Core Cellular Processes Research Articles

Cancer Proliferation Gene Discovery Through Functional Genomics

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

A Chemogenomic Screen in Saccharomyces cerevisiae Uncovers a Primary Role for the Mitochondria in Farnesol Toxicity and Its Regulation by the Pkc1 Pathway

The isoprenoid farnesol has been shown to preferentially induce apoptosis in cancerous cells; however, the mode of action of farnesol-induced death is not established. We used chemogenomic profiling using Saccharomyces cerevisiae to probe the core cellular processes targeted by farnesol. This screen revealed 48 genes whose inactivation increased sensitivity to farnesol. The gene set indicated a role for the generation of oxygen radicals by the Rieske iron-sulfur component of complex III of the electron transport chain as a major mediator of farnesol-induced cell death. Consistent with this, loss of mitochondrial DNA, which abolishes electron transport, resulted in robust resistance to farnesol. A genomic interaction map predicted interconnectedness between the Pkc1 signaling pathway and farnesol sensitivity via regulation of the generation of reactive oxygen species. Consistent with this prediction (i) Pkc1, Bck1, and Mkk1 relocalized to the mitochondria upon farnesol addition, (ii) inactivation of the only non-essential and non-redundant member of the Pkc1 signaling pathway, BCK1, resulted in farnesol sensitivity, and (iii) expression of activated alleles of PKC1, BCK1, and MKK1 increased resistance to farnesol and hydrogen peroxide. Sensitivity to farnesol was not affected by the presence of the osmostabilizer sorbitol nor did farnesol affect phosphorylation of the ultimate Pkc1-responsive kinase responsible for controlling the cell wall integrity pathway, Slt2. The data indicate that the generation of reactive oxygen species by the electron transport chain is a primary mechanism by which farnesol kills cells. The Pkc1 signaling pathway regulates farnesol-mediated cell death through management of the generation of reactive oxygen species.

A bacterial dynamin-like protein

Dynamins form a superfamily of large mechano-chemical GTPases that includes the classical dynamins and dynamin-like proteins (DLPs). They are found throughout the Eukarya, functioning in core cellular processes such as endocytosis and organelle division. Many bacteria are predicted by sequence to possess large GTPases with the same multidomain architecture that is found in DLPs. Mechanistic dissection of dynamin family members has been impeded by a lack of high-resolution structural data currently restricted to the GTPase and pleckstrin homology domains, and the dynamin-related human guanylate-binding protein. Here we present the crystal structure of a cyanobacterial DLP in both nucleotide-free and GDP-associated conformation. The bacterial DLP shows dynamin-like qualities, such as helical self-assembly and tubulation of a lipid bilayer. In vivo, it localizes to the membrane in a manner reminiscent of FZL, a chloroplast-specific dynamin-related protein with which it shares sequence similarity. Our results provide structural and mechanistic insight that may be relevant across the dynamin superfamily. Concurrently, we show compelling similarity between a cyanobacterial and chloroplast DLP that, given the endosymbiotic ancestry of chloroplasts, questions the evolutionary origins of dynamins.

Prenylation is not necessary for endogenous Ras activation in non‐malignant cells

Ras monomeric GTPases are pivotal to many core cellular processes such as proliferation and differentiation. The post-translational prenylation of Ras with a farnesyl or a geranylgeranyl moiety is thought to be critical for its membrane binding and consequent signaling activity. Inhibitors of Ras prenylation have an anti-proliferative effect in some Ras-transformed cells. We present a study of the effects of prenylation inhibitors on endogenous, wild-type Ras in three renal cell types, namely primary adult human renal fibroblasts, primary adult human mesangial cells, and a primate renal fibroblast cell line (Vero cells). We have previously demonstrated that Ras is necessary for normal proliferation in these cells. Here we show that Ras is farnesylated and not geranylgeranylated in all three cell types. Furthermore, inhibiting Ras farnesylation has no effect on cell proliferation or Ras activation. Although inhibiting geranylgeranylation in these cells does inhibit proliferation, this is through an Ras-independent mechanism. Non-prenylated Ras is able to localize to the plasma membrane, bind Raf when cells are stimulated by epidermal growth factor or platelet-derived growth factor, and activate the Ras downstream effectors mitogen-activated protein kinase and phosphotidylinositol 3-kinase. We conclude that in wild-type cells, endogenous Ras does not need to be prenylated to be active.

On the Temporal Self-Organisation of Saccharomyces cerevisae

The budding yeast Saccharomyces cerevisiae has been one of the premier models for the study of eukaryote molecular biology for over 50 years. These studies have revealed a complex and robust yeast phenome and elucidated many of the underlying principles common to all eukaryotes including DNA, RNA, protein and metabolite interaction networks. However the degree complexity and integration of the cellular network has made dissecting the temporal dynamics of the phenotype a rather daunting task. Here I review work on glycolytic oscillation, oscillation observed in continuous culture and colony pattern formation, and find that redox is a central thread underpinning these phenomena. The outputs of the systems involve sub-networks that are at the core of the cellular network, e.g., glycolysis, stress response, respiration, cell cycle, amino acid biosynthesis, leading to the conclusion that a fundamental redox attractor underpins these core cellular processes. Keywords: Saccharomyces cerevisae, phenotype, glycolytic, oscillation

Infantile peri-osteitis.

Viruses are one of the major causes of various acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how...