The physical separation of daughter cells during cytokinesis represents the final step of cell division and provides the basis for cell multiplication during proliferation and development. Cytokinesis also plays a key role in preventing genomic instability, a hallmark of cancer cells. Our group investigates the mechanisms that orchestrate cytokinesis in mammalian cells and explores the consequences of cell division failure and aneuploidy in the context of cancer.
Eukaryotic cells faithfully partition their genetic information in the form of chromosomes to daughter cells during cell division. Defects in cell division are inevitably associated with the emergence of aneuploid genomes, a feature common to most cancer cells that is characterised by the missing or supernumerary chromosomes. Given the importance of cell division for the development of malignancies, mitosis is a central target in antiproliferative cancer therapy.
Cell division involves dramatic changes in cellular architecture. After the mitotic spindle has segregated sister chromatids to opposite poles of the cell, contraction of the cell membrane separates the cytoplasm of the two nascent daughter cells. This process is referred to as cytokinesis. In animal cells, cytokinesis is accomplished by the constriction of an actomyosin-based structure, called the contractile ring. Local activation of the GTPase RhoA at the equatorial cortex in anaphase promotes the assembly of the contractile ring (Figure 1c). Following cleavage furrow ingression, membrane fusion generates two physically distinct daughter cells.
In animal cells, the mitotic spindle plays an important role in positioning the cleavage plane. At anaphase an array of overlapping microtubules forms a structure referred to as the central spindle (Figure 1b and 1c). This structure is located midway between the two masses of segregated DNA and serves as a signaling platform for the initiation of cytokinesis at the overlying cell cortex. Recruitment of the conserved RhoGEF protein Ect2 to the central spindle in anaphase is thought to elicit the local activation of RhoA at the equatorial cortex, which in turn leads to the initiation of cytokinesis (Figure 1c).
Figure 1. Phospho-regulation of cytokinesis by Polo-like kinase 1 (Plk1). a) Identification of Plk1 phosphorylation target sites within HsCyk-4 using recombinant Plk1 and a peptide array. b) Plk1-dependent phosphorylation of HsCyk-4 (Ser157) at the central spindle during anaphase. c) Model for Plk1 function in regulating Ect2 recruitment to the central spindle and triggering the initiation of cytokinesis in human cells.
Phospho-regulation of cytokinesis
The highly conserved mitotic kinase Plk1 (Polo-like kinase 1) controls a multitude of processes during cell division. Using a potent and selective Plk1 inhibitor, we have discovered a key role for Plk1 in triggering the initiation of cytokinesis in human cells. Acute inhibition of Plk1 at anaphase abolishes RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. We found that Plk1 regulates RhoA by promoting the interaction of the RhoGEF Ect2 with its central spindle anchor protein HsCyk-4, a subunit of the central spindl in complex (HsCyk-4/Mklp1). Our data suggested that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle (Figure 1c). Consistent with this hypothesis, Plk1 localises to the central spindle at anaphase.
Over the last year we have been investigating the molecular basis for how Plk1 induces formation of the Ect2/Hs-Cyk4 complex. Using in vitro kinase assays and peptide array experiments we were able to identify a region within HsCyk-4 that contains a cluster of Plk1 phosphorylation target sites (Figure 1a). Phospho-specific antibodies raised against one of these sites confirmed that HsCyk-4 is phosphorylated by Plk1 at the central spindle during anaphase (Figure 1b). In collaboration with Michael Glotzer (University of Chicago), we demonstrated that simultaneous mutation of Plk1 target sites within HsCyk-4 blocks Ect2 recruitment to the central spindle and prevents the onset of cytokinesis thereby mimicking the Plk1 inhibition phenotype. Ect2's N-terminus shares homology with BRCT domains that can act as phospho-peptide binding modules. Mutation of conserved residues, which are predicted to coordinate the phosphate within Ect2's BRCT domains, also abolishes the recruitment of Ect2 to the central spindle. In summary, our data suggest that phosphorylation of HsCyk-4 by Plk1 at the central spindle during anaphase provides a landing platform for Ect2's BRCT domains (Figure 1c). Currently, we are exploring the dynamics and requirements of this Plk1- dependent signaling pathway that controls the activity of the key cytokinetic switch RhoA and lies at the heart of cleavage furrow induction in animal cells.
Molecular mechanisms regulating and executing cytokinesis in mammalian cells
We use a combination of mass spectrometry and functional genomics to identify molecules involved in different aspects of cytokinesis. Subsequently, we employ cell biology, biochemistry, and time-lapse microscopy to unearth the molecular function of these factors. This will help us to gain insights at the molecular level into how cells orchestrate microtubule, actin, and membrane action to bring about cell division at the right time and at the right place.
The causes and consequences of tetraploidy
A growing body of evidence links cytokinesis defects and tetraploidy to tumorigenesis. Pharmacological or genetic inhibition of cytokinesis in murine cells leads to progressive aneuploidy and tumorigenesis. Furthermore, lesions in several tumor suppressor genes perturb the successful completion of cytokinesis. These and other observations have led to the hypothesis that tetraploid cells could be a transient intermediate on the road to aneuploidy and cancer. While tetraploid cells face several challenges including a duplicated set of chromosomes and spindle poles, recent data show that polyploidy can provide an evolutionary advantage in the acquisition of new phenotypic traits.
We are using small-molecule compounds and RNA interference to generate tetraploid mammalian cells with the aim to investigate the effects of tetraploidy on proliferation, genomic stability, mitotic dynamics, and tumorigenesis (Figure 2). We plan to use our findings in cultured cells to extend our studies to animal models. Furthermore, we are interested in determining how tumor suppressor genes influence cytokinesis. Characterising the origin and fate of tetraploid cells will help us to understand the impact of cell division failure on tumorigenesis and might be useful to locate points of vulnerability of aneuploid cancer cells.
Figure 2. Efficient S-phase entry and DNA replication in bi-nucleated and tetraploid human epithelial cells.Figure 2. Efficient S-phase entry and DNA replication in bi-nucleated and tetraploid human epithelial cells.
For a list of refereed research papers, see Publications (in navigation on left).
