RESUMO
To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.
Assuntos
Proteômica , Imagem Individual de Molécula , DNA , Microscopia de Fluorescência/métodos , Neurônios , ProteínasRESUMO
Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.
Assuntos
Mecanotransdução Celular , Imagem Individual de Molécula , Animais , Humanos , Camundongos , Fenômenos Biomecânicos , Adesão Celular , DNA/química , DNA/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Sobrevivência Celular , Pareamento de Bases , CalibragemRESUMO
Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45â Mcm2-7â GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMGâ DONSONâ DONSONâ CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Imagem Individual de Molécula , Humanos , Proteínas de Ciclo Celular/metabolismo , Origem de Replicação , Animais , DNA Helicases/metabolismo , RecQ Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Assuntos
Integrinas , Talina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adesão Celular , Células CHO , Cricetulus , Integrinas/metabolismo , Integrinas/química , Ligantes , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Imagem Individual de Molécula , Talina/metabolismo , Talina/químicaRESUMO
Accurate protein synthesis (translation) relies on translation factors that rectify ribosome fluctuations into a unidirectional process. Understanding this process requires structural characterization of the ribosome and translation-factor dynamics. In the 2000s, crystallographic studies determined high-resolution structures of ribosomes stalled with translation factors, providing a starting point for visualizing translation. Recent progress in single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution of numerous structures sampled in heterogeneous complexes (ensembles). Ensemble and time-resolved cryo-EM have now revealed unprecedented views of ribosome transitions in the three principal stages of translation: initiation, elongation, and termination. This review focuses on how translation factors help achieve high accuracy and efficiency of translation by monitoring distinct ribosome conformations and by differentially shifting the equilibria of ribosome rearrangements for cognate and near-cognate substrates.
Assuntos
Ribossomos , Imagem Individual de Molécula , Microscopia Crioeletrônica , Ribossomos/metabolismoRESUMO
Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.
Assuntos
COVID-19 , Pandemias , Microscopia Crioeletrônica/métodos , Humanos , SARS-CoV-2 , Imagem Individual de MoléculaRESUMO
We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.
Assuntos
Técnicas Biossensoriais , Peptídeos/química , Imagem Individual de Molécula , Animais , Adesão Celular , Linhagem Celular , Sobrevivência Celular , Embrião de Mamíferos/citologia , Ativação Enzimática , Fibroblastos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Camundongos , Nanopartículas/química , Conformação Proteica , Quinases da Família src/metabolismoRESUMO
Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Resistência à Doença/imunologia , Imunidade Vegetal , Transdução de Sinais , Animais , Morte Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Ácido Glutâmico/metabolismo , Bicamadas Lipídicas/metabolismo , Oócitos/metabolismo , Células Vegetais/metabolismo , Multimerização Proteica , Protoplastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Imagem Individual de Molécula , Vacúolos/metabolismo , XenopusRESUMO
This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.
Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , DNA/ultraestrutura , Regulação da Expressão Gênica , RNA Mensageiro/ultraestrutura , Pequeno RNA não Traduzido/ultraestrutura , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Replicação do DNA , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Microscopia de Fluorescência , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Coloração e Rotulagem/métodos , Telômero/metabolismo , Telômero/ultraestrutura , Transcrição GênicaRESUMO
P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H+, Na+, K+, Ca2+, and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca2+-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.
Assuntos
Trifosfato de Adenosina/química , ATPases Transportadoras de Cobre/química , ATPase Trocadora de Hidrogênio-Potássio/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPase Trocadora de Sódio-Potássio/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Cátions Bivalentes , Cátions Monovalentes , ATPases Transportadoras de Cobre/genética , ATPases Transportadoras de Cobre/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Humanos , Transporte de Íons , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Prótons , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Imagem Individual de Molécula , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Especificidade por SubstratoRESUMO
RNA viruses are among the most prevalent pathogens and are a major burden on society. Although RNA viruses have been studied extensively, little is known about the processes that occur during the first several hours of infection because of a lack of sensitive assays. Here we develop a single-molecule imaging assay, virus infection real-time imaging (VIRIM), to study translation and replication of individual RNA viruses in live cells. VIRIM uncovered a striking heterogeneity in replication dynamics between cells and revealed extensive coordination between translation and replication of single viral RNAs. Furthermore, using VIRIM, we identify the replication step of the incoming viral RNA as a major bottleneck of successful infection and identify host genes that are responsible for inhibition of early virus replication. Single-molecule imaging of virus infection is a powerful tool to study virus replication and virus-host interactions that may be broadly applicable to RNA viruses.
Assuntos
Biossíntese de Proteínas , Vírus de RNA/fisiologia , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Interferons/metabolismo , Transporte de RNA , RNA Viral/genética , Reprodutibilidade dos Testes , Imagem Individual de Molécula , Fatores de TempoRESUMO
Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Biossíntese de Proteínas/genética , Transporte de RNA/genética , Imagem Individual de Molécula , Estresse Fisiológico , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Replicação do DNA/genética , DNA/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromossomos/metabolismo , Ciprofloxacina/farmacologia , DNA/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas Recombinantes , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Imagem Individual de Molécula , Espectrometria de Massas em Tandem , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/ultraestrutura , Inibidores da Topoisomerase II/farmacologiaRESUMO
In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.
Assuntos
Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Animais , Humanos , Cinética , Imagem Óptica/métodosRESUMO
Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Fatores de Transcrição/química , Vaccinia virus/ultraestrutura , Proteínas Virais/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Imagem Individual de Molécula , Vaccinia virus/genética , Vaccinia virus/metabolismoRESUMO
Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events-RF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics-reveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.
Assuntos
Nanotecnologia , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Cinética , Mutagênese , RNA Polimerase II/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Assuntos
Corantes Fluorescentes/química , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Genes Reporter , Células HEK293 , Humanos , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Ribossomos/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologiaRESUMO
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
Assuntos
DNA Helicases/metabolismo , DNA Helicases/fisiologia , Reparo do DNA/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA/fisiologia , Feminino , Masculino , Proteólise , Imagem Individual de Molécula/métodos , Xenopus laevis/metabolismoRESUMO
Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Metiltransferases/química , Complexos Multienzimáticos/química , Nucleotidiltransferases/química , Monoéster Fosfórico Hidrolases/química , Vaccinia virus/ultraestrutura , Proteínas Virais/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/ultraestrutura , RNA Mensageiro/química , Imagem Individual de Molécula , Transcrição Gênica , Vaccinia virus/genética , Vaccinia virus/metabolismoRESUMO
Super-resolution optical imaging based on the switching and localization of individual fluorescent molecules [photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), etc.] has evolved remarkably over the last decade. Originally driven by pushing technological limits, it has become a tool of biological discovery. The initial demand for impressive pictures showing well-studied biological structures has been replaced by a need for quantitative, reliable data providing dependable evidence for specific unresolved biological hypotheses. In this review, we highlight applications that showcase this development, identify the features that led to their success, and discuss remaining challenges and difficulties. In this context, we consider the complex topic of defining resolution for this imaging modality and address some of the more common analytical methods used with this data.