Expression and Characterization of the Flocculin Flo11/Muc1, a Yeast Mannoprotein with Homotypic Properties of Adhesion

The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticusstrains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion. The fungal adhesins are a family of cell surface proteins that mediate adherence to environmental substrates or to other cells (7, 45). Adhesins are critically important in the initial steps of fungal pathogenicity, when fungal cells must adhere to host tissue. For the common human pathogens Candida albicans and Candida glabrata, the involvement of multiple adhesins in the adherence of fungal cells to host tissue has been demonstrated (4, 5, 18, 26, 43). Among the adhesins is the flocculin family of Saccharomyces cerevisiae cell wall proteins that mediate flocculation, which is asexual calcium-dependent cell-cell aggregation. The most recently described member of the yeast flocculin gene family, FLO11/MUC1 (24, 30), is the only flocculin expressed in the Σ1278b strain of S. cerevisiae (17), and it exhibits a wide variety of phenotypes. Some of these phenotypes are strain specific. Yeast cells of strain background Σ1278b have been shown to require FLO11 for invasive growth (23, 30), the development of pseudohyphae (24, 29), and the formation of biofilms on plastic (36), but they do not flocculate. On the other hand, the variant strain S. cerevisiae var. diastaticus, which is highly flocculent, has been shown to require FLO11 for flocculation (30). FLO11 is also required in Σ1278b strains for the formation of mats with hub and spoke structures on semisolid agar (36). The common laboratory strain background S288C does not express FLO11 due to a nonsense mutation in the transcriptional activator FLO8(28). In some industrial strains, FLO11 mediates formation of the specialized biofilms called flors that are necessary for the production of sherry wine (19, 48). The common feature of all these phenotypes is adhesion. Commensurate with the many different pathways that regulate its expression, FLO11 has been shown to have a promoter that is among the largest described for yeast, at over 3 kb (38). Much more is known about gene regulation of FLO11 (for reviews, see references 11, 25, and 32) than about the structure and function of the protein. We further investigated the FLO11-dependent phenotypes of S. cerevisiae var. diastaticus and found that it also differs from Σ1278b in that the haploids do not invade agar and the diploids do not form pseudohyphae. In order to investigate these strain differences in the phenotypes of FLO11we expressed and purified the Flo11 proteins from S. cerevisiae var. diastaticus and from Σ1278b and examined their properties. An in vitro system was created for studying the adhesive characteristics of the expressed Flo11 by attaching the protein to microscopic beads and testing the adhesive properties of the beads. Microscopic beads that can be coated covalently with proteins or ligands have been used to simplify several complex biological processes. For example, Gaur and Klotz used microscopic magnetic beads coated with extracellular matrix proteins to isolate a C. albicans adhesin gene, ALS5, by expression cloning in S. cerevisiae (13). Further work using such beads resulted in characterization of the adhesion properties of Ala1p and Als1p (12, 14, 15, 21, 22, 35). In this study, we have used this approach to study the in vitro properties of purified Flo11 proteins from two different strains

The 3D Facies Architecture and Petrophysical Properties of Hyaloclastite Delta Deposits : An Integrated Photogrammetry and Petrophysical Study from southern Iceland

ACKNOWLEDGEMENTS Dougal Jerram is partly funded through a Norwegian Research Council Centres of Excellence project (project number 223272, CEED). Adam Soule, Kirstie Wright and an anonymous reviewer are thanked for their extensive comments which helped to improve the final manuscript. We thank Cynthia Ebinger for clear editorial guidance and handing of the manuscript.Peer reviewedPostprin

Loss of Tumor Suppressor TMEM127 Drives Ret-Mediated Transformation Through Disrupted Membrane Dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation

A Longitudinal Study of Medicaid Coverage for Tobacco Dependence Treatments in Massachusetts and Associated Decreases in Hospitalizations for Cardiovascular Disease

Thomas Land and colleagues show that among Massachusetts Medicaid subscribers, use of a comprehensive tobacco cessation pharmacotherapy benefit was followed by a substantial decrease in claims for hospitalizations for acute myocardial infarction and acute coronary heart disease

Collective Management Organisations, Creativity and Cultural Diversity

Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Generation of Trophoblast Stem Cells from Rabbit Embryonic Stem Cells with BMP4

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-β receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-β signalings in maintenance of rabbit TS cell self-renewal