Exploring the regulation and function of the human guanine nucleotide exchange factor Ect2 (epithelial cell transforming protein 2) in cytokinesis

Cytokinesis is the process that divides the cytoplasm of a parent
cell into two. In animal cells, cytokinesis requires the formation
of the central spindle and the contractile ring structures. The
onset of cytokinesis is marked during anaphase with the
specification of the division site, followed by cleavage furrow
formation and ingression, midbody formation and abscission. The
astral microtubules that originate from the centrosomes and the
anti-parallel microtubules of the central spindle are proposed to
determine the site of cleavage furrow formation (Bringmann and
Hyman, 2005). The acto-myosin based contractile ring assembles at
the division site and constricts the cytoplasm which is supported
by the fusion of membrane vesicles to the ingressing plasma
membrane. All these processes together result in the formation of
two daughter cells. The small GTPase RhoA is one of the most
upstream regulators of contractile ring assembly at the cortex. Rho
proteins are activated by GEF’s (guanine nucleotide exchange
factors) and one GEF that is required for cytokinesis is Ect2
(epithelial cell transforming protein2) (Tatsumoto et al., 1999).
The Drosophila pebble (pbl) gene product is the founding member of
the Ect2 protein family and has been shown to be required for
cytokinesis (Lehner, 1992). In mammals, Ect2 was originally
identified as a transforming protein in an expression cloning assay
(Miki et al., 1993) and subsequently shown to be essential for
cytokinesis. In this study, we have explored the temporal and
spatial mechanisms that regulate Ect2 function. In agreement with
previous studies, we show that Ect2 is a cell cycle regulated
protein and is phosphorylated during mitosis. We identify a number
of potentially interesting endogenous phosphorylation sites in
Ect2, including potential Plk1 and Cdk1 sites. Although we have not
been able to determine the function of these phosphorylation sites,
their strong conservation among different species implies that they
accomplish evolutionarily conserved roles.The identification of
these phosphorylation sites sets the stage for future functional
analyses. In complementary studies, we have shown that the central
spindle and cell cortex localizations of Ect2 are facilitated by
the BRCT and PH domains, respectively. The targeting of Ect2 to the
central spindle is mediated by the MKlp1/MgcRacGAP and
MKlp2/Aurora-B complexes. Of the two complexes, we show that Ect2
interacts and colocalizes only with the MKlp1/MgcRacGAP complex in
telophase and propose that this interaction is mediated by a
phosphorylation dependent docking mechanism that targets Ect2 to
the central spindle. Interestingly, the displacement of Ect2 from
the central spindle did not prevent cytokinesis, suggesting that
localized GEF activity is not absolutely essential for cleavage
furrow ingression and cytokinesis. In the second part of this
thesis, we have explored the role of Ect2 during cytokinesis and
show that, in Ect2 depleted cells, levels of RhoA and Citron kinase
are diminished at the cleavage site, concomitant with the
impairment of cleavage furrow formation and ingression.
Additionally, overexpression of appropriate amino-terminal Ect2
fragments in cells also hinders cytokinesis. In these cells, RhoA
and Citron kinase localize to the cortex and cleavage furrow
ingression occurs, but, the subsequent abscission fails. Taken
together, these results suggest that proper function of Ect2 is not
only important for cleavage furrow ingression, but also for cell
abscission. Finally, we investigate the overexpression phenotypes
of different Ect2 truncation mutants. We show that abscission
failure correlates with the persistence of amino-terminal Ect2
fragments at striking ring-like structures surrounding the midbody,
indicating that completion of cell division requires the
displacement of Ect2 from the contractile ring and its re-import
into the reforming cell nucleus. Collectively, our data indicate
that multiple mechanisms cooperate to regulate Ect2 in a
spatial-temporal manner.