The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to dev... more The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.
In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal c... more In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potass...
Ca 2+ entry in neuronal cells is modulated by the activation of numerous G protein coupled recept... more Ca 2+ entry in neuronal cells is modulated by the activation of numerous G protein coupled receptors (GPCR). Much effort has been invested in studying direct G protein inhibition of voltage-dependent Ca V 2 type calcium channels. This inhibition occurs through a series of landmark convergent modifications in channel biophysical properties. An integrated view of the structural organization of the G βγ dimer binding site pocket on the channel is progressively emerging. In this review, it is shown how a variable geometry of the G βγ binding pocket can yield distinct sets of channel inhibition. In addition, specific mechanisms are proposed for the channe l regulation by G proteins which take into account the regulatory input of each G βγ binding element.
Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biolog... more Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically ...
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered... more The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.
T-type calcium channels (the Ca V 3 channel family) are involved in defining the resting membrane... more T-type calcium channels (the Ca V 3 channel family) are involved in defining the resting membrane potential and in neuronal activities such as oscillations and rebound depolarization. Their physiological roles depend upon the channel activation and inactivation kinetics. A fast inactivation that stops the ionic flux of calcium in tens of milliseconds has already been described in both native and heterologously expressed channels. Here, using HEK 293 cells expressing the rat Ca V 3.1 channel and whole-cell voltage clamp, we investigate an additional inactivation process, which can be distinguished from the previously described fast inactivation by its slow time course of recovery from inactivation (τ = 1 s) and by its sensitivity to external calcium. Steady-state slow inactivation is voltage dependent around the resting membrane potential (the potential of half-inactivation (V 0.5) = −70 mV, slope factor = 7.4 mV) and can reduce the calcium current by up to 50%. Near resting potential, the slow inactivation displays a half-time of induction of tens of seconds. The slow inactivation therefore modulates the availability of T-type calcium channels depending upon recent cell history, providing a mechanism to store information in a time scale of seconds.
Contribution of the kinetics of G proteins dissociation to the characteristic modifications of N-... more Contribution of the kinetics of G proteins dissociation to the characteristic modifications of N-type calcium channel activity
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly b... more Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants...
Recently, two members of a new family of Ca 2+ channel α1 subunits, α1G (or Ca v T. 1) and α1H (o... more Recently, two members of a new family of Ca 2+ channel α1 subunits, α1G (or Ca v T. 1) and α1H (or Ca v T. 2), have been cloned and expressed. These α1 subunits generate Ba 2+ currents similar to the T-type Ca 2+ currents present in sensory neurons. Here, we ...
The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to dev... more The great demand for long-wavelength and high signal-to-noise Ca(2+) indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.
In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal c... more In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potass...
Ca 2+ entry in neuronal cells is modulated by the activation of numerous G protein coupled recept... more Ca 2+ entry in neuronal cells is modulated by the activation of numerous G protein coupled receptors (GPCR). Much effort has been invested in studying direct G protein inhibition of voltage-dependent Ca V 2 type calcium channels. This inhibition occurs through a series of landmark convergent modifications in channel biophysical properties. An integrated view of the structural organization of the G βγ dimer binding site pocket on the channel is progressively emerging. In this review, it is shown how a variable geometry of the G βγ binding pocket can yield distinct sets of channel inhibition. In addition, specific mechanisms are proposed for the channe l regulation by G proteins which take into account the regulatory input of each G βγ binding element.
Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biolog... more Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically ...
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered... more The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.
T-type calcium channels (the Ca V 3 channel family) are involved in defining the resting membrane... more T-type calcium channels (the Ca V 3 channel family) are involved in defining the resting membrane potential and in neuronal activities such as oscillations and rebound depolarization. Their physiological roles depend upon the channel activation and inactivation kinetics. A fast inactivation that stops the ionic flux of calcium in tens of milliseconds has already been described in both native and heterologously expressed channels. Here, using HEK 293 cells expressing the rat Ca V 3.1 channel and whole-cell voltage clamp, we investigate an additional inactivation process, which can be distinguished from the previously described fast inactivation by its slow time course of recovery from inactivation (τ = 1 s) and by its sensitivity to external calcium. Steady-state slow inactivation is voltage dependent around the resting membrane potential (the potential of half-inactivation (V 0.5) = −70 mV, slope factor = 7.4 mV) and can reduce the calcium current by up to 50%. Near resting potential, the slow inactivation displays a half-time of induction of tens of seconds. The slow inactivation therefore modulates the availability of T-type calcium channels depending upon recent cell history, providing a mechanism to store information in a time scale of seconds.
Contribution of the kinetics of G proteins dissociation to the characteristic modifications of N-... more Contribution of the kinetics of G proteins dissociation to the characteristic modifications of N-type calcium channel activity
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly b... more Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants...
Recently, two members of a new family of Ca 2+ channel α1 subunits, α1G (or Ca v T. 1) and α1H (o... more Recently, two members of a new family of Ca 2+ channel α1 subunits, α1G (or Ca v T. 1) and α1H (or Ca v T. 2), have been cloned and expressed. These α1 subunits generate Ba 2+ currents similar to the T-type Ca 2+ currents present in sensory neurons. Here, we ...
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Papers by Anne Feltz