Dataset Information

A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

ABSTRACT: Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). To demonstrate its utility, we have applied CPX to study (1) trafficking of biologically relevant synthetic vesicles and (2) intracellular processes involved in transmission of ?-synuclein (?S) pathology. Our results demonstrate that CPX can serve as a minimally perturbing probe for tracking the dynamics of biomolecules.

SUBMITTER: Jun JV

PROVIDER: S-EPMC6732186 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

Jun Joomyung V JV Haney Conor M CM Karpowicz Richard J RJ Giannakoulias Sam S Lee Virginia M-Y VM Petersson E James EJ Chenoweth David M DM

Journal of the American Chemical Society 20190128 5

Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). T ...[more]

PMID: 30657670

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Project description:Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI's uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.

Dataset Information

A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

Publications

A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

Similar Datasets

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