14 research outputs found
Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles
Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter
Development of Strategies for Targeting Voltage-Sensitive Fluorescent Dyes to Subcellular Organelles
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Development of Strategies for Targeting Voltage-Sensitive Fluorescent Dyes to Subcellular Organelles
Membrane potential is a crucial signaling factor in many biological processes and understanding these processes requires measurements of membrane potential in a normal physiological environment. Voltage-sensitive fluorophores provide a non-invasive tool for high throughput optical measurements of membrane potential. In the Miller lab, we focus on a class of voltage-sensitive fluorophores known as VoltageFluors which rapidly modulate their fluorescence in response to changes in membrane potential via changes in the rate of photoinduced electron transfer from an electron-rich aniline. Prior to this work, VoltageFluors had only been targeted to the extracellular surface of plasma membranes. This work reports the development of new strategies for targeting of VoltageFluors to specific membranes including those of subcellular organelles. Chapter 1 provides background material on methods of targeting fluorescent probes to organelles. Chapter 2 describes the targeting and methods for use of a novel VoltageFluor, SPIRIT RhoVR, that localizes to the mitochondrial inner membrane via electrostatic accumulation followed by unmasking of an ester for retention in mitochondria. Chapter 3 describes a tetrazine-transcyclooctene click chemistry strategy for targeting of a novel VoltageFluor, LUnAR RhoVR, to multiple cellular locations with the primary focus being the endoplasmic reticulum (ER). Using LUnAR RhoVR, we were able to monitor ER membrane potential during calcium release and establish the directionality of ER-plasma membrane electrical coupling. Chapter 4 focuses on the physics behind energy transfer in fluorescent dyes, provides a computational description of VoltageFluor sensitivity, and proposes an approach for using VoltageFluors as ratiometric sensors of membrane potential. This dissertation follows this main body of work with two appendices describing initial steps towards synthesizing water-soluble and enzymatically uncageable dyes that would enhance targeting of fluorescent dyes to specific membranes in complex samples
Development of Strategies for Targeting Voltage-Sensitive Fluorescent Dyes to Subcellular Organelles
Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Small Molecule, Permeable, Internally Redistributing for Inner Membrane Targeting Rhodamine Voltage Reporter
Mitochondria are the site of aerobic respiration, producing
ATP via oxidative phosphorylation as protons flow down their electrochemical
gradient through ATP synthase. This negative membrane potential across the
inner mitochondrial membrane (ΔΨm) represents a fundamental
biophysical parameter central to cellular life. Traditional, electrode-based
methods for recording membrane potential are impossible to implement on
mitochondria within intact cells. Fluorescent ΔΨm indicators based
on cationic, lipophilic dyes are a common alternative, but these indicators are
complicated by concentration-dependent artifacts and the requirement to
maintain dye in the extracellular solution to visualize reversible ΔΨm
dynamics. Here, we report the first example of a fluorescent ΔΨm
reporter that does not rely on ΔΨm-dependent accumulation. We
re-directed the localization of a photoinduced electron transfer (PeT)-based
indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the
carboxylate of RhoVR 1 as an acetoxy methyl (AM) ester. Once within
mitochondria, esterases remove the AM-ester, trapping RhoVR inside of the
mitochondrial matrix, where it can incorporate within the inner membrane and
reversibly report on changes in ΔΨm. We show that this Small molecule,
Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage
reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of
different cell lines and responds reversibly to changes in ΔΨm
induced by exceptionally low concentrations of the uncoupler FCCP without the
need for exogenous pools of dye (unlike traditional, accumulation-based
rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging,
enabling simultaneous, real-time observation of cytosolic Ca2+,
plasma membrane potential, and reversible ΔΨm dynamics.</p
Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter
Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨm) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨm indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-dependent accumulation. We redirected the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxymethyl (AM) ester. Once within mitochondria, esterases remove the AM ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨm. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage Reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨm induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca2+, plasma membrane potential, and reversible ΔΨm dynamics
A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence
We report a small molecule enzyme pair for optical voltage
sensing via quenching of bioluminescence. This Quenching Bioluminescent
Voltage Indicator, or Q-BOLT, pairs the dark absorbing,
voltage-sensitive dipicrylamine with membrane-localized bioluminescence from
the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through
resonance energy transfer (QRET) as a function of membrane potential. Fusion of
HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy
transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is
ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct
visualization of changes in membrane potential in live cells via three distinct
readouts: change in QRET, BRET, and the ratio between bioluminescence emission
and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and
>100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for
excitation light. In cardiac monolayers derived from human induced pluripotent
stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations.
Q-BOLT is the first example of a hybrid small molecule – protein voltage
indicator that does not require excitation light and may be useful in contexts
where excitation light is limiting.
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Bioorthogonal, fluorogenic targeting of voltage-sensitive fluorophores for visualizing membrane potential dynamics in cellular organelles
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 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 LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine based Voltage Reporter) for optically monitoring membrane potential changes in the endoplasmic reticulum (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 LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of LUnAR RhoVR and Cer-TCO is voltage sensitive and that LUnAR RhoVR can be targeted to intact ER in living cells. Using 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 LUnAR RhoVR to directly visualize functional coupling between plasma-ER membrane 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 LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology
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Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles
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