Biology subjects

Leong, L. M.

Publications and source records attributed to Leong, L. M..

7 recordsLinked to original sources

bioRxiv2026

Imaging mitral/tufted glomeruli in the mouse olfactory bulb using the genetically encoded voltage indicator ArcLight

In the mammalian olfactory bulb, each olfactory receptor neuron type maps to receptor-specific channels called glomeruli. These input axons interact with the apical dendrites of mitral/tufted cells, which are projection neurons that send axons to the olfactory cortex. Therefore, each glomerulus reflects the input-output relationship for a different olfactory receptor type, which is transformed by a complex synaptic network. While prior 2-photon Ca2+ imaging experiments have shown that the glomerular output is heterogeneous with respect to how odor concentration information is processed, the nature of voltage dynamics remains unclear. Therefore, we used the genetically encoded voltage indicator (GEVI) ArcLight to image the glomerular output in vivo in anesthetized mice. We found that ArcLight could resolve both excitatory and suppressive odor-evoked signals from mitral/tufted glomeruli. ArcLight expression remained stable over multi-week imaging sessions and successfully captured odor- and concentration-specific patterns of activation across a large concentration range. Importantly, this approach allowed us to resolve heterogeneous concentration-response sensitivities and respiratory-coupled dynamics. We also demonstrate that several newer GEVIs can similarly measure odor-evoked signals using epifluorescence imaging. Our study establishes ArcLight and other emerging GEVIs as powerful tools for dissecting how sensory information is encoded and transformed through the mouse olfactory bulb.

neuroscience↗

bioRxiv2026

Turning the Fluorescent Protein Barrel into a Programmable Electrostatic Device Reveals Differential Bleaching States of the Chromophore

Genetically encoded voltage indicators (GEVIs) based on fluorescent proteins (FPs) report membrane potential changes through voltage-driven conformational rearrangements of a voltage-sensing domain that perturb the electrostatic and hydrogen-bonding environment of a fused FP chromophore. While the chromophore is largely protected from the external environment by the FP {beta}-can, previous results suggest that this structure can also act as a programmable electrostatic grid that influences chromophore flexibility and fluorescence transition states. Here, we show that varying the external polar offset of {beta}-sheet residues flanking a chromophore-proximal position systematically reshapes fluorescence transitions, altering response polarity and kinetics. Importantly, the influence of these external residues depends strongly on the internal sidechain chemistry, revealing reciprocal coupling between {beta}-sheet electrostatics and chromophore-proximal residues. We further show that the functional effects of glutamine and asparagine substitutions at this position are strongly context-dependent and can be tuned by appropriate external polar offsets, consistent with electrostatic control of internal sidechain orientation and expanding the molecular switch repertoire. Disrupting steady-state fluorescence further revealed that the secondary component of the voltage-dependent fluorescence transition is preferentially light-sensitive and can be selectively diminished by repeated stimulation, indicating that it arises from a photophysically distinct process separable from the primary response. In addition, altering the chromophore protonation equilibrium (e.g., T65S) substantially changes response kinetics, indicating that chromophore state influences how {beta}-can electrostatics couple to fluorescence transitions. Together, these results define general programming rules by which the electrostatic grid of the FP {beta}-barrel can be tuned to control chromophore optical properties both at rest and during perturbation of the steady state.

biophysics↗

bioRxiv2024

Recent odor experience selectively modulates olfactory sensitivity across the glomerular output in the mouse olfactory bulb

Although animals can reliably locate and recognize odorants embedded in complex environments, the neural circuits for accomplishing these tasks remain incompletely understood. Adaptation is likely to be important as it could allow neurons in a brain area to adjust to the broader sensory environment. Adaptive processes must be flexible enough to allow the brain to make dynamic adjustments, while maintaining sufficient stability so that organisms do not forget important olfactory associations. Processing within the mouse olfactory bulb is likely involved in generating adaptation, although there are conflicting models of how it transforms the glomerular output of the mouse olfactory bulb. Here we performed 2-photon Ca2+ imaging from mitral/tufted glomeruli in awake mice to determine the time course of recovery from adaptation, and whether it acts broadly or selectively across the glomerular population. Individual glomerular responses, as well as the overall population odor representation was similar across imaging sessions. However, odor-concentration pairings presented with interstimulus intervals upwards of 30-s evoked heterogeneous adaptation that was concentration-dependent. We demonstrate that this form of adaptation is unrelated to variations in respiration, and olfactory receptor neuron glomerular measurements indicate that it is unlikely to be inherited from the periphery. Our results indicate that the olfactory bulb output can reliably transmit stable odor representations, but recent odor experiences can selectively shape neural responsiveness for upwards of 30 seconds. We propose that neural circuits that allow for non-uniform adaptation across mitral/tufted glomerular could be important for making dynamic adjustments in complex odor environments.

neuroscience↗

bioRxiv2023

S1 hydrophobic residues alter the voltage sensing phosphatase enzymatic function and voltage sensing

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

biophysics↗

bioRxiv2022

Precise temporal control of GEVI conformations enables the visualization of charge migration in a fluorescent protein resulting in an improved optical response

Rapid and reproducible optical transitions of a fluorescent protein (FP) can be achieved with a Genetically Encoded Voltage Indicator (GEVI) via manipulation of the membrane potential. These transitions revealed novel effects of internal mutations near the chromophore that would not be detected under steady state conditions. Mutating an internal threonine (T203) affected the speed of the voltage-dependent fluorescence transition suggesting a conformational change inside the protein. These optical transitions also demonstrated interplay between internal and externally oriented sidechains of the {beta}-can structure. Replacing the steric hindrance of a phenylalanine near the chromophore with threonine (F165T) did not alter the resting fluorescence but resulted in a more complex fluorescent transition providing evidence for a flexible chromophore undergoing conformational changes. F165T orientation was influenced by the flanking external amino acids at positions 164 and 166 with 164F/165T/166T exacerbating the complexity of the voltage-dependent transition while 164T/165T/166F reduced the flexibility of the chromophore resembling the transition pattern of the original F165 version. Alphafold predictions reveal a threonine switch with different orientations of the F165T internal side chain depending on the direction of the offset in polarity at external positions 164 and 166. The crystal structures of the pH-sensitive FP, Super Ecliptic pHluorin and two derivatives solved in varying pH conditions also indicate interactions between the external protein surface and the internal environment providing another example of a threonine switch near the chromophore at T203. This ability to orient internal sidechains has led to the development of a novel GEVI that gets brighter upon depolarization of the plasma membrane, works at low light levels, is less susceptible to physiological pH, and provides in vivo signals. These observations affecting fluorescent transitions should also prove valuable to the development of any FP-based biosensor.

neuroscience↗

bioRxiv2020

Proton wires mediate the optical signal for ArcLight-type Genetically Encoded Voltage Indicators

The genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of hot spots capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the {beta}-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp. Statement of SignificanceArcLight-type GEVIs utilize proton pathways that send charge information outside of the FP to the internal chromophore enabling voltage induced conformational changes to affect fluorescence. These pathways are excitation wavelength specific suggesting that different external positions affect the protonated and deprotonated states of the chromophore.

biophysics↗

bioRxiv2020

Improving the flexibility of Genetically Encoded Voltage Indicators via intermolecular FRET

A new family of Genetically Encoded Voltage Indicators (GEVIs) has been developed based on inter-molecular Forster Resonance Energy Transfer (FRET). To test the hypothesis that the GEVI, ArcLight, functions via interactions between the fluorescent protein (FP) domain of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were co-transfected with both the FRET donor and acceptor GEVIs. Using a CFP donor and an RFP acceptor, we were able to observe a voltage dependent signal with a Stokes shift of over 200 nm. The intermolecular FRET strategy also works for rhodopsin-based probes potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. First, the signals are larger. Apparently the voltage-induced conformational change moves the two FPs independently thereby increasing the dynamic range. Second, the expression of the FRET donor and acceptor can be restricted independently enabling greater cell type specificity as well as refined subcellular voltage reporting.

neuroscience↗