RESUMO
Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.
Assuntos
Proteínas , Transdução de Sinais , Animais , Humanos , Camundongos , Linhagem Celular , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Fosforilação , Sobrevivência CelularRESUMO
Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.
Assuntos
Vias Biossintéticas , Chlamydomonas reinhardtii , Proteínas de Cloroplastos , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , FotossínteseRESUMO
Genetically encoded fluorescent biosensors are powerful tools for monitoring biochemical activities in live cells, but their multiplexing capacity is limited by the available spectral space. We overcome this problem by developing a set of barcoding proteins that can generate over 100 barcodes and are spectrally separable from commonly used biosensors. Mixtures of barcoded cells expressing different biosensors are simultaneously imaged and analyzed by deep learning models to achieve massively multiplexed tracking of signaling events. Importantly, different biosensors in cell mixtures show highly coordinated activities, thus facilitating the delineation of their temporal relationship. Simultaneous tracking of multiple biosensors in the receptor tyrosine kinase signaling network reveals distinct mechanisms of effector adaptation, cell autonomous and non-autonomous effects of KRAS mutations, as well as complex interactions in the network. Biosensor barcoding presents a scalable method to expand multiplexing capabilities for deciphering the complexity of signaling networks and their interactions between cells.
Assuntos
Técnicas Biossensoriais/métodos , Células/ultraestrutura , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , HumanosRESUMO
Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Lisossomos/metabolismo , Mitofagia/fisiologia , Animais , Autofagia/fisiologia , Imunofluorescência/métodos , Corantes Fluorescentes/química , Humanos , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitofagia/genéticaRESUMO
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
Assuntos
Células Enteroendócrinas/metabolismo , RNA Mensageiro/genética , Células Cultivadas , Hormônios Gastrointestinais/genética , Trato Gastrointestinal/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Organoides/metabolismo , Fatores de Transcrição/genética , Transcriptoma/genéticaRESUMO
In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.
Assuntos
Corantes Fluorescentes/metabolismo , Genes Reporter , Imagem Óptica , Transdução de Sinais , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Células Piramidais/metabolismoRESUMO
Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.
Assuntos
Técnicas Biossensoriais/métodos , Espécies Reativas de Oxigênio/análise , Corantes Fluorescentes , Imagem Óptica , Oxirredução , Estresse OxidativoRESUMO
Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.
Assuntos
Bactérias/metabolismo , Parede Celular/metabolismo , Peptidoglicano/biossíntese , Aminoácidos/química , Bactérias/ultraestrutura , Parede Celular/ultraestrutura , Corantes Fluorescentes/química , Microscopia de Força Atômica , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular ß-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.
Assuntos
Arginina/química , Chaperonas Moleculares/química , Proteína FUS de Ligação a RNA/química , Esclerose Lateral Amiotrófica/metabolismo , Animais , Cátions , Metilação de DNA , Demência Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteína FUS de Ligação a RNA/metabolismo , Tirosina/química , Xenopus laevisRESUMO
Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Edição de Genes/métodos , Fatores de Transcrição/genética , Animais , Núcleo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Fatores de Transcrição/metabolismoRESUMO
Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.
Assuntos
Escherichia coli/metabolismo , Transdução de Sinais , Aerobiose , Anaerobiose , Sequência de Bases , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilaminas/metabolismo , Metilaminas/farmacologia , Oxigênio/metabolismo , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Regulação para CimaRESUMO
Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14 days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.
Assuntos
Bacteroides/classificação , Bacteroides/genética , Microbioma Gastrointestinal , Engenharia Genética , Animais , Bacteroides/isolamento & purificação , Vida Livre de Germes , Proteínas de Fluorescência Verde/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.
Assuntos
Vibrio cholerae/citologia , Vibrio cholerae/patogenicidade , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Locomoção , Camundongos , Peptidoglicano/metabolismo , Periplasma/metabolismo , Alinhamento de Sequência , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , VirulênciaRESUMO
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
Assuntos
Epigenômica , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula , Células Clonais/citologia , Fluorescência , Hematopoese , Inflamação/patologia , Camundongos , Transcrição GênicaRESUMO
Hydrogen peroxide (H2O2) is a prime member of the reactive oxygen species (ROS) family of molecules produced during normal cell function and in response to various stimuli, but if left unchecked, it can inflict oxidative damage on all types of biological macromolecules and lead to cell death. In this context, a major source of H2O2 for redox signaling purposes is the NADPH oxidase (Nox) family of enzymes, which were classically studied for their roles in phagocytic immune response but have now been found to exist in virtually all mammalian cell types in various isoforms with distinct tissue and subcellular localizations. Downstream of this tightly regulated ROS generation, site-specific, reversible covalent modification of proteins, particularly oxidation of cysteine thiols to sulfenic acids, represents a prominent posttranslational modification akin to phosphorylation as an emerging molecular mechanism for transforming an oxidant signal into a dynamic biological response. We review two complementary types of chemical tools that enable (a) specific detection of H2O2 generated at its sources and (b) mapping of sulfenic acid posttranslational modification targets that mediate its signaling functions, which can be used to study this important chemical signal in biological systems.
Assuntos
Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/metabolismo , Transdução de Sinais , Animais , Humanos , Oxirredução , Ácidos Sulfênicos/metabolismoRESUMO
Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.
Assuntos
Técnicas Biossensoriais , Epigênese Genética , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Lentivirus/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Montagem e Desmontagem da Cromatina , Epigenoma , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Lentivirus/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.
Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/fisiologia , Animais , Bioquímica/métodos , Biomarcadores/química , Caspases/química , Química Farmacêutica/métodos , Desenho de Fármacos , Escherichia coli/enzimologia , Humanos , Peptídeos/química , Proteômica/métodos , Especificidade por SubstratoRESUMO
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína Vermelha FluorescenteRESUMO
Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.
Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Complexo Shelterina , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.