Cell Polarity In Fission Yeast Research Articles

Local and global Cdc42 guanine nucleotide exchange factors for fission yeast cell polarity are coordinated by microtubules and the Tea1-Tea4-Pom1 axis.

ABSTRACTThe conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeast Schizosaccharomyces pombe has two Cdc42 guanine nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, we show here that when scd1 function is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4 or Pom1 leads to disruption of polarized growth. Instead, cells adopt an isotropic-like pattern of growth, which we term PORTLI growth. Surprisingly, PORTLI growth is caused by spatially inappropriate activity of Gef1. Although most Cdc42 GEFs are membrane associated, we find that Gef1 is a broadly distributed cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1–Tea4–Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results suggest a new model of fission yeast cell polarity regulation, involving coordination of ‘local’ (Scd1) and ‘global’ (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks.

Mechanisms Connecting the Conserved Protein Kinases Ssp1, Kin1, and Pom1 in Fission Yeast Cell Polarity and Division

Connections between the protein kinases that function within complex cell polarity networks are poorly understood. Rod-shaped fission yeast cells grow in ahighly polarized manner, and genetic screens haveidentified many protein kinases, including theCaMKK-like Ssp1 and the MARK/PAR-1 family kinase Kin1, that are required for polarized growth and cell shape, but their functional mechanisms and connections have been unknown [1-5]. We found that Ssp1 promotes cell polarity by phosphorylating the activation loop of Kin1. Kin1 regulates cell polarity and cytokinesis through unknown mechanisms [4-7]. We performed a large-scale phosphoproteomic screen and found that Kin1 phosphorylates itself and Pal1 to promote growth at cell tips, and these proteins are interdependent for localization to growing cell tips. Additional Kin1 substrates for cell polarity and cytokinesis (Tea4, Mod5, Cdc15, and Cyk3) were also phosphorylated by asecond kinase, the DYRK family member Pom1 [8]. Kin1 and Pom1 were enriched at opposite ends of growing cells, and they phosphorylated largely non-overlapping sites on shared substrates. Combined inhibition of both Kin1and Pom1 led to synthetic defects in their shared substrates Cdc15 andCyk3, confirming a non-redundant functional connection through shared substrates. These findings uncover a new Ssp1-Kin1 signaling pathway, and define its functional and mechanistic connection with Pom1 signaling for cellpolarity and cytokinesis. These kinases are conserved in many eukaryotes including humans, suggesting that similar connections and mechanisms might operate in a broad range of cells.

Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast.

Surface charges at the inner leaflet of the plasma membrane may contribute to regulate the surface recruitment of key signaling factors. Phosphatidylserine (PS) is an abundant charged lipid that may regulate charge distribution in different cell types. Here we characterize the subcellular distribution and function of PS in the rod-shaped, polarized fission yeast. We find that PS preferably accumulates at cell tips and defines a gradient of negative charges along the cell surface. This polarization depends on actin-mediated endocytosis and contributes to the subcellular partitioning of charged polarity-regulating Rho GTPases like Rho1 or Cdc42 in a protein charge-dependent manner. Cells depleted of PS have altered cell dimensions and fail to properly regulate growth from the second end, suggesting a role for PS and membrane charge in polarized cell growth.

In vitro systems for the study of microtubule-based cell polarity in fission yeast.

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries.

Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity.

Microtubules and the protein tea1p have important roles in regulating cell polarity in the fission yeast Schizosaccharomyces pombe. Here, using combinations of drugs, environmental perturbations and genetic mutants, we demonstrate that once a cell polarity axis is established, microtubules have at best a minor role in maintaining the cortical actin cytoskeleton and the rate and direction of cell growth. In addition, we find that after perturbations that disrupt cell polarity and the cortical actin cytoskeleton, microtubules are not required for re-establishment of polarity per se. However, after such perturbations, the distribution of cytoplasmic microtubules plays an important role in dictating the position of sites of polarity re-establishment. Furthermore, this influence of microtubule distribution on site selection during polarity re-establishment requires the presence of tea1p, suggesting that tea1p is crucial for coupling microtubule distribution to the regulation of cell polarity. Our results suggest a model in which, at the cellular level, two distinct and separable mechanisms contribute to how tea1p regulates site selection during polarity re-establishment. First, tea1p remaining at cell tips after cortical depolarization can serve as a cortical landmark for microtubule-independent site selection; second, tea1p newly targeted to the cell cortex by association with microtubules can promote the formation of polarity axes de novo.

Control of Cell Polarity in Fission Yeast by Association of Orb6p Kinase with the Highly Conserved Protein Methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.

Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast

Background: In many cell types, microtubules are thought to direct the spatial distribution of F-actin in cell polarity. Schizosaccharomyces pombe cells exhibit a regulated program of polarized cell growth: after cell division, they grow first in a monopolar manner at the old end, and in G2 phase, initiate growth at the previous cell division site (the new end). The role of microtubule ends in cell polarity is highlighted by the finding that the cell polarity factor, tea1p, is present on microtubule plus ends and cell tips [1].Results: Here, we characterize S. pombe bud6p/fat1p, a homolog of S. cerevisiae Bud6/Aip3. bud6Δ mutant cells have a specific defect in the efficient initiation of growth at the new end and like tea1Δ cells, form T-shaped cells in a cdc11 background. Bud6-GFP localizes to both cell tips and the cytokinesis ring. Maintenance of cell tip localization is dependent upon actin but not microtubules. Bud6-GFP localization is tea1p dependent, and tea1p localization is not bud6p dependent. tea1Δ and bud6Δ cells generally grow in a monopolar manner but exhibit different growth patterns. tea1Δbud6Δ mutants resemble tea1Δ mutants. Tea1p and bud6p coimmunoprecipitate and comigrate in large complexes.Conclusions: Our studies show that tea1p (a microtubule end-associated factor) and bud6p (an actin-associated factor) function in a common pathway, with bud6p downstream of tea1p. To our knowledge, bud6p is the first protein shown to interact physically with tea1p. These studies delineate a pathway for how microtubule plus ends function to polarize the actin cytoskeleton through actin-associated polarity factors.

Mis-specification of cortical identity in a fission yeast PAK mutant

The regulation of cell polarity in the fission yeast Schizosaccharomyces pombe is apparent in the restriction of extensile growth to the two ends of a cylindrically shaped cell, and in a specific transition - termed 'new-end take-off' (NETO) - between monopolar and bipolar growth mid-way through the cell cycle [1]. Several genes have been identified that affect one or more aspects of cell polarity (reviewed in [2] [3]), and the molecular pathways regulating cell polarity in fission yeast appear to be conserved among eukaryotes [3] [4] [5] [6] [7] [8] [9], but it is less clear how the proteins involved organize polarity at the level of the entire cell. Here, we describe novel cytological markers of cell polarity in fission yeast and their unusual localization in the monopolar growth mutant orb2-34, which carries a non-lethal mutation in the essential gene shk1(+)/pak1(+)/orb2(+), which encodes a p21-activated kinase (PAK) family member [8] [9] [10] [11] [12]. Our results suggest that, in contrast to other monopolar-growing mutants, the monopolar phenotype of the orb2-34 mutant might not be due to a defect in activating end growth per se, but rather reflects a failure of one of the cell ends to maintain the molecular properties that identify an end. Thus, one role of the Shk1/Pak1 kinase in vivo might be to contribute to how a cell recognizes its ends as sites for growth.