Microtubule nucleation in mitosis by a RanGTP-dependent protein complex

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The gamma-tubulin ring complex (γTuRC) nucleates microtubules (MTs) in eukaryotic cells.
• RanGTP regulates MT nucleation around chromosomes during mitosis, crucial for spindle assembly.
• The precise mechanism of RanGTP-mediated MT nucleation activation remains unclear.

Purpose of the Study:

• To elucidate the mechanism by which RanGTP activates microtubule nucleation.
• To identify the key molecular players and interactions involved in this pathway.

Main Methods:

• Utilized Xenopus egg extracts and in vitro reconstitution assays.
• Employed depletion/add-back experiments with mutant TPX2 and NEDD1 proteins.
• Investigated protein-protein interactions and phosphorylation events.

Main Results:

• RanGTP triggers MT nucleation via a specific γTuRC subcomplex involving XRHAMM.
• RanGTP directly interacts with TPX2 and XRHAMM, mediating complex recruitment.
• Activation necessitates NEDD1 phosphorylation at S405 by Aurora A, itself activated by TPX2.
• TPX2 acts as the scaffold for recruiting the activated complex.

Conclusions:

• The XRHAMM-γTuRC complex is the direct target of RanGTP activation.
• A TPX2-RHAMM-γTuRC supracomplex is formed, requiring NEDD1 phosphorylation and TPX2-dependent recruitment.
• TPX2 is identified as the sole direct RanGTP target, and NEDD1 as the essential Aurora A substrate in this pathway.