RESUMO
Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB-promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II-TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.
Assuntos
Regiões Promotoras Genéticas/fisiologia , Multimerização Proteica/fisiologia , Fatores de Transcrição TFII/metabolismo , Ativação Transcricional/fisiologia , Linhagem Celular Tumoral , Humanos , Microscopia de Interferência , Ligação Proteica , RNA Polimerase II/metabolismo , Deleção de Sequência , Fatores de TempoRESUMO
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.
Assuntos
DNA/metabolismo , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia/métodos , Coloração Cromossômica , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Processamento de Imagem Assistida por Computador , Análise de Sequência de DNA/métodosRESUMO
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.
Assuntos
Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Processos Fotoquímicos , Imagem Individual de Molécula/métodos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Células COS , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Células-Tronco Embrionárias , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/efeitos da radiação , Humanos , Ligantes , Luz , Camundongos , Microscopia de Fluorescência , Estrutura Molecular , Fotoquímica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/efeitos da radiação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Coloração e RotulagemRESUMO
Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapse-specific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single ß-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcode-binding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of ß-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.
Assuntos
Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Transporte de RNA/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/biossíntese , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular , Citoplasma/metabolismo , Dendritos/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Neurônios/citologia , Transporte Proteico/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismoRESUMO
Live-cell imaging and particle tracking provide rich information on mechanisms of intracellular transport. However, trajectory analysis procedures to infer complex transport dynamics involving stochastic switching between active transport and diffusive motion are lacking. We applied Bayesian model selection to hidden Markov modeling to infer transient transport states from trajectories of mRNA-protein complexes in live mouse hippocampal neurons and metaphase kinetochores in dividing human cells. The software is available at http://hmm-bayes.org/.
Assuntos
Actinas/metabolismo , Hipocampo/metabolismo , Modelos Biológicos , Imagem Molecular/métodos , Neurônios/citologia , Neurônios/metabolismo , Animais , Teorema de Bayes , Células Cultivadas , Simulação por Computador , Feminino , Células HeLa , Hipocampo/citologia , Humanos , Cadeias de Markov , Camundongos , MicroRNAs/metabolismo , Microscopia de Fluorescência/métodos , Modelos Estatísticos , Reconhecimento Automatizado de Padrão/métodos , Transporte Proteico/fisiologia , SoftwareRESUMO
Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.
Assuntos
Corantes Fluorescentes/química , Microscopia Ultravioleta/métodos , Imagem Molecular/métodos , Azetidinas/química , Técnicas de Química Sintética , Cumarínicos/química , Fluoresceína/química , Corantes Fluorescentes/análise , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Modelos Moleculares , Teoria Quântica , Rodaminas/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta/métodos , Relação Estrutura-AtividadeRESUMO
We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.
Assuntos
Proteínas Luminescentes/química , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Animais , Antígenos , Mapeamento Encefálico , Drosophila , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios , Conformação ProteicaRESUMO
Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide- or alkyne-tagged glycans, a sufficiently low spatial density of dye-labeled glycans was achieved, enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye-labeled glycans in mammary cancer cells revealed constrained diffusion of both N- and O-linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.
Assuntos
Neoplasias/metabolismo , Polissacarídeos/metabolismo , Corantes Fluorescentes , Células HeLa , Humanos , Neoplasias/patologiaRESUMO
During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechanism of enzyme activation. By integrating the output of individual RelA molecules and ppGpp degradation pathways, this regulatory circuit contributes to a fast and coordinated transition to stringency.
Assuntos
Escherichia coli/enzimologia , Guanosina Tetrafosfato/biossíntese , Ligases/metabolismo , Regulação Alostérica , Ativação Enzimática , Escherichia coli/metabolismo , Ribossomos/metabolismoRESUMO
The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ligases/metabolismo , Ribossomos/metabolismo , Aminoácidos/farmacologia , Biocatálise , Western Blotting , Divisão Celular/efeitos dos fármacos , Simulação por Computador , Citosol/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Citometria de Fluxo , Guanosina Tetrafosfato/metabolismo , Cinética , Ligases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metionina/farmacologia , Microscopia Confocal , Fatores de Tempo , Triptofano/farmacologiaRESUMO
Inside the cell, proteins essential for signaling, morphogenesis, and migration navigate complex pathways, typically via vesicular trafficking or microtubule-driven mechanisms 1-3 . However, the process by which soluble cytoskeletal monomers maneuver through the cytoplasm's ever-changing environment to reach their destinations without using these pathways remains unknown. 4-6 Here, we show that actin cytoskeletal treadmilling leads to the formation of a semi-permeable actin-myosin barrier, creating a specialized compartment separated from the rest of the cell body that directs proteins toward the cell edge by advection, diffusion facilitated by fluid flow. Contraction at this barrier generates a molecularly non-specific fluid flow that transports actin, actin-binding proteins, adhesion proteins, and even inert proteins forward. The local curvature of the barrier specifically targets these proteins toward protruding edges of the leading edge, sites of new filament growth, effectively coordinating protein distribution with cellular dynamics. Outside this compartment, diffusion remains the primary mode of protein transport, contrasting sharply with the directed advection within. This discovery reveals a novel protein transport mechanism that redefines the front of the cell as a pseudo-organelle, actively orchestrating protein mobilization for cellular front activities such as protrusion and adhesion. By elucidating a new model of protein dynamics at the cellular front, this work contributes a critical piece to the puzzle of how cells adapt their internal structures for targeted and rapid response to extracellular cues. The findings challenge the current understanding of intracellular transport, suggesting that cells possess highly specialized and previously unrecognized organizational strategies for managing protein distribution efficiently, providing a new framework for understanding the cellular architecture's role in rapid response and adaptation to environmental changes.
RESUMO
Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of "glitter bomb" labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.
RESUMO
Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.
RESUMO
mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5'cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.
Assuntos
Fatores de Iniciação de Peptídeos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Fatores de Iniciação de Peptídeos/genética , Capuzes de RNA/metabolismo , Iniciação Traducional da Cadeia PeptídicaRESUMO
The translation initiation complex 4F (eIF4F) is a rate-limiting factor in protein synthesis. Alterations in eIF4F activity are linked to several diseases, including cancer and infectious diseases. To this end, coronaviruses require eIF4F complex activity to produce proteins essential for their life cycle. Efforts to target coronaviruses by abrogating translation have been largely limited to repurposing existing eIF4F complex inhibitors. Here, we report the results of a high throughput screen to identify small molecules that disrupt eIF4F complex formation and inhibit coronavirus RNA and protein levels. Of 338,000 small molecules screened for inhibition of the eIF4F-driven, CAP-dependent translation, we identified SBI-1232 and two structurally related analogs, SBI-5844 and SBI-0498, that inhibit human coronavirus OC43 (HCoV-OC43; OC43) with minimal cell toxicity. Notably, gene expression changes after OC43 infection of Vero E6 or A549 cells were effectively reverted upon treatment with SBI-5844 or SBI-0498. Moreover, SBI-5844 or SBI-0498 treatment effectively impeded the eIF4F complex assembly, with concomitant inhibition of newly synthesized OC43 nucleocapsid protein and OC43 RNA and protein levels. Overall, we identify SBI-5844 and SBI-0498 as small molecules targeting the eIF4F complex that may limit coronavirus transcripts and proteins, thereby representing a basis for developing novel therapeutic modalities against coronaviruses.
RESUMO
Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.
Assuntos
Proteínas Nucleares , Fatores de Transcrição , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromossomos/genética , Cromossomos/metabolismo , Mamíferos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.
Assuntos
Histonas/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transcrição Gênica , Montagem e Desmontagem da Cromatina , Histonas/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula , Sítio de Iniciação de TranscriçãoRESUMO
The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.
Assuntos
Corantes Fluorescentes/síntese química , Microscopia de Fluorescência/métodos , Animais , Corantes Fluorescentes/química , Humanos , Ligantes , Mamíferos , Estrutura Molecular , Fenômenos de Química Orgânica , Imagem Individual de Molécula , Coloração e RotulagemRESUMO
Enzymes are dynamic entities: both their conformation and catalytic activity fluctuate over time. When such fluctuations are relatively fast, it is not surprising that the classical Michaelis-Menten (MM) relationship between the steady-state enzymatic velocity and the substrate concentration still holds. However, recent single-molecule experiments have shown that this is the case even for an enzyme whose catalytic activity fluctuates on the 10(-4)-10 s range. The purpose of this paper is to examine various scenarios in which slowly fluctuating enzymes would still obey the MM relationship. Specifically, we consider (1) the quasi-static condition (e.g., the conformational fluctuation of the enzyme-substrate complex is much slower than binding, catalysis, and the conformational fluctuations of the free enzyme), (2) the quasi-equilibrium condition (when the substrate dissociation is much faster than catalysis, irrespective of the time scales or amplitudes of conformational fluctuations), and (3) the conformational-equilibrium condition (when the dissociation and catalytic rates depend on the conformational coordinate in the same way). For each of these scenarios, the physical meaning of the apparent Michaelis constant and catalytic rate constant is provided. Finally, as an example, the theoretical analysis of a recent single-molecule enzyme assay is considered in light of the perspectives presented in this paper.
Assuntos
Biofísica/métodos , Físico-Química/métodos , Enzimas/química , Catálise , Difusão , Cinética , Modelos Químicos , Modelos Moleculares , Modelos Estatísticos , Modelos Teóricos , Conformação Molecular , Termodinâmica , beta-Galactosidase/químicaRESUMO
Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single ß-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, ß-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.