dc.description.abstract | The Chromosomal Passenger Complex (CPC) is a major regulator of
mitosis composed of the catalytic subunit Aurora B, the inner centromere protein
INCENP, Survivin and Borealin/Dasra B. The CPC controls aspects of mitosis,
ranging from chromosome and spindle structure to the correction of kinetochore-microtubule
attachment errors, regulation of mitotic progression and completion of
cytokinesis (Carmena et al., 2012). Knocking down any one CPC component
induces delocalization of the others and disrupts mitotic progression (Adams et al.,
2001 ; Carvalho et al., 2003; Lens et al., 2003 ; Gassmann et al., 2004; Vader et al.,
2006).
Telophase Disc (TD-60), also known as RCC2, is a putative Guanine
Exchange Factor (GEF) that has been suggested to be involved in completion of
cytokinesis through GTPase activation (Mollinari et al., 2003). However, its
mechanism of action is still unclear. Interestingly, TD-60 has a typical
Chromosomal Passenger Complex (CPC) localization (Andreassen et al., 1991) and
its down-regulation alters CPC localisation during early mitosis. However, it is not
a member of the CPC immunoprecipitated from mitotic cells (Gassmann et al.,
2004).
Here, I improved human TD-60 recombinant protein production by
expressing a synthetic cDNA in the baculovirus expression system. This allowed me
to characterize TD-60-associated GEF activity in vitro and study its possible
influence on core CPC activity in vivo. I tested purified human TD-60 against a
broad selection of GTPase targets, representing each GTPase family, in an
established GEF assay. My data demonstrated that TD-60 has consistent high GEF
activity in vitro towards the Ras-like protein A, RalA.
To understand if TD-60 links RalA GTPase function to the CPC in
vivo, I performed TD-60 and RalA RNAi experiments in HeLa cells. Interestingly,
both TD-60 and RalA-depleted cells exhibit destabilized kinetochore fibers, a
similar defective prometaphase-like bipolar spindle structure, and an abnormal
centromeric accumulation of the CPC in early mitosis. In order to confirm that
phenotypes seen after TD-60 depletion were due to lack of RalA activation in vivo,
I generated a constitutively active RalA mutant that I transfected into TD-60-
deficient cells. Strikingly, the RalA Q72L active mutant (mimicking the GTP-bound
form) rescued the abnormal bipolar spindle structure, corrected the defective
kinetochore-microtubules attachments, and rescued the atypical CPC distributions
observed at centromeres after TD-60 depletion.
These results suggest that TD-60-associated RalA GEF activity stabilizes
kinetochore-microtubule attachments in early mitosis and that, TD-60 links RalA
GTPase function to the CPC during mitosis. | en |