Visualizing multimerization of plasticity-related gene 5 at the plasma membrane using FLIM-FRET

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• Plasticity-related gene 5 (PRG5) is a vertebrate-specific membrane protein in the lipid-phosphate phosphatase (LPP) family.
• PRG5 is highly expressed in neurons and influences growth-cone guidance and spine formation.
• PRG5 overexpression induces filopodia and plasma membrane protrusions, impacting neuronal spine density and morphology.

Purpose of the Study:

• To elucidate the precise mechanisms by which PRG5 induces membrane protrusions and orchestrates cellular processes.
• To investigate the oligomeric state and localization of PRG5 within cells.

Main Methods:

• In vitro biochemical assays to detect PRG5 homo-dimers and multimers in HEK293T cells.
• Fluorescence Lifetime Imaging (FLIM) coupled with Förster Resonance Energy Transfer (FRET) to quantify PRG5 multimer localization.
• Visualization in living HEK293T cells and fixed primary hippocampal neurons.

Main Results:

• A significant fraction of PRG5 exists as homo-dimers and larger multimers in HEK293T cells.
• PRG5 multimers were specifically localized in non-neuronal filopodia and neuronal spine-like structures.
• FLIM-FRET successfully quantified the specific localization of PRG5 multimers in cellular protrusions.

Conclusions:

• PRG5 multimerization is demonstrated for the first time.
• PRG5 multimers specifically localize to cellular protrusions, including neuronal spines.
• PRG5 multimerization may play a functional role in interacting with extracellular matrix molecules or stabilizing membrane protrusions.