Protocol to alter a protein's phase separation capacity to control cell fate transitions

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Protein phase separation is a key regulator of biological processes, including transcription.
• Understanding and manipulating protein phase separation is critical for deciphering cellular functions.
• Intrinsically disordered regions (IDRs) are known to drive protein phase separation.

Purpose of the Study:

• To develop and present a protocol for manipulating the phase separation capacity of proteins.
• To investigate the role of protein phase separation in cell fate transitions.
• To establish a method for disrupting and rescuing protein phase separation.

Main Methods:

• Mutation of residues within intrinsically disordered regions (IDRs) to disrupt phase separation.
• Fusion of a known phase separation-driving IDR to rescue disrupted phase separation.
• Assessment of the impact of altered phase separation on cell fate transitions.

Main Results:

• The developed protocol effectively allows for the manipulation of protein phase separation capacity.
• Disrupting protein phase separation by mutating IDRs attenuates cell fate transitions.
• Rescuing phase separation by fusing a functional IDR restores the transitions.

Conclusions:

• Protein phase separation is essential for promoting cell fate transitions.
• The presented protocol provides a tool to study the functional consequences of modulating protein phase separation.
• Predicting effective mutation sites within IDRs remains a challenge for precise manipulation.