Mapping the cell cycle-dependent centrosomal interactome

A series of highly regulated and precisely ordered events must occur for cells to multiply. Understanding the dynamic interactions of proteins driving this process are crucial for deciphering cell fate. The centrosome is a non-membrane-bound organelle that acts as the primary microtubule organizing centre and is believed to act as a central hub for numerous cell cycle regulators. Consistent with this view, multiple cell cycle regulatory proteins have been reported to facilitate faithful cell division processes through their centrosomal localisation. However, a detailed mechanistic understanding of how these signalling proteins regulate cell cycle transitions from the centrosome remains elusive. To better understand the regulatory signalling networks anchored on the centrosome, an endogenous proximity labelling system called miniTurbo was fused with centrosomal proteins Centrin 2, PCNT and Cep192 to detect the centrosome interactome. The cell cycle-dependent interactome of the centrosome was mapped by combining proximity biotinylation and a reversible drug-mediated cell cycle synchronisation method based on the CDK4/6 inhibitor palbociclib. Differential analysis of the centrosome interactome resulted in the identification of proteins associated with numerous biological processes, including gene expression, DNA/RNA processing, and cell cycle regulation. Functional analysis of three enriched interactors, AATF, PPP1CB, and TRIM25 was performed. Initial analysis on AATF revealed that this protein participates in the G1/S and G2/M transition when depleted using the auxin-inducible degradation system. Unfortunately contrary to the enrichment results, fluorescently tagged AATF-AID2-mNeonGreen was found to be predominantly localised on the nucleolus. Similarly, PPP1CB-AID2-mNeonGreen could not be detected on the centrosome, and its degradation showed no effects on cell cycle progression. For TRIM25-AID2-mNeonGreen, it was observed to selectively associate with the centrosome during the G2 phase, and degron mediated depletion experiments suggest that TRIM25 may be involved in G2 phase regulation. This work presents an initial study of protein dynamics at centrosomes during distinct cell cycle stages and ongoing validation of enriched interactors is anticipated to reveal additional centrosomal cell cycle regulatory proteins.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2025

Author dc:creator

• Renzhi, Yao

Advisors dc:contributor.advisor

• Chan, Chan
• Von Kriegsheim, Alex
• Björklund, Mikael

• Centrosome
• Cell cycle
• Interactome
• Proximity labelling
• TRIM25

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.7488/era/6293

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/43760