Rae1 interaction with NuMA is required for bipolar spindle formation

In eukaryotic cells, the faithful segregation of daughter chromosomes during cell division depends on formation of a microtubule (MT)-based bipolar spindle apparatus. The Nuclear Mitotic Apparatus protein (NuMA) is recruited from interphase nuclei to spindle MTs during mitosis. The carboxy terminal domain of NuMA binds MTs, allowing a NuMA dimerto function as a "divalent" crosslinker that bundles MTs. The messenger RNA export factor, Rael, also binds to MTs. Lowering Rael or increasing NuMA levels in cells results in spindle abnormalities. We have identified a mitotic-specific interaction between Rael and NuMA and have explored the relationship between Rael and NuMA in spindle formation. We have mapped a specific binding site for Rael on NuMA that would convert a NuMA dimer to a "tetravalent" crosslinker of MTs. In mitosis, reducing Rael or increasing NuMA concentration would be expected to alter the valency of NuMA toward MTs; the "density" of NuMA-MT crosslinks in these conditions would be diminished, even though a threshold number of crosslinks sufficient to stabilize aberrant multipolar spindles may form. Consistent with this interpretation, we found that coupling NuMA overexpression to Rae1 overexpression or coupling Rael depletion to NuMA depletion prevented the formation of aberrant spindles. Likewise, we found that overexpression of the specific Rae1-binding domain of NuMA in HeLa cells led to aberrant spindle formation. These data point to the Rae1-NuMA interaction as a critical element for normal spindle formation in mitosis.