Search

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. From ultrastructural analysis, it is known that presynaptic boutons along single axons differ in the number of vesicles docked at the active zone. It is not clear whether the probability of these vesicles to get released (pves) is homogenous or also varies between individual boutons. Here, we optically measure evoked transmitter release at individual Schaffer collateral synapses at different calcium concentrations, using the genetically encoded glutamate sensor iGluSnFR. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. We find that Schaffer collateral boutons typically release single vesicles under low pves conditions and switch to multivesicular release in high calcium saline. The potency of individual boutons is highly correlated with their vesicular release probability while the number of releasable vesicles affects synaptic output only under high pves conditions.

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that interaction of both Ca2+/CaM-N53I and Ca2+/CaM-A102V with the RyR2 binding domain is decreased. Ca2+/CaM–RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild type (WT). We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events; CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ (also known as CAMK2D) phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate the development of strategies for future therapies.

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca

Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca2+) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca2+ signals, by flash photolysis of the caged Ca2+ chelator diazo‐2‐acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca2+ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca2+ signal as a functionally essential input into mitosis.—Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome‐localized calcium signal is essential for mammalian cell mitosis.

Information processing in the brain is controlled by quantal release of neurotransmitters, a tightly regulated process. Even in a single axon, presynaptic boutons differ in the number of docked vesicles, but it is not known if the vesicular release probability (pves) is homogenous or variable between individual boutons. We optically measured evoked transmitter release at individual Schaffer collateral synapses using the genetically encoded glutamate sensor iGluSnFR, localizing the fusion site on the bouton with high spatiotemporal precision. Fitting a binomial model to measured response amplitude distributions allowed us to extract the quantal parameters N, pves, and q. Schaffer collateral boutons typically released only a single vesicle under low pves conditions and switched to multivesicular release in high calcium saline. We found that pves was highly variable between individual boutons and had a dominant impact on presynaptic output.

Protein-based fluorescent glutamate sensors have the potential for real-time monitoring of synaptic and cellular glutamate concentration changes, however even the fastest currently available sensors’ response times of 2-3 ms are too slow for accurate reporting of the post-synaptic AMPA receptor function in physiological conditions. We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding: affinity variants of iGluSnFR termed iGluh, iGlumand iGlulcovering a range ofKd−s (5.8 μM, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP with aKdof 9.7 μM are presented. The fluorescence response kinetics of all the probes are consistent with two-step mechanisms involving ligand binding and rate limiting isomerisation, however the contribution in each step to the total fluorescence enhancement and kinetic paths to the final state are diverse. In contrast to the previously characterised ultrafast indicators iGluuand iGluf, for which fluorescence enhancement occurred only in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with a significant fraction of the fluorescence increase evoked by glutamate binding which, in the case of iGluhand Fl-GluBP, occurs with a diffusion limited rate constant. The above genetically encoded and chemically labelled fluorescent glutamate sensor variants demonstrate how single amino acid changes around the binding site introduce structural heterogeneity affecting the kinetic mechanism of interactions with glutamate. Through their broad affinity range and mechanistic variety, the probes contribute to a novel toolkit for monitoring processes of glutamate neurotransmission and cellular homeostasis.STATEMENT OF SIGNIFICANCEGlutamate is a major excitatory neurotransmitter, important in synaptic plasticity e.g. memory formation. Although predicted to clear rapidly from the synaptic cleft following presynaptic release, optical monitoring of glutamate neurotransmission has only become possible with the advent of fluorescent, protein-based indicators. Understanding their biophysical properties is important for quantification of the observed processes. Here we report the biophysical characterisation of a number of glutamate indicator variants based on the bacterial periplasmic glutamate/aspartate binding protein, revealing the subtle differences in their kinetic pathway caused by structural alteration of the glutamate binding protein by point mutations. Diffusion limited glutamate binding indicated by a novel chemically labelled probe hints at the mechanism that underlies the rapid response of the AMPA receptor.

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses during development, homeostasis, and disease. However, non-invasive investigation of cellular mechanosensing in complex and intact live tissue remains challenging. Here, we developed GenEPi, a genetically-encoded fluorescent intensiometric reporter for mechanical stimuli based on Piezo1, an essential mechanosensitive ion channel found in vertebrates. We show that GenEPi has high specificity and spatiotemporal resolution for Piezo1-dependent mechanical stimuli, exemplified by resolving repetitive mechanical stimuli of spontaneously contracting cardiomyocytes within microtissues, in a non-invasive manner.

Calcium signaling in neurons as in other cell types can lead to varied changes in cellular function. Neuronal Ca(2+) signaling processes have also become adapted to modulate the function of specific pathways over a wide variety of time domains and these can have effects on, for example, axon outgrowth, neuronal survival, and changes in synaptic strength. Ca(2+) also plays a key role in synapses as the trigger for fast neurotransmitter release. Given its physiological importance, abnormalities in neuronal Ca(2+) signaling potentially underlie many different neurological and neurodegenerative diseases. The mechanisms by which changes in intracellular Ca(2+) concentration in neurons can bring about diverse responses is underpinned by the roles of ubiquitous or specialized neuronal Ca(2+) sensors. It has been established that synaptotagmins have key functions in neurotransmitter release, and, in addition to calmodulin, other families of EF-hand-containing neuronal Ca(2+) sensors, including the neuronal calcium sensor (NCS) and the calcium-binding protein (CaBP) families, play important physiological roles in neuronal Ca(2+) signaling. It has become increasingly apparent that these various Ca(2+) sensors may also be crucial for aspects of neuronal dysfunction and disease either indirectly or directly as a direct consequence of genetic variation or mutations. An understanding of the molecular basis for the regulation of the targets of the Ca(2+) sensors and the physiological roles of each protein in identified neurons may contribute to future approaches to the development of treatments for a variety of human neuronal disorders.

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGlu(u). We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGlu(u) single-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGlu(u) amplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, ∼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGlu(u). Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention. SIGNIFICANCE STATEMENT Alterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, the sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor extracellular glutamate concentration ([Glu]) at individual corticostriatal synapses, we can now quantify the time constant of perisynaptic [Glu] decay (as an indicator of uptake) and the maximal [Glu] elevation next to the active zone (as an indicator of Glu release). The results provide a positive answer to the hitherto unresolved question of whether neurodegeneration (e.g., Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

Changes in the balance between glutamate (Glu) release and uptake may stimulate synaptic reorganization and even synapse loss. In the case of neurodegeneration, a mismatch between astroglial Glu uptake and presynaptic Glu release could be detected if both parameters were assessed independently and at a single-synapse level. This has now become possible due to a new imaging assay with the genetically encoded ultrafast Glu sensor iGluu. We report findings from individual corticostriatal synapses in acute slices prepared from mice of either sex that were >1 year of age. Contrasting patterns of short-term plasticity and a size criterion identified two classes of terminals, presumably corresponding to the previously defined IT (intratelencephalic) and PT (pyramidal tract) synapses. The latter exhibited a higher degree of frequency potentiation/residual Glu accumulation and were selected for our first iGluusingle-synapse study in Q175 mice, a model of Huntington's disease (HD). In HD mice, the decay time constant of the perisynaptic Glu concentration (TauD), as an indicator of uptake, and the peak iGluuamplitude, as an indicator of release, were prolonged and reduced, respectively. Treatment of WT preparations with the astrocytic Glu uptake blocker TFB-TBOA (100 nm) mimicked the TauD changes in homozygotes. Considering the largest TauD values encountered in WT, ∼40% of PT synapses tested in Q175 heterozygotes can be classified as dysfunctional. Moreover, HD but not WT synapses exhibited a positive correlation between TauD and the peak amplitude of iGluu. Finally, EAAT2 (excitatory amino acid transport protein 2) immunoreactivity was reduced next to corticostriatal terminals. Thus, astrocytic Glu transport remains a promising target for therapeutic intervention.SIGNIFICANCE STATEMENTAlterations in astrocytic Glu uptake can play a role in synaptic plasticity and neurodegeneration. Until now, the sensitivity of synaptic responses to pharmacological transport block and the resulting activation of NMDA receptors were regarded as reliable evidence for a mismatch between synaptic uptake and release. But the latter parameters are interdependent. Using a new genetically encoded sensor to monitor extracellular glutamate concentration ([Glu]) at individual corticostriatal synapses, we can now quantify the time constant of perisynaptic [Glu] decay (as an indicator of uptake) and the maximal [Glu] elevation next to the active zone (as an indicator of Glu release). The results provide a positive answer to the hitherto unresolved question of whether neurodegeneration (e.g., Huntington's disease) associates with a glutamate uptake deficit at tripartite excitatory synapses.

To generate a complex multicellular organism like a human requires enormous expansion in cell numbers and this is achieved predominantly through mitosis. Defects in mitosis can lead to premature ageing and cancer so understanding how it is regulated has important implications for human disease. Early data from plant and invertebrate model systems indicated that calcium (Ca2+) could influence mitosis. Here we explore this key question in the cell biology of mammalian cells by targeting high affinity genetically encoded Ca2+sensors to mitosis specific subcellular locations. We reveal a prolonged yet spatially restricted Ca2+signal at the centrosomes of mitotic cells using an actin-targeted Ca2+sensor. Local depletion of Ca2+at centrosomes using flash-photolysis of the caged Ca2+chelator diazo-2 arrests mitosis and we provide evidence that this signal emanates from the endoplasmic reticulum. In summary, we characterize a centrosomal Ca2+signal as a functionally essential input into mitosis. This extends our understanding of the complex regulatory network controlling cell division and pinpoints Ca2+as an important controller of this fundamental process.

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. Here we show by biochemical and imaging approaches that lysosomes undergo exocytosis at telophase and that this requires the activity of phosphatidylinositol 4-kinase-IIIβ. Inhibition of lysosome exocytosis resulted in mitotic failure in a significant proportion of cells suggesting that this facet of lysosome physiology is essential and represents a new regulatory mechanism in mitosis.

Glutamatergic synapses display a rich repertoire of plasticity mechanisms on many different time scales, involving dynamic changes in the efficacy of transmitter release as well as changes in the number and function of postsynaptic glutamate receptors. The genetically encoded glutamate sensor iGluSnFR enables visualization of glutamate release from presynaptic terminals at frequencies up to ∼10 Hz. However, to resolve glutamate dynamics during high frequency bursts, faster indicators are required. Here we report the development of fast (iGluf) and ultrafast (iGluu) variants with comparable brightness, but increased Kd for glutamate (137 μM and 600 μM, respectively). Compared to iGluSnFR, iGluu has a 6-fold faster dissociation rate in vitro and 5-fold faster kinetics in synapses. Fitting a three-state model to kinetic data, we identify the large conformational change after glutamate binding as the rate-limiting step. In rat hippocampal slice culture stimulated at 100 Hz, we find that iGluu is sufficiently fast to resolve individual glutamate release events, revealing that glutamate is rapidly cleared from the synaptic cleft. Depression of iGluu responses during 100 Hz trains correlates with depression of postsynaptic EPSPs, indicating that depression during high frequency stimulation is purely presynaptic in origin. At individual boutons, the recovery from depression could be predicted from the amount of glutamate released on the second pulse (paired pulse facilitation/depression), demonstrating differential frequency-dependent filtering of spike trains at Schaffer collateral boutons.Significance StatementExcitatory synapses convert presynaptic action potentials into chemical signals that are sensed by postsynaptic glutamate receptors. To eavesdrop on synaptic transmission, genetically encoded fluorescent sensors for glutamate have been developed. However, even the best available sensors lag behind the very fast glutamate dynamics in the synaptic cleft. Here we report the development of an ultrafast genetically encoded glutamate sensor, iGluu, which allowed us to image glutamate clearance and synaptic depression during 100 Hz spike trains. We found that only boutons showing paired-pulse facilitation were able to rapidly recover from depression. Thus, presynaptic boutons act as frequency-specific filters to transmit select features of the spike train to specific postsynaptic cells.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f

Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm2) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R–BADAN (Kd 0.86±0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R–BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung.

The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including β-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of penicillin-binding protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.

Faithful reporting of temporal patterns of intracellular Ca(2+) dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca(2+) signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca(2+) affinities and accelerated kinetics by weakening the Ca(2+)-calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3fast is 40-fold faster than GCaMP3 with Ca(2+) decay and rise times, t1/2, of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3fast revealed discreet transients with significantly faster Ca(2+) dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca(2+) at 940 nm, GCaMP3fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca(2+) kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probes.

In intracellular calcium signaling studies genetically encoded probes offer greater intracellular domain specificity than traditional fluorescent signaling molecules, with the ability to be targeted to specific organelles and micro-domains. To this end we evaluated the ability of seven novel mutagenic derivatives of GCaMP3 calcium probe (EF-3, EF-4, EF-34, RS-1, RS-1 EF-3, RS-1 EF-4, and RS-1 EF-34) to rapidly detect intracellular calcium compared to the chemical calcium probe Fura-2 AM. GCaMP3 derived probes were transiently transfected into HEK293T cells which were subsequently incubated in 1 µM Fura-2 AM for 20 minutes prior to experimentation. Fura-2 and GCaMP3-derived probe fluorescence was measured at 380 nm and 488 nm respectively as HEK293T cells were rapidly exposed to 100 µM ATP for 5 s. While mutated probes did not exhibit a greater change in fluorescence than GCaMP3 precursor, mutants EF-3 and EF-4 fluorescence changes were 128.4 and 58.2 % ΔF/Fo respectively, significantly greater than Fura-2 response in the same cells. Additionally, many probes demonstrated faster rise and decay kinetics, with rise time and decay time values up to 150 % faster than Fura-2. Our results suggest that mutations in GCaMP3 can produce genetically encoded probes capable of measuring calcium with equal or larger fluorescence responses as traditional chemical dyes. Additionally, several probes such as GCaMP3 EF-4 demonstrate not only increased fluorescence but dramatically improved kinetic responses - facilitating increasingly sensitive measurements of intracellular calcium signaling.

Green Fluorescent-Calmodulin Proteins (GCaMPs) have been the reporters of choice for visualizing neuronal network activity in vivo. With the GCaMP6 generation sufficient brightness was achieved for single action potential (AP) detection1. However, the Ca2+-response kinetics of GCaMP6 probes remained limiting to separating individual APs. The aim was to accelerate the Ca2+ response kinetics of GCaMP6 probes by point mutations in the Ca2+.CaM.RS20 complex2. A series of probes retaining high fluorescence dynamic ranges was generated with in vitro dissociation constants (Kd) for Ca2+ in the μM range (0.1-3.3 μM) and Hill coefficients of 1.7 to 4.2. Two highlighted probes termed GCaMP6fast and GCaMP6bright had in vitro half times (t1/2) at 37°C for Ca2+ rise of 1.3 ms and 3.3 ± 0.2 ms and decay t1/2-s of 2.8 ± 0.1 and 3.5 ± 0.1 ms, respectively, compared to the 10 ± 1 ms rise and 63 ± 6 ms decay times for GCaMP6f. Fluorescence changes on Ca2+ association were highly cooperative and characterized by a rate limiting conformational change. In vivo Ca2+ responses associated with AP firing patterns were tested in cultured hippocampal slices by two-photon imaging at 28°C. The kinetic performance of GCaMP6fast and GCaMP6bright was compared with that of GCaMP6f. Ca2+ decay kinetics were determined by monitoring fluorescence changes evoked by 5 APs fired at 100 Hz. t1/2 values for GCaMP6fast and GCaMP6bright were 58 ms and 126 ms, respectively. With an up to 7-fold faster in vivo decay kinetics than GCaMP6f, GCaMP6fast and GCaMP6bright are promising tools for monitoring brain activity.1Podor et al., Neurophotonics, Epub 2015 Apr 30.2Helassa et al., Scientific Reports, accepted.This work was funded by Wellcome Trust (094385/Z/10/Z to KT), BBSRC (BB/M02556X/1 to KT), CIHR (MOP-123514 to AF) and NSERC (RGPIN-170421 to AF).

The pneumococcus is an important Gram-positive pathogen, which shows increasing resistance to antibiotics, including β-lactams that target peptidoglycan assembly. Understanding cell-wall synthesis, at the molecular and cellular level, is essential for the prospect of combating drug resistance. As a first step towards reconstituting pneumococcal cell-wall assembly in vitro, we present the characterization of the glycosyltransferase activity of penicillin-binding protein (PBP)2a from Streptococcus pneumoniae. Recombinant full-length membrane-anchored PBP2a was purified by ion-exchange chromatography. The glycosyltransferase activity of this enzyme was found to differ from that of a truncated periplasmic form. The full-length protein with its cytoplasmic and transmembrane segment synthesizes longer glycan chains than the shorter form. The transpeptidase active site was functional, as shown by its reactivity towards bocillin and the catalysis of the hydrolysis of a thiol-ester substrate analogue. However, PBP2a did not cross-link the peptide stems of glycan chains in vitro. The absence of transpeptidase activity indicates that an essential component is missing from the in vitro system.

International audience; The adsorption of the insecticidal Cry1Aa protein from Bacillus thuringiensis (Br-toxin) on a model clay surface was studied to understand the structural changes of the protein induced by the clay surface. We studied the adsorption of the monomeric and soluble oligomeric forms of the Cry1Aa toxin as a function of pH and ionic strength conditions on montmorillonite, which is an electronegative phyllosilicate. Cry1Aa secondary structure was determined from the amide I' FTIR absorption profiles. Accessibility to the solvent was determined by NH/ND exchange to characterize conformational flexibility of the different states of the Cry1Aa protein. The size distribution of Cry1Aa solutions was obtained by dynamic light scattering (DLS). From combined DLS and FTIR measurements, we conclude that montmorillonite traps the Cry1Aa toxin in its monomeric state, preventing the oligomerization of the protein. The oligomeric forms were adsorbed onto the clay without significant structural changes.

International audience; We studied the kinetics of adsorption of alexa-labeled Bt toxin CrylAa, in monomer and oligomer states, on muscovite mica, acid-treated hydrophilic glass, and hydrophobized glass, in the configuration of laminar flow of solution in a slit. Normal confocal fluorescence through the liquid volume allows the visualization of the concentration in solution over the time of adsorption, in addition to the signal due to the adsorbed molecules at the interface. The solution signal is used as calibration for estimation of interfacial concentration. We found low adsorption of the monomer compared to oligomers on the three types of surface. The kinetic adsorption barrier for oligomers increases in the order hydrophobized glass, muscovite mica, acid-treated hydrophilic glass. This suggests enhanced immobilization in soil if toxin is under oligomer state.

Genetically encoded calmodulin-based calcium probes (GCaMPs) have become the reporters of choice for visualising the calcium flux associated with action potentials in vivo. A major limitation of currently available GCaMPs is the slow kinetics of fluorescence changes induced by calcium association and dissociation. We have addressed this issue by generating a series of mutants of GCaMP3 in the calcium binding sites of calmodulin alone1 and in combination with mutations in the RS20 target peptide sequence2 with the view of lowering the affinity for calcium and accelerating the calcium response kinetics. The calcium association kinetics for the resulting GCaMP3 EF-hand and peptide mutants were highly cooperative and characterized by a rate limiting conformational change. Fluorescence changes on calcium association were up to 7-fold faster compared to GCaMP3. Calcium dissociation rates were up to 60-fold faster than GCaMP3 and 25-fold faster than the newly developed GCaMP6 fast (GCaMP6f). Dissociation constants (Kd) for calcium were in the μM range with Hill coefficients from 2 to 5. Two-photon cross-sections of mutants were comparable to GCaMP3. Fluorescence responses of mutated GCaMP3s to calcium transients in endothelial cells were similar to those seen with small molecule indicators. The principles employed proved to accelerate the calcium kinetics of GCaMP3 and can be applied to the new generations of GCaMPs to generate low affinity probes.1Jama A et al. JBC, 2011, 286:12308-12316.2Torok K and Trentham DR. Biochemistry, 1994, 33:12807-12820.This work is funded by the Wellcome Trust grant number 094385/Z/10/Z to KT.

The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.