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
March 10th, 2010 / No Comments » / by Ossipova O, Sokol SY
Cell polarity, Notch signaling and neurogenesis.
Cell Cycle. 2010 Jan 1;9(1):1-2
Authors: Ossipova O, Sokol SY
PMID: 20016263 [PubMed - indexed for MEDLINE]
Posted in: Cell Cycle
March 10th, 2010 / No Comments » / by Marques JT, Kim K, Wu PH, Alleyne TM, Jafari N, Carthew RW
Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila.
Nat Struct Mol Biol. 2010 Jan;17(1):24-30
Authors: Marques JT, Kim K, Wu PH, Alleyne TM, Jafari N, Carthew RW
In Drosophila melanogaster, the small interfering RNA (siRNA) pathway is triggered by exogenous double-stranded RNA (dsRNA) or upon viral infection. This pathway requires Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) called R2D2. A potentially distinct siRNA pathway, which requires Dcr-2 in association with a different dsRBP, called Loquacious (Loqs), is activated by endogenous dsRNA derived from transposons, structured loci and overlapping transcripts. Here we show that different sources of dsRNA enter a common siRNA pathway that requires R2D2 and Loqs. R2D2 and loqs mutants show impaired silencing triggered by injection of exogenous dsRNA or by artificial and natural expression of endogenous dsRNA. In addition, we show that these dsRBPs function sequentially and nonredundantly in collaboration with Dcr-2. Loqs is primarily required for dsRNA processing, whereas R2D2 is essential for the subsequent loading of siRNAs into effector Ago-RISC complexes.
PMID: 20037596 [PubMed - indexed for MEDLINE]
Posted in: Nat Struct Mol Biol
March 10th, 2010 / No Comments » / by Paquette N, Broemer M, Aggarwal K, Chen L, Husson M, Ertürk-Hasdemir D, Reichhart JM, Meier P, Silverman N
Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF-kappaB signaling.
Mol Cell. 2010 Jan 29;37(2):172-82
Authors: Paquette N, Broemer M, Aggarwal K, Chen L, Husson M, Ertürk-Hasdemir D, Reichhart JM, Meier P, Silverman N
Innate immune responses are critical for the immediate protection against microbial infection. In Drosophila, infection leads to the rapid and robust production of antimicrobial peptides through two NF-kappaB signaling pathways-IMD and Toll. The IMD pathway is triggered by DAP-type peptidoglycan, common to most Gram-negative bacteria. Signaling downstream from the peptidoglycan receptors is thought to involve K63 ubiquitination and caspase-mediated cleavage, but the molecular mechanisms remain obscure. We now show that PGN stimulation causes caspase-mediated cleavage of the imd protein, exposing a highly conserved IAP-binding motif (IBM) at its neo-N terminus. A functional IBM is required for the association of cleaved IMD with the ubiquitin E3-ligase DIAP2. Through its association with DIAP2, IMD is rapidly conjugated with K63-linked polyubiquitin chains. These results mechanistically connect caspase-mediated cleavage and K63 ubiquitination in immune-induced NF-kappaB signaling.
PMID: 20122400 [PubMed - indexed for MEDLINE]
Posted in: Mol Cell
March 10th, 2010 / No Comments » / by Deas E, Dunn L
Unraveling LRRK2 pathogenesis: common pathways for complex genes?
J Neurosci. 2010 Feb 3;30(5):1577-9
Authors: Deas E, Dunn L
PMID: 20130167 [PubMed - indexed for MEDLINE]
Posted in: J Neurosci
March 10th, 2010 / No Comments » / by Capelson M, Liang Y, Schulte R, Mair W, Wagner U, Hetzer MW
Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes.
Cell. 2010 Feb 5;140(3):372-83
Authors: Capelson M, Liang Y, Schulte R, Mair W, Wagner U, Hetzer MW
Nuclear pore complexes have recently been shown to play roles in gene activation; however their potential involvement in metazoan transcription remains unclear. Here we show that the nucleoporins Sec13, Nup98, and Nup88, as well as a group of FG-repeat nucleoporins, bind to the Drosophila genome at functionally distinct loci that often do not represent nuclear envelope contact sites. Whereas Nup88 localizes to silent loci, Sec13, Nup98, and a subset of FG-repeat nucleoporins bind to developmentally regulated genes undergoing transcription induction. Strikingly, RNAi-mediated knockdown of intranuclear Sec13 and Nup98 specifically inhibits transcription of their target genes and prevents efficient reactivation of transcription after heat shock, suggesting an essential role of NPC components in regulating complex gene expression programs of multicellular organisms.
PMID: 20144761 [PubMed - indexed for MEDLINE]
Posted in: Cell
