Download Analyne Manzano Schroeder

Analyne Manzano Schroeder
Proseminar 200
February 24, 2004
News and Views Draft
Piecing Together Pathways: Mitotic Exit
Mitotic exit is tightly regulated by multiple-ordered pathways of biochemical
switches that ultimately signal a dividing cell to undergo cytokinesis. These checkpoint
pathways are interconnected; this ensures proper distribution of fully replicated sisterchromatids, but also contributes to the complexity of cell division.
The mechanism of mitotic regulation involves an ordered pattern of activation and
degradation of proteins that determines the mitotic phase of the cell. The functional
specificity of the anaphase-promoting complex (APC), a key multi-subunit protein in
mitotic regulation, is dictated by the alternating coactivators that are bound to it. Cdc20,
for example, is bound to APC during metaphase and activated by mitotic Cdk1 and Plk1
that triggers the degradation of cyclins that allow separation of sister chromatids. Cdc20
is in turn degraded and replaced by cdh1 during anaphase that maintains cyclin
suppression, allowing cytokinesis to occur and establishes G1 phase of the cell cycle.
These events are further regulated by proteins that function in other pathways, that turn
on and off APC function, in order to delay or proceed with cell division, depending on
whether the genome has been fully replicated and are ready to segregate. For example,
APC binding to Cdh1 is antagonized by Bub-2, a factor in the spindle checkpoint
pathway that delays anaphase when chromosomes are not properly attached to
kinetochores. These checkpoints are further complicated by the redundancy in the
pathways. Methods must be applied to organize and outline the series of events that
regulate mitosis.
The functions of many proteins involved in mitosis have primarily been studied in
vitro, but their physiological roles in the cell have not been elucidated. Fluorescent
tagging (FP-tagging) has enabled scientists to observe the behavior of proteins in vivo
through live-cell assays. By tagging two different proteins in a single cell, one can
determine relative timing of activity, localization, interactions and degradation leading to
inactivation of the proteins. In this manner, by comparing the timing of activation
between different pairs of proteins, a pathway of mitotic events can be constructed.
Furthermore, by tying together data from loss-of-function and gain-of-function studies,
the regulatory role of these proteins in vivo can be determined.
The application of these techniques have led Lindon et al. (2004) to demonstrate
ordered proteolysis of APC/Ccdh1 substrates that are required for normal mitotic exit.
Furthermore, through FP-tagging of Plk1, Aurora, Cyclin B1, and securin, they were able
to show that contrary to previous findings, Plk1 inactivation is necessary for chromatid
separation and cytokinesis. Studies using a non-degradable (mutated ubiquitination site)
Plk1 species found that mitotic exit was affected. Inhibiting Plk1 inactivation either by
preventing ubiquitination or inhibiting a parallel, alternative pathway that
dephosphorylates Plk1, results in delayed mitotic exit.
These methods have the potential to be applied to other cellular pathways, an
example of which is endocytosis (dynamin and actin tagging). The power of these
techniques lies in the ability to follow cellular processes in real time, within one cell.
Furthermore, studies of cellular process in vivo can lead to observations that differ
compared to studies in vitro, as reflected by the investigations by Lindon et al. (2004).
Reference:
Lindon, C. and Pines, J. Ordered proteolysis in anaphase inactivates Plk1 to contribute to
proper mitotic exit in human cells. Cell Biol. 164, 233-241 (2004).
Morgan, D.O. Regulation of the APC and the exit from mitosis. Nature Cell Biol. 1,
E47-E53 (1999).