A red-emitting carborhodamine for monitoring and measuring membrane potential.

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.

A red-emitting carborhodamine for monitoring and measuring membrane potential.

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.

Flipping the Switch: Reverse-Demand Voltage-Sensitive Fluorophores.

Fluorescence microscopy with fluorescent reporters that respond to environmental cues is a powerful method for interrogating biochemistry and biophysics in living systems. Photoinduced electron transfer (PeT) is commonly used as a trigger to modulate fluorescence in response to changes in the biological environment. PeT-based indicators rely on PeT either into the excited state (acceptor PeT) or out of the excited state (donor PeT). Our group has been developing voltage-sensitive fluorophores (VF dyes) that respond to changes in biological membrane potential (Vm). We hypothesize that the mechanism of voltage sensitivity arises from acceptor PeT (a-PeT) from an electron-rich aniline-containing molecular wire into the excited-state fluorophore, resulting in decreased fluorescence at negative Vm. In this work, we reversed the direction of electron flow to access donor-excited PeT (d-PeT) VF dyes by introducing electron-withdrawing rather than electron-rich molecular wires. VF dyes containing electron-withdrawing groups show voltage-sensitive fluorescence, but with the opposite polarity: hyperpolarizing Vm now gives fluorescence increases. We used a combination of computation and experiment to design and synthesize five d-PeT VF targets, two of which are voltage-sensitive.

VoltageFluor dyes and fluorescence lifetime imaging for optical measurement of membrane potential.

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.

Net Charge and Nonpolar Content Guide the Identification of Folded and Prion Proteins.

The degree of hydrophobicity and net charge per residue are physical properties that enable the discrimination of folded from intrinsically disordered proteins (IDPs) solely on the basis of amino acid sequence. Here, we improve upon the existing classification of proteins and IDPs based on the parameters mentioned above by adopting the scale of nonpolar content of Rose et al. and by taking amino acid side-chain acidity and basicity into account. The resulting algorithm, denoted here as net charge nonpolar or NECNOP, enables the facile prediction of the folded and disordered status of proteins under physiologically relevant conditions with >95% accuracy, based on amino-acid sequence alone. The NECNOP approach displays a much-enhanced performance for proteins with >140 residues, suggesting that small proteins are more likely to have irregular charge and hydrophobicity features. NECNOP analysis of the entire Escherichia coli proteome identifies specific net charge and nonpolar regions peculiar to soluble, integral membrane, and non-integral membrane proteins. Surprisingly, protein net charge and hydrophobicity are found to converge to specific values as chain length increases, across the E. coli proteome. In addition, NECNOP plots enable the straightforward identification of protein sequences corresponding to prion proteins and promise to serve as a powerful predictive tool for the design of large proteins. In summary, NECNOP plots are a straightforward approach that improves our understanding of the relation between the amino acid sequence and three-dimensional structure of proteins as a function of molecular mass.