Engineering an effective Mn-binding MRI reporter protein by subcellular targeting

Area of Science:

• Biophysics
• Molecular Biology
• Medical Imaging

Background:

• Manganese (Mn) is a biologically active metal and effective contrast agent for Mn-enhanced MRI (MEMRI).
• Genetic reporters are valuable tools for MEMRI, enabling targeted Mn accumulation and enhanced imaging contrast.

Purpose of the Study:

• To develop and evaluate a novel Mn-binding protein, MntR, as a genetic reporter for MEMRI.
• To engineer MntR for expression in mammalian cells and target it to specific subcellular locations.

Main Methods:

• The bacterial Mn-binding protein MntR was engineered for mammalian cell expression and subcellular targeting (cytosol, ER, Golgi).
• HEK293 and B16 melanoma cells were transfected and analyzed using immunocytochemistry, MR imaging, and relaxometry.
• In vivo studies were conducted using a B16 tumor model.

Main Results:

• Immunocytochemistry confirmed successful MntR targeting to the cytosol, endoplasmic reticulum, and Golgi apparatus.
• Targeting MntR to the Golgi apparatus resulted in significant R1 changes and T1 contrast in vitro and in vivo.
• Co-expression with DMT1 enhanced contrast in cultured B16 cells but not significantly in the in vivo tumor model.

Conclusions:

• This second-generation reporter system expands the utility of genetically encoded reporters for MEMRI.
• The study provides insights into Mn biology and the mechanisms underlying endogenous MEMRI contrast.