RESUMO
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⺠transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⺠probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⺠or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca(2+) and pH transients using live-cell fluorescence imaging.
Assuntos
Técnicas Biossensoriais/instrumentação , Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência/instrumentação , Espaço Intracelular/metabolismo , Imagem Molecular/métodos , Prótons , Animais , Fenômenos Biofísicos , Linhagem Celular , Endocitose , Endossomos/metabolismo , Corantes Fluorescentes/química , Íons , Lisossomos/metabolismo , Nanopartículas , Pontos Quânticos/química , Rodaminas/química , TitulometriaRESUMO
In the developing brain, cortical GABAergic interneurons migrate long distances from the medial ganglionic eminence (MGE) in which they are generated, to the cortex in which they settle. MGE cells express the cell adhesion molecule N-cadherin, a homophilic cell-cell adhesion molecule that regulates numerous steps of brain development, from neuroepithelium morphogenesis to synapse formation. N-cadherin is also expressed in embryonic territories crossed by MGE cells during their migration. In this study, we demonstrate that N-cadherin is a key player in the long-distance migration of future cortical interneurons. Using N-cadherin-coated substrate, we show that N-cadherin-dependent adhesion promotes the migration of mouse MGE cells in vitro. Conversely, mouse MGE cells electroporated with a construct interfering with cadherin function show reduced cell motility, leading process instability, and impaired polarization associated with abnormal myosin IIB dynamics. In vivo, the capability of electroporated MGE cells to invade the developing cortical plate is altered. Using genetic ablation of N-cadherin in mouse embryos, we show that N-cadherin-depleted MGEs are severely disorganized. MGE cells hardly exit the disorganized proliferative area. N-cadherin ablation at the postmitotic stage, which does not affect MGE morphogenesis, alters MGE cell motility and directionality. The tangential migration to the cortex of N-cadherin ablated MGE cells is delayed, and their radial migration within the cortical plate is perturbed. Altogether, these results identify N-cadherin as a pivotal adhesion substrate that activates cell motility in future cortical interneurons and maintains cell polarity over their long-distance migration to the developing cortex.
Assuntos
Caderinas/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Córtex Cerebral/metabolismo , Interneurônios/metabolismo , Neurogênese/fisiologia , Animais , Caderinas/deficiência , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Feminino , Previsões , Humanos , Masculino , Camundongos , Camundongos Transgênicos , GravidezRESUMO
Array tomography (AT) allows one to localize sub-cellular components within the structural context of cells in 3D through the imaging of serial sections. Using this technique, the z-resolution can be improved physically by cutting ultra-thin sections. Nevertheless, conventional immunofluorescence staining of those sections is time consuming and requires relatively large amounts of costly antibody solutions. Moreover, epitopes are only readily accessible at the section's surface, leaving the volume of the serial sections unlabeled. Localization of receptors at neuronal synapses in 3D in their native cellular ultrastructural context is important for understanding signaling processes. Here, we present in vivo labeling of receptors via fluorophore-coupled tags in combination with super-resolution AT. We present two workflows where we label receptors at the plasma membrane: first, in vivo labeling via microinjection with a setup consisting of readily available components and self-manufactured microscope table equipment and second, live receptor labeling by using a cell-permeable tag. To take advantage of a near-to-native preservation of tissues for subsequent scanning electron microscopy (SEM), we also apply high-pressure freezing and freeze substitution. The advantages and disadvantages of our workflows are discussed.
Assuntos
Sinapses , Tomografia , Animais , Sinapses/metabolismo , Sinapses/ultraestrutura , Tomografia/métodos , Imageamento Tridimensional/métodos , Coloração e Rotulagem/métodos , Camundongos , Microscopia Eletrônica de Varredura/métodos , Corantes Fluorescentes/química , Microinjeções/métodos , Neurônios/metabolismo , RatosRESUMO
Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca2+] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.
Assuntos
Cálcio/química , Indicadores e Reagentes/química , Quelantes/síntese química , Quelantes/química , Ácido Egtázico/análogos & derivados , Ácido Egtázico/síntese química , Ácido Egtázico/química , Eletroquímica , Fluorescência , Indicadores e Reagentes/síntese química , Espectrofotometria Infravermelho , Ácido Trifluoracético/químicaRESUMO
Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.
Assuntos
Microscopia de Fluorescência/métodos , Pontos Quânticos , Absorção , Biotinilação , Citoplasma/metabolismo , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Modelos Estatísticos , Nanopartículas , Nanotecnologia/métodos , Semicondutores , Estreptavidina/química , Fatores de TempoRESUMO
The embryonic development of the cortex involves a phase of long distance migration of interneurons born in the basal telencephalon. Interneurons first migrate tangentially and then reorient their trajectories radially to enter the developing cortex. We have shown that migrating interneurons can assemble a primary cilium, which maintains the centrosome to the plasma membrane and processes signals to control interneuron trajectory (Baudoin et al., 2012). In the developing cortex, N-cadherin is expressed by migrating interneurons and by cells in their migratory pathway. N-cadherin promotes the motility and maintains the polarity of tangentially migrating interneurons (Luccardini et al., 2013). Because N-cadherin is an important factor that regulates the migration of medial ganglionic eminence (MGE) cells in vivo, we further characterized the motility and polarity of MGE cells on a substrate that only comprises this protein. MGE cells migrating on a N-cadherin substrate were seven times faster than on a laminin substrate and two times faster than on a substrate of cortical cells. A primary cilium was much less frequently observed on MGE cells migrating on N-cadherin than on laminin. Nevertheless, the mature centriole (MC) frequently docked to the plasma membrane in MGE cells migrating on N-cadherin, suggesting that plasma membrane docking is a basic feature of the centrosome in migrating MGE cells. On the N-cadherin substrate, centrosomal and nuclear movements were remarkably synchronous and the centrosome remained near the nucleus. Interestingly, MGE cells with cadherin invalidation presented centrosomal movements no longer coordinated with nuclear movements. In summary, MGE cells migrating on a pure substrate of N-cadherin show fast, coordinated nuclear and centrosomal movements, and rarely present a primary cilium.
RESUMO
The role of the microfilaments and microtubules cytoskeleton in the stability of the subcellular distribution and function of GABAA receptors has been studied in rat cerebellar granule cells in culture. The disruption of either the microfilaments or the microtubules structures did not result in detectable changes in the receptors distribution, as assessed by immunocytochemistry, or in their function, as assessed by the whole-cell patch-clamp approach. A distinct disruption of both the subcellular distribution and the function of the GABAA receptors was found only if both microfilaments and microtubules were destroyed. The results suggest that, in the short term, the plasma membrane localization/stabilization and function of these receptors in granule cells are largely independent from microfilaments and microtubules individually, although they obviously depend on the presence of an organized cellular framework.
Assuntos
Citoesqueleto de Actina/química , Cerebelo/química , Cerebelo/citologia , Microtúbulos/química , Receptores de GABA-A/análise , Animais , Animais Recém-Nascidos , Células Cultivadas , Ratos , Ratos Sprague-DawleyRESUMO
In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.
Assuntos
Movimento Celular/fisiologia , Centrossomo/fisiologia , Córtex Cerebral/embriologia , Cílios/fisiologia , Proteínas Hedgehog/fisiologia , Neurônios/fisiologia , Animais , Gânglios da Base/citologia , Gânglios da Base/embriologia , Polaridade Celular/fisiologia , Centríolos/fisiologia , Córtex Cerebral/citologia , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Neurogênese/fisiologia , Neurônios/citologia , Transdução de Sinais/fisiologiaRESUMO
The limited choice and poor performance of red-emitting calcium (Ca(2+)) indicators have hampered microfluorometric measurements of the intracellular free Ca(2+) concentration in cells expressing yellow- or green-fluorescent protein constructs. A long-wavelength Ca(2+) indicator would also permit a better discrimination against cellular autofluorescence than the commonly used fluorescein-based probes. Here, we report an improved synthesis and characterization of Calcium Ruby, a red-emitting probe consisting of an extended rhodamine chromophore (578/602 nm peak excitation/emission) conjugated to BAPTA and having an additional NH(2) linker arm. The low-affinity variant (K(D,Ca) approximately 30 microM) with a chloride in meta position that was specifically designed for the detection of large and rapid Ca(2+) transients. While Calcium Ruby is a mitochondrial Ca(2+)probe, its conjugation, via the NH(2) tail, to a 10,000 MW dextran abolishes the sub-cellular compartmentalization and generates a cytosolic Ca(2+) probe with an affinity matched to microdomain Ca(2+) signals. As an example, we show depolarization-evoked Ca(2+) signals triggering the exocytosis of individual chromaffin granules. Calcium Ruby should be of use in a wide range of applications involving dual- or triple labeling schemes or targeted sub-cellular Ca(2+) measurements.
Assuntos
Cálcio/metabolismo , Células Cromafins/metabolismo , Citoplasma/metabolismo , Dextranos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/metabolismo , Rodaminas/metabolismo , Medula Suprarrenal/citologia , Animais , Sinalização do Cálcio , Bovinos , Dextranos/química , Imageamento Tridimensional , Microdomínios da Membrana/metabolismo , Rodaminas/química , Espectrometria de Fluorescência , Coloração e RotulagemRESUMO
Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Nanopartículas/química , Nanotecnologia/métodos , Cicloexanos/química , Difusão , Emulsões , Etanol/química , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Micelas , Microscopia Eletrônica de Transmissão/métodos , Modelos Estatísticos , Óleos , TemperaturaRESUMO
A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and multiple organic fluorophores as acceptors is reported and the photophysics of the system is characterized. Most nanoparticle-based biosensors reported so far use the detection of specific changes of the donor/acceptor distance under the influence of analyte binding. Our nanoparticle design on the other hand leads to sensors that detect spectral changes of the acceptor (under the influence of analyte binding) at fixed donor/acceptor distance by the introduction of the acceptor into the polymer coating. This approach allows for short acceptor-donor separation and thus for high-energy transfer efficiencies. Advantageously, the binding properties of the hydrophilic polymer coating further allows for addition of poly(ethylene glycol) shells for improved colloidal stability.
Assuntos
Cristalização/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Polietilenoglicóis/química , Pontos Quânticos , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Nanotecnologia/métodos , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Semiconductor nanocrystals (NCs) are increasingly being used as photoluminescen markers in biological imaging. Their brightness, large Stokes shift, and high photostability compared to organic fluorophores permit the exploration of biological phenomena at the single-molecule scale with superior temporal resolution and spatial precision. NCs have predominantly been used as extracellular markers for tagging and tracking membrane proteins. Successful internalization and intracellular labelling with NCs have been demonstrated for both fixed immunolabelled and live cells. However, the precise localization and subcellular compartment labelled are less clear. Generally, live cell studies are limited by the requirement of fairly invasive protocols for loading NCs and the relatively large size of NCs compared to the cellular machinery, along with the subsequent sequestration of NCs in endosomal/lysosomal compartments. For long-period observation the potential cytotoxicity of cytoplasmically loaded NCs must be evaluated. This review focuses on the challenges of intracellular uses of NCs.
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We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.
Assuntos
Cinesinas/fisiologia , Proteínas Motores Moleculares/fisiologia , Pontos Quânticos , Transporte Biológico , Fenômenos Biomecânicos , Células HeLa , HumanosAssuntos
Cerebelo/citologia , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular , Movimento Celular , Cerebelo/embriologia , Imunoglobulinas/metabolismo , Camundongos , Morfogênese , Neurônios/metabolismo , Domínios PDZ , Ligação Proteica , Células-Tronco/fisiologia , UbiquitinaçãoRESUMO
Neurons were free-hand isolated under the stereomicroscope from the bovine trigeminal mesencephalic nucleus. These neurons were challenged with monoclonal antibodies against the alpha1 and the beta2/3 subunits of the GABA(A) receptor. The neurons showed a strong reaction to both antibodies. The reaction was mainly intracellular in the round cell bodies and in the axoplasm of the axon emerging from these pseudounipolar cells. This suggests the synthesis of these subunits in the cell body and their transport along the axon.
Assuntos
Mesencéfalo/fisiologia , Neurônios Aferentes/fisiologia , Receptores de GABA-A/metabolismo , Animais , Transporte Axonal , Bovinos , Subunidades Proteicas/metabolismo , Frações Subcelulares/metabolismoRESUMO
Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.
Assuntos
Neurônios/imunologia , Receptores de GABA-A/imunologia , Núcleo Vestibular Lateral/imunologia , Animais , Bovinos , Imuno-Histoquímica , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Núcleo Vestibular Lateral/metabolismoRESUMO
Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.