Sumoylation regulates FMRP-mediated dendritic spine elimination and maturation.

Fragile X syndrome (FXS) is the most frequent inherited cause of intellectual disability and the best-studied monogenic cause of autism. FXS results from the functional absence of the fragile X mental retardation protein (FMRP) leading to abnormal pruning and consequently to synaptic communication defects. Here we show that FMRP is a substrate of the small ubiquitin-like modifier (SUMO) pathway in the brain and identify its active SUMO sites. We unravel the functional consequences of FMRP sumoylation in neurons by combining molecular replacement strategy, biochemical reconstitution assays with advanced live-cell imaging. We first demonstrate that FMRP sumoylation is promoted by activation of metabotropic glutamate receptors. We then show that this increase in sumoylation controls the homomerization of FMRP within dendritic mRNA granules which, in turn, regulates spine elimination and maturation. Altogether, our findings reveal the sumoylation of FMRP as a critical activity-dependent regulatory mechanism of FMRP-mediated neuronal function.

The energy disruptor metformin targets mitochondrial integrity via modification of calcium flux in cancer cells.

Mitochondrial integrity is critical for the regulation of cellular energy and apoptosis. Metformin is an energy disruptor targeting complex I of the respiratory chain. We demonstrate that metformin induces endoplasmic reticulum (ER) stress, calcium release from the ER and subsequent uptake of calcium into the mitochondria, thus leading to mitochondrial swelling. Metformin triggers the disorganization of the cristae and inner mitochondrial membrane in several cancer cells and tumors. Mechanistically, these alterations were found to be due to calcium entry into the mitochondria, because the swelling induced by metformin was reversed by the inhibition of mitochondrial calcium uniporter (MCU). We also demonstrated that metformin inhibits the opening of mPTP and induces mitochondrial biogenesis. Altogether, the inhibition of mPTP and the increase in mitochondrial biogenesis may account for the poor pro-apoptotic effect of metformin in cancer cells.

Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy.

Here we demonstrate that in a niche-like coculture system, cells from both primary and cultured acute myeloid leukemia (AML) sources take up functional mitochondria from murine or human bone marrow stromal cells. Using different molecular and imaging approaches, we show that AML cells can increase their mitochondrial mass up to 14%. After coculture, recipient AML cells showed a 1.5-fold increase in mitochondrial adenosine triphosphate production and were less prone to mitochondrial depolarization after chemotherapy, displaying a higher survival. This unidirectional transfer enhanced by some chemotherapeutic agents required cell-cell contacts and proceeded through an endocytic pathway. Transfer was greater in AML blasts compared with normal cord blood CD34(+) cells. Finally, we demonstrate that mitochondrial transfer was observed in vivo in an NSG immunodeficient mouse xenograft model and also occurred in human leukemia initiating cells and progenitors. As mitochondrial transfer provides a clear survival advantage following chemotherapy and a higher leukemic long-term culture initiating cell potential, targeting mitochondrial transfer could represent a future therapeutic target for AML treatment.

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.

Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging.

Living cells are chemical mixtures of exceptional interest and significance, whose investigation requires the development of powerful analytical tools fulfilling the demanding constraints resulting from their singular features. In particular, multiplexed observation of a large number of molecular targets with high spatiotemporal resolution appears highly desirable. One attractive road to address this analytical challenge relies on engaging the targets in reactions and exploiting the rich kinetic signature of the resulting reactive module, which originates from its topology and its rate constants. This review explores the various facets of this promising strategy. We first emphasize the singularity of the content of a living cell as a chemical mixture and suggest that its multiplexed observation is significant and timely. Then, we show that exploiting the kinetics of analytical processes is relevant to selectively detect a given analyte: upon perturbing the system, the kinetic window associated to response read-out has to be matched with that of the targeted reactive module. Eventually, we introduce the state-of-the-art of cell imaging exploiting protocols based on reaction kinetics and draw some promising perspectives.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature.

Single-particle tracking methods for the study of membrane receptors dynamics.

Single-particle tracking (SPT) applications have been growing rapidly in the field of cell biology, and in particular in neurobiology, as a means of unravelling the involvement of diffusion dynamics of neurotransmitter receptors and other synaptic proteins in the regulation of neuronal activity. Suitable probes and technological improvements make SPT more accessible than it used to be and open up broad applications in cellular biology. In this technical highlight, we give an overview of the experimental approach in SPT. The concepts and results in neurobiology have already been the object of detailed reviews. Here, we focus on a qualitative description of the implementation of SPT, from molecule labelling to acquisition, data treatment and analysis of protein diffusion properties. Constraints, limitations and future developments are discussed.

Gephyrin oligomerization controls GlyR mobility and synaptic clustering.

High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N- and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N- and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time single-particle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

Revealing signaling in single cells by single- and two-photon fluorescence lifetime imaging microscopy.

Lipids are actively involved in many cellular processes. Their roles pivot toward determining membrane structure, compartment targeting, and membrane fusion but also regulation of cell signaling via their interactions with proteins and the production of second messengers. As they play a key role in cell signaling, the study of protein-protein interaction and protein conformation change in relationship with their interaction with lipids is of major importance. Until recently, the ability to detect in situ and in real time the dynamics of various biological events and signals without perturbing the cellular environment has been a real challenge. However, the emergence of fluorescence imaging of cells and tissues has allowed the dynamic aspects of the cell to be investigated in a more physiological context than the disassembled model systems employed in traditional biochemical analysis. This chapter highlights some of the many biological applications and uses of frequency- and time-domain fluorescence lifetime imaging microscopy (FLIM) applied to the detection of Förster resonance energy transfer (FRET). The first part describes a FRET system, the second part discusses its study by FLIM, and the third part describes the application of these methods to a panel of biological questions such as (1) spatio-temporal interaction of protein kinase B (PKB) with 3-phosphoinositide dependent protein kinase-1 (PDK1), (2) PKB conformation change, (3) dynamics of PKB activation, (4) interaction of phosphatidylinositol transfer protein (PITP) and phospholipase D (PLD) with lipids.

Intramolecular and intermolecular interactions of protein kinase B define its activation in vivo.

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide-dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a "PH-in" conformer. This domain-domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a "PH-out" conformer and enabling PDK1 access the activation loop. The active, phosphorylated, "PH-out" conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.

Photophysics of a series of efficient fluorescent pH probes for dual-emission-wavelength measurements in aqueous solutions.

This paper evaluates the 5-aryl-2-pyridyloxazole backbone to engineer donor-acceptor fluorescent pH probes after one- or two-photon absorption. Parent fluorophores, as well as derivatives that can be used to label biomolecules, can be easily obtained in good yields. These molecules exhibit a large one-photon absorption in the near-UV range, and a strong fluorescence emission that covers the whole visible domain. The 5-aryl-2-pyridyloxazole derivatives also possess significant cross sections for two-photon absorption. Upon pyridine protonation, large shifts were observed in the absorption spectra after one- and two-photon excitation, as well as in the emission spectra. This feature was used to measure the pK(a) of the investigated compounds that range between 2 and 8. In most of the investigated derivatives, the pK(a) increased upon light excitation and protonation exchanges took place during the lifetime of the excited state, as shown by phase-modulation fluorometry analysis. Several 5-aryl-2-pyridyloxazole derivatives are suggested as efficient probes to reliably measure the pH of aqueous solutions by means of ratiometric methods that are dependent on fluorescence emission.

Reactant concentrations from fluorescence correlation spectroscopy with tailored fluorescent probes. An example of local calibration-free pH measurement.

The present account is concerned with the measurement of local reactant concentrations by observing specific fluorescent probes in fluorescence correlation spectroscopy (FCS). The Theoretical Analysis section revisits the photophysical, thermodynamic, and kinetic information that is contained in the corresponding FCS correlation curves. In particular, we examine the conditions under which FCS is revealed as a superior tool to measure concentrations of reactive species. Careful molecular engineering of the specific fluorescent probes that simultaneously integrates photophysical, thermodynamic, and kinetic constraints will be required to benefit most from FCS. We illustrate the FCS titration approach with a series of fluorescent probes that we tailored to measure pH at around 4-6 by FCS after two-photon excitation. We show that an optimal design allows one to access pH without any preliminary calibrations such as the determination of the protonation constant or the photophysical properties of the fluorescent probe.

An approach to extract rate constants from reaction--diffusion dynamics in a microchannel.

A theoretical model is proposed to extract rate constants of second-order chemical reactions down to the millisecond time scale from the observation of reaction-diffusion processes in a microchannel. We validate this theoretical approach by examining an appropriate model reaction. The measured rate constant is in excellent agreement with this obtained from nuclear magnetic resonance experiments.

Stochastic resonance to control diffusive motion in chemistry.

This paper reports on a novel procedure to tune the effective diffusion coefficient of a field-sensitive reactant in the presence of a periodic external field. We investigate the motion of two negatively charged azo dyes interacting with alpha-cyclodextrin (alpha-CD) upon action of a periodic square wave electrical field. We show that the dyes exhibit an effective diffusion coefficient D(eff) that depends on the rate constants for dye complexation within alpha-CD, the period and the amplitude of the field. UV-vis absorption, gradient field (1)H NMR, and fluorescence correlation spectroscopy (FCS) after two photon excitation are used to evidence that D(eff) may be increased far beyond its intrinsic value when specific relations interpreted as a stochastic resonance are fulfilled. The present results may find useful applications in chemical kinetics as well as for molecular sorting.

Molecular sorting by stochastic resonance.

To sort a targeted species from a mixture, we introduce a procedure that relies on the enhancement of its effective diffusion coefficient. We use the formation of a host-guest complex between alpha-cyclodextrin and a dye to evidence the dye dispersion when the medium is submitted to an oscillating field. In particular, we demonstrate that the effective diffusion coefficient of the dye may be increased far beyond its intrinsic value by tuning the driving field frequency in the stochastic resonance regime. We use this effect to selectively sort from a mixture a dye that is addressed by its rate constants for association with alpha-cyclodextrin.