Biology subjects

Laughlin, S. T.

Publications and source records attributed to Laughlin, S. T..

4 recordsLinked to original sources

bioRxiv2025

Chemogenetic and optogenetic strategies for spatiotemporal control of split-enzyme-based calcium recording

Methods for monitoring physiological changes in cellular Ca2+ levels have been in high demand for their utility in monitoring neuronal signaling. Recently, we introduced SCANR (Split-Tobacco Etch Virus (TEV) protease Calcium-regulated Neuron Recorder), which reports on Ca2+ changes in cells through the binding of calmodulin and M13 to reconstitute an active TEV protease. First-generation SCANR marked all of the Ca2+ spikes that occur throughout the lifetime of the cell, but it did not have a mechanism for controlling the time window in which recording of physiological changes in Ca2+ occurred. Here, we explore both chemical and light-based strategies for controlling the time and place in which Ca2+ recording occurs. We describe the adaptation of six popular chemo- and opto-genetics methods for controlling protein activity and subcellular localization to the SCANR system. We report two successful strategies, one that leverages the LOV-J optogenetics system for sterically controlling protein interactions and another that employs chemogenetic manipulation of subcellular protein distribution using the FKBP/FRB rapamycin binding pair.

neuroscience↗

bioRxiv2025

Blood-brain-barrier permeable fluorescent astrocyte probes.

The brains astrocytes play pivotal roles in brain homeostasis, but their contributions to neural circuit function are only beginning to be explored. In order to study astrocytes in their native environment, the field needs imaging probes that are cell-specific, non-toxic, and complementary to existing genetic techniques. We recently described a panel of cationic fluorophores that selectively label rodent and zebrafish astrocytes at low concentrations and short incubation times. Here, we expand on those first-generation astrocyte probes with a new class of brighter and more photostable dyes that allow for imaging across various wavelengths. We demonstrate these probes subcellular localization to the mitochondria and ability to cross the blood-brain barrier in zebrafish. This new class of astrocyte markers will aid researchers in visualizing and identifying subpopulations of astrocytes in both rodent and zebrafish model systems.

neuroscience↗

bioRxiv2023

Redox Activated Substrates for Enhancing Activatable Cyclopropene Bioorthogonal Reactions

Bioorthogonal chemistry has become a mainstay in chemical biology and is making inroads in the clinic with recent advances in protein targeting and drug release. Since the fields beginning, a major focus has been on designing bioorthogonal reagents with good selectivity, reactivity, and stability in complex biological environments. More recently, chemists have imbued reagents with new functionalities like click-and-release or light/enzyme-controllable reactivity. We have previously developed a controllable cyclopropene-based bioorthogonal ligation, which has excellent stability in physiological conditions and can be triggered to react with tetrazines by exposure to enzymes, biologically significant small molecules, or light spanning the visual spectrum. Here, to improve reactivity and gain a better understanding of this system, we screened diene reaction partners for the cyclopropene. We found that a cyclopropene-quinone pair is 26 times faster than reactions with 1,2,4,5-tetrazines. Additionally, we showed that the reaction of the cyclopropene-quinone pair can be activated by two orthogonal mechanisms, caging group removal on the cyclopropene and oxidation/reduction of the quinone. Finally, we demonstrated that this caged cyclopropene-quinone can be used as a bioorthogonal imaging tool to label the membranes of cultured cells.

biochemistry↗

bioRxiv2023

Pyranthiones/Pyrones: Click and Release Donors for Subcellular Hydrogen Sulfide Delivery and Labeling

Hydrogen sulfide (H2S), one of the most important gasotransmitters, plays a critical role in endogenous signaling pathways of many diseases. However, developing H2S donors with both tunable release kinetics and high release efficiency for subcellular delivery has been challenging. Here, we describe a click and release reaction between pyrone/pyranthiones and bicyclononyne (BCN). This reaction features a release of CO2/COS with second-order rate constants comparable to Strain-Promoted Azide-Alkyne Cycloaddition reactions (SPAACs). Interestingly, pyranthiones showed enhanced reaction rates compared to their pyrone counterparts. We investigated pyrone biorthogonality and demonstrated their utility in protein labeling applications. Moreover, we synthesized substituted pyranthiones with H2S release kinetics that can address the range of physiologically relevant H2S dynamics in cells and achieved quantitative H2S release efficiency in vitro. Finally, we explored the potential of pyranthiones as H2S/COS donors for mitochondrial-targeted H2S delivery in living cells.

biochemistry↗