Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in mitosis and required for the proper completion of abscission
Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis wherein the intercellular membrane is cleaved between daughter cells. LARG co-localizes with &agr;-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of siRNA-resistant LARG prevented this apoptosis. LARG is phosphorylated in early mitosis through a Cdk1-dependent pathway and is dephosphorylated upon anaphase transition. With the onset of cytokinesis, LARG translocates to membrane-associated structures and interacts with contractile ring components. Live cell imaging of LARG-depleted cells revealed greatly delayed fission kinetics in abscission wherein a population of cells with persistent bridges undergo apoptosis; however, these delayed fission kinetics appear to be rescued by Aurora B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just prior to the abscission event. In contrast to studies of other RhoGEFs, particularly ECT2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. Further, mutation of the conserved tyrosine residue in the DH core of LARG results in an inability to bind RhoA and apoptosis. These results establish LARG as a novel and temporally distinct RhoGEF required for the completion of abscission and mitotic viability. Moreover, the importance of LARG just prior to abscission suggests RhoA is critical downstream of contractile ring ingression in a secondary activation event.
Molecular biology|Cellular biology|Biochemistry
Martz, Matthew K, "Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in mitosis and required for the proper completion of abscission" (2013). ETD Collection for Thomas Jefferson University. AAI3562537.