March 10th, 2010 / No Comments » / by McLellan J, O'Neil N, Tarailo S, Stoepel J, Bryan J, Rose A, Hieter P
Synthetic lethal genetic interactions that decrease somatic cell proliferation in Caenorhabditis elegans identify the alternative RFC CTF18 as a candidate cancer drug target.
Mol Biol Cell. 2009 Dec;20(24):5306-13
Authors: McLellan J, O'Neil N, Tarailo S, Stoepel J, Bryan J, Rose A, Hieter P
Somatic mutations causing chromosome instability (CIN) in tumors can be exploited for selective killing of cancer cells by knockdown of second-site genes causing synthetic lethality. We tested and statistically validated synthetic lethal (SL) interactions between mutations in six Saccharomyces cerevisiae CIN genes orthologous to genes mutated in colon tumors and five additional CIN genes. To identify which SL interactions are conserved in higher organisms and represent potential chemotherapeutic targets, we developed an assay system in Caenorhabditis elegans to test genetic interactions causing synthetic proliferation defects in somatic cells. We made use of postembryonic RNA interference and the vulval cell lineage of C. elegans as a readout for somatic cell proliferation defects. We identified SL interactions between members of the cohesin complex and CTF4, RAD27, and components of the alternative RFC(CTF18) complex. The genetic interactions tested are highly conserved between S. cerevisiae and C. elegans and suggest that the alternative RFC components DCC1, CTF8, and CTF18 are ideal therapeutic targets because of their mild phenotype when knocked down singly in C. elegans. Furthermore, the C. elegans assay system will contribute to our knowledge of genetic interactions in a multicellular animal and is a powerful approach to identify new cancer therapeutic targets.
PMID: 19846659 [PubMed - indexed for MEDLINE]
Posted in: Mol Biol Cell
March 10th, 2010 / No Comments » / by Li XB, Xu D, Wang XL, Huang GQ, Luo J, Li DD, Zhang ZT, Xu WL
Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.
J Exp Bot. 2010;61(1):41-53
Authors: Li XB, Xu D, Wang XL, Huang GQ, Luo J, Li DD, Zhang ZT, Xu WL
To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.
PMID: 19861654 [PubMed - indexed for MEDLINE]
Posted in: J Exp Bot
March 10th, 2010 / No Comments » / by Coffman VC, Nile AH, Lee IJ, Liu H, Wu JQ
Roles of formin nodes and myosin motor activity in Mid1p-dependent contractile-ring assembly during fission yeast cytokinesis.
Mol Biol Cell. 2009 Dec;20(24):5195-210
Authors: Coffman VC, Nile AH, Lee IJ, Liu H, Wu JQ
Two prevailing models have emerged to explain the mechanism of contractile-ring assembly during cytokinesis in the fission yeast Schizosaccharomyces pombe: the spot/leading cable model and the search, capture, pull, and release (SCPR) model. We tested some of the basic assumptions of the two models. Monte Carlo simulations of the SCPR model require that the formin Cdc12p is present in >30 nodes from which actin filaments are nucleated and captured by myosin-II in neighboring nodes. The force produced by myosin motors pulls the nodes together to form a compact contractile ring. Live microscopy of cells expressing Cdc12p fluorescent fusion proteins shows for the first time that Cdc12p localizes to a broad band of 30-50 dynamic nodes, where actin filaments are nucleated in random directions. The proposed progenitor spot, essential for the spot/leading cable model, usually disappears without nucleating actin filaments. alpha-Actinin ain1 deletion cells form a normal contractile ring through nodes in the absence of the spot. Myosin motor activity is required to condense the nodes into a contractile ring, based on slower or absent node condensation in myo2-E1 and UCS rng3-65 mutants. Taken together, these data provide strong support for the SCPR model of contractile-ring formation in cytokinesis.
PMID: 19864459 [PubMed - indexed for MEDLINE]
Posted in: Mol Biol Cell
March 10th, 2010 / No Comments » / by Johnson D
New connections identify Sch9 as a central node in ribosome biosynthesis.
Cell Cycle. 2010 Jan 1;9(1):26-7
Authors: Johnson D
PMID: 20016255 [PubMed - indexed for MEDLINE]
Posted in: Cell Cycle
March 10th, 2010 / No Comments » / by Steczkiewicz K, Knizewski L, Rychlewski L, Ginalski K
TOS1 is circularly permuted 1,3-beta-glucanase.
Cell Cycle. 2010 Jan 1;9(1):201-4
Authors: Steczkiewicz K, Knizewski L, Rychlewski L, Ginalski K
PMID: 20016268 [PubMed - indexed for MEDLINE]
Posted in: Cell Cycle
March 10th, 2010 / No Comments » / by Dibya D, Sander S, Smith EA
Identifying cytoplasmic proteins that affect receptor clustering using fluorescence resonance energy transfer and RNA interference.
Anal Bioanal Chem. 2009 Dec;395(7):2303-11
Authors: Dibya D, Sander S, Smith EA
Unraveling the complex, dynamic organization of the cell membrane can provide vital information about many aspects of cellular functions. Reported herein is a method for identifying cytoplasmic proteins that affect cell membrane protein organization. RNA interference (RNAi) is used to reduce the expression of select cytoplasmic proteins and a fluorescence resonance energy transfer (FRET) assay is used to measure changes in receptor microclustering. The advantage of this assay is that it does not require attaching fluorescent tags to the receptor. A change in energy transfer after reducing the expression of a cytoplasmic protein provides information about the protein's role in altering receptor organization. As a demonstration of the method, cytoplasmic proteins involved in integrin microclustering have been identified. The cytoplasmic proteins targeted in this study include: dreadlock, integrin-linked kinase, paxillin, steamer duck, vinculin, rhea, focal adhesion kinase, and actin 42A. Reducing the expression of vinculin, paxillin, rhea, and focal adhesion kinase increased integrin microclustering, as measured by an increase in energy transfer in cells expressing alphaPS2CbetaPS integrins. No change in integrin microclustering was measured in a control cell line. Integrin mutants exhibited different microclustering properties compared to the wild-type integrins after reducing the expression of the listed cytoplasmic proteins. The results demonstrate the utility of this assay format, and provide insight into the function of cytoplasmic proteins in integrin microclustering.
PMID: 19806349 [PubMed - indexed for MEDLINE]
Posted in: Anal Bioanal Chem
March 10th, 2010 / No Comments » / by Sharp DJ, Rath U
Mitosis: KLP61F goes wee!
Curr Biol. 2009 Oct 13;19(19):R899-901
Authors: Sharp DJ, Rath U
Kinesin-5s help assemble the bipolar spindle by crosslinking and sliding apart antiparallel microtubules. A recent study has uncovered a novel pathway for the phospho-regulation of these motors.
PMID: 19825352 [PubMed - indexed for MEDLINE]
Posted in: Curr Biol
March 10th, 2010 / No Comments » / by Badouel C, Garg A, McNeill H
Herding Hippos: regulating growth in flies and man.
Curr Opin Cell Biol. 2009 Dec;21(6):837-43
Authors: Badouel C, Garg A, McNeill H
Control of cell number requires the coordinate regulation of cell proliferation and cell death. Studies in both the fly and mouse have identified the Hippo kinase pathway as a key signaling pathway that controls cell proliferation and apoptosis. Several studies have implicated the Hippo pathway in a variety of cancers. Recent studies have also revealed a role for the Hippo pathway in the control of cell fate decisions during development. In this review, we will cover the current model of Hippo signaling in development. We will explore the differences between the Hippo pathway in invertebrates and mammals, and focus on recent advances in understanding how this conserved pathway is regulated.
PMID: 19846288 [PubMed - indexed for MEDLINE]
Posted in: Curr Opin Cell Biol
March 10th, 2010 / No Comments » / by Sabogal A, Lyubimov AY, Corn JE, Berger JM, Rio DC
THAP proteins target specific DNA sites through bipartite recognition of adjacent major and minor grooves.
Nat Struct Mol Biol. 2010 Jan;17(1):117-23
Authors: Sabogal A, Lyubimov AY, Corn JE, Berger JM, Rio DC
THAP-family C(2)CH zinc-coordinating DNA-binding proteins function in diverse eukaryotic cellular processes, such as transposition, transcriptional repression, stem-cell pluripotency, angiogenesis and neurological function. To determine the molecular basis for sequence-specific DNA recognition by THAP proteins, we solved the crystal structure of the Drosophila melanogaster P element transposase THAP domain (DmTHAP) in complex with a natural 10-base-pair site. In contrast to C(2)H(2) zinc fingers, DmTHAP docks a conserved beta-sheet into the major groove and a basic C-terminal loop into the adjacent minor groove. We confirmed specific protein-DNA interactions by mutagenesis and DNA-binding assays. Sequence analysis of natural and in vitro-selected binding sites suggests that several THAPs (DmTHAP and human THAP1 and THAP9) recognize a bipartite TXXGGGX(A/T) consensus motif; homology suggests THAP proteins bind DNA through a bipartite interaction. These findings reveal the conserved mechanisms by which THAP-family proteins engage specific chromosomal target elements.
PMID: 20010837 [PubMed - indexed for MEDLINE]
Posted in: Nat Struct Mol Biol
March 10th, 2010 / No Comments » / by Stolz A, Bastians H
A novel role for Chk2 after DNA damage in mitosis?
Cell Cycle. 2010 Jan 1;9(1):25-6
Authors: Stolz A, Bastians H
PMID: 20016261 [PubMed - indexed for MEDLINE]
Posted in: Cell Cycle
