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1.
Proc Natl Acad Sci U S A ; 121(14): e2315264121, 2024 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-38551837

RESUMEN

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.


Asunto(s)
Colorantes Fluorescentes , Potenciales de la Membrana , Potenciales de Acción , Membrana Celular , Microscopía Fluorescente/métodos
2.
J Am Chem Soc ; 144(27): 12138-12146, 2022 07 13.
Artículo en Inglés | MEDLINE | ID: mdl-35776693

RESUMEN

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma-ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.


Asunto(s)
Colorantes Fluorescentes , Orgánulos , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/metabolismo , Ionóforos/metabolismo , Lisosomas/metabolismo , Potenciales de la Membrana , Orgánulos/metabolismo , Rodaminas/metabolismo
3.
Org Biomol Chem ; 15(48): 10160-10163, 2017 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-29182189

RESUMEN

An indicator displacement assay has been adapted to detect the diol products of the aldol reaction between 4-nitrobenzaldehyde and hydroxyacetone in crude reaction mixtures. This provides a rapid colorimetric method of detecting product formation and thus evaluating potential catalysts, which is demonstrated using multiple catalytic peptides.


Asunto(s)
Colorimetría , Péptidos/química , Catálisis , Estructura Molecular , Péptidos/síntesis química
4.
Bioelectricity ; 6(1): 34-41, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38516638

RESUMEN

Introduction: Membrane potential (Vm), the voltage across a cell membrane, is an important biophysical phenomenon, central to the physiology of cells, tissues, and organisms. Voltage-sensitive fluorescent indicators are a powerful method for interrogating membrane potential in living systems, but most indicators are best suited for detecting changes in membrane potential rather than measuring values of the membrane potential. One promising approach is to use fluorescence lifetime imaging microscopy (FLIM) in combination of chemically synthesized dyes to estimate a value of membrane potential. However, a drawback is that chemically synthesized dyes show poor specificity of staining. Objectives: To address this problem, we applied a chemical-genetic voltage imaging approach to FLIM to enable optical estimation of membrane potential values from genetically defined cells. Results: In this report, we detail the characterization and evaluation of two of these systems in mammalian cells. We further validate the use of a FLIM-based chemical genetic voltage indicator in mammalian neurons. Conclusions: Finally, we discuss opportunities for future improvements to chemical-genetic FLIM-based voltage indicators.

5.
bioRxiv ; 2023 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-37873283

RESUMEN

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes, but also measure values of membrane potentials. This study discloses a new fluorescent indicator which can address both. We describe the synthesis of a new sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials with greater signal-to-noise than state-of-the-art BeRST (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of action potentials in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

6.
ACS Cent Sci ; 8(1): 118-121, 2022 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-35111902

RESUMEN

Voltage-sensitive fluorescent reporters can reveal fast changes in the membrane potential in neurons and cardiomyocytes. However, in many cases, illumination in the presence of the fluorescent reporters results in disruptions to the action potential shape that limits the length of recording sessions. We show here that a molecular prosthetic approach, previously limited to fluorophores, rather than indicators, can be used to substantially prolong imaging in neurons and cardiomyocytes.

7.
Annu Rev Biophys ; 50: 447-468, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33651949

RESUMEN

Membrane potential (Vmem) is a fundamental biophysical signal present in all cells. Vmem signals range in time from milliseconds to days, and they span lengths from microns to centimeters. Vmem affects many cellular processes, ranging from neurotransmitter release to cell cycle control to tissue patterning. However, existing tools are not suitable for Vmem quantification in many of these areas. In this review, we outline the diverse biology of Vmem, drafting a wish list of features for a Vmem sensing platform. We then use these guidelines to discuss electrode-based and optical platforms for interrogating Vmem. On the one hand, electrode-based strategies exhibit excellent quantification but are most effective in short-term, cellular recordings. On the other hand, optical strategies provide easier access to diverse samples but generally only detect relative changes in Vmem. By combining the respective strengths of these technologies, recent advances in optical quantification of absolute Vmem enable new inquiries into Vmem biology.


Asunto(s)
Potenciales de la Membrana , Animales , Humanos , Espacio Intracelular , Factores de Tiempo
8.
Methods Enzymol ; 653: 267-293, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34099175

RESUMEN

Membrane potential is a fundamental biophysical parameter common to all of cellular life. Traditional methods to measure membrane potential rely on electrodes, which are invasive and low-throughput. Optical methods to measure membrane potential are attractive because they have the potential to be less invasive and higher throughput than classic electrode based techniques. However, most optical measurements rely on changes in fluorescence intensity to detect changes in membrane potential. In this chapter, we discuss the use of fluorescence lifetime imaging microscopy (FLIM) and voltage-sensitive fluorophores (VoltageFluors, or VF dyes) to estimate the millivolt value of membrane potentials in living cells. We discuss theory, application, protocols, and shortcomings of this approach.


Asunto(s)
Colorantes Fluorescentes , Imagen Óptica , Potenciales de la Membrana , Microscopía Fluorescente
9.
Sci Adv ; 5(12): eaay3771, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31897432

RESUMEN

Imaging neuromodulation with synthetic probes is an emerging technology for studying neurotransmission. However, most synthetic probes are developed through conjugation of fluorescent signal transducers to preexisting recognition moieties such as antibodies or receptors. We introduce a generic platform to evolve synthetic molecular recognition on the surface of near-infrared fluorescent single-wall carbon nanotube (SWCNT) signal transducers. We demonstrate evolution of molecular recognition toward neuromodulator serotonin generated from large libraries of ~6.9 × 1010 unique ssDNA sequences conjugated to SWCNTs. This probe is reversible and produces a ~200% fluorescence enhancement upon exposure to serotonin with a K d = 6.3 µM, and shows selective responsivity over serotonin analogs, metabolites, and receptor-targeting drugs. Furthermore, this probe remains responsive and reversible upon repeat exposure to exogenous serotonin in the extracellular space of acute brain slices. Our results suggest that evolution of nanosensors could be generically implemented to develop other neuromodulator probes with synthetic molecular recognition.


Asunto(s)
Rayos Infrarrojos , Neurotransmisores/química , Serotonina/química , Serotonina/metabolismo , Transmisión Sináptica/fisiología , Animales , Secuencia de Bases , Encéfalo/citología , ADN de Cadena Simple/química , Espacio Extracelular/diagnóstico por imagen , Ligandos , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Nanotubos de Carbono/química , Imagen Óptica , Polinucleótidos/química , Transductores
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