On growth and form: control of cell morphogenesis in fission yeast

In the past year, we have gained considerable insight into the process of cell morphogenesis and the establishment of positional information in fission yeast. The highlights include a better understanding of the role of the microtubule cytoskeleton in the control of cell shape, as well as the identification of novel genes essential for the establishment of cell polarity and for the positioning of the site of cell division.     

Regulation of fission yeast morphogenesis by PP2A activator pta2

Cell polarization is key for the function of most eukaryotic cells, and regulates cell shape, migration and tissue architecture. Fission yeast, Schizosaccharomyces pombe cells are cylindrical and polarize cell growth to one or both cell tips dependent on the cell cycle stage. Whereas microtubule cytoskeleton contributes to the positioning of the growth sites by delivering polarity factors to the cell ends, the Cdc42 GTPase polarizes secretion via actin-dependent delivery and tethering of secretory vesicles to plasma membrane. How growth is restricted to cell tips and how re-initiation of tip growth is regulated in the cell cycle remains poorly understood. In this work we investigated the function of protein phosphatase type 2A (PP2A) in S. pombe morphogenesis by deleting the evolutionary conserved PTPA-type regulatory subunit that we named pta2. pta2-deleted cells showed morphological defects and altered growth pattern. Consistent with this, actin patches and active Cdc42 were mislocalized in the pta2 deletion. These defects were additive to the lack of Cdc42-GAP Rga4. pta2Δ cells show upregulated Cdc42 activity and pta2 interacts genetically with polarisome components Tea1, Tea4 and For3 leading to complete loss of cell polarity and rounded morphology. Thus, regulation of polarity by PP2A requires the polarisome and involves Pta2-dependent control of Cdc42 activity.     

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP₂-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.     

TOR signaling in fission yeast

Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.     

Ruptured fission yeast walls

Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis. As shown previously (Johnson et al.,Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length. The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986). The median length of cells was found to decline through the log phase in an unbalanced manner.     

Smashed fission yeast walls

Twenty-three samples of fission yeast cells (Schizosaccharomyces pombe) were smashed by shaking them with glass beads. The samples represented all phases of the culture cycle, with the lag and log phases emphasized. Ruptured walls of the smashed cells were observed by phase-contrast and electron microscopy. Ruptures were tabulated with respect to their magnitudes and locations. Ruptures occurred not at random, nor at sites directed by geometry, but predominated in certain definable wall regions. These discontinuities were correlated with morphogenetic activities of the cell. Thus, the extensile end was found to be most fragile through most of the culture cycle. Also fragile was the nonextensile end, its edge more than its middle. Further, the data were applied to the testing of predictions from extant models (Johnson endohydrolytic softening model and Wessels presoftened-posthardened and crosslinking model) for hyphal tip extension. The frequency of rupture at the extensile (old) end of the cell was qualitatively predicted by both models; the frequency at the nonextensile (new) end was not predictable by either. Rupture frequencies and characteristics at other regions conformed to predictions by one or the other model, but rarely by both.     

High-throughput knockout screen in fission yeast

We have designed the most efficient strategy to knock out genes in fission yeast Schizosaccharomyces pombe on a large scale. Our technique is based on knockout constructs that contain regions homologous to the target gene cloned into vectors carrying dominant drug-resistance markers. Most of the steps are carried out in a 96-well format, allowing simultaneous deletion of 96 genes in one batch. Based on our knockout technique, we designed a strategy for cloning knockout constructs for all predicted fission yeast genes, which is available in a form of a searchable database http://mendel.imp.ac.at/Pombe_deletion/. We validated this technique in a screen where we identified novel genes required for chromosome segregation during meiosis. Here, we present our protocol with detailed instructions. Using this protocol, one person can knock out 96 S. pombe genes in 8 days.     

Oxygen toxicity in a fission yeast

Continuous exposure of synchronous cultures of Schizosaccharomyces pombe to 2.0 atmospheres oxygen beginning at any point in the first two-thirds of the cell cycle prevented subsequent cell division. Similar exposure during the last one-third of the cell cycle did not prevent cell division. The inhibition of division was totally reversible. Exposure to 2.0 atmospheres oxygen for 2.5 hours did not affect oxygen consumption. Oxygen at 1.0 atmospheres reduced growth rate and protein synthesis by 44%. Similar exposure to 1.0 atmospheres reduced transport of glycine-¹⁴C, L-leucine-¹⁴C, and uracil-¹⁴C by 95%, 73%, and 89% respectively. Analysis of the kinetics of uptake of these materials showed noncompetitive inhibition of transport by oxygen. The primary effect in rapidly appearing oxygen toxicity apparently involved interference with the transport capabilities of the cell membrane.     

Programmed cell death in fission yeast

Recently a metacaspase, encoded by YCA1, has been implicated in a primitive form of apoptosis or programmed cell death in yeast. Previously it had been shown that over-expression of mammalian pro-apoptotic proteins can induce cell death in yeast, but the mechanism of how cell death occurred was not clearly established. More recently, it has been shown that DNA or oxidative damage, or other cell cycle blocks, can result in cell death that mimics apoptosis in higher cells. Also, in fission yeast deletion of genes required for triacylglycerol synthesis leads to cell death and expression of apoptotic markers. A metacaspase sharing greater than 40% identity to budding yeast Yca1 has been identified in fission yeast, however, its role in programmed cell death is not yet known. Analysis of the genetic pathways that influence cell death in yeast may provide insights into the mechanisms of apoptosis in all eukaryotic organisms.     

Nuclear size control in fission yeast

Along-standing biological question is how a eukaryotic cell controls the size of its nucleus. We report here that in fission yeast, nuclear size is proportional to cell size over a 35-fold range, and use mutants to show that a 16-fold change in nuclear DNA content does not influence the relative size of the nucleus. Multi-nucleated cells with unevenly distributed nuclei reveal that nuclei surrounded by a greater volume of cytoplasm grow more rapidly. During interphase of the cell cycle nuclear growth is proportional to cell growth, and during mitosis there is a rapid expansion of the nuclear envelope. When the nuclear/cell (N/C) volume ratio is increased by centrifugation or genetic manipulation, nuclear growth is arrested while the cell continues to grow; in contrast, low N/C ratios are rapidly corrected by nuclear growth. We propose that there is a general cellular control linking nuclear growth to cell size.     

Phosphoproteome analysis of fission yeast

Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.     

Physiological aspects of conjugation in fission yeast

Summary Conjugation was studied in Schizosaccharomyces pombe using liquid media. Nitrogen, which was growth-limiting in a synthetic medium, had to be consumed completely before conjugation could start. Conjugation was preceded by sexual agglutination. Agglutinability was not constitutive in heterothallic strains. It only developed when cells of h ⁺ and h ^- mating type were grown in mixed culture for at least 2.5 hr before the start of conjugation.