RESUMO
We introduce a framework for end-to-end integrative modeling of 3D single-cell multi-channel fluorescent image data of diverse subcellular structures. We employ stacked conditional ß-variational autoencoders to first learn a latent representation of cell morphology, and then learn a latent representation of subcellular structure localization which is conditioned on the learned cell morphology. Our model is flexible and can be trained on images of arbitrary subcellular structures and at varying degrees of sparsity and reconstruction fidelity. We train our full model on 3D cell image data and explore design trade-offs in the 2D setting. Once trained, our model can be used to predict plausible locations of structures in cells where these structures were not imaged. The trained model can also be used to quantify the variation in the location of subcellular structures by generating plausible instantiations of each structure in arbitrary cell geometries. We apply our trained model to a small drug perturbation screen to demonstrate its applicability to new data. We show how the latent representations of drugged cells differ from unperturbed cells as expected by on-target effects of the drugs.
Assuntos
Núcleo Celular/fisiologia , Forma Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Espaço Intracelular , Modelos Biológicos , Células Cultivadas , Biologia Computacional , Humanos , Imageamento Tridimensional , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Microscopia de Fluorescência , Análise de Célula ÚnicaRESUMO
This protocol measures the 3D Euclidean distance (Δ3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ3D is corrected through a Bayesian algorithm, with ΔEC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure ΔEC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espaço Intracelular/fisiologia , Microscopia de Fluorescência/métodos , Algoritmos , Células Cultivadas , Corantes Fluorescentes/química , Humanos , Cinetocoros/fisiologia , SoftwareRESUMO
Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.
Assuntos
Ciclo Celular/fisiologia , Espaço Intracelular/fisiologia , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Tamanho Celular , Citocinese/fisiologia , Espaço Intracelular/química , Imagem com Lapso de TempoRESUMO
Macromolecule condensates, phase separation, and membraneless compartments have become an important area of cell biology research where new biophysical concepts are emerging. This article discusses the possibility that condensates assemble on multivalent surfaces such as DNA, microtubules, or lipid bilayers by multilayer adsorption. Langmuir isotherm theory conceptualized saturable surface binding and deeply influenced physical biochemistry. Brunauer-Emmett-Teller (BET) theory extended Langmuir's ideas to multilayer adsorption. A BET-inspired biochemical model predicts that surface-binding proteins with a tendency to self-associate will form multilayered condensates on binding surfaces. These "bound condensates" are expected to assemble well below the saturation concentration for liquid-liquid phase separation, so they can compete subunits away from phase-separated droplets and are thermodynamically pinned to the binding surface. Tau binding to microtubules is an interesting test case. The nonsaturable binding isotherm is reminiscent of BET predictions, but assembly of Tau-rich domains at low concentrations requires a different model. Surface-bound condensates may find multiple biological uses, particularly in situations where it is important that condensate assembly is spatially constrained, such as gene regulation.
Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Organelas/metabolismo , Fenômenos Biofísicos , DNA/metabolismo , Espaço Intracelular/fisiologia , Bicamadas Lipídicas/metabolismo , Organelas/química , Propriedades de Superfície , TermodinâmicaRESUMO
The remote actuation of cellular processes such as migration or neuronal outgrowth is a challenge for future therapeutic applications in regenerative medicine. Among the different methods that have been proposed, the use of magnetic nanoparticles appears to be promising, since magnetic fields can act at a distance without interactions with the surrounding biological system. To control biological processes at a subcellular spatial resolution, magnetic nanoparticles can be used either to induce biochemical reactions locally or to apply forces on different elements of the cell. Here, we show that cell migration and neurite outgrowth can be directed by the forces produced by a switchable parallelized array of micro-magnetic pillars, following the passive uptake of nanoparticles. Using live cell imaging, we first demonstrate that adherent cell migration can be biased toward magnetic pillars and that cells can be reversibly trapped onto these pillars. Second, using differentiated neuronal cells we were able to induce events of neurite outgrowth in the direction of the pillars without impending cell viability. Our results show that the range of forces applied needs to be adapted precisely to the cellular process under consideration. We propose that cellular actuation is the result of the force on the plasma membrane caused by magnetically filled endo-compartments, which exert a pulling force on the cell periphery.
Assuntos
Movimento Celular/efeitos dos fármacos , Magnetismo/métodos , Nanopartículas de Magnetita/uso terapêutico , Espaço Intracelular/fisiologia , Campos Magnéticos , Nanopartículas de Magnetita/análise , Fenômenos Mecânicos , Crescimento Neuronal/efeitos dos fármacos , Fenômenos Físicos , Medicina Regenerativa/métodosRESUMO
Intracellular recording of spinal motoneurons in vivo provides a "gold standard" for determining the cells' electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.
Assuntos
Terapia por Estimulação Elétrica , Espaço Intracelular/fisiologia , Neurônios Motores/citologia , Medula Espinal/citologia , Potenciais de Ação/fisiologia , Animais , Eletrodos , Masculino , Ratos Wistar , Reprodutibilidade dos TestesRESUMO
Spatial organization is a characteristic of all cells, achieved in eukaryotic cells by utilizing both membrane-bound and membrane-less organelles. One of the key processes in eukaryotes is RNA splicing, which readies mRNA for translation. This complex and highly dynamical chemical process involves assembly and disassembly of many molecules in multiple cellular compartments and their transport among compartments. Our goal is to model the effect of spatial organization of membrane-less organelles (specifically nuclear speckles) and of organelle heterogeneity on splicing particle biogenesis in mammalian cells. Based on multiple sources of complementary experimental data, we constructed a spatial model of a HeLa cell to capture intracellular crowding effects. We then developed chemical reaction networks to describe the formation of RNA splicing machinery complexes and splicing processes within nuclear speckles (specific type of non-membrane-bound organelles). We incorporated these networks into our spatially-resolved human cell model and performed stochastic simulations for up to 15 minutes of biological time, the longest thus far for a eukaryotic cell. We find that an increase (decrease) in the number of nuclear pore complexes increases (decreases) the number of assembled splicing particles; and that compartmentalization is critical for the yield of correctly-assembled particles. We also show that a slight increase of splicing particle localization into nuclear speckles leads to a disproportionate enhancement of mRNA splicing and a reduction in the noise of generated mRNA. Our model also predicts that the distance between genes and speckles has a considerable effect on the mRNA production rate, with genes located closer to speckles producing mRNA at higher levels, emphasizing the importance of genome organization around speckles. The HeLa cell model, including organelles and sub-compartments, provides a flexible foundation to study other cellular processes that are strongly modulated by spatiotemporal heterogeneity.
Assuntos
Modelos Biológicos , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , Spliceossomos , Biologia Computacional , Simulação por Computador , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Espaço Intracelular/fisiologia , Cinética , RNA Mensageiro/química , Spliceossomos/química , Spliceossomos/metabolismo , Spliceossomos/fisiologiaRESUMO
The investigation of complex biological processes in vivo often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide-alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein-protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.
Assuntos
Conjugação Genética/fisiologia , Espaço Intracelular/metabolismo , Engenharia Metabólica/métodos , Biologia Sintética/métodos , Conjugação Genética/genética , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Espaço Intracelular/fisiologia , Modelos Biológicos , Proteínas/genética , Proteínas/metabolismoRESUMO
The transmembrane protein Crumbs (Crb), a key regulator of apical polarity, has a known involvement in establishment of the apical zonula adherens in epithelia, although the precise mechanism remains elusive. The zonula adherens are required to maintain the integrity and orderly arrangement of epithelia. Loss of the zonula adherens leads to morphogenetic defects in the tissues derived from epithelium. In this study, we revealed that the intracellular tail of Crb2a promoted the apical distribution of adherens junctions (AJs) in zebrafish retinal and lens epithelia, but caused assembly into unstable punctum adherens-like adhesion plaques. The extracellular region of Crb2a guided the transformation of AJs from the punctum adherens into stable zonula adherens. Accordingly, a truncated form of Crb2a lacking the extracellular region (Crb2aΔEX) could only partially rescue the retinal patterning defects in crb2a null mutant zebrafish (crb2am289). By contrast, constitutive over-expression of Crb2aΔEX disrupted the integrity of the outer limiting membrane in photoreceptors, which is derived from the zonula adherens of the retinal neuroepithelium. This study demonstrated that both the extracellular region and the intracellular tail of Crb2a are required to guide the formation of the apical zonula adherens.
Assuntos
Junções Aderentes/fisiologia , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Epitélio/metabolismo , Espaço Extracelular/fisiologia , Espaço Intracelular/fisiologia , Cristalino/metabolismo , Proteínas de Membrana/genética , Mutação , Retina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Cells are dynamic structures that continually generate and sustain mechanical forces within their environments. Cells respond to mechanical forces by changing their shape, moving, and differentiating. These reactions are caused by intracellular skeletal changes, which induce changes in cellular mechanical properties such as stiffness, elasticity, viscoelasticity, and adhesiveness. Interdisciplinary research combining molecular biology with physics and mechanical engineering has been conducted to characterize cellular mechanical properties and understand the fundamental mechanisms of mechanotransduction. In this review, we focus on the role of cytoskeletal proteins in cellular mechanics. The specific role of each cytoskeletal protein, including actin, intermediate filaments, and microtubules, on cellular elasticity is summarized along with the effects of interactions between the fibers.
Assuntos
Osso e Ossos/fisiologia , Elasticidade , Espaço Intracelular/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismoRESUMO
The conduction of electrical signals through cardiac tissue is essential for maintaining the function of the heart, and conduction abnormalities are known to potentially lead to life-threatening arrhythmias. The properties of cardiac conduction have therefore been the topic of intense study for decades, but a number of questions related to the mechanisms of conduction still remain unresolved. In this paper, we demonstrate how the so-called EMI model may be used to study some of these open questions. In the EMI model, the extracellular space, the cell membrane, the intracellular space and the cell connections are all represented as separate parts of the computational domain, and the model therefore allows for study of local properties that are hard to represent in the classical homogenized bidomain or monodomain models commonly used to study cardiac conduction. We conclude that a non-uniform sodium channel distribution increases the conduction velocity and decreases the time delays over gap junctions of reduced coupling in the EMI model simulations. We also present a theoretical optimal cell length with respect to conduction velocity and consider the possibility of ephaptic coupling (i.e. cell-to-cell coupling through the extracellular potential) acting as an alternative or supporting mechanism to gap junction coupling. We conclude that for a non-uniform distribution of sodium channels and a sufficiently small intercellular distance, ephaptic coupling can influence the dynamics of the sodium channels and potentially provide cell-to-cell coupling when the gap junction connection is absent.
Assuntos
Sistema de Condução Cardíaco/fisiologia , Modelos Cardiovasculares , Animais , Arritmias Cardíacas/fisiopatologia , Membrana Celular/fisiologia , Biologia Computacional , Simulação por Computador , Fenômenos Eletrofisiológicos , Espaço Extracelular/fisiologia , Junções Comunicantes/fisiologia , Humanos , Espaço Intracelular/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Canais de Sódio/fisiologiaRESUMO
Robust and precise ratio control of heterogeneous phenotypes within an isogenic population is an essential task, especially in the development and differentiation of a large number of cells such as bacteria, sensory receptors, and blood cells. However, the mechanisms of such ratio control are poorly understood. Here, we employ experimental and mathematical techniques to understand the combined effects of signal induction and gene expression stochasticity on phenotypic multimodality. We identify two strategies to control phenotypic ratios from an initially homogeneous population, suitable roughly to high-noise and low-noise intracellular environments, and we show that both can be used to generate precise fractional differentiation. In noisy gene expression contexts, such as those found in bacteria, induction within the circuit's bistable region is enough to cause noise-induced bimodality within a feasible time frame. However, in less noisy contexts, such as tightly controlled eukaryotic systems, spontaneous state transitions are rare and hence bimodality needs to be induced with a controlled pulse of induction that falls outside the bistable region. Finally, we show that noise levels, system response time, and ratio tuning accuracy impose trade-offs and limitations on both ratio control strategies, which guide the selection of strategy alternatives.
Assuntos
Diferenciação Celular/fisiologia , Espaço Intracelular/fisiologia , Modelos Biológicos , Biologia Sintética , Algoritmos , Escherichia coli/fisiologia , Expressão Gênica/fisiologia , Processos EstocásticosRESUMO
The microscopic assessment of the colocalization of fluorescent signals has been widely used in cell biology. Although imaging techniques have drastically improved over the past decades, the quantification of colocalization by measures such as the Pearson correlation coefficient or Manders overlap coefficient, has not changed. Here, we report the development of an R-based application that allows to (i) automatically segment cells and subcellular compartments, (ii) measure morphology and texture features, and (iii) calculate the degree of colocalization within each cell. Colocalization can thus be studied on a cell-by-cell basis, permitting to perform statistical analyses of cellular populations and subpopulations. ColocalizR has been designed to parallelize tasks, making it applicable to the analysis of large data sets. Its graphical user interface makes it suitable for researchers without specific knowledge in image analysis. Moreover, results can be exported into a wide range of formats rendering post-analysis adaptable to statistical requirements. This application and its source code are freely available at https://github.com/kroemerlab/ColocalizR.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Espaço Intracelular , Microscopia de Fluorescência/métodos , Software , Linhagem Celular Tumoral , Humanos , Espaço Intracelular/diagnóstico por imagem , Espaço Intracelular/fisiologia , Biologia de Sistemas , Interface Usuário-ComputadorRESUMO
Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an "optimum" [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.
Assuntos
Espaço Intracelular/fisiologia , Auto-Splicing de RNA Ribossômico/fisiologia , Catálise/efeitos dos fármacos , Biologia Celular , Biologia Computacional/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Magnésio/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Polietilenoglicóis , Dobramento de Proteína/efeitos dos fármacos , RNA Catalítico/metabolismo , RNA Catalítico/fisiologia , Auto-Splicing de RNA Ribossômico/metabolismoRESUMO
Absence of screening pigment in insect compound eyes has been linked to visual dysfunction. We investigated how its loss in a white-eyed mutant (W-E) alters the photoreceptor electrophysiological properties, opsin gene expression, and the behavior of the cockroach, Periplaneta americana. Whole-cell patch-clamp recordings of green-sensitive photoreceptors in W-E cockroaches gave reduced membrane capacitance, absolute sensitivity to light, and light-induced currents. Decreased low-pass filtering increased voltage-bump amplitudes in W-E photoreceptors. Intracellular recordings showed that angular sensitivity of W-E photoreceptors had two distinct components: a large narrow component with the same acceptance angle as wild type, plus a relatively small wide component. Information processing was evaluated using Gaussian white-noise modulated light stimulation. In bright light, W-E photoreceptors demonstrated higher signal gain and signal power than wild-type photoreceptors. Expression levels of the primary UV- and green-sensitive opsins were lower and the secondary green-sensitive opsin significantly higher in W-E than in wild-type retinae. In behavioral experiments, W-E cockroaches were significantly less active in dim green light, consistent with the relatively low light sensitivity of their photoreceptors. Overall, these differences can be related to the loss of screening pigment function and to a compensatory decrease in the rhabdomere size in W-E retinae.
Assuntos
Olho Composto de Artrópodes/fisiologia , Periplaneta/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Visão Ocular/fisiologia , Animais , Comportamento Animal/fisiologia , Capacitância Elétrica , Expressão Gênica , Proteínas de Insetos/metabolismo , Espaço Intracelular/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Atividade Motora , Opsinas/metabolismo , Técnicas de Patch-Clamp , Estimulação Luminosa , Pigmentação , Potássio/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Microcirculation is a network of perfused capillaries that connects macrocirculation with the cells. Although research has provided insight into microcirculatory blood flow, our knowledge remains limited. In this article, we propose a new role of microcirculation in physiological and shock states. In healthy individuals, microcirculation maintains cellular homeostasis via preconditioning. When blood volume decreases, the ensuing microcirculatory changes result in heterogeneity of perfusion and tissue oxygenation. Initially, this is partly compensated by the preserved autoregulation and the increase in the metabolism rate of cells, but at later stages, the loss of autoregulation activates the cascade of intracellular hypothermia.
Assuntos
Hipotermia/fisiopatologia , Microcirculação/fisiologia , Volume Sanguíneo , Temperatura Corporal , Hemodinâmica , Homeostase , Humanos , Espaço Intracelular/fisiologia , Precondicionamento Isquêmico , Modelos Cardiovasculares , Choque Hemorrágico/fisiopatologiaRESUMO
Single, isolated epithelial cells move randomly; however, during wound healing, organism development, cancer metastasis, and many other multicellular phenomena, motile cells group into a collective and migrate persistently in a directed manner. Recent work has examined the physics and biochemistry that coordinates the motions of these groups of cells. Of late, two mechanisms have been touted as being crucial to the physics of these systems: leader cells and jamming. However, the actual importance of these to collective migration remains circumstantial. Fundamentally, collective behavior must arise from the actions of individual cells. Here, we show how biophysical activity of an isolated cell impacts collective dynamics in epithelial layers. Although many reports suggest that wound closure rates depend on isolated cell speed and/or leader cells, we find that these correlations are not universally true, nor do collective dynamics follow the trends suggested by models for jamming. Instead, our experimental data, when coupled with a mathematical model for collective migration, shows that intracellular contractile stress, isolated cell speed, and adhesion all play a substantial role in influencing epithelial dynamics, and that alterations in contraction and/or substrate adhesion can cause confluent epithelial monolayers to exhibit an increase in motility, a feature reminiscent of cancer metastasis. These results directly question the validity of wound-healing assays as a general means for measuring cell migration, and provide further insight into the salient physics of collective migration.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Simulação por Computador , Cães , Células Epiteliais/citologia , Espaço Intracelular/fisiologia , Células Madin Darby de Rim Canino , Microscopia , Modelos Biológicos , Cicatrização/fisiologiaRESUMO
Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.
Assuntos
Técnicas Eletroquímicas/métodos , Miócitos Cardíacos/fisiologia , Nanoestruturas/química , Neurônios/fisiologia , Potenciais de Ação , Animais , Citoplasma/metabolismo , Espaço Extracelular/fisiologia , Espaço Intracelular/fisiologia , Microeletrodos , Fenômenos Físicos , Ratos Sprague-Dawley , Razão Sinal-RuídoRESUMO
Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/química , Substâncias Luminescentes/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Transporte Proteico/fisiologia , Animais , Biomarcadores/química , Complexo de Golgi/metabolismo , Espaço Intracelular/fisiologia , Camundongos , Vesículas Transportadoras/fisiologiaRESUMO
BACKGROUND: Investigation of neural circuit functioning often requires statistical interpretation of events in subthreshold electrophysiological recordings. This problem is non-trivial because recordings may have moderate levels of structured noise and events may have distinct kinetics. In addition, novel experimental designs that combine optical and electrophysiological methods will depend upon statistical tools that combine multimodal data. NEW METHOD: We present a Bayesian approach for inferring the timing, strength, and kinetics of post-synaptic currents (PSCs) from voltage-clamp electrophysiological recordings on a per event basis. The simple generative model for a single voltage-clamp recording flexibly extends to include additional structure to enable experiments designed to probe synaptic connectivity. RESULTS: We validate the approach on simulated and real data. We also demonstrate that extensions of the basic PSC detection algorithm can handle recordings contaminated with optically evoked currents, and we simulate a scenario in which calcium imaging observations, available for a subset of neurons, can be fused with electrophysiological data to achieve higher temporal resolution. COMPARISON WITH EXISTING METHODS: We apply this approach to simulated and real ground truth data to demonstrate its higher sensitivity in detecting small signal-to-noise events and its increased robustness to noise compared to standard methods for detecting PSCs. CONCLUSIONS: The new Bayesian event analysis approach for electrophysiological recordings should allow for better estimation of physiological parameters under more variable conditions and help support new experimental designs for circuit mapping.