Abstract
The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe “semisynthetic” pH-sensitive protein conjugates with organic fluorophores, carbofluorescein, and Virginia Orange that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.
Highlights
The green fluorescent protein (GFP)-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds
To circumvent the problems with genetically encoded red fluorophores, we develop a “semisynthetic” sensor platform using red-shifted chemical fluorophores derived from fluorescein— carbofluorescein (CFl) and Virginia Orange (VO)—with pH sensitivities similar to SEP
We found that the propensity of the CFl and VO fluorophores to adopt the neutral lactone form (Fig. 1c, d) allows for efficient intracellular labeling (Fig. 2a) with SNAP-tag ligands 4 or 5 without the use of other masking groups, which are typically required for fluorescein-based compounds
Summary
The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Upon fusion with the plasma membrane, the contents of the vesicle rapidly equilibrate with the extracellular environment (pH 7.4) This large change in pH allows for the visualization of exocytosis using a pH-sensitive variant of green fluorescent protein (GFP) that is expressed as a fusion with a vesicular membrane protein[2]. To circumvent the problems with genetically encoded red fluorophores, we develop a “semisynthetic” sensor platform using red-shifted chemical fluorophores derived from fluorescein— carbofluorescein (CFl) and Virginia Orange (VO)—with pH sensitivities similar to SEP We have adapted these fluorophores to serve as probes for exo- and endocytosis in two ways. We label an antibody targeted to the extracellular epitope of a vesicular protein, synaptotagmin[1], which allow the imaging of the exo-/endocytosis cycle of an endogenous protein
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