Cytokinesis Mechanisms in Yeast

Nobody can deny that yeasts are anything but a good friend to man. The nonpathogenic, unicellular budding yeast Saccharomyces cerevisiae (commonly known as baker's or brewer's yeast) produces carbon dioxide to leaven bread and supplies the alcohol in beer and wine. Leftover yeast extract is turned into an edible spread that is popular in Australia (as "vegemite") and among fans of World War II rations in the UK (as "marmite"). Budding yeast also provides a well-established model system for cell biologists. In addition to being an ingredient in African millet beer, Schizosaccharomyces pombe (a fission yeast) is another powerful model system for cell biologists.

As model organisms, yeast have been an important tool in biological research for studying fundamental cellular processes. In the 1970s, Lee Hartwell and Paul Nurse pioneered research on the cell cycle using both types of yeast, receiving a Nobel Prize for their efforts. Here we consider the last act of the cell cycle, known as cytokinesis. This event follows M phase, when chromosome segregation is complete and the daughter cells are ready to separate. Our understanding of this complex process has, not surprisingly, benefited from a number of studies conducted in yeast.

Landmark experiments in the 1970s unveiled the role of cytoskeletal proteins in cytokinesis. Researchers discovered that a belt of actin and class II myosin forms a contractile ring at the cleavage furrow of animal cells that drives cytokinesis (Fujiwara & Pollard 1976; Mabuchi & Okuno 1977; Schroeder 1973). However, it is still unclear how this extremely complex structure works — in fission yeast it's made up of more than fifty proteins. How is the ring positioned and assembled at the cortex? How are rings organized, and how do they constrict? How does the cell cycle control the timing of assembly and constriction? We know that microtubules direct the segregation of chromosomes, and evidence from a number of systems indicates that communication between the microtubule and actin cytoskeletons contributes to the regulation of ring assembly and constriction during mitosis. However, understanding how signals from the mitotic apparatus influence key ring components remains a major question in the field.

From a medical viewpoint, it is critical we know how the cytokinesis machinery works. Abnormal cytokinesis leads to unequal division of chromosomes (aneuploidy) and supernumerary centrosomes, both characteristic of many human tumors. Key components of the contractile ring that drive rapid cell proliferation are actually potential drug targets in certain cancers. Thus, establishing the fundamental features of contractile rings in robust model organisms is vital so that the knowledge can be applied to more complex cells that are harder to study. What follows is a focus on several recent studies performed with fission yeast that are providing new insights into contractile ring function.

Fission yeast provide a convenient model system to study the actin cytoskeleton. The system draws its strength from its relative simplicity. While vertebrate cytoskeletons are complex, with actin filaments assembling into more than fifteen different actin structures, fission yeast rely on only three: actin cables, actin patches, and contractile rings (Figure 1).

Because fission yeast have a small genome and very little redundancy among their genes, scientists can easily identify key cytokinesis genes. One technique for doing this is complementation, in which fission yeast cytokinesis mutants are transformed by the insertion of DNA segments. Specifically, the cytokinesis mutants are transformed with plasmid DNA derived from a broad plasmid library of fission yeast genomic DNA. Many colonies are created with different transformations. Subsequent isolation of yeast cells whose cytokinesis mutant effect was rescued by transformation help identify the exact plasmids carrying the genes of interest. These plasmids are then characterized further with DNA sequencing

Another way to identify key cytokinesis genes is to manipulate the fission yeast genome using targeted homologous recombination. This is a standard procedure that allows (among other things) introduction of colored tags; for example, green fluorescent protein (GFP) protein can be fused with the gene of interest, and both can be expressed at the same time, at true endogenous levels. In this way, sophisticated imaging techniques can be used to track gene expression by following a fluorescent protein. These imaging techniques permit high spatial and temporal resolution, and therefore sensitive tracking of protein movements. Such technological advances, combined with an increasing knowledge base of genetic function, should ensure continued rapid progress in this field.

Careful analysis of assembling rings and elegant genetic studies in fission yeast have led to the proposal of two independent yet complementary mechanisms of ring assembly. The first is the "search, capture, pull, and release" mechanism, which is based on the arrival and dynamics of different components at assembling rings (Figure 2; Vavylonis et al. 2008). In this model, the preparation for cytokinesis begins before the cell enters mitosis. The anillin-like adaptor protein Mid1p surrounds the nucleus in the midcell region as about sixty-five cortical nodes and provides the spatial cue for ring placement, emerging about an hour before cells enter mitosis (Celton-Morizur et al. 2004; Chang et al. 1996; Sohrmann et al. 1996; Wu et al. 2003). Just before the onset of mitosis, these nodes recruit additional Mid1p from the nucleus, followed by a number of highly conserved cytoskeletal proteins (fission yeast protein names in parentheses): myosin II (Myo2p), IQGAP (Rng2p), F-BAR protein (Cdc15p), formin (Cdc12p), tropomyosin (Cdc8p), and a-actinin (Ain1p) (Sohrmann et al. 1996; Wu et al. 2003; Bahler et al. 1998; Coffman et al. 2009; Skau et al. 2009).

Upon entering mitosis, Cdc12p activates the growth of actin filaments, which associate with tropomyosin and elongate rapidly (Skau et al. 2009). These filaments contact other nodes and their Myo2p motors, which repeatedly bind, tug on, and break up the interconnecting filaments, enabling ring compaction (Vavylonis et al. 2008; Coffman et al. 2009; Stark et al. 2010). Multiple layers of regulation appear to converge on Myo2p. The conserved UCS protein Rng3p works with the Hsp90 (heat shock protein 90) chaperone to stabilize Myo2p motor activity upon its recruitment to the division site (Lord & Pollard 2004; Lord et al. 2008; Mishra, D'souza, et al. 2005; Wong et al. 2000). Phosphorylation of Myo2p's regulatory light chain (RLC/Rlc1p) and association of actin filaments with tropomyosin enhance Myo2p motor activity favoring ring compaction (Stark et al. 2010; Sladewski et al. 2009). Ain1p and Rng2p cross-link actin filaments into bundles during ring assembly (Takaine et al. 2009; Wu et al. 2001).

Evidence for the second model of ring assembly, the "leading cable" model, lies in the fact that cytokinesis and growth is still supported in cells lacking Mid1p and nodes (Chang et al. 1996; Sohrmann et al. 1996; Hachet & Simanis 2008; Huang et al. 2008). Ring assembly in the absence of nodes relies on the septation initiation network (SIN; Hachet & Simanis 2008; Huang et al. 2008; Schmidt et al. 1997). The "leading cable" model stems from the observation that under certain conditions actomyosin cables coalesce into rings following growth from a single spot on the cortex (Figure 3; Arai & Mabuchi 2002; Mishra & Oliferenko 2008). The SIN is a conserved signal transduction pathway that triggers ring constriction and septum formation at the end of anaphase (Wolfe & Gould 2005). While SIN activity peaks at the time of ring constriction, significant activity is evident earlier in mitosis and ensures that rings formed are compact and homogenous (Hachet & Simanis 2008; Sparks et al. 1999). In the absence of Mid1p (and nodes), rings are misplaced and disorganized (Chang et al. 1996; Sohrmann et al. 1996). However, defects in organization can be suppressed by delaying septum formation (Figure 3). Rings fail to assemble in the absence of both Mid1 and SIN function, highlighting the overlapping roles of the two proposed mechanisms (Hachet & Simanis 2008).

Key ring components activate a cytokinesis checkpoint when compromised. Two such components (Cdc15p and Myo2p) are known substrates of the SIN-dependent Cdc14 family phosphatase (Clp1p; Clifford et al. 2008; Wachtler et al. 2006). These phosphatases move from the nucleolus to the cytoplasm, where they dephosphorylate cyclin-dependent kinase (CDK) substrates, causing the cell to shift toward cytokinesis and mitotic exit (Wolfe & Gould 2005). However, phosphorylation of Clp1p by the terminal SIN kinase Sid2p promotes the retention of Clp1p in the cytoplasm via an interaction with another protein (the 14-3-3 protein Rad24p; Chen et al. 2008; Mishra, Karagiannis, et al. 2005). When ring integrity is compromised, Clp1p activates a cytokinesis checkpoint, during which Clp1p activity is also utilized to stabilize ring structures (Mishra et al. 2004).

During ring assembly, the N-terminal F-BAR domain of Cdc15p recruits formin (Cdc12p) and self-assembles into filaments that are thought to deform membranes — a hint of cytokinesis morphology (Carnahan & Gould 2003; Roberts-Galbraith et al. 2010). In addition, the C-terminal SH3 domain of Cdc15p recruits more proteins that stabilize the ring (Ge & Balasubramanian 2008; Pinar et al. 2008; Roberts-Galbraith et al. 2009). Consistent with being a downstream SIN target, Cdc15p is dephosphorylated upon SIN activation and relies on the SIN for localization at the ring. Interestingly, cdc15 mutants show a phenotype similar to that of SIN mutants, as they fail to form homogenous rings (Hachet & Simanis 2008).

Overall, the central region of Cdc15p contains more than thirty-three different phosphorylation sites. Eleven of them are likely Clp1p targets (Roberts-Galbraith et al. 2010). Dephosphorylation of this region (by Clp1p and unidentified phosphatases) promotes an open conformation facilitating self-assembly and association with binding partners (Figure 3; Roberts-Galbraith et al. 2010). In another line of evidence, mutagenesis of phosphorylation sites showed that premature dephosphorylation favors interphase assembly of Cdc15p and other key components (e.g., Rng2p and Myo2p) at midcell. Thus, cell cycle-specific dephosphorylation of Cdc15p during mitosis appears to drive ring assembly in the "leading cable" model. Although the Myo2p tail is phosphorylated at a single serine (S1444), mutagenesis at this site revealed only minor defects in ring dynamics, implying that tail phosphorylation (at least at S1444 alone) is not critical to cytokinesis (Sladewski et al. 2009).

Contractile ring assembly relies on two overlapping pathways. One pathway is built on the spatial organization of ring components provided by the nodes; the second pathway relies on signaling from the SIN, which appears to promote the function of the key node component Cdc15p. The fact that Mid1p anchors Clp1p at assembled rings suggests that direct cooperation between the two pathways may also occur at the preassembled nodes. Many major questions need answers before we can generate meaningful models of contractile ring function. To begin crafting such models, we need to understand the molecular function and regulation (in space and time) of a number of key ring components. Fission yeast provides us with a simple platform to answer these questions, using a variety of experimental approaches.