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To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

International audience; Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Small-molecule fluorescent stains enable the imaging of cellular structures without the need for genetic manipulation. Here, we introduce 2,7-diaminobenzopyrylium (DAB) dyes as live-cell mitochondrial stains excited with violet light. This amalgam of the coumarin and rhodamine fluorophore structures yields dyes with high photostability and tunable spectral properties.

SummaryChemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.

SUMMARY Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure., In Brief Yan et al. examine how functional activity of nicotinic cholinergic receptors is distributed in diverse VTA cell types, revealing nAChR activity in VTA glutamate neurons. These receptors modulate local glutamate transmission in VTA, suggesting mechanisms by which nicotine influences mesolimbic circuitry.

Access to cis-regulatory elements packaged in chromatin is essential for directing gene expression and cell viability. Here, we report a super-resolution imaging strategy, 3D ATAC-PALM, that enables direct visualization of the entire accessible genome. We found that active chromosomal segments are organized into spatially-segregated accessible chromatin domains (ACDs). Rapid depletion of CTCF or Cohesin (RAD21 subunit) induced enhanced ACD clustering, reduced physical separation between intrachromosomal ACDs, and differentially regulated ACD compaction. Experimental perturbations and polymer modeling suggest that dynamic protein-protein and protein-DNA interactions within ACDs couple with loop extrusion to organize ACD topology. Dysorganization of ACDs upon CTCF or Cohesin loss alters transcription factor binding and target search dynamics in living cells. These results uncover fundamental mechanisms underpinning the formation of 3D genome architecture and its pivotal function in transcriptional regulation.

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

INTRODUCTION. DNA binding transcription factors (TFs) are quintessential regulators of eukaryotic gene expression. Early studies of TFs revealed their well-structured DNA binding domains (DBDs) and identified functionally critical activation domains (ADs) required for transcription. It later became evident that many ADs contain intrinsically disordered low-complexity sequence domains (LCDs), but how LCDs activate transcription has remained unclear. Although it is known that transcriptional activation by LCDs requires selective interaction with binding partners, it has been challenging to directly measure selective LCD-LCD recognition in vivo and unravel its mechanism of action. RATIONALE. Traditional biochemical reconstitution and genetics studies have identified most of the molecular players central to transcription regulation. However, the mechanism by which weak, dynamic protein-protein interactions drive gene activation in living cells has remained unknown. Advances in live-cell single-molecule imaging have opened a new frontier for studying transcription in vivo. In this study, we used synthetic LacO (Lac operator) arrays as well as endogenous GGAA microsatellite loci to study LCD-LCD interactions of TFs such as EWS/FLI1, TAF15, and Sp1 in live cells. To probe the dynamic behavior of TF LCDs at target genomic loci, we have combined CRISPR-Cas9 genome editing, mutagenesis, gene activation, cell transformation assays, and various high-resolution imaging approaches including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, lattice light-sheet microscopy, three-dimensional DNA fluorescence in situ hybridization, and live-cell single-particle tracking. RESULTS. Live-cell single-molecule imaging revealed that TF LCDs interact to form local high-concentration hubs at both synthetic DNA arrays and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic (seconds to minutes), selective for binding partners, and differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid, reversible, and selective multivalent LCD-LCD interactions occur between TFs and the RNA Pol II machinery to activate transcription. We observed formation of functional TF LCD hubs at a wide range of intranuclear TF concentrations. Although we detected apparent liquid-liquid phase separation with gross overexpression of LCDs, transcriptionally competent TF LCD hubs were observed at physiological TF levels at endogenous chromosomal loci in the absence of detectable phase separation. In addition, mutagenesis, gene expression, and cell transformation assays in Ewing’s sarcoma cells revealed a functional link between LCD-LCD interactions, transactivation capacity, and oncogenic potential. CONCLUSION. The use of various imaging methods in live cells powerfully complements in vitro studies and provides new insights into the nature of LCD interactions and their role in gene regulation. We propose that transactivation domains function by forming local high-concentration hubs of TFs via dynamic, multivalent, and specific LCD-LCD interactions. It also seems likely that weak, dynamic, and transient contacts between TFs play a role in disease-causing dysregulation of gene expression (i.e., EWS/FLI1 in Ewing’s sarcoma), suggesting that LCD-LCD interactions may represent a new class of viable drug targets. Although we examined a small subset of TF LCDs, the principles uncovered regarding the dynamics and mechanisms driving LCD-LCD interactions may be applicable to other classes of proteins and biomolecular interactions occurring in many cell types.

Photoactivatable (‘caged’) pharmacological agents have revolutionized neuroscience but the palette of available ligands is limited. We describe a general method for caging tertiary amines using an unconventional quaternary ammonium linkage that is chemically stable and elicits a desirable red-shift in activation wavelength. A photoactivatable nicotine (PA-Nic) prepared using this strategy could be uncaged via 1- or 2-photon excitation, making it useful for optopharmacology experiments to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs) but how they perform transactivation functions remains unclear. Recent studies report that TF-LCDs can undergo hydrogel formation or liquid-liquid phase separation in vitro. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II) and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid reversible and multivalent LCD-LCD interactions occur between TFs and the Pol II machinery, which underpins a central mechanism for transactivation and plays a key role in gene expression and disease.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Detection of antibodies is essential for the diagnosis of many disease states, including infectious diseases, autoimmune diseases and allergies. Most current antibody detection assays involve multistep detection schemes in which molecular recognition and signal generation are separate processes. A well-known example is the enzyme-linked immunosorbent assay (ELISA), which combines high sensitivity and specificity with strong signal amplification. However, ELISA and other heterogeneous methods require multiple, time-consuming washing and incubation steps, which limits their applicability in point-of-care diagnostics and high-throughput applications. In recent years, several new antibody detection strategies have been developed in which antibody binding and signal generation are integrated within a single biomolecular reporter. These strategies aim to rival ELISA in terms of sensitivity and specificity, while decreasing the time and effort required to perform an assay. Here, we review recent developments in this field according to their mechanism of action and discuss their advantages and limitations.

A caged rhodamine 110 derivative for the specific labeling of SNAP-tag fusion proteins is introduced. The caged rhodamine 110 derivative permits the labeling of cell surface proteins in living cells and of intracellular proteins in fixed cells. The probe requires only a single caging group to maintain the fluorophore in a non-fluorescent state and becomes highly fluorescent after uncaging. The high contrast ratio is confirmed both in bulk and at the single molecule level. This property, together with its high photon yield makes it an excellent dye for photoactivated localization microscopy (PALM), as we demonstrate here.

Photosensitive probes are powerful tools to study cellular processes with high temporal and spatial resolution. However, most synthetic fluorophores suited for biomolecular imaging have not been converted yet to appropriate photosensitive analogues. Here we describe a generally applicable strategy for the generation of photoactivatable and photoconvertible fluorescent probes that can be selectively coupled to SNAP-tag fusion proteins in living cells. Photoactivatable versions of fluorescein and Cy3 as well as a photoconvertible Cy5-Cy3 probe were prepared and coupled to selected proteins on the cell surface, in the cytosol, and in the nucleus of cells. In proof-of-principle experiments, the photoactivatable Cy3 probe was used to characterize the mobility of a lipid-anchored cell surface protein and of a G protein coupled receptor (GPCR). This work establishes a generally applicable strategy for the generation of a large variety of different photosensitive fluorophores with tailor-made properties for biomolecular imaging.

SNAP‐tag: We introduce a photoswitchable O6‐benzylguanine derivative and demonstrate its use for super‐resolution microscopy of SNAP‐tagged proteins based on single fluorophore localization. Stochastic Optical Reconstruction Microscopy (STORM) reveals SNAP‐tagged microtubule structures with ∼25 nm resolution. The described probe in combination with the versatile SNAP‐tag labeling opens new possibilities for imaging biological structures at the nanoscale.

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP-tag technology and O-6-benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long-term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O-6-benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo-deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti-fluorescein antibodies. Developmental Dynamics 240:820-827, 2011. (C) 2011 Wiley-Liss, Inc.

Reference LIP-ARTICLE-2008-010doi:10.1002/cbic.200700472View record in Web of Science Record created on 2008-06-10, modified on 2017-05-12