INTRODUCTIONAequorin is a small protein produced by the genus Aequorea that was widely used in th... more INTRODUCTIONAequorin is a small protein produced by the genus Aequorea that was widely used in the 1960s and 1970s as a probe to measure Ca(2+) in living cells. The invention of the carboxylate Ca(2+) indicators, which are much simpler to load into intact living cells and to calibrate and image at the single-cell level, has led most groups to abandon aequorin. Yet, this latter Ca(2+) indicator still offers some advantages over the fluorescent probes. In particular, the use of molecular biological techniques for expressing recombinant aequorin in mammalian cells, thus eliminating the need for microinjection, has opened new possibilities for this probe. Among the new uses of aequorin, one of the most interesting is the potential for targeting it specifically to different cellular locations, thus opening the possibility of monitoring selectively the dynamics of [Ca(2+)] with unprecedented spatial resolution. This article briefly discusses the problems concerned with targeting aequorin to different locations, the advantages and disadvantages offered by the steep dependence of luminescence on [Ca(2+)], and the instruments needed to obtain reliable measurements.
ThemechanismofcGMPproductioninolfactorysensoryneurons(OSNs)ispoorlyunderstood,althoughthismesseng... more ThemechanismofcGMPproductioninolfactorysensoryneurons(OSNs)ispoorlyunderstood,althoughthismessengertakespartinseveralkey processes such as adaptation, neuronal development, and long-term cellular responses to odorant stimulation. Many aspects of the regulation of cGMP in OSNs are still unknown or highly controversial, such as its subcellular heterogeneity, mechanism of coupling to odorant receptors and downstream targets. Here, we have investigated the dynamics and the intracellular distribution of cGMP in living rat OSNs in culture transfected with a genetically encoded sensor for cGMP. We demonstrate that OSNs treated with pharmacological stimuli able to activate membrane or soluble guanylyl cyclase (sGC) presented an increase in cGMP in the entire neuron, from cilia-dendrite to the axon terminus-growth cone. Upon odorant stimulation, a rise in cGMP was again found in the entire neuron, including the axon terminus, where it is locally synthesized. The odorant-dependent rise in cGMP is due to sGC activation by nitric oxide (NO) and requires an increase of cAMP. The link between cAMP and NO synthase appears to be the rise in cytosolic Ca 2ϩ concentration elicited by either plasma membrane Ca 2ϩ channel activation or Ca 2ϩ mobilization from stores via the guanine nucleotide exchange factor Epac. Finally, we show that a cGMP rise can elicit both in vitro and in vivo the phosphorylation of nuclear CREB, suggesting that this signaling pathway may be relevant for both local events (pathfinding, neurotransmitter release) and more distal processes involving gene expression regulation.
Over recent decades, diverse intracellular organelles have been recognized as key determinants of... more Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca 2⍣ signaling in eukaryotes. In yeast however, information on intra-organellar Ca 2⍣ concentrations is scarce, despite the demonstrated importance of Ca 2⍣ signals for this microorganism. Here, we directly monitored free Ca 2⍣ in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca 2⍣-sensitive photoprotein aequorin. Ca 2⍣ uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steadystate, the free Ca 2⍣ concentration in the ER lumen was limited to~10 μM, and ER Ca 2⍣ sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca 2⍣ pumps. In pmr1 null mutants, free Ca 2⍣ in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca 2⍣ uptake activity in yeast.
Proceedings of the National Academy of Sciences, 1984
The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intrace... more The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximat...
PML/RARa is the abnormal protein product of the Acute Promyelocytic Leukemia-speci®c 15;17 transl... more PML/RARa is the abnormal protein product of the Acute Promyelocytic Leukemia-speci®c 15;17 translocation. Both the PML and RARa components are required for the PML/RARa biological activities, namely its capacity to block dierentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within speci®c nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARa protein contains the RING ®nger, two newly de®ned cystein/histidine-rich motifs called Bboxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring+B1-B2 and coiled-coil regions are both indispensable and sucient to target PML to the NBs; (ii) individual deletions of the various PML domains have no eect on its growth suppressor activity; (iii) the Ring+B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sucient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring+B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the eects of PML/RARa on survival derive from the activation of the incorporated PML sequence.
We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal ant... more We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected. In contrast, surface modulation of CD2 molecules did not prevent the [Ca2+]i increase induced by anti-TCR mAb. Similarly, InsP3 increase was strongly reduced only after modulation of CD3-TCR complex (but not of CD2 molecules). Therefore, it appears that surface modulation of CD3-TCR complex causes T cell refractoriness by inhibiting the very early metabolic events that follow receptor-ligand interactions.
Two routes to neurotoxicity? Tetanus and botulinum toxins cause death by preventing small synapti... more Two routes to neurotoxicity? Tetanus and botulinum toxins cause death by preventing small synaptic vesicle release. Recent results suggest mechanisms for these toxins and may provide new insights into the cell biology of synapses.
Proceedings of the National Academy of Sciences, 2018
Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleio... more Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleiotropy of the cAMP cascade. This characteristic of the pathway is currently attributed to the ability of phosphodiesterases or adenylate cyclases to create subcellular sites (microdomains) where the concentration of cAMP is distinct from that of the surrounding areas. The role of phosphatases in this process has not been tested. Here we show that limited access of phosphatases to the PKA targets present at the outer mitochondrial membrane generates distinct microdomains of PKA phosphorylated proteins despite there being no differences in the local cAMP levels. These results describe an alternative mechanism capable of generating functional cAMP/PKA-dependent microdomains and may be extrapolated to the compartmentalization of other kinase-dependent events.
Contact sites are discrete areas of organelle proximity that coordinate essential physiological p... more Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER-mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER-mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses ...
Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the... more Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential a...
Cellular signaling networks are composed of multiple pathways, often interconnected, that form co... more Cellular signaling networks are composed of multiple pathways, often interconnected, that form complex networks with great potential for cross-talk. Signal decoding depends on the nature of the message as well as its amplitude, temporal pattern, and spatial distribution. In addition, the existence of membrane-bound organelles, which are both targets and generators of messages, add further complexity to the system. The availability of sensors that can localize to specific compartments in live cells and monitor their targets with high spatial and temporal resolution is thus crucial for a better understanding of cell pathophysiology. For this reason, over the last four decades, a variety of strategies have been developed, not only to generate novel and more sensitive probes for ions, metabolites, and enzymatic activity, but also to selectively deliver these sensors to specific intracellular compartments. In this review, we summarize the principles that have been used to target organic ...
Calcium ion (Ca 2+) is a ubiquitous intracellular messenger and changes in its concentration impa... more Calcium ion (Ca 2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca 2+ store and the free Ca 2+ concentration ([Ca 2+ ]) within its lumen ([Ca 2+ ] ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca 2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca 2+ probes are plagued by different drawbacks, such as a double dissociation constant (K d) for Ca 2+ , low dynamic range, and an affinity for the cation that is too high for the levels of [Ca 2+ ] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca 2+ affinity and dynamic range for monitoring [Ca 2+ ] variations within the ER. As an example, resting [Ca 2+ ] ER have been evaluated in a known paradigm of altered ER Ca 2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca 2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca 2+ content in cells expressing mutated PS2, compared to controls.
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2016
The distribution and function of sympathetic innervation in skeletal muscle have largely remained... more The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.
Ca(2+) homeostasis in peroxisomes has been an unsolved problem for many years. Recently novel pro... more Ca(2+) homeostasis in peroxisomes has been an unsolved problem for many years. Recently novel probes to monitor Ca(2+) levels in the lumen of peroxisomes in living cells of both animal and plant cells have been developed. Here we discuss the contrasting results obtained in mammalian cells with chemiluminecsent (aequorin) and fluorescent (cameleon) probes targeted to peroxisomes. We briefly discuss the different characteristics of these probes and the possible pitfalls of the two approaches. We conclude that the contrasting results obtained with the two probes may reflect a heterogeneity among peroxisomes in mammalian cells. We also discuss the results obtained in plant peroxisomes. In particular we demonstrate that Ca(2+) increases in the cytoplasm are mirrored by similar rises of Ca(2+) concentration the lumen of peroxisomes. The increases in peroxisome Ca(2+) level results in the activation of a catalase isoform, CAT3. Other functional roles of peroxisomal Ca(2+) changes in plant ...
INTRODUCTIONAequorin is a small protein produced by the genus Aequorea that was widely used in th... more INTRODUCTIONAequorin is a small protein produced by the genus Aequorea that was widely used in the 1960s and 1970s as a probe to measure Ca(2+) in living cells. The invention of the carboxylate Ca(2+) indicators, which are much simpler to load into intact living cells and to calibrate and image at the single-cell level, has led most groups to abandon aequorin. Yet, this latter Ca(2+) indicator still offers some advantages over the fluorescent probes. In particular, the use of molecular biological techniques for expressing recombinant aequorin in mammalian cells, thus eliminating the need for microinjection, has opened new possibilities for this probe. Among the new uses of aequorin, one of the most interesting is the potential for targeting it specifically to different cellular locations, thus opening the possibility of monitoring selectively the dynamics of [Ca(2+)] with unprecedented spatial resolution. This article briefly discusses the problems concerned with targeting aequorin to different locations, the advantages and disadvantages offered by the steep dependence of luminescence on [Ca(2+)], and the instruments needed to obtain reliable measurements.
ThemechanismofcGMPproductioninolfactorysensoryneurons(OSNs)ispoorlyunderstood,althoughthismesseng... more ThemechanismofcGMPproductioninolfactorysensoryneurons(OSNs)ispoorlyunderstood,althoughthismessengertakespartinseveralkey processes such as adaptation, neuronal development, and long-term cellular responses to odorant stimulation. Many aspects of the regulation of cGMP in OSNs are still unknown or highly controversial, such as its subcellular heterogeneity, mechanism of coupling to odorant receptors and downstream targets. Here, we have investigated the dynamics and the intracellular distribution of cGMP in living rat OSNs in culture transfected with a genetically encoded sensor for cGMP. We demonstrate that OSNs treated with pharmacological stimuli able to activate membrane or soluble guanylyl cyclase (sGC) presented an increase in cGMP in the entire neuron, from cilia-dendrite to the axon terminus-growth cone. Upon odorant stimulation, a rise in cGMP was again found in the entire neuron, including the axon terminus, where it is locally synthesized. The odorant-dependent rise in cGMP is due to sGC activation by nitric oxide (NO) and requires an increase of cAMP. The link between cAMP and NO synthase appears to be the rise in cytosolic Ca 2ϩ concentration elicited by either plasma membrane Ca 2ϩ channel activation or Ca 2ϩ mobilization from stores via the guanine nucleotide exchange factor Epac. Finally, we show that a cGMP rise can elicit both in vitro and in vivo the phosphorylation of nuclear CREB, suggesting that this signaling pathway may be relevant for both local events (pathfinding, neurotransmitter release) and more distal processes involving gene expression regulation.
Over recent decades, diverse intracellular organelles have been recognized as key determinants of... more Over recent decades, diverse intracellular organelles have been recognized as key determinants of Ca 2⍣ signaling in eukaryotes. In yeast however, information on intra-organellar Ca 2⍣ concentrations is scarce, despite the demonstrated importance of Ca 2⍣ signals for this microorganism. Here, we directly monitored free Ca 2⍣ in the lumen of the endoplasmic reticulum (ER) of yeast cells, using a specifically targeted version of the Ca 2⍣-sensitive photoprotein aequorin. Ca 2⍣ uptake into the yeast ER displayed characteristics distinctly different from the mammalian ER. At steadystate, the free Ca 2⍣ concentration in the ER lumen was limited to~10 μM, and ER Ca 2⍣ sequestration was insensitive to thapsigargin, an inhibitor specific for mammalian ER Ca 2⍣ pumps. In pmr1 null mutants, free Ca 2⍣ in the ER was reduced by 50%. Our findings identify the secretory pathway pump Pmr1, predominantly localized in the Golgi, as a major component of ER Ca 2⍣ uptake activity in yeast.
Proceedings of the National Academy of Sciences, 1984
The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intrace... more The cytosolic concentration of free Ca2+ in bovine neutrophils was monitored by using the intracellular Ca2+ indicator quin2, 2-[[2-bis(acetylamino)-5-methylphenoxy]methyl-6-methoxy-8- bis(acetylamino)]quinoline. Neutrophils at rest have a cytosolic Ca2+ concentration of 85 +/- 5 nM, which in 2-4 min increases to 300-400 nM upon interaction with the complement fragment C5a in a concentration range of 35 pM to 1.2 microM. In the same concentration range, C5a also sequentially activates neutrophil directional migration (ED50 less than 0.5 nM), O-2 production (ED50 = 9 nM), and secretion of the contents of specific granules (ED50 = 39 nM). The selective Ca2+ ionophore ionomycin also increases cytosolic Ca2+ concentration above 1 microM under conditions where it stimulates neutrophil functions. Conversely, phorbol 12-myristate 13-acetate markedly activates secretion and O-2 production without modifying the average cytosolic Ca2+ concentration. In the presence of EGTA (Ca2+out approximat...
PML/RARa is the abnormal protein product of the Acute Promyelocytic Leukemia-speci®c 15;17 transl... more PML/RARa is the abnormal protein product of the Acute Promyelocytic Leukemia-speci®c 15;17 translocation. Both the PML and RARa components are required for the PML/RARa biological activities, namely its capacity to block dierentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within speci®c nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARa protein contains the RING ®nger, two newly de®ned cystein/histidine-rich motifs called Bboxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring+B1-B2 and coiled-coil regions are both indispensable and sucient to target PML to the NBs; (ii) individual deletions of the various PML domains have no eect on its growth suppressor activity; (iii) the Ring+B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sucient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring+B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the eects of PML/RARa on survival derive from the activation of the incorporated PML sequence.
We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal ant... more We investigated the mechanism involved in T cell unresponsiveness that follows the monoclonal antibody-induced surface modulation of the CD3-TCR complex. We determined whether modulation of CD3-TCR affected the early metabolic steps such as [Ca2+]i rise and InsP3 formation. A strong inhibition of the increase on [Ca2+]i mediated by either anti-TCR or anti-CD2 mAbs was detected. In contrast, surface modulation of CD2 molecules did not prevent the [Ca2+]i increase induced by anti-TCR mAb. Similarly, InsP3 increase was strongly reduced only after modulation of CD3-TCR complex (but not of CD2 molecules). Therefore, it appears that surface modulation of CD3-TCR complex causes T cell refractoriness by inhibiting the very early metabolic events that follow receptor-ligand interactions.
Two routes to neurotoxicity? Tetanus and botulinum toxins cause death by preventing small synapti... more Two routes to neurotoxicity? Tetanus and botulinum toxins cause death by preventing small synaptic vesicle release. Recent results suggest mechanisms for these toxins and may provide new insights into the cell biology of synapses.
Proceedings of the National Academy of Sciences, 2018
Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleio... more Significance The selective phosphorylation of spatially distinct PKA targets is key for the pleiotropy of the cAMP cascade. This characteristic of the pathway is currently attributed to the ability of phosphodiesterases or adenylate cyclases to create subcellular sites (microdomains) where the concentration of cAMP is distinct from that of the surrounding areas. The role of phosphatases in this process has not been tested. Here we show that limited access of phosphatases to the PKA targets present at the outer mitochondrial membrane generates distinct microdomains of PKA phosphorylated proteins despite there being no differences in the local cAMP levels. These results describe an alternative mechanism capable of generating functional cAMP/PKA-dependent microdomains and may be extrapolated to the compartmentalization of other kinase-dependent events.
Contact sites are discrete areas of organelle proximity that coordinate essential physiological p... more Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER-mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER-mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses ...
Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the... more Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential a...
Cellular signaling networks are composed of multiple pathways, often interconnected, that form co... more Cellular signaling networks are composed of multiple pathways, often interconnected, that form complex networks with great potential for cross-talk. Signal decoding depends on the nature of the message as well as its amplitude, temporal pattern, and spatial distribution. In addition, the existence of membrane-bound organelles, which are both targets and generators of messages, add further complexity to the system. The availability of sensors that can localize to specific compartments in live cells and monitor their targets with high spatial and temporal resolution is thus crucial for a better understanding of cell pathophysiology. For this reason, over the last four decades, a variety of strategies have been developed, not only to generate novel and more sensitive probes for ions, metabolites, and enzymatic activity, but also to selectively deliver these sensors to specific intracellular compartments. In this review, we summarize the principles that have been used to target organic ...
Calcium ion (Ca 2+) is a ubiquitous intracellular messenger and changes in its concentration impa... more Calcium ion (Ca 2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca 2+ store and the free Ca 2+ concentration ([Ca 2+ ]) within its lumen ([Ca 2+ ] ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca 2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca 2+ probes are plagued by different drawbacks, such as a double dissociation constant (K d) for Ca 2+ , low dynamic range, and an affinity for the cation that is too high for the levels of [Ca 2+ ] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca 2+ affinity and dynamic range for monitoring [Ca 2+ ] variations within the ER. As an example, resting [Ca 2+ ] ER have been evaluated in a known paradigm of altered ER Ca 2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer's Disease-linked protein Presenilin 2 (PS2). The lower Ca 2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca 2+ content in cells expressing mutated PS2, compared to controls.
Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2016
The distribution and function of sympathetic innervation in skeletal muscle have largely remained... more The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic β2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.
Ca(2+) homeostasis in peroxisomes has been an unsolved problem for many years. Recently novel pro... more Ca(2+) homeostasis in peroxisomes has been an unsolved problem for many years. Recently novel probes to monitor Ca(2+) levels in the lumen of peroxisomes in living cells of both animal and plant cells have been developed. Here we discuss the contrasting results obtained in mammalian cells with chemiluminecsent (aequorin) and fluorescent (cameleon) probes targeted to peroxisomes. We briefly discuss the different characteristics of these probes and the possible pitfalls of the two approaches. We conclude that the contrasting results obtained with the two probes may reflect a heterogeneity among peroxisomes in mammalian cells. We also discuss the results obtained in plant peroxisomes. In particular we demonstrate that Ca(2+) increases in the cytoplasm are mirrored by similar rises of Ca(2+) concentration the lumen of peroxisomes. The increases in peroxisome Ca(2+) level results in the activation of a catalase isoform, CAT3. Other functional roles of peroxisomal Ca(2+) changes in plant ...
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