Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior re... more Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we developed the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio (SNR) of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also targeted kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppressed seizure symptoms, suggesting its applicability in controlling disease-related neurons. Fu...
Proceedings of the National Academy of Sciences of the United States of America, Aug 16, 2016
Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic stre... more Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein-protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1-containing AMPARs. Our results support the large body of research indicating a pr...
In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, wh... more In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exo-cytic vesicles of AMPA and GABA A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesi-cles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABA A receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exo-cytosis of AMPA and GABA A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons. AMPA receptor | GABA A receptor | constitutive exocytosis | TIRFM | SNARE I n the mammalian central nervous system, neurons receive ex-citatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neuro-transmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA A receptors, which are predominantly heter-opentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a " plasma membrane sorting model, " different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a " vesicle sorting model, " different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the exo-cytic pathways of AMPA and GABA A receptors have similar but also distinct properties (1, 2). Increasing evidence has suggested roles for the SNARE protein family in vesicular trafficking of AMPA and GABA A receptors (4– 17). SNAREs are a large family of membrane-associated proteins critical for many intracellular membrane trafficking events. The family is subdivided into v-SNAREs (synaptobrevin/VAMP, vesi-cle-associated membrane proteins) and t-SNAREs (syntaxins and SNAP25, synaptosomal-associated protein of 25 kDa) based on their localization on trafficking vesicles or target membranes, respectively. To mediate vesicle fusion with target membranes, SNARE proteins form a four-helix bundle (SNARE complex) consisting of two coiled-coil domains from SNAP25, one coiled-coil domain from syntaxin, and a coiled-coil domain from VAMPs (18). Formation of the helical bundle can be disrupted by neurotoxins, which specifically cleave different SNARE proteins (19). Each SNARE subfamily is composed of genes with high homology but different tissue specificity and subcellular localization. It remains to be determined whether individual SNAREs play specific roles in regulating the membrane trafficking of individual proteins. To address how AMPA and GABA A receptors are sorted in the exocytic pathway and what molecules are involved in regulating exocytosis of these receptors, we specifically studied con-stitutive exocytosis of AMPA and GABA A receptor subunits using total internal reflection fluorescence microscopy (TIRFM) in combination with immunocytochemistry, electrophysiology, and electron microscopy methods. Together, we revealed that AMPA and GABA A receptors are initially sorted into different vesicles in the Golgi apparatus and delivered to different domains at the plasma membrane and are regulated by specific Rab proteins and Significance In neurons most fast excitatory neurotransmission is mediated by AMPA receptors, which cluster at excitatory synapses primarily on dendritic spines. Fast synaptic inhibition is largely mediated by GABA A receptors, which cluster at inhibitory syn-apses mainly on the soma and dendritic shafts. It is unclear how these receptors are segregated and delivered to specific locations on the plasma membranes. Here we directly studied the constitutive exocytosis of AMPA and GABA A receptors and demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. Their exocytosis is dependent on distinct Rab GTPases and SNARE complexes. Our results reveal fundamental mechanisms underlying the sorting of excitatory and inhibitory neurotransmitter receptors in neurons.
Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial memb... more Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ~30-70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB interacting proteins--function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.
Proceedings of the National Academy of Sciences of the United States of America, Jan 1, 2012
The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at d... more The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, chi (5'-GCTGGTGG), which is read as a single-stranded DNA sequence while the enzyme is unwinding DNA at over approximately 1,000 bp/s. Previous studies implicated the RecC subunit as the "chi-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for chi recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, 10.1073/pnas.1206076109]. The genetic analyses revealed two classes of mutants. Here we use ensemble and single-molecule criteria to biochemically establish that one class of mutants (type 1) has lost the capacity to recognize chi (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical chi, but they have gained the capacity to precociously recognize single-nucleotide variants of chi. Based on the RecBCD structure, these mutant classes define an alpha-helix responsible for chi recognition that is allosterically coupled to a structural latch. When opened, we propose that the latch permits access to an alternative exit channel for the single-stranded DNA downstream of chi, thereby avoiding degradation by the nuclease domain. These findings provide a unique perspective into the mechanism by which recognition of a single-stranded DNA sequence switches the translocating RecBCD from a destructive nuclease to a constructive component of recombinational DNA repair.
In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemb... more In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.
Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior re... more Verifying causal effects of neural circuits is essential for proving direct a circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we developed the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio (SNR) of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also targeted kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppressed seizure symptoms, suggesting its applicability in controlling disease-related neurons. Fu...
Proceedings of the National Academy of Sciences of the United States of America, Aug 16, 2016
Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic stre... more Regulation of AMPA receptor (AMPAR) function is a fundamental mechanism controlling synaptic strength during long-term potentiation/depression and homeostatic scaling. AMPAR function and membrane trafficking is controlled by protein-protein interactions, as well as by posttranslational modifications. Phosphorylation of the GluA1 AMPAR subunit at S845 and S831 play especially important roles during synaptic plasticity. Recent controversy has emerged regarding the extent to which GluA1 phosphorylation may contribute to synaptic plasticity. Here we used a variety of methods to measure the population of phosphorylated GluA1-containing AMPARs in cultured primary neurons and mouse forebrain. Phosphorylated GluA1 represents large fractions from 12% to 50% of the total population under basal and stimulated conditions in vitro and in vivo. Furthermore, a large fraction of synapses are positive for phospho-GluA1-containing AMPARs. Our results support the large body of research indicating a pr...
In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, wh... more In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exo-cytic vesicles of AMPA and GABA A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesi-cles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABA A receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exo-cytosis of AMPA and GABA A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons. AMPA receptor | GABA A receptor | constitutive exocytosis | TIRFM | SNARE I n the mammalian central nervous system, neurons receive ex-citatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neuro-transmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA A receptors, which are predominantly heter-opentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a " plasma membrane sorting model, " different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a " vesicle sorting model, " different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the exo-cytic pathways of AMPA and GABA A receptors have similar but also distinct properties (1, 2). Increasing evidence has suggested roles for the SNARE protein family in vesicular trafficking of AMPA and GABA A receptors (4– 17). SNAREs are a large family of membrane-associated proteins critical for many intracellular membrane trafficking events. The family is subdivided into v-SNAREs (synaptobrevin/VAMP, vesi-cle-associated membrane proteins) and t-SNAREs (syntaxins and SNAP25, synaptosomal-associated protein of 25 kDa) based on their localization on trafficking vesicles or target membranes, respectively. To mediate vesicle fusion with target membranes, SNARE proteins form a four-helix bundle (SNARE complex) consisting of two coiled-coil domains from SNAP25, one coiled-coil domain from syntaxin, and a coiled-coil domain from VAMPs (18). Formation of the helical bundle can be disrupted by neurotoxins, which specifically cleave different SNARE proteins (19). Each SNARE subfamily is composed of genes with high homology but different tissue specificity and subcellular localization. It remains to be determined whether individual SNAREs play specific roles in regulating the membrane trafficking of individual proteins. To address how AMPA and GABA A receptors are sorted in the exocytic pathway and what molecules are involved in regulating exocytosis of these receptors, we specifically studied con-stitutive exocytosis of AMPA and GABA A receptor subunits using total internal reflection fluorescence microscopy (TIRFM) in combination with immunocytochemistry, electrophysiology, and electron microscopy methods. Together, we revealed that AMPA and GABA A receptors are initially sorted into different vesicles in the Golgi apparatus and delivered to different domains at the plasma membrane and are regulated by specific Rab proteins and Significance In neurons most fast excitatory neurotransmission is mediated by AMPA receptors, which cluster at excitatory synapses primarily on dendritic spines. Fast synaptic inhibition is largely mediated by GABA A receptors, which cluster at inhibitory syn-apses mainly on the soma and dendritic shafts. It is unclear how these receptors are segregated and delivered to specific locations on the plasma membranes. Here we directly studied the constitutive exocytosis of AMPA and GABA A receptors and demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. Their exocytosis is dependent on distinct Rab GTPases and SNARE complexes. Our results reveal fundamental mechanisms underlying the sorting of excitatory and inhibitory neurotransmitter receptors in neurons.
Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial memb... more Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ~30-70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB interacting proteins--function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.
Proceedings of the National Academy of Sciences of the United States of America, Jan 1, 2012
The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at d... more The RecBCD enzyme is a complex heterotrimeric helicase/nuclease that initiates recombination at double-stranded DNA breaks. In Escherichia coli, its activities are regulated by the octameric recombination hotspot, chi (5'-GCTGGTGG), which is read as a single-stranded DNA sequence while the enzyme is unwinding DNA at over approximately 1,000 bp/s. Previous studies implicated the RecC subunit as the "chi-scanning element" in this process. Site-directed mutagenesis and phenotypic analyses identified residues in RecC responsible for chi recognition [Handa N, et al., (2012) Proc Natl Acad Sci USA, 10.1073/pnas.1206076109]. The genetic analyses revealed two classes of mutants. Here we use ensemble and single-molecule criteria to biochemically establish that one class of mutants (type 1) has lost the capacity to recognize chi (lost-recognition), whereas the second class (type 2) has a lowered specificity for recognition (relaxed-specificity). The relaxed-specificity mutants still recognize canonical chi, but they have gained the capacity to precociously recognize single-nucleotide variants of chi. Based on the RecBCD structure, these mutant classes define an alpha-helix responsible for chi recognition that is allosterically coupled to a structural latch. When opened, we propose that the latch permits access to an alternative exit channel for the single-stranded DNA downstream of chi, thereby avoiding degradation by the nuclease domain. These findings provide a unique perspective into the mechanism by which recognition of a single-stranded DNA sequence switches the translocating RecBCD from a destructive nuclease to a constructive component of recombinational DNA repair.
In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemb... more In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual proteins or assemblies of proteins acting on single molecules of DNA. The general experimental design involves attaching a single DNA molecule to a polystyrene microsphere that is then used as a microscopic handle to manipulate individual DNA molecules with a laser trap. Visualization is achieved by fluorescently labeling either the DNA or the protein of interest, followed by direct imaging using high-sensitivity fluorescence microscopy. We describe the sample preparation and instrumentation used to visualize the interaction of individual proteins with single molecules of DNA. As examples, we describe the application of these methods to the study of proteins involved in recombination-mediated DNA repair, a process essential for the maintenance of genomic integrity.
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