TOG-tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• XMAP215, CLASP, and Crescerin proteins utilize Tumor Overexpressed Gene (TOG) domains to regulate microtubule dynamics.
• These TOG domains are known to bind tubulin, but their distinct architectures and specific roles in microtubule polymerization or pausing remain incompletely understood.

Purpose of the Study:

• To investigate the distinct architectures and tubulin-binding properties of TOG domains within the XMAP215 family.
• To elucidate the specific roles of these TOG domains in modulating microtubule polymerization and mitotic spindle function.

Main Methods:

• Utilized a pentameric TOG array from Drosophila melanogaster Msps as a model system.
• Employed protein engineering to create and test an engineered TOG1-2-5 array.
• Investigated the role of TOG5-microtubule binding through genetic manipulation (deletion and mutation) and assessed its impact on spindle architecture and the spindle assembly checkpoint via Mad2 knockdown.

Main Results:

• Msps TOG domains exhibit distinct architectures, binding either free or polymerized tubulin, with a polarized array driving microtubule polymerization.
• An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity.
• A TOG5-specific N-terminal HEAT repeat was identified as essential for binding microtubule lattice-incorporated tubulin, maintaining mitotic spindle formation. Deletion or mutation of TOG5 disrupted spindle architecture and increased the mitotic index.

Conclusions:

• TOG domains possess distinct architectures and binding properties crucial for their function in microtubule regulation.
• TOG5 plays a critical, specific role in maintaining mitotic fidelity by directly interacting with the microtubule lattice.
• The sequence-specific arrangement of architecturally distinct TOG domains underlies the regulatory activity of TOG arrays on microtubules.