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Detecting protein-protein interaction during liquid-liquid phase separation using fluorogenic protein sensors.

Yanan Huang1, Junlin Chen1, Chia-Heng Hsiung1

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Dimerization-dependent fluorescent proteins (ddFPs) enable detection of protein interactions during liquid-liquid phase separation (LLPS). This chemical biology approach revealed RNA

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Cellular condensates, resembling membraneless organelles, form via liquid-liquid phase separation (LLPS).
  • Understanding protein and RNA interactions is crucial for elucidating LLPS mechanisms and associated diseases.
  • Traditional fluorescence microscopy struggles to reliably detect protein interactions due to diffraction limits.

Purpose of the Study:

  • To develop a novel chemical biology method for detecting protein interactions during LLPS.
  • To investigate the role of RNA in mediating interactions between RNA-binding proteins (RBPs) within condensates.

Main Methods:

  • Utilized dimerization-dependent fluorescent proteins (ddFPs) as a novel chemical biology approach.
  • Applied ddFPs to detect protein-protein interactions during LLPS in vitro and in living cells.
  • Examined the interactions between FUS and TDP-43, two key proteins involved in LLPS.

Main Results:

  • Successfully employed ddFPs to visualize and confirm protein interactions during LLPS.
  • Demonstrated the interaction between FUS and TDP-43 within forming cellular condensates.
  • Revealed that the interaction between FUS and TDP-43 is associated with RNA binding during LLPS.

Conclusions:

  • ddFPs provide a robust method for detecting protein interactions in LLPS.
  • RNA plays a critical role in mediating interactions between RBPs during condensate formation.
  • ddFPs have broad potential for uncovering novel protein interactions and their functions in biomolecular condensate dynamics.