Influence of human Ect2 depletion and overexpression on cleavage furrow formation and abscission

Abstract

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.

Novel Functions of Ect2 in Polar Lamellipodia Formation and Polarity Maintenance during “Contractile Ring-Independent” Cytokinesis in Adherent Cells

“…RhoA was immunostained in cells fixed with TCA, which has been known to preserve active RhoA (Yonemura et al, 2004;Yuce et al, 2005), whereas Ect2 was immunostained in cells fixed with formaldehyde. We confirmed the findings of an earlier study (Chalamalasetty et al, 2006) showing that RhoA accumulated at the equatorial cortex during anaphase in both HeLa and HT1080 cells ( Figure 1A, left panels). Ect2 localized at the cell cortex as well as the central spindle in HeLa cells but predominantly at the central spindle in HT1080 cells ( Figure 1A, middle and right panels).…”

“…Still, these PRC1-depleted cells fail to carry out abscission (Mollinari et al, 2005;Nishimura and Yonemura, 2006), which is consistent with the observation made in HeLa cells that accumulation of Ect2 at the central spindle is necessary for abscission (Chalamalasetty et al, 2006). Contrary to this model, the Ect2-depleted HT1080 cells efficiently completed division.…”

“…Recently, Chalamalasetty et al (2006) showed that in HeLa cells an N-terminal fragment of Ect2 (N-Ect2) that acts as a dominant negative form of Ect2 (Tatsumoto et al, 1999;Kimura et al, 2000;Yoshizaki et al, 2004) does not prevent furrow ingression, but does prevent abscission, whereas RNA interference (RNAi)-dependent Ect2 depletion does prevent furrow formation. They concluded that a physiological level of Ect2 is not required at the central spindle for cytokinesis in HeLa cells, based on the observation that N-Ect2 displaced endogenous Ect2 incompletely from the central spindle.…”

LPS-induced nuclear translocation of RhoA is dependent on NF-κB in the human lung cancer cell line A549

“…However, the activity of GTPases is also regulated by three types of proteins: guanine nucleotide exchange factors (GEFs) catalyze the exchange of GDP for GTP and thereby activate Rho (16); GTPase activating proteins (GAPs) enhance the hydrolytic ability of GTPases thereby inactivating the proteins (17); and guanine nucleotide dissociation inhibitors (GDIs) also inactivate the GTPases by removing them from the plasma membrane (PM) and maintaining them in an inactive form in the cytosol (18). The majority of Rho-GEFs are localized to the cytoplasm or the PM (16), but two GEFs, Net1 and Ect2, have been reported to localize to the nucleus at a steady state (19,20). These two GEFs contain an NLS that is necessary for the proteins to be targeted to the nucleus (21,22).…”

Cleavage Furrow Formation for Avascular Tumor Growth based on Finite Element Modeling

“…Although researches, such as [9,11,13], have shown that the spindle plays a key role not only in bringing about the mechanical separation of chromosomes, but also in determining the position of the cleavage furrow in animal cells, the exact nature of this role, such as whether spindle inhibits furrow formation close to the spindle poles or delivers a inducing signal of furrow formation at the equatorial cortex, has been debated. In this paper, we proposed a finite element based cleavage furrow formation approach, which tries to understand the mechanisms of avascular tumor growth not only from the chemical aspect, such as modeling the role of cell cycle control factors and reaction diffusion substances, but aslo from the physical aspect, such as modeling the role of cell surface and cell volume.…”