Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Nuclear import receptors importin β and transportin regulate spatial organization during mitosis.
• Importin β represses assembly factors away from chromatin, with RanGTP facilitating localized assembly.
• The regulatory mechanism of transportin remained unclear, with competing models proposed.

Purpose of the Study:

• To elucidate the mechanism by which transportin regulates assembly factors during mitosis.
• To determine if transportin acts directly or indirectly via RanGTP sequestration.
• To investigate the role of transportin in the spatial regulation of mitotic processes.

Main Methods:

• Experiments using Xenopus assembly extracts with M9M and TLB mutants.
• In vitro binding assays with ELYS and the nucleoporin 107-160 complex.
• In vivo studies involving M9M-transfected cells.

Main Results:

• Evidence supports a model of direct inhibition by transportin, rather than RanGTP sequestration.
• M9M addition to mitotic cytosol induced microtubule aster assembly.
• Transportin blocked binding of ELYS and Nup107-160 to kinetochores, reversible by M9M.
• M9M transfection led to spindle and cytokinesis defects in 30% of cells.

Conclusions:

• Transportin directly inhibits assembly factors, acting in parallel to importin β.
• Both importin β and transportin function as "global positioning system" (GPS) pathways.
• These parallel pathways ensure precise spatial regulation of mitotic events around chromatin.