A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis.

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Imaging

Background:

• Fluorescent protein biosensors are crucial for tracking pH dynamics in cellular events like exocytosis.
• Chemogenetic reporters offer a versatile alternative, combining chemical probe diversity with genetic encoding.
• Existing methods face challenges with off-target signals from unbound organic fluorophores.

Purpose of the Study:

• To develop a novel fluorogenic and chemogenetic pH sensor with enhanced signal selectivity for live-cell imaging.
• To create a wash-free imaging solution for monitoring pH variations during exocytosis.
• To establish a versatile alternative to fluorescent proteins for studying protein dynamics.

Main Methods:

• Screening of four fluorogenic molecular rotor structures based on protein chromophore analogues.
• Selection and characterization of pHluo-Halo-1, a cell-permeable molecular pH indicator.
• Application of pHluo-Halo-1 with HaloTag for wash-free imaging of CD63-HaloTag fusion protein secretion via TIRF microscopy.

Main Results:

• pHluo-Halo-1 exhibits good pH sensitivity (pKa of 6.3), suitable for monitoring exocytosis-related pH changes.
• The sensor demonstrates excellent labeling selectivity in cells through local fluorescence activation via HaloTag reaction.
• Successful application in tracking CD63-HaloTag secretion dynamics using TIRF microscopy.

Conclusions:

• The pHluo-Halo-1 sensor provides a highly selective, wash-free method for studying pH dynamics in live cells.
• This chemogenetic strategy offers a powerful alternative to fluorescent proteins for elucidating protein trafficking and regulatory mechanisms.
• The combination of tunable fluorogenic probes and protein tags opens new avenues for advanced cellular imaging.