Dual SLIPT-A Lipid Mimic to Enable Spatiotemporally Defined, Sequential Protein Dimerization

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Signaling

Background:

• Spatiotemporal protein regulation is vital for cellular processes like signal integration and crosstalk management.
• Lipids play a key role in membrane-associated signaling by mediating sequential protein recruitment.
• Understanding these dynamic recruitment events is crucial for deciphering complex cellular communication.

Purpose of the Study:

• To develop a novel tool, dual SLIPT (self-localizing ligand-induced protein translocation), that emulates lipid-mediated sequential protein recruitment.
• To enable precise spatiotemporal control over the recruitment and dimerization of two proteins of interest at the plasma membrane.
• To investigate dynamic cytosol-to-plasma membrane recruitment events and their impact on cellular signaling.

Main Methods:

• Development of dual SLIPT, a lipid-analog tool localizing to the plasma membrane inner leaflet.
• Utilizing trimethoprim (TMP) and HaloTag ligand (HTL) to recruit cytosolic proteins fused to specific tags (e.g., ^iK6eDHFR, HOB).
• Systematic linker extension to prevent steric hindrance and Förster resonance energy transfer (FRET) for verifying protein binding and dimerization.

Main Results:

• Dual SLIPT successfully recruits two proteins of interest to the plasma membrane, facilitating their simultaneous binding and dimerization.
• The tool functions effectively at physiologically relevant concentrations, mimicking recruitment by transient lipid species.
• A photocontrollable variant, dual SLIPT^NVOC, was developed, allowing light-induced spatiotemporal protein dimerization.

Conclusions:

• Dual SLIPT provides a versatile platform for studying lipid-mediated protein recruitment and dynamic signaling events.
• The photocontrollable dual SLIPT^NVOC offers unprecedented spatiotemporal control, closely mimicking physiological processes.
• This technology advances the ability to interrogate complex signaling pathways at the cell membrane.