Mark Petronczki : Cell Division and Aneuploidy

Every second several hundred thousand cells in our body duplicate themselves through a process known as cell division. The accurate partitioning of all 46 chromosomes during cell division is essential for cellular health and survival. Errors in the process of cell division give rise to cells that have gained or lost copies of chromosomes. These genomic imbalances also referred to as aneuploidy, are a hallmark of cancer and have important implications for the development and treatment of malignancies. Our lab uses animal cell systems to investigate the consequences of aneuploidy and the molecular mechanisms that underlie the process of cell division. We are particularly interested in the process of cytokinesis, the final step of cell division that partitions segregated sister genomes and leads to the birth of new daughter cells. Last year, we identified an important step in the delivery of the cytokinetic signal to the cell envelope.

Cytokinesis – plasma membrane versus mitotic spindle

Animal cells divide by segregating sister chromatids to opposite poles before redrawing their boundaries during cytokinesis. At anaphase, the constriction of the plasma membrane leads to the formation of a cleavage furrow that separates the cytoplasm of the two nascent daughter cells (Figure 1). Cleavage furrow ingression is driven by a membrane-associated and actomyosin-based structure, called the contractile ring. Local activation of the small GTPase RhoA at the equatorial cell cortex in anaphase plays a key role in the assembly and constriction of the contractile ring. Following furrow ingression a membrane fusion reaction, called abscission, completes cytokinesis and yields two physically distinct daughter cells.

Figure 1 Localisation of the Rho guanine exchange factor Ect2 during cytokinesis in human cells Confocal live cell imaging of a monoclonal cell line stably coexpressing histone H2B-mCherry (red) and GFP-Ect2 (white). Time point t = 0 min was set to the metaphase-to-anaphase transition. The open and filled arrowheads indicate localisation of Ect2 to the spindle midzone and plasmamembrane, respectively. (Reprinted from Developmental Cell, Vol. 21, Issue 6, Kuan-Chung Su, Tohru Takaki and Mark Petronczki, Targeting of the RhoGEF Ect2 to the Equatorial Membrane Controls Cleavage Furrow Formation during Cytokinesis, p. 1104-1115, © 2011, with permission from Elsevier)

Formation of the cleavage furrow has to be tightly coordinated with chromosome segregation so that cytokinesis occurs only after anaphase onset and only at the equator. Temporal control is exerted by cyclin-dependent kinase 1 (CDK1), which inhibits cytokinesis prior to anaphase onset. Although cleavage furrow formation occurs at the plasma membrane, research over the last three decades has established that the mitotic spindle positions the cleavage furrow and the zone of active RhoA in animal cells. The ability of microtubule-associated protein complexes to  control cytokinetic events at the cell cortex and plasma membrane is poorly understood. One element of the anaphase spindle that plays a pivotal role in controlling the formation and positioning of the cleavage furrow at the equator is the spindle midzone (Figure 2). The spindle midzone is a stable array of interdigitated microtubules that assembles at anaphase onset midway between segregating chromatids.

Figuee 2 Model for the initiation of cytokinesis in human cells Metaphase: CDK1-dependent phosphorylation of Ect2 inhibits membrane association of Ect2. Anaphase: After inactivation of CDK1, Ect2’s pleckstrin homology domain (PH) and polybasic cluster mediate membrane association of the protein. Binding to centralspindlin recruits Ect2 to the spindle midzone and directs the enrichment of Ect2 at the equatorial plasma membrane. At the equatorial membrane Ect2 can activate RhoA to initiate the formation of a cleavage furrow. (Reprinted from Developmental Cell, Vol. 21, Issue 6, Kuan-Chung Su, Tohru Takaki and Mark Petronczki, Targeting of the RhoGEF Ect2 to the Equatorial Membrane Controls Cleavage Furrow Formation during Cytokinesis, p. 1104-1115, © 2011, with permission from Elsevier) (http://www.sciencedirect.com/science/article/pii/S1534580711005132).

Delivery of the cytokinetic signal to the plasma membrane

At the heart of cleavage furrow formation in animal cells lies the conserved Rho guanine nucleotide exchange factor Ect2 (Figure 1 and 2). Ect2 and its orthologs in other animal species, such as Pebble in Drosophila, are essential for RhoA activation and cytokinesis. Ect2 is recruited to spindle midzone by virtue of binding to centralspindlin, an important structural component of the midzone (Figure 2). The molecular basis for how Ect2 delivers the cytokinetic signal from microtubules to the plasma membrane
remained unknown.

We have developed a gene replacement system in human cells that allowed us to track Ect2 through cell division in live human cells (Figure 1). We found that Ect2 not only localises to the spindle midzone after anaphase onset (Figure 1, open arrowhead) but also accumulates at the equatorial plasma membrane (Figure 1, filled arrowhead) during cytokinesis in live cells. We discovered that a pleckstrin homology (PH) domain and a polybasic cluster (PBC) within Ect2 cooperate to target the protein to the plasma membrane (Figure 2). Importantly, we could demonstrate that guanine nucleotide exchange activity and membrane targeting are two separable but essential properties of Ect2 during formation of the cytokinetic furrow. Ect2 alleles lacking guanine nucleotide exchange function are unable to activate RhoA despite accumulating at the equatorial membrane. Conversely, Ect2 alleles lacking the ability to interact with the membrane are unable to locally activate RhoA at the equatorial membrane despite retaining exchange activity in vitro. Membrane localisation of Ect2 is spatially confined to the equator by centralspindlin, Ect2’s spindle midzone anchor complex, and is temporally coordinated with chromosome segregation through the activation state of CDK1 (Figure 2).

Our analysis suggests that targeting of Ect2 to the equatorial membrane represents a key step in the delivery of the cytokinetic signal to the cortex (Figure 2) (Su et al., 2011;Developmental Cell. 21: 1104-1115). The spindle midzone might act as a spatial cue to break the isotropic localisation of Ect2 at the plasma membrane and to direct the protein to the equatorial cleavage plane.

We are currently focussing on the identification of the lipid species that are involved in the association of Ect2 with the plasma membrane during cytokinesis. Furthermore, we are dissecting the mechanism underlying the peripheral accumulation of Ect2 at the cell equator by using perturbation experiments and by tracking Ect2, and its dynamic behaviour at the spindle midzone and plasma membrane. Lastly, we are characterising a direct molecular link between the mitotic spindle and plasma membrane that we have recently identified and that could play a key role in the final abscission stage of cytokinesis.