Comparative Analysis of Cytokinesis in Budding Yeast, Fission Yeast and Animal Cells

AbstractCytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity

Particle-based model of cellular morphogenesis in budding yeast

We apply Lagrangian particle method combined with the level-set method to model morphogenesis of budding yeast on the subcellular level. We model the biochemical reactions, anisotropic diffusion, membrane-cytoplasmic transport of proteins and introduction of new membrane material (exocytosis) that occur on the plasma membrane. Exocytosis results in protrusion of the membrane surface. Hence, to model these phenomena we need to solve a system of reaction-diffusion-advection equations on the evolving surface

Daughter Cell Identity Emerges from the Interplay of Cdc42, Septins, and Exocytosis

Asymmetric cell division plays a crucial role in cell differentiation, unequal replicative senescence, and stem cell maintenance. In budding yeast, the identities of mother and daughter cells begin to diverge at bud emergence when distinct plasma-membrane domains are formed and separated by a septin ring. However, the mechanisms underlying this transformation remain unknown. Here, we show that septins recruited to the site of polarization by Cdc42-GTP inhibit Cdc42 activity in a negative feedback loop, and this inhibition depends on Cdc42 GTPase-activating proteins. Combining live-cell imaging and computational modeling, we demonstrate that the septin ring is sculpted by polarized exocytosis, which creates a hole in the accumulating septin density and relieves the inhibition of Cdc42. The nascent ring generates a sharp boundary that confines the Cdc42 activity and exocytosis strictly to its enclosure and thus clearly delineates the daughter cell identity. Our findings define a fundamental mechanism underlying eukaryotic cell fate differentiation

Mitotic exit kinase Dbf2 directly phosphorylates chitin synthase Chs2 to regulate cytokinesis in budding yeast

How cell cycle machinery regulates extracellular matrix (ECM) remodeling during cytokinesis remains poorly understood. In the budding yeast Saccharomyces cerevisiae, the primary septum (PS), a functional equivalent of animal ECM, is synthesized during cytokinesis by the chitin synthase Chs2. Here, we report that Dbf2, a conserved mitotic exit kinase, localizes to the division site after Chs2 and directly phosphorylates Chs2 on several residues, including Ser-217. Both phosphodeficient (chs2‑S217A) and phosphomimic (chs2‑S217D) mutations cause defects in cytokinesis, suggesting that dynamic phosphorylation–dephosphorylation of Ser-217 is critical for Chs2 function. It is striking that Chs2‑S217A constricts asymmetrically with the actomyosin ring (AMR), whereas Chs2-S217D displays little or no constriction and remains highly mobile at the division site. These data suggest that Chs2 phosphorylation by Dbf2 triggers its dissociation from the AMR during the late stage of cytokinesis. Of interest, both chs2‑S217A and chs2‑S217D mutants are robustly suppressed by increased dosage of Cyk3, a cytokinesis protein that displays Dbf2‑dependent localization and also stimulates Chs2‑mediated chitin synthesis. Thus Dbf2 regulates PS formation through at least two independent pathways: direct phosphorylation and Cyk3‑mediated activation of Chs2. Our study establishes a mechanism for direct cell cycle control of ECM remodeling during cytokinesis

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80–100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane

Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein–mediated zone of inhibition

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site

Hof1 and Chs4 Interact via F-BAR Domain and Sel1-like Repeats to Control Extracellular Matrix Deposition during Cytokinesis

Localized extracellular matrix (ECM) remodeling is thought to stabilize the cleavage furrow and maintain cell shape during cytokinesis [1-14]. This remodeling is spatiotemporally coordinated with a cytoskeletal structure pertaining to a kingdom of life, for example the FtsZ ring in bacteria [15], the phragmoplast in plants [16], and the actomyosin ring in fungi and animals [17, 18]. Although the cytoskeletal structures have been analyzed extensively, the mechanisms of ECM remodeling remain poorly understood. In the budding yeast Saccharomyces cerevisiae, ECM remodeling refers to sequential formations of the primary and secondary septa that are catalyzed by chitin synthase-II (Chs2) and chitin synthase-III (the catalytic subunit Chs3 and its activator Chs4), respectively [18, 19]. Surprisingly, both Chs2 and Chs3 are delivered to the division site at the onset of cytokinesis [6, 20]. What keeps Chs3 inactive until secondary septum formation remains unknown. Here, weshow that Hof1 binds to the Sel1-like repeats (SLRs) of Chs4 via its F-BAR domain and inhibits Chs3-mediated chitin synthesis during cytokinesis. In addition, Hof1 is required for rapid accumulation as well as efficient removal of Chs4 at the division site. This study uncovers a mechanism by which Hof1 controls timely activation of Chs3 during cytokinesis and defines a novel interaction and function for the conserved F-BAR domain and SLR that are otherwise known for their abilities to bind membrane lipids [21, 22] and scaffold protein complex formation [23]

Identification of Novel, Evolutionarily Conserved Cdc42p-interacting Proteins and of Redundant Pathways Linking Cdc24p and Cdc42p to Actin Polarization in Yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p–Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures

Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.

We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p

Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

The tail of yeast myosin II is localized to the division site by two distinct molecular pathways and sufficient for promoting actomyosin ring assembly, furrow ingression, and guidance in ECM remodeling