Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission.
The guanine nucleotide-exchange factor (GEF) Ect2 is essential for cytokinesis. Here we studied the subcellular localization of Ect2 and examined the consequences of either depleting or overexpressing Ect2 in human cells. We show that in mitotic cells Ect2 localizes to the central spindle and to the cell cortex. The latter association is mediated through a PH domain in Ect2 and central spindle localization requires the MKlp1-MgcRacGAP and MKlp2-Aurora-B complexes. Ect2 directly interacts with MKlp1-MgcRacGAP through its BRCT domain, whereas MKlp2-Aurora-B probably exerts a regulatory role in Ect2 central spindle targeting. Depletion of Ect2 impaired cleavage furrow formation and RhoA and Citron kinase failed to accumulate at the cleavage furrow. Ect2 displacement from the central spindle revealed that physiological levels of this protein in this location are not crucial for RhoA activation and cytokinesis. In cells overexpressing appropriate N-terminal Ect2 fragments, RhoA and Citron kinase localized to the cleavage furrow and ingression occurred, but abscission failed. This failure could be correlated with the persistence of these fragments at structures surrounding the midbody, suggesting that abscission requires the displacement of Ect2 from the contractile ring and its re-import into the nucleus.
Chalamalasetty
Ravindra B
RB
Max Planck Institute for Biochemistry, Department of Cell Biology, Am Klopferspitz 18, 82152 Martinsried, Germany.
Hümmer
Stefan
S
Nigg
Erich A
EA
Silljé
Herman H W
HH
D064107:Aurora Kinase B; D064127:Aurora Kinases; D002467:Cell Nucleus / Q000378:metabolism; D048749:Cytokinesis; D020690:GTPase-Activating Proteins / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
C489850:ECT2 protein, human; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; C092935:KIF23 protein, human; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; C115995:mgcRacGAP; C115635:citron-kinase; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D020742:rhoA GTP-Binding Protein