Showing papers by "Michael Snyder published in 1998"

Cell polarity and morphogenesis in budding yeast

Abstract: ▪ Abstract Eukaryotic cells respond to intracellular and extracellular cues to direct asymmetric cell growth and division. The yeast Saccharomyces cerevisiae undergoes polarized growth at several times during budding and mating and is a useful model organism for studying asymmetric growth and division. In recent years, many regulatory and cytoskeletal components important for directing and executing growth have been identified, and molecular mechanisms have been elucidated in yeast. Key signaling pathways that regulate polarization during the cell cycle and mating response have been described. Since many of the components important for polarized cell growth are conserved in other organisms, the basic mechanisms mediating polarized cell growth are likely to be universal among eukaryotes.

Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

Abstract: The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2D, pea2D, and bud6D mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2pinteracting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.

Pheromone-regulated Genes Required for Yeast Mating Differentiation

Abstract: Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for β-galactosidase (β-gal) expression in the presence and absence of α factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: β-gal and Fig2::β-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.

Ursodiol Prophylaxis against Hepatic Complications of Allogeneic Bone Marrow Transplantation: A Randomized, Double-Blind, Placebo-Controlled Trial

Abstract: Background Hepatic complications are a major cause of illness and death after bone marrow transplantation. Objective To confirm the results of a pilot study that indicated that ursodiol prophylaxis could reduce the incidence of veno-occlusive disease of the liver. Design Randomized, double-blind, placebo-controlled study. Setting Tertiary care teaching hospital. Patients 67 consecutive patients undergoing transplantation with allogeneic bone marrow (donated by a relative) in whom busulfan plus cyclophosphamide was used as the preparative regimen and cyclosporine plus methotrexate was used to prevent graft-versus-host disease. Intervention Before the preparative regimen was started, patients were randomly assigned to receive ursodiol, 300 mg twice daily (or 300 mg in the morning and 600 mg in the evening if body weight was > 90 kg), or placebo. Measurements Patients were prospectively evaluated for the clinical diagnosis of veno-occlusive disease, the occurrence of acute graft-versus-host disease, and survival. Results The incidence of veno-occlusive disease was 40% (13 of 32 patients) in placebo recipients and 15% (5 of 34 patients) in ursodiol recipients (P = 0.03). Assignment to placebo was the only pretransplantation characteristic that predicted the development of veno-occlusive disease. The most significant predictor of 100-day mortality was the diagnosis of veno-occlusive disease. The difference in actuarial risk for hematologic relapse in patients with chronic myelogenous leukemia and nonhepatic toxicities between the two groups was not statistically significant (13% in the ursodiol group and 20% in the placebo group; P > 0.2). Conclusion Ursodiol prophylaxis seemed to decrease the incidence of hepatic complications after allogeneic bone marrow transplantation in patients who received a preparative regimen with busulfan plus cyclophosphamide.

The spa2-related protein, sph1p, is important for polarized growth in yeast

Abstract: The Saccharomyces cerevisiae protein Sph1p is both structurally and functionally related to the polarity protein, Spa2p. Sph1p and Spa2p are predicted to share three 100-amino acid domains each exceeding 30% sequence identity, and the amino-terminal domain of each protein contains a direct repeat common to Homo sapiens and Caenorhabditis elegans protein sequences. sph1- and spa2-deleted cells possess defects in mating projection morphology and pseudohyphal growth. sph1(Delta) spa2(Delta) double mutants also exhibit a strong haploid invasive growth defect and an exacerbated mating projection defect relative to either sph1(Delta) or spa2(Delta) single mutants. Consistent with a role in polarized growth, Sph1p localizes to growth sites in a cell cycle-dependent manner: Sph1p concentrates as a cortical patch at the presumptive bud site in unbudded cells, at the tip of small, medium and large buds, and at the bud neck prior to cytokinesis. In pheromone-treated cells, Sph1p localizes to the tip of the mating projection. Proper localization of Sph1p to sites of active growth during budding and mating requires Spa2p. Sph1p interacts in the two-hybrid system with three mitogen-activated protein (MAP) kinase kinases (MAPKKs): Mkk1p and Mkk2p, which function in the cell wall integrity/cell polarization MAP kinase pathway, and Ste7p, which operates in the pheromone and pseudohyphal signaling response pathways. Sph1p also interacts weakly with STE11, the MAPKKK known to activate STE7. Moreover, two-hybrid interactions between SPH1 and STE7 and STE11 occur independently of STE5, a proposed scaffolding protein which interacts with several members of this MAP kinase module. We speculate that Spa2p and Sph1p may function during pseudohyphal and haploid invasive growth to help tether this MAP kinase module to sites of polarized growth. Our results indicate that Spa2p and Sph1p comprise two related proteins important for the control of cell morphogenesis in yeast.

Ursodiol Prophylaxis against Hepatic Complications of Allogeneic Bone Marrow Transplantation

Abstract: Background: Hepatic complications are a major cause of illness and death after bone marrow transplantation. Objective: To confirm the results of a pilot study that indicated that ursodiol prophylax...

10 Transposon Tagging I: A Novel System for Monitoring Protein Production, Function and Localization

Abstract: Publisher Summary The use of transposons allows the rapid construction of a large number of the alleles of a gene of interest. Transposon insertion libraries can be used for both the mutagenesis and identification of genes regulated by particular growth conditions and strain backgrounds. This chapter presents an overview of a newly developed multipurpose transposon mutagenesis system that allows the monitoring of protein production, function, and localization in yeast. The system uses two basic types of transposon, designated as mTn-3×HA/lacZ and mTn 3×HA/GFP. The transposon system was tested by the mutagenesis of several individual yeast genes. The HAT tag was successfully used to analyze the localization of the Spa2, Arp100, and Sao1 proteins. The new transposons expand the repertoire of insertions that may be generated to include GFT fusions and epitope tags. It is expected that shuttle mutagenesis will continue to be an important tool for the characterization of individual genes and their products and for systematic analysis of the entire yeast genome.

Cell Polarity in the Budding Yeast Saccharomyces Cerevisiae

Abstract: Publisher Summary This chapter reviews the different steps and components involved in polarized growth in vegetative, pseudohyphal, and mating yeast cells. Polarized cell growth and polarized cell division are two basic processes important for both unicellular and multicellular organisms. Polarized cell growth is crucial for producing precise cellular structures and shapes that help mediate the specialized functions of distinct cell types. Polarized cell divisions occur at critical times in development and are responsible for directing appropriate cell–cell contacts, mediating growth in a specific direction, and establishing cytoplasmic differences between two daughter cells. Thus, both polarized growth and polarized divisions are essential for the development of tissues and entire organisms. Bud formation and projection formation are distinct mechanisms for polarized cell growth, and in the past years, a wealth of information about how these processes occur has been generated. Many of the components involved in polarized cell growth in yeast, including both structural components such as actin and regulatory components, such as mitogen-activated protein-kinase pathways and Rho proteins, are remarkably conserved in structure and in function with other eucaryotic cell types.