The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

Area of Science:

• Cell Biology
• Molecular Biology
• Structural Biology

Background:

• The centrosome, a key microtubule-organizing center, has poorly understood protein organization principles.
• The structural transition of the centrosome between interphase and mitosis involves uncharacterized protein interactions.
• Centrosomin (CM2) in Drosophila melanogaster undergoes significant cell cycle-dependent structural changes.

Purpose of the Study:

• To investigate the protein-protein interactions governing centrosomin (CM2) organization and structural transitions.
• To elucidate the role of CM2 dimerization and intramolecular interactions in centrosome structure.
• To identify novel binding partners and regulatory mechanisms of CM2.

Main Methods:

• In-cell microscopy to observe protein localization and dynamics.
• In vitro oligomer assessment to evaluate protein assembly.
• Nuclear Magnetic Resonance (NMR) binding assays to confirm molecular interactions.
• Biochemical assays to test interactions with other proteins and calcium.

Main Results:

• CM2 dimerization is not essential for centrosome recruitment; CM2 alone exhibits cell cycle-dependent rearrangement.
• NMR confirms intramolecular CM2 interaction, identifying L1137 as critical for binding.
• CM2 interacts in vitro with Drosophila pericentrin-like-protein (PLP) using the same residues as intramolecular binding.
• A calcium-sensitive interaction between CM2 and calmodulin was discovered.

Conclusions:

• CM2's intramolecular interactions and binding to PLP are key to centrosome structure.
• Calcium may regulate centrosomin-mediated assemblies, similar to mammalian pericentrin.
• An expanded model suggests CM2-PLP interaction forms an interphase layer, nucleating intramolecular interactions for mitotic expansion.