In vitro BioID: mapping the CENP-A microenvironment with high temporal and spatial resolution

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The centromere is essential for chromosome segregation, with CENP-A histone variant forming the kinetochore foundation.
• Characterizing the dynamic molecular neighborhood of CENP-A during mitosis is challenging with existing methods.
• BioID uses biotin ligase to identify proximal proteins, but traditional methods require long incubation times.

Purpose of the Study:

• To develop a rapid method for characterizing the CENP-A interactome.
• To overcome temporal limitations in studying dynamic protein neighborhoods during the cell cycle.
• To establish a versatile technique for analyzing protein interactions in frozen physiological states.

Main Methods:

• Developed in vitro BioID (ivBioID), a modification of the BioID proximity labeling technique.
• Utilized a promiscuous biotin ligase (BirA*) fused to CENP-A in lysed cells.
• Performed the reaction in vitro with cells in a fixed physiological state, enabling rapid analysis within minutes.

Main Results:

• Successfully characterized CENP-A interactors using ivBioID in a significantly reduced timeframe (minutes).
• Demonstrated the ability to study protein neighborhoods in 'frozen' cellular states.
• Established ivBioID as a powerful tool for temporal proteomic analysis.

Conclusions:

• In vitro BioID (ivBioID) provides a rapid and efficient method for identifying proximal proteins, such as CENP-A interactors.
• This technique overcomes the temporal limitations of traditional BioID, enabling the study of dynamic molecular neighborhoods.
• ivBioID has broad potential for studying protein interactions that change during the cell cycle or other physiological conditions.