Fluorogenic probes for imaging cellular phosphatase activity

Abstract

The ability to visualize enzyme activity in a cell, tissue, or living organism can greatly enhance our understanding of the biological roles of that enzyme. While many aspects of cellular signaling are controlled by reversible protein phosphorylation, our understanding of the biological roles of the protein phosphatases involved is limited. Here, we provide an overview of progress toward the development of fluorescent probes that can be used to visualize the activity of protein phosphatases. Significant advances include the development of probes with visible and near-infrared (near-IR) excitation and emission profiles, which provides greater tissue and whole-animal imaging capabilities. In addition, the development of peptide-based probes has provided some selectivity for a phosphatase of interest. Key challenges involve the difficulty of achieving sufficient selectivity for an individual member of a phosphatase enzyme family and the necessity of fully validating the best probes before they can be adopted widely.

Keywords: Cellular imaging; Fluorescent probe; Protein phosphatase.

Figure 1.

(a) Structures of visible light organic fluorophores. The number below each probe refers to the corresponding reference. (b) Cellular fluorescence resulting from two photon excitation of TP-Phos in HeLa cells. Image adapted from reference . (c) Cellular fluorescence from TCF-ALP in the absence (left) or presence (right) of the alkaline phosphatase inhibitor levamisole. Images adapted from reference under a CC BY license. Copyright 2019 Gwynne, Sedgwick, Gardiner, Williams, Kim, Lowe, Maillard, Jenkins, Bull, Sessler, Yoon and James

Figure 2.

(a) Structures of C-Sox probes used to measure phosphatase activity. Upon dephosphorylation, Mg²⁺ binding is significantly reduced, leading to a decrease in fluorescence. (b) Structure of the CD45 selective probe SP1. Fluorescence resulting from probe dephosphorylation in RAW 264.7 cells in the absence (top) or presence (bottom) of the PTP inhibitor orthovanadate. Green fluorescence indicates cell nuclei. Number below each probe is the corresponding reference. Images adapted from reference . Copyright 2012 National Academy of Sciences

Figure 3.

Structures of the (a) dihydroxanthene-hemicyanine probes and (b) cyanine dye probes. (c) Fluorescence imaging of HepG2 cells with QcyP (left), a mitochondria tracking dye (middle), and the merged images (right) demonstrating its mitochondria localization. Overlap between the two fluorescent probes is indicated by the yellow color. Images adapted from reference . (d) Near IR imagining of NALP up to 2 hours after injection into BALB/c mice with a tumor xenograph. Number below each probe is the corresponding reference. Images adapted from reference .