The meeting will explore the relationship between form and function in biological macromolecules.... more The meeting will explore the relationship between form and function in biological macromolecules. Sessions on the structural analysis of proteins and the means by which form is attained will lead into sessions on how this form determines function. Talks at the meeting will cover the gamut from studies on single molecules in solution through membrane proteins to macromolecular assemblies examined using a variety of theoretical as well as experimental techniques.
Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone ph... more Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensi...
We describe the development of the photoswitchable agonistLOGO, which activates GIRK channels in ... more We describe the development of the photoswitchable agonistLOGO, which activates GIRK channels in the dark and is rapidly deactivated upon exposure to long wavelength UV irradiation.LOGOcan be used to optically silence action potential firing in dissociated hippocampal neurons and exhibits activityin vivo, controlling the motility of zebrafish larvae in a light-dependent fashion.
Methods in molecular biology (Clifton, N.J.), 2015
Understanding the function of ion channels is a major goal of molecular neurophysiology. While st... more Understanding the function of ion channels is a major goal of molecular neurophysiology. While standard electrophysiological methods are invaluable tools to investigate the gating of ion channels, the structural rearrangements that mediate the way a channel senses physiological signals and opens and closes its gates cannot be measured electrically in a direct way. Here, we describe a method, based on site-specific labeling of a channel of interest with an environmentally sensitive fluorophore, which makes it possible to monitor conformational changes of ion channels in biological membranes in real time.
The properties of LiGluR resemble in several ways those of ChR2, the naturally photosensitive ion... more The properties of LiGluR resemble in several ways those of ChR2, the naturally photosensitive ion channel found in green algae [Boyden et al., 2005]. LiGluR and ChR2 are both cation selective channels that permeate monovalent ions and Ca2+, both need to be introduced by transfection or transgenesis, and both require a chemical photoswitch. In the case of LiGluR this is MAG, which needs to be infused, while in ChR2 this is retinal, which is naturally available in the retina and possibly in other parts of the nervous system. We compared photostimulation of neurons using our engineered LiGluR and the naturally light sensitive ChR2. The two channels, both under CMV promoter, were transfected into neurons cultured from the same animals, and recordings were made on the same day after transfection. We found light-evoked LiGluR currents to be ~5-fold larger than those of ChR2 (Supplemental Fig. 1A, B) and to fire APs with shorter illumination (1- 5 ms) than reported for ChR2 under similar culture conditions (5-15 ms) [Boyden et al., 2005; Li et al., 2005; Zhang et al., 2007]. While ChR2 currents desensitize by ~50%, LiGluR currents remain stable
Genetically encoded fluorescent probes of neural activity represent new promising tools for syste... more Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparative in vivo analysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of the Drosophila larval neuromuscular junction. Robust neural activity did not result in any or noteworthy fluorescence changes when Flash-Pericam, Camgaroo-1, and Camgaroo-2 were expressed. However, calculated on the raw data, fractional fluorescence changes up to 18% were reported by synapto-pHluorin, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, GCaMP1.3, GCaMP1.6, and the troponin C-based calcium sensor TN-L15. The response characteristics of all of these indicators differed considerably from each other, with GCaMP1.6 reporting high rates of neural activity with the largest and fastest fluorescence changes. However, GCaMP1.6 suffered from photobleaching, whereas the fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible, with TN-L15 showing the fastest rise of the signals at lower activity rates. We show for GCaMP1.3 and YC3.3 that an expanded range of neural activity evoked fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR). The expression level of the indicator biased the signal kinetics and SNR, whereas the signal amplitude was independent. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.
In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from ... more In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containing a VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 in the transmembrane VSD to form the ion selectivity filter in the channel's open conformation.
At the Drosophila larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-... more At the Drosophila larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created SynapCam, a version of the Ca 2+ reporter Cameleon. SynapCam localizes to the postsynapse and selectively reveals Ca 2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca 2+ signals were uniform within a given connection (i.e., bouton and postsynapse pair), but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging revealed a matching axonal gradient, with higher Ca 2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient.
Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2009
Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light... more Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparat...
Canadian Journal of Physiology and Pharmacology, 1988
In C1-spinal, pentobarbital-anaesthetized or anemically decerebrated cats, the preganglionic inpu... more In C1-spinal, pentobarbital-anaesthetized or anemically decerebrated cats, the preganglionic input to the acutely decentralized right stellate ganglion was stimulated with 10- to 30-s trains at 20–40 Hz. Electrical stimulation consistently produced an increase in heart rate in the presence of blocking doses of hexamethonium and atropine or after depletion of acetylcholine from the preganglionic axons by prolonged low frequency stimulation in the presence of hemicholinium. The increase in heart rate had a delayed slow onset, lasted several minutes, and was abolished by propranolol or by section of the inferior cardiac nerve. The magnitude and duration of the heart rate increase were related to intensity, frequency, and duration of preganglionic stimulation. The response to stimulation of a given white ramus was progressively attenuated, and eventually irreversibly lost, during prolonged continuous stimulation of that ramus, while the response to stimulation of a different unstimulate...
Accumulation of amyloid-b peptides (Ab), the proteolytic products of the amyloid precursor protei... more Accumulation of amyloid-b peptides (Ab), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Ab40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Ab40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/G i/oprotein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Ab40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.
Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/E... more Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by themec-4(d)mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on theXenopusoocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 ac...
Anaerobic archaea M. acetivorans, a versatile saltwater adapted methanogen, is capable of convert... more Anaerobic archaea M. acetivorans, a versatile saltwater adapted methanogen, is capable of converting seven different organic molecules into methane. Utilization of these substrates involves three main reaction pathways that are differentially regulated by the organism depending on growth conditions. We have developed a kinetic model for methanogenesis using RNA-seq data, single molecule enumeration of protein abundance (SiMPull) and kinetic parameters from literature the. RNA-seq data generated on different growth conditions was also used to create a transcriptional regulatory model. Stochasticity in gene expression is known to create heterogeneity in an isogenic population. Such heterogeneity in transcriptional regulators, which are known to be present in small copy numbers, can effect formation of different cell phenotypes allowing cells in a colony to utilize different energy sources. Integrating the kinetic model of methanogenesis with a transcriptional regulation model results in a most comprehensive model for methanogenesis to date. We simulate this model using reaction-diffusion master equation (RDME) based Lattice Microbes software package to simulate monoclonal cells and study the variability in pathway and substrate usage under environmental and industrial conditions. Simulated behaviors allow identification and analysis of phenotypes that arise and the resulting sensitivity of methanogenesis to protein copy number, substrate availability and transcriptional regulation noise. These results lay the groundwork necessary for studying the individual behaviors to ultimately simulate a colony of methanogens sharing space and resources.
Measuring signal transduction in large numbers of cells with high spatial and temporal resolution... more Measuring signal transduction in large numbers of cells with high spatial and temporal resolution is fundamental to studying information processing in the nervous system. DNA-encoded sensors have an advantage in that they can be introduced into an organism noninvasively and targeted to specific brain regions, cell types, or subcellular compartments. A variety of chimeric proteins that report transmembrane voltage have been developed. The prototype sensor, FlaSh, is a green fluorescent protein fused to a voltage-sensitive K(+) channel, where voltage-dependent rearrangements in the channel induce changes in the protein's fluorescence. Subsequent sensors have refined this basic design using a monomeric voltage-sensing phosphatase domain from Ciona intestinalis and pairs of fluorescent proteins to produce a larger fluorescent signal. These sensors and their uses are discussed here.
... [ 19] Green Fluorescent Protein-Based Sensors for Detecting Signal 7ransduction and Monitorin... more ... [ 19] Green Fluorescent Protein-Based Sensors for Detecting Signal 7ransduction and Monitoring Ion Channel Function By MICAH S. SIEGEL and EHUD Y. ISACOFF Introduction ... This mutation blocks conduction by locking a gate in the pore into a closed conformation. ...
K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations i... more K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the select...
The meeting will explore the relationship between form and function in biological macromolecules.... more The meeting will explore the relationship between form and function in biological macromolecules. Sessions on the structural analysis of proteins and the means by which form is attained will lead into sessions on how this form determines function. Talks at the meeting will cover the gamut from studies on single molecules in solution through membrane proteins to macromolecular assemblies examined using a variety of theoretical as well as experimental techniques.
Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone ph... more Inherited and age-related retinal degenerative diseases cause progressive loss of rod and cone photoreceptors, leading to blindness, but spare downstream retinal neurons, which can be targeted for optogenetic therapy. However, optogenetic approaches have been limited by either low light sensitivity or slow kinetics, and lack adaptation to changes in ambient light, and not been shown to restore object vision. We find that the vertebrate medium wavelength cone opsin (MW-opsin) overcomes these limitations and supports vision in dim light. MW-opsin enables an otherwise blind retinitis pigmenotosa mouse to discriminate temporal and spatial light patterns displayed on a standard LCD computer tablet, displays adaption to changes in ambient light, and restores open-field novel object exploration under incidental room light. By contrast, rhodopsin, which is similar in sensitivity but slower in light response and has greater rundown, fails these tests. Thus, MW-opsin provides the speed, sensi...
We describe the development of the photoswitchable agonistLOGO, which activates GIRK channels in ... more We describe the development of the photoswitchable agonistLOGO, which activates GIRK channels in the dark and is rapidly deactivated upon exposure to long wavelength UV irradiation.LOGOcan be used to optically silence action potential firing in dissociated hippocampal neurons and exhibits activityin vivo, controlling the motility of zebrafish larvae in a light-dependent fashion.
Methods in molecular biology (Clifton, N.J.), 2015
Understanding the function of ion channels is a major goal of molecular neurophysiology. While st... more Understanding the function of ion channels is a major goal of molecular neurophysiology. While standard electrophysiological methods are invaluable tools to investigate the gating of ion channels, the structural rearrangements that mediate the way a channel senses physiological signals and opens and closes its gates cannot be measured electrically in a direct way. Here, we describe a method, based on site-specific labeling of a channel of interest with an environmentally sensitive fluorophore, which makes it possible to monitor conformational changes of ion channels in biological membranes in real time.
The properties of LiGluR resemble in several ways those of ChR2, the naturally photosensitive ion... more The properties of LiGluR resemble in several ways those of ChR2, the naturally photosensitive ion channel found in green algae [Boyden et al., 2005]. LiGluR and ChR2 are both cation selective channels that permeate monovalent ions and Ca2+, both need to be introduced by transfection or transgenesis, and both require a chemical photoswitch. In the case of LiGluR this is MAG, which needs to be infused, while in ChR2 this is retinal, which is naturally available in the retina and possibly in other parts of the nervous system. We compared photostimulation of neurons using our engineered LiGluR and the naturally light sensitive ChR2. The two channels, both under CMV promoter, were transfected into neurons cultured from the same animals, and recordings were made on the same day after transfection. We found light-evoked LiGluR currents to be ~5-fold larger than those of ChR2 (Supplemental Fig. 1A, B) and to fire APs with shorter illumination (1- 5 ms) than reported for ChR2 under similar culture conditions (5-15 ms) [Boyden et al., 2005; Li et al., 2005; Zhang et al., 2007]. While ChR2 currents desensitize by ~50%, LiGluR currents remain stable
Genetically encoded fluorescent probes of neural activity represent new promising tools for syste... more Genetically encoded fluorescent probes of neural activity represent new promising tools for systems neuroscience. Here, we present a comparative in vivo analysis of 10 different genetically encoded calcium indicators, as well as the pH-sensitive synapto-pHluorin. We analyzed their fluorescence changes in presynaptic boutons of the Drosophila larval neuromuscular junction. Robust neural activity did not result in any or noteworthy fluorescence changes when Flash-Pericam, Camgaroo-1, and Camgaroo-2 were expressed. However, calculated on the raw data, fractional fluorescence changes up to 18% were reported by synapto-pHluorin, Yellow Cameleon 2.0, 2.3, and 3.3, Inverse-Pericam, GCaMP1.3, GCaMP1.6, and the troponin C-based calcium sensor TN-L15. The response characteristics of all of these indicators differed considerably from each other, with GCaMP1.6 reporting high rates of neural activity with the largest and fastest fluorescence changes. However, GCaMP1.6 suffered from photobleaching, whereas the fluorescence signals of the double-chromophore indicators were in general smaller but more photostable and reproducible, with TN-L15 showing the fastest rise of the signals at lower activity rates. We show for GCaMP1.3 and YC3.3 that an expanded range of neural activity evoked fairly linear fluorescence changes and a corresponding linear increase in the signal-to-noise ratio (SNR). The expression level of the indicator biased the signal kinetics and SNR, whereas the signal amplitude was independent. The presented data will be useful for in vivo experiments with respect to the selection of an appropriate indicator, as well as for the correct interpretation of the optical signals.
In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from ... more In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containing a VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 in the transmembrane VSD to form the ion selectivity filter in the channel's open conformation.
At the Drosophila larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-... more At the Drosophila larval neuromuscular junction (NMJ), a motor neuron releases glutamate from 30-100 boutons onto the muscle it innervates. How transmission strength is distributed among the boutons of the NMJ is unknown. To address this, we created SynapCam, a version of the Ca 2+ reporter Cameleon. SynapCam localizes to the postsynapse and selectively reveals Ca 2+ influx through glutamate receptors (GluRs) with single-impulse and single-bouton resolution. GluR-based Ca 2+ signals were uniform within a given connection (i.e., bouton and postsynapse pair), but differed considerably among connections of an NMJ. A steep gradient of transmission strength was observed along axonal branches, from weak proximal connections to strong distal ones. Presynaptic imaging revealed a matching axonal gradient, with higher Ca 2+ influx and exocytosis at distal boutons. The results suggest that transmission strength is mainly determined presynaptically at the level of individual boutons, possibly by one or more factors existing in a gradient.
Proceedings of the National Academy of Sciences of the United States of America, Jan 21, 2009
Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light... more Photoswitched tethered ligands (PTLs) can be used to remotely control protein function with light. We have studied the geometric and conformational factors that determine the efficacy of PTL gating in the ionotropic glutamate receptor iGluR6 using a family of photoiosomerizable MAG (maleimide-azobenzene-glutamate) PTLs that covalently attach to the clamshell ligand-binding domain. Experiments and molecular dynamics simulations of the modified proteins show that optical switching depends on 2 factors: (i) the relative occupancy of the binding pocket in the 2 photoisomers of MAG and (ii) the degree of clamshell closure that is possible given the disposition of the MAG linker. A synthesized short version of MAG turns the channel on in either the cis or trans state, depending on the point of attachment. This yin/yang optical control makes it possible for 1 wavelength of light to elicit action potentials in one set of neurons, while deexciting a second set of neurons in the same preparat...
Canadian Journal of Physiology and Pharmacology, 1988
In C1-spinal, pentobarbital-anaesthetized or anemically decerebrated cats, the preganglionic inpu... more In C1-spinal, pentobarbital-anaesthetized or anemically decerebrated cats, the preganglionic input to the acutely decentralized right stellate ganglion was stimulated with 10- to 30-s trains at 20–40 Hz. Electrical stimulation consistently produced an increase in heart rate in the presence of blocking doses of hexamethonium and atropine or after depletion of acetylcholine from the preganglionic axons by prolonged low frequency stimulation in the presence of hemicholinium. The increase in heart rate had a delayed slow onset, lasted several minutes, and was abolished by propranolol or by section of the inferior cardiac nerve. The magnitude and duration of the heart rate increase were related to intensity, frequency, and duration of preganglionic stimulation. The response to stimulation of a given white ramus was progressively attenuated, and eventually irreversibly lost, during prolonged continuous stimulation of that ramus, while the response to stimulation of a different unstimulate...
Accumulation of amyloid-b peptides (Ab), the proteolytic products of the amyloid precursor protei... more Accumulation of amyloid-b peptides (Ab), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Ab40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Ab40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/G i/oprotein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Ab40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.
Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/E... more Caenorhabditis eleganssenses gentle touch via a mechanotransduction channel formed from the DEG/ENaC proteins MEC-4 and MEC-10. An additional protein, the paraoxonase-like protein MEC-6, is essential for transduction, and previous work suggested that MEC-6 was part of the transduction complex. We found that MEC-6 and a similar protein, POML-1, reside primarily in the endoplasmic reticulum and do not colocalize with MEC-4 on the plasma membrane in vivo. As with MEC-6, POML-1 is needed for touch sensitivity, for the neurodegeneration caused by themec-4(d)mutation, and for the expression and distribution of MEC-4 in vivo. Both proteins are likely needed for the proper folding or assembly of MEC-4 channels in vivo as measured by FRET. MEC-6 detectably increases the rate of MEC-4 accumulation on theXenopusoocyte plasma membrane. These results suggest that MEC-6 and POML-1 interact with MEC-4 to facilitate expression and localization of MEC-4 on the cell surface. Thus, MEC-6 and POML-1 ac...
Anaerobic archaea M. acetivorans, a versatile saltwater adapted methanogen, is capable of convert... more Anaerobic archaea M. acetivorans, a versatile saltwater adapted methanogen, is capable of converting seven different organic molecules into methane. Utilization of these substrates involves three main reaction pathways that are differentially regulated by the organism depending on growth conditions. We have developed a kinetic model for methanogenesis using RNA-seq data, single molecule enumeration of protein abundance (SiMPull) and kinetic parameters from literature the. RNA-seq data generated on different growth conditions was also used to create a transcriptional regulatory model. Stochasticity in gene expression is known to create heterogeneity in an isogenic population. Such heterogeneity in transcriptional regulators, which are known to be present in small copy numbers, can effect formation of different cell phenotypes allowing cells in a colony to utilize different energy sources. Integrating the kinetic model of methanogenesis with a transcriptional regulation model results in a most comprehensive model for methanogenesis to date. We simulate this model using reaction-diffusion master equation (RDME) based Lattice Microbes software package to simulate monoclonal cells and study the variability in pathway and substrate usage under environmental and industrial conditions. Simulated behaviors allow identification and analysis of phenotypes that arise and the resulting sensitivity of methanogenesis to protein copy number, substrate availability and transcriptional regulation noise. These results lay the groundwork necessary for studying the individual behaviors to ultimately simulate a colony of methanogens sharing space and resources.
Measuring signal transduction in large numbers of cells with high spatial and temporal resolution... more Measuring signal transduction in large numbers of cells with high spatial and temporal resolution is fundamental to studying information processing in the nervous system. DNA-encoded sensors have an advantage in that they can be introduced into an organism noninvasively and targeted to specific brain regions, cell types, or subcellular compartments. A variety of chimeric proteins that report transmembrane voltage have been developed. The prototype sensor, FlaSh, is a green fluorescent protein fused to a voltage-sensitive K(+) channel, where voltage-dependent rearrangements in the channel induce changes in the protein's fluorescence. Subsequent sensors have refined this basic design using a monomeric voltage-sensing phosphatase domain from Ciona intestinalis and pairs of fluorescent proteins to produce a larger fluorescent signal. These sensors and their uses are discussed here.
... [ 19] Green Fluorescent Protein-Based Sensors for Detecting Signal 7ransduction and Monitorin... more ... [ 19] Green Fluorescent Protein-Based Sensors for Detecting Signal 7ransduction and Monitoring Ion Channel Function By MICAH S. SIEGEL and EHUD Y. ISACOFF Introduction ... This mutation blocks conduction by locking a gate in the pore into a closed conformation. ...
K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations i... more K+ channels can be occupied by multiple permeant ions that appear to bind at discrete locations in the conduction pathway. Neither the molecular nature of the binding sites nor their relation to the activation or inactivation gates that control ion flow are well understood. We used the permeant ion Ba2+ as a K+ analog to probe for K+ ion binding sites and their relationship to the activation and inactivation gates. Our data are consistent with the existence of three single-file permeant-ion binding sites: one deep site, which binds Ba2+ with high affinity, and two more external sites whose occupancy influences Ba2+ movement to and from the deep site. All three sites are accessible to the external solution in channels with a closed activation gate, and the deep site lies between the activation gate and the C-type inactivation gate. We identify mutations in the P-region that disrupt two of the binding sites, as well as an energy barrier between the sites that may be part of the select...
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Papers by E. Isacoff