatine 8 (TRPM8) channel isoforms as functional ER Ca2+ release channels expressed in mitochondria-associated ER membranes (MAMs). These TRPM8 isoforms exhibit an unconventional structure with 4 transmembrane domains (TMs) instead of 6 TMs characteristic of the TRP channel archetype. We show that these 4TM-TRPM8 isoforms form functional channels in the ER and participate in regulation of the steady-state Ca2+ concentration ([Ca2+]) in mitochondria and the ER. Thus, our study identifies 4TM-TRPM8 isoforms as ER Ca2+ release mechanism distinct from classical Ca2+ release channels.

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.

FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin.

Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of EFRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.

Impairment of GABAB receptor dimer by endogenous 14-3-3ζ in chronic pain conditions.

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein-coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de-dimerization in pathological conditions. We show here that 14-3-3ζ, a GABAB1-binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14-3-3ζ is overexpressed and weakens GABAB inhibition. Using anti-14-3-3ζ siRNA or competing peptides disrupts 14-3-3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti-nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.

Label-free microscopy and stress responses reveal the functional organization of Pseudodiaptomus marinus copepod myofibrils.

Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4 °C or salinity jumps from 30 to 15 psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0 psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18 °C/30 psu to 4° C/0 psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation-contraction coupling and adequate power supply in copepods muscles.

PRC1 components exhibit different binding kinetics in Polycomb bodies.

BACKGROUND INFORMATION: Polycomb group (PcG) proteins keep the memory of cell identity by maintaining the repression of numerous target genes. They accumulate into nuclear foci called Polycomb bodies, which function in Drosophila cells as silencing compartments where PcG target genes convene. PcG proteins also exert their activities elsewhere in the nucleoplasm. In mammalian cells, the dynamic organisation and function of Polycomb bodies remain to be determined. RESULTS: Fluorescently tagged PcG proteins CBXs and their partners BMI1 and RING1 form foci of different sizes and intensities in human U2OS cells. Fluorescence recovery after photobleaching (FRAP) analysis reveals that PcG dynamics outside foci is governed by diffusion as complexes and transient binding. In sharp contrast, recovery curves inside foci are substantially slower and exhibit large variability. The slow binding component amplitudes correlate with the intensities and sizes of these foci, suggesting that foci contained varying numbers of binding sites. CBX4-green fluorescent protein (GFP) foci were more stable than CBX8-GFP foci; yet the presence of CBX4 or CBX8-GFP in the same focus had a minor impact on BMI1 and RING1 recovery kinetics. CONCLUSION: We propose that FRAP recovery curves provide information about PcG binding to their target genes outside foci and about PcG spreading onto chromatin inside foci.

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.

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.

NS2 protein of hepatitis C virus interacts with structural and non-structural proteins towards virus assembly.

Growing experimental evidence indicates that, in addition to the physical virion components, the non-structural proteins of hepatitis C virus (HCV) are intimately involved in orchestrating morphogenesis. Since it is dispensable for HCV RNA replication, the non-structural viral protein NS2 is suggested to play a central role in HCV particle assembly. However, despite genetic evidences, we have almost no understanding about NS2 protein-protein interactions and their role in the production of infectious particles. Here, we used co-immunoprecipitation and/or fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy analyses to study the interactions between NS2 and the viroporin p7 and the HCV glycoprotein E2. In addition, we used alanine scanning insertion mutagenesis as well as other mutations in the context of an infectious virus to investigate the functional role of NS2 in HCV assembly. Finally, the subcellular localization of NS2 and several mutants was analyzed by confocal microscopy. Our data demonstrate molecular interactions between NS2 and p7 and E2. Furthermore, we show that, in the context of an infectious virus, NS2 accumulates over time in endoplasmic reticulum-derived dotted structures and colocalizes with both the envelope glycoproteins and components of the replication complex in close proximity to the HCV core protein and lipid droplets, a location that has been shown to be essential for virus assembly. We show that NS2 transmembrane region is crucial for both E2 interaction and subcellular localization. Moreover, specific mutations in core, envelope proteins, p7 and NS5A reported to abolish viral assembly changed the subcellular localization of NS2 protein. Together, these observations indicate that NS2 protein attracts the envelope proteins at the assembly site and it crosstalks with non-structural proteins for virus assembly.

Combined SPT and FCS methods reveal a mechanism of RNAP II oversampling in cell nuclei.

Gene expression orchestration is a key question in fundamental and applied research. Different models for transcription regulation were proposed, yet the dynamic regulation of RNA polymerase II (RNAP II) activity remains a matter of debate. To improve our knowledge of this topic, we investigated RNAP II motility in eukaryotic cells by combining single particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) techniques, to take advantage of their different sensitivities in order to analyze together slow and fast molecular movements. Thanks to calibrated samples, we developed a benchmark for quantitative analysis of molecular dynamics, to eliminate the main potential instrumental biases. We applied this workflow to study the diffusion of RPB1, the catalytic subunit of RNAP II. By a cross-analysis of FCS and SPT, we could highlight different RPB1 motility states and identifyed a stationary state, a slow diffusion state, and two different modes of subdiffusion. Interestingly, our analysis also unveiled the oversampling by RPB1 of nuclear subdomains. Based on these data, we propose a novel model of spatio-temporal transcription regulation. Altogether, our results highlight the importance of combining microscopy approaches at different time scales to get a full insight into the real complexity of molecular kinetics in cells.

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm.

Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress. To minimize exposure time, we have developed an original approach that statistically inflates the number of collected photons. Our approach, called Adaptive Monte Carlo Data Inflation (AMDI), combines the well-known bootstrap technique with an adaptive Parzen kernel. We here demonstrate using both Monte Carlo simulations and live cells that our robust method accurately estimate fluorescence lifetimes with exposure time reduced up to 50 times for monoexponential decays (corresponding to a minimum of 20 photons/pixel), and 10 times for biexponential decays (corresponding to a minimum of 5,000 photons/pixel), compared to standard fitting method. Thanks to AMDI, in Förster resonance energy transfer experiments, it is possible to estimate all fitting parameters accurately without constraining any parameters. By reducing the commonly used spatial binning factor, our technique also improves the spatial resolution of FLIM images.

Simple phasor-based deep neural network for fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to probe the molecular environment of fluorophores. The analysis of FLIM images is usually performed with time consuming fitting methods. For accelerating this analysis, sophisticated deep learning architectures based on convolutional neural networks have been developed for restrained lifetime ranges but they require long training time. In this work, we present a simple neural network formed only with fully connected layers able to analyze fluorescence lifetime images. It is based on the reduction of high dimensional fluorescence intensity temporal decays into four parameters which are the phasor coordinates, the mean and amplitude-weighted lifetimes. This network called Phasor-Net has been applied for a time domain FLIM system excited with an 80 MHz laser repetition frequency, with negligible jitter and afterpulsing. Due to the restricted time interval of 12.5 ns, the training range of the lifetimes was limited between 0.2 and 3.0 ns; and the total photon number was lower than 106, as encountered in live cell imaging. From simulated biexponential decays, we demonstrate that Phasor-Net is more precise and less biased than standard fitting methods. We demonstrate also that this simple architecture gives almost comparable performance than those obtained from more sophisticated networks but with a faster training process (15 min instead of 30 min). We finally apply successfully our method to determine biexponential decays parameters for FLIM experiments in living cells expressing EGFP linked to mCherry and fused to a plasma membrane protein.

Characteristic ERK1/2 signaling dynamics distinguishes necroptosis from apoptosis.

ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Cell adhesion properties on chemically micropatterned boron-doped diamond surfaces.

The adhesion properties of living cells were investigated on a range of chemically modified boron-doped diamond (BDD) surfaces. We studied the influence of oxidized, H-, amine- (NH(2)-), methyl- (CH(3)-), trifluoromethyl- (CF(3)-) and vinyl- (CH(2)═CH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells behavior. Cell-surface interactions were analyzed by fluorescence microscopy in terms of cell attachment, spreading and proliferation. U2OS cells poorly adhered on hydrophobic surfaces and their growth was blocked. In contrast, L929 cells were mainly influenced by the presence of perfluoroalkyl chains in regard to their morphology. The results were subsequently applied to selectively micropattern U2OS cells on dual hydrophobic/hydrophilic surfaces prepared by a UV/ozone lithographic approach. U2OS cells colonized preferentially hydrophilic (oxide-terminated) motifs, forming confluent arrays with distinguishable edges separating the alkyl regions.

Control of cell death/survival balance by the MET dependence receptor.

Control of cell death/survival balance is an important feature to maintain tissue homeostasis. Dependence receptors are able to induce either survival or cell death in presence or absence of their ligand, respectively. However, their precise mechanism of action and their physiological importance are still elusive for most of them including the MET receptor. We evidence that pro-apoptotic fragment generated by caspase cleavage of MET localizes to the mitochondria-associated membrane region. This fragment triggers a calcium transfer from endoplasmic reticulum to mitochondria, which is instrumental for the apoptotic action of the receptor. Knock-in mice bearing a mutation of MET caspase cleavage site highlighted that p40MET production is important for FAS-driven hepatocyte apoptosis, and demonstrate that MET acts as a dependence receptor in vivo. Our data shed light on new signaling mechanisms for dependence receptors' control of cell survival/death balance, which may offer new clues for the pathophysiology of epithelial structures.

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours.

BACKGROUND: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks. RESULTS: To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin. CONCLUSION: FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.

Functional cooperation between Stat-1 and ets-1 to optimize icam-1 gene transcription.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the immune system, enabling the interactions between effector cells and target cells. It is also known to be involved in tumor growth and metastasis. Its expression is transcriptionally regulated by several proinflammatory cytokines including IFN-gamma, which induces ICAM-1 transcription via the JAK-STAT signaling pathway in a Stat1-dependent fashion. The ICAM-1 promoter contains several cis-active regulatory elements including 2 Ets binding sites (EBSs) located at positions -158 and -138 relatively to the AUG, which were previously shown to play a role in the constitutive activity of the ICAM-1 promoter. In the present study, we have determined whether the EBSs are also involved in the regulation of ICAM-1 gene transcription by pro-inflammatory cytokines. Transient transfection assays were performed with reporter genes containing ICAM-1 promoter constructions cloned upstream from the firefly luciferase gene. Site-specific mutations of the EBS diminished the promoter activity stimulated by IFN-gamma, although the IFN-gamma responsive element (pIgammaRE), which binds Stat1, was intact. Stimulation of the transcriptional activity following IFN-gamma treatment was significantly reduced when both EBSs were inactivated. Co-immunoprecipitation experiments provided evidence of a physical interaction involving Ets1 and Stat1. In COS-1 and HEK 293 cells cotransfected with CFP-Stat1 and YFP-Ets fusion protein, fluorescence resonance energy transfer experiments confirmed the close proximity of these 2 proteins in living cells following treatment with IFN-gamma.

Apolipoprotein A-V modulates insulin secretion in pancreatic beta-cells through its interaction with midkine.

Apolipoprotein A-V is an important determinant of plasma triglyceride level in both humans and mice. This study showed the physiological impact of apoA-V on insulin secretion in rat pancreatic beta-cells (INS-1 cells). In order to precise the mechanism of action, binding experiments coupled to mass spectrometry were performed to identify a potential membrane receptor. Results showed an interaction between apoA-V and midkine protein. Confocal microscopy confirmed the plasma membrane co-localisation of this two-proteins after the treatment of INS-1 cells with the apo-AV recombinant protein and indicated that the cell surface midkine could be involved in apoA-V endocytosis, since these two proteins were co-translocated at the plasma membrane or in the cytosol compartment. This co-localisation is correlated with an increase in insulin secretion in a dose dependant manner during short incubation period. Reduction of midkine expression by small interfering RNA duplexes revealed a decrease in the ability of these transfected cells to secrete insulin in presence of apoA-V. Competition experiments for the apoA-V-midkine binding at the cell surface using antibody directed against midkine is able to influence INS-1 cell function as insulin secretion. Our results showed apoA-V ability to enhance insulin secretion in beta-cells and provide evidence of an internalization pathway involving the midkine as partner.

Three-dimensional polar representation for multispectral fluorescence lifetime imaging microscopy.

Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent.