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PHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps.

ABSTRACT: pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).

SUBMITTER: Liu A

PROVIDER: S-EPMC7930027 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Chemically synthesized near-infrared aza-BODIPY dyes displayed off-on fluorescence at acidic pH (pKa = 6.2-6.6) through the suppression of the photoinduced electron transfer and/or internal charge transfer process. The apparent pKas of the dyes were shifted well above physiological pH in a hydrophobic microenvironment, which led to "turned-on" fluorescence in micelles and liposomes at neutral and basic pH. Bovine serum albumin also activated the fluorescence, though to a much lesser extent. When these small molecular dyes entered cells, instead of being fluorescent only in acidic organelles, the whole cytoplasm exhibited fluorescence, with a signal/background ratio as high as ?10 in no-wash live-cell imaging. The dye 1-labeled cells remained highly fluorescent even after 3 days. Moreover, slight variations of the dye structure resulted in significantly different intracellular fluorescence behaviors, possibly because of their different cellular uptake and intracellular activation capabilities. After the separation of cellular components, the fraction of plasma membrane and endoplasmic reticulum showed the highest fluorescence, further confirming the fluorescence activation by membrane structures. The fluorescence intensity of these dyes at different intracellular pHs (6.80 and 8.00) did not differ significantly, indicating that intracellular pH did not play a critical role. Altogether, we showed here for the first time that the fluorescence of pH-sensitive aza-BODIPY dyes was switched intracellularly not by acidic pH, but by intracellular membranes (and proteins as well). The excellent membrane permeability, ultrahigh fluorescence contrast ratio, persistent fluorescent signal, and minimal biological interference of dye 1 make it an ideal choice for live-cell imaging and in vivo cell tracking. These findings also imply that the intracellular fluorescence properties of pH-sensitive dyes should be carefully examined before they are used as pH indicators.

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PHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps.

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