bluuebunny/crossref_metadata_2025 huggingface dataset:
Studying Integrin-Mediated Cell Adhesion at the Single-Molecule Level Using AFM Force Spectroscopy
Authors: Franz Clemens M. , Taubenberger Anna , Puech Pierre-Henri , Muller Daniel J.
Published: December 2007

The establishment of cell adhesion involves specific recognition events between individual cell-surface receptors and molecules of the cellular environment. However, characterizing single-molecule adhesion events in the context of a living cell presents an experimental challenge. The atomic force microscope (AFM) operated in force spectroscopy mode provides an ultrasensitive method to investigate cell adhesion forces at the level of single receptor-ligand bonds. With a living cell attached to the AFM cantilever, the number of cell-substrate interactions can be controlled and limited to the formation of single receptor-ligand bonds. From force-distance (F-D) curves recorded during cell detachment, the strength of single receptor-ligand bonds can be determined. Furthermore, by varying the rate of force application during bond rupture, a dynamic force spectrum (DFS) can be generated from which additional parameters that describe the energy landscape of the interaction, such as dissociation rate and energy barrier width, can be obtained. Using the example of α
2
β
1
integrin–mediated adhesion to type I collagen, we provide a detailed description of how dynamic AFM single-cell force spectroscopy (SCFS) adhesion measurements can be performed with single-molecule sensitivity, and how specific energy landscape parameters of the integrin-collagen bond can be extracted from the DFS.
Doping the Bone Marrow
Authors: Foley John F.
Published: December 2007

Repopulation of the bone marrow (BM) with transplanted hematopoietic stem cells is crucial for treatment of immunodeficiencies and malignancies. BM stem cells, which express CD34, are mobilized by treatment with the myeloid cytokines granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The bone marrow is a highly innervated tissue containing stores of dopamine, so Spiegel
et al
. studied the effects of dopamine on hematopoiesis. Flow cytometry revealed that treatment of human CD34
+
cells with G-CSF increased the abundance of the dopamine receptors DR3 and DR5. In vitro migration assays demonstrated that GM-CSF treatment led to an increase in the number of CD34
+
cells that migrated to dopamine. By measuring colony formation, the authors found that dopamine treatment increased the proliferation of CD34
+
cells. Moreover, engraftment of human G-CSF-mobilized CD34
+
cells in susceptible mouse BM was increased by pretreatment with dopamine agonists. Similar results were found with the β
2
-adrenergic receptor (β
2
-AR) agonist epinephrine. Flow cytometry demonstrated that treatment of CD34
+
cells with GM-CSF and epinephrine resulted in the accumulation of β-catenin, a downstream mediator of the Wnt signaling pathway, which is known to play a role in stem cell proliferation. Engraftment of mouse BM by GM-CSF and epinephrine-treated human CD34
+
cells was inhibited by blocking Wnt signaling. This study illustrates an important role for neurotransmitters in regulating the functions of hematopoietic progenitor cells and suggests that combining neurotransmitters with myeloid cytokines may be clinically relevant in enhancing the effectiveness of stem cell transplantation.

A. Spiegel, S. Shivtiel, A. Kalinkovich, A. Ludin, N. Netzer, P. Goichberg, Y. Azaria, I. Resnick, I. Hardan, H. Ben-Hur, A. Nagler, M. Rubinstein, T. Lapidot, Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34
+
cells through Wnt signaling.
Nat. Immunol.
8
, 1123-1131 (2007).
[PubMed]
The TCR in the Dock
Authors: Foley John F.
Published: December 2007

The Rho family guanosine triphosphatase Rac regulates the actin cytoskeleton and microtubule dynamics. Tanaka
et al
. investigated a role for Dock2, a guanine nucleotide exchange factor and activator of Rac in the regulation of CD4
+
T cell differentiation. The authors first established that deficiency in Dock2 in mouse CD4
+
T cells resulted in increased T helper 2 (T
H
2) responses, such as secretion of interleukin-4 (IL-4). Antigenic stimulation of

Dock2
–/–

CD4
+
T cells produced a higher proportion of T
H
2 cells than did similar stimulation of

Dock2
+/+

cells, whose response was dependent on IL-4. IL-4 signaling is necessary both for stimulating T
H
2 differentiation and for inhibiting T
H
1 differentiation. Flow cytometry revealed that, whereas T cell receptor (TCR) stimulation of

Dock2
+/+

CD4
+
T cells resulted in a gradual loss of the IL-4 receptor α subunit (IL-4Rα) from the cell surface, this response was inhibited in

Dock2
–/–

cells. Treatment of

Dock2
–/–

CD4
+
T cells with a cell-permeable dominant negative Rac mutant inhibited the TCR-stimulated decrease in IL-4Rα abundance. Western blotting showed that TCR stimulation of

Dock2
–/–

CD4
+
T cells resulted in increased and more sustained phosphorylation of signal transducer and activator of transcription 6 (STAT6), a downstream target of IL-4Rα that is necessary for T
H
2 differentiation. Confocal microscopy showed that TCR stimulation of

Dock2
+/+

CD4
+
T cells resulted in the Rac-dependent internalization of the IL-4Rα and its degradation in lysosomes, and this process was inhibited in

Dock2
–/–

cells. Together these data suggest a new mechanism by which the TCR regulates T cell differentiation. TCR signals through Dock2 and Rac leading to the internalization of IL-4Rα would inhibit IL-4-stimulated T
H
2 differentiation and thus favor T
H
1 differentiation.

Y. Tanaka, S. Hamano, K. Gotoh, Y. Murata, Y. Kunisaki, A. Nishikimi, R. Takii, M. Kawaguchi, A. Inayoshi, S. Masuko, K. Himeno, T. Sasazuki, Y. Fukui, T helper type 2 differentiation and intracellular trafficking of the interleukin 4 receptor-α subunit controlled by the Rac activator Dock2.
Nat. Immunol.
8
, 1067-1075 (2007).
[PubMed]
PI3K Class IB Pathway
Authors: Andrews Simon , Stephens Len R. , Hawkins Phillip T.
Published: December 2007

Class I phosphoinositide 3-kinases (PI3Ks) are well-established signal transduction enzymes that play an important role in the mechanisms by which a wide variety of cell surface receptors control several cellular functions, including cellular growth, division, survival, and movement. Class IB PI3K (also known as PI3Kγ) allows fast-acting, heterotrimeric GTP-binding protein–coupled receptors to access this pathway. Activation of class IB PI3K results in the rapid synthesis of phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P
3
] and its dephosphorylation product, PI(3,4)P
2
, in the plasma membrane. These two lipid messengers bind to multiple, pleckstrin homology (PH) domain–containing effectors, which together regulate a complex signaling web downstream of receptor activation. This pathway regulates the activity of protein kinases and small guanosine triphosphatases that control cellular movement, adhesion, contraction, and secretion. Most of the ligands that have been established to activate class IB PI3K are involved in coordinating the body’s response to injury and infection through the regulation of multiple cell types in the immune system and vascular lining. Mice lacking the catalytic subunit of class IB PI3K are remarkably resistant to the development of several inflammatory pathologies in mouse models of human inflammatory disease. These results suggest small molecule inhibitors of class IB PI3K may represent a novel class of therapeutic agents that may complement existing anti-inflammatory treatments.
Cytokinin Signaling Pathway
Authors: Müller Bruno , Sheen Jen
Published: December 2007

Cytokinins are key regulators of a large number of processes in plant development, which is highly plastic and adaptive, and remarkably resilient and self-perpetuating. Cytokinin signaling involves a multistep two-component system, also called a phosphorelay signaling system. Genes encoding the central components to build the cytokinin phosphorelay system are found in diverse plant species, resulting in the establishment of a canonical model of cytokinin signaling that is likely representative and conserved in plants. The cytokinin signaling pathway can be modulated by nutrients, stress, and other hormones, and is integrated into a signaling network in both shoots and roots.
Arabidopsis
Cytokinin Signaling Pathway
Authors: Müller Bruno , Sheen Jen
Published: December 2007

Cytokinins are essential plant hormones that control cell division, shoot meristem initiation, leaf and root differentiation, vasculature patterning, chloroplast biogenesis, photomorphogenesis, fertility, seed development, senescence, and stress tolerance. The
Arabidopsis
cytokinin signal transduction pathway involves hybrid histidine protein kinases [AHK2, AHK3, and AHK4 (also known as CRE1or WOL)] as cytokinin receptors, histidine phosphotransfer proteins (AHPs), and nuclear response regulators (ARRs) that serve as transcriptional regulators. There are four major steps in the cytokinin phosphorelay: (i) AHK sensing and signaling, (ii) AHP nuclear translocation, (iii) ARR-dependent transcriptional activation, and (iv) a negative-feedback loop through cytokinin-inducible ARR gene products. Each step is executed by components encoded by multigene families. The effects of cytokinin depend on cell type, environment, and developmental stage. The response is frequently the outcome of interactions with other plant signaling pathways.
Hedgehog Signaling Pathway
Authors: Jacob Leni , Lum Lawrence
Published: December 2007

The Hedgehog (Hh) signaling pathway is essential for development and tissue homeostasis in metazoans. Probably as a result of its potent influence on cell-fate outcomes, the Hh pathway when corrupted results in malformations and diseases such as cancer. Many of the pathway components that contribute to Hh-mediated signal transduction are presented.
Hedgehog Signaling Pathway in
Drosophila
Authors: Jacob Leni , Lum Lawrence
Published: December 2007

Studies in
Drosophila
have provided the framework for understanding human development and disease processes governed by the Hedgehog (Hh) family of secreted signaling molecules. The
Drosophila
proteins that participate in Hh-mediated signal transduction are highlighted in a Connections Map pathway.
How Clocks See the Light
Authors: Ray L. Bryan
Published: December 2007

The circadian clocks that regulate daily rhythms in various processes in living organisms are entrained to a 24-hour cycle by mechanisms that detect daily changes in the amount of light in the environment. Hirayama
et al
. show that hydrogen peroxide (H
2
O
2
) can function as a required signaling molecule to transmit the sensation of light to changes in timing of the biochemical clock. In zebrafish, oscillators are present in peripheral tissues and organs and are sensitive to exposure to light. Hirayama
et al
. used Z3 cells (a cell line from zebrafish embryos) to show that exposure of the cells to light caused increased production of H
2
O
2
(which was detected in cells transfected with a fluorescent probe molecule sensitive to oxidation by reactive oxygen species). Exposure of the cells to H
2
O
2
increased expression of zebrafish
Cryptochrome
and
Period
genes (which encode components of the core clock machinery) and did so with a time course similar to that observed when cells were exposed to light. Catalase is an antioxidant enzyme that can degrade H
2
O
2
, and the authors confirmed that light stimulated expression of the
zCat
gene, but it did so with a delayed time course consistent with its possible function in a negative feedback loop to cyclically suppress expression of the clock genes that initially resulted from light-induced generation of H
2
O
2
. In mammalian cells, H
2
O
2
did not influence expression of the clock genes, but then, mammalian peripheral tissues are not responsive to light. What remains unknown is the identity of the actual phototransducer in the zebrafish system.

J. Hirayama, S. Cho, P. Sassone-Corsi, Circadian control by the reduction/oxidation pathway: Catalase represses light-dependent clock gene expression in the zebrafish.
Proc. Natl. Acad. Sci. U.S.A.
104
, 15747-15752 (2007).
[Abstract]
[Full Text]
Harming Thyself
Authors: Foley John F.
Published: December 2007

Psoriasis is an inflammatory autoimmune disorder of the skin characterized by excessive proliferation of keratinocytes and the presence of plasmacytoid dendritic cells (pDCs). Whereas pDCs normally sense microbial DNA in a Toll-like receptor 9 (TLR9)-dependent fashion, in psoriatic lesions pDCs respond to self-DNA and secrete interferon-α (IFN-α), which exacerbates the condition. Lande
et al
. investigated the mechanism involved and screened extracts of normal and psoriatic human skin (fractionated by reversed-phase high-performance liquid chromatography) for factors that could stimulate IFN-α production by cultured human pDCs. Extracts of normal skin did not induce a response; however, a fraction from psoriatic skin stimulated robust production of IFN-α as measured by enzyme-linked immunosorbent assay. Mass spectrometry identified the cathelicidin LL37, an antimicrobial peptide, as the stimulating factor. The addition of an antibody against LL37 blocked the stimulation by psoriatic skin extract of IFN-α secretion by pDCs. Western blotting and real-time reverse transcription polymerase chain reaction assays showed that LL37 protein and mRNA were more abundant in psoriatic skin than in healthy skin. LL37 is produced in the skin as an antibacterial defense after tissue damage, which often precedes psoriatic episodes. Treatment of pDC cultures with DNase, which removed DNA from dead pDCs, blocked the ability of LL37 to stimulate IFN-α secretion. Atomic force microscopy showed that LL37 formed aggregated complexes with DNA, and confocal microscopy and flow cytometry showed that these LL37-DNA complexes entered pDCs and were localized to early endosomes, where TLR9 is found. As Baumgarth and Bevins suggest, this study, which shows that LL37 can complex with self-DNA (released from dead keratinocytes) to activate pDCs in psoriasis and so disrupt tolerance (the nonresponsiveness to self) has implications for the treatment of autoimmunity.

R. Lande, J. Gregorio, V. Facchinetti, B. Chatterjee, Y.-H. Wang, B. Homey, W. Cao, Y.-H. Wang, B. Su, F. O. Nestle, T. Zal, I. Mellman, J.-M. Schröder, Y.-J. Liu, M. Gilliet, Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
Nature
449
, 564-569 (2007).
[PubMed]

N. Baumgarth, C. L. Bevins, Skin deep but complex.
Nature
,
449
, 551-553 (2007).
[PubMed]
Keeping p53 in the Nucleus
Authors: Gough Nancy R.
Published: December 2007

One of the functions of the tumor suppressor p53 is as a transcriptional regulator, and this activity is stimulated in response to DNA damage. Among the many types of posttranslational modifications that control p53’s various functions is the addition of poly(ADP-ribose), mediated by poly(ADP-ribose) polymerase 1 (PARP-1), the activity of which is stimulated by DNA damage. Kanai
et al
. identified the residues in mouse p53 that were the major sites of poly(ADP-ribosyl)ation as Glu
255
, Asp
256
, and Glu
268
and then studied how expression of a p53 fusion protein in which these residues were changed to alanine (GFP-p53EDE/A) influenced the localization and transcriptional activity of the protein compared with expression of a wild-type GFP p53 fusion protein (GFP-WT p53) in mouse embryo fibroblasts (MEFs) deficient for p53. In p53
–/–
MEFs, GFP-WT p53 was predominantly nuclear, whereas GFP-p53EDE/A was predominantly cytosolic, even when the cells were subjected to the DNA-damaging agent doxorubicin, which suggests that poly(ADP-ribosyl)ation is important for nuclear accumulation of p53. In response to DNA damage, cells expressing GFP-WT p53 showed increased abundance of p21, which is encoded by a p53-target gene, whereas cells expressing GFP-p53EDE/A did not. Consistent with PARP-1 as the enzyme responsible for the poly(ADP-ribosyl)ation of p53, MEFs deficient for PARP-1 showed decreased stability of p53 and failed to up-regulate p21 in response to DNA damage. In cells in which nuclear export was pharmacologically inhibited, the GFP-p53EDE/A mutant increased the abundance of p21 as effectively as WT p53, thus showing that poly(ADP-ribosyl)ation was not required for transactivation activity but that its effect on transactivation activity of p53 was due to decreased accumulation of p53 in the nucleus. The interaction of tagged Crm1, a nuclear export receptor, with tagged p53 or p53EDE/A was enhanced for the p53EDE/A mutant, suggesting that poly(ADP-ribosyl)ation interferes with the binding of p53 with Crm1. In vitro binding assays with poly(ADP-ribosyl)ated p53 and nonmodified p53 and Crm1 supported this interpretation. Thus, the authors propose a model whereby DNA damage promotes the hyperactivation of PARP-1, which targets p53, and poly(ADP-ribosyl)ated p53 then accumulates in the nucleus because it fails to interact with the nuclear export receptor Crm1.

M. Kanai, K. Hanashiro, S.-H. Kim, S. Hanai, A. H. Boulares, M. Miwa, K. Fukasawa, Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation.
Nat. Cell Biol.
9
, 1175-1183 (2007).
[PubMed]
Learning, Autism, and the Synapse
Authors: Kiberstis Paula A.
Published: December 2007

A small number of individuals with autism harbor mutations in genes encoding neuroligins and neurexins, cell adhesion proteins that facilitate neuronal communication across synapses. Tabuchi
et al
. (see the Perspective by Crawley) studied the functional consequences of one of these mutations, an R451C substitution in neuroligin-3, by introducing the mutant protein into mice. The mice displayed enhanced spatial learning skills but impaired social interactions, and these behavioral changes were accompanied by a selective increase in inhibitory synaptic transmission. Thus, alterations in the balance of excitatory and inhibitory synapses can affect learning, and such alterations may be a contributing factor in the pathogenesis of autism.

K. Tabuchi, J. Blundell, M. R. Etherton, R. E. Hammer, X. Liu, C. M. Powell, T. C. Südhof, A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice.
Science
318
, 71-76 (2007).
[Abstract]
[Full Text]

J. N. Crawley, Testing hypotheses about autism.
Science
318
, 56-57 (2007).
[Summary]
[Full Text]
Receptors Look Outward: Revealing Signals That Bring Excitation to Synapses
Authors: Gerrow Kimberly , El-Husseini Alaa
Published: December 2007

Defining the molecular mechanisms that govern the trafficking of glutamate receptors to excitatory synaptic contacts is fundamental to understanding the mechanisms that regulate synapse maturation and neuronal excitability. Previous studies have identified several scaffolding molecules and adaptor proteins that regulate glutamate receptor trafficking and retention at the synapse. Recent work, however, has elucidated new players such as the N-cadherin adhesion complex, and members of the pentraxin family that regulate clustering of glutamate receptors through extracellular protein interactions. Here, we highlight recently identified modes that regulate glutamate receptor clustering, and discuss their relevance to synapse maturation.
P.S. to PS (Phosphatidylserine)–Pertinent Proteins in Apoptotic Cell Clearance
Authors: Schlegel Robert A. , Williamson Patrick
Published: December 2007

The psr protein has been proposed as the critical receptor that detects phosphatidylserine (PS) on the surface of apoptotic cells. However, for some time there has been evidence that this protein is not at the cell surface but in the nucleus. Now, the phenotype of a knockout of the
Drosophila
psr protein (dPSR) has discredited the identification altogether, lending impetus both to uncovering the real function of the protein and to identifying the real PS receptor. Interpretations of studies of two other genes supposedly involved in PS transport may be built on similarly shaky foundations.
An Active Player
Authors: Adler Elizabeth M.
Published: December 2007

Scaffolding proteins that bind to and organize components of signaling pathways into macromolecular complexes are crucial to the efficiency, specificity, and speed of various signaling pathways. For instance, INAD (inactivation, no after-potential D), a PDZ domain-containing scaffolding protein that, together with three of its binding partners, constitutes the core complex of the
Drosophila
phototransduction cascade, is critical to the electrical response to light (see Montell). Using x-ray crystallography, Mishra
et al
. found that, in the oxidized state, the fifth INAD PDZ protein interaction domain (PDZ5) assumed a distorted conformation inconsistent with canonical ligand binding. This conformation depended on an intramolecular disulfide bond and, in the reduced state, PDZ5 structurally resembled other PDZ domains. In transgenic flies expressing wild-type INAD, light exposure led to formation of the disulfide bond (and thus the oxidized conformation), whereas subsequent darkness was associated with return to the reduced form; development of the oxidized form was attenuated in flies lacking the major isoform of rhodopsin. Electrophysiological analysis of isolated photoreceptor cells expressing a mutant form of INAD in which PDZ5 only existed in the reduced conformation indicated that the sensitivity of the mutant form to light was normal but that loss of a refractory period led to defective termination of the response to bright light. Moreover, flies with the mutant INAD showed a defective escape response to a brief exposure to darkness (mimicking the shadow of a predator). Thus, rather than performing a purely passive function in organizing signaling proteins, INAD appears to play a dynamic role in the
Drosophila
phototransduction pathway.

P. Mishra, M. Socolich, M. A. Wall, J. Graves, Z. Wang, R. Ranganathan, Dynamic scaffolding in a G protein-coupled signaling system.
Cell
131
, 80-92 (2007).
[PubMed]

C. Montell, Dynamic regulation of the INAD signaling scaffold becomes crystal clear.
Cell
131
, 19-21 (2007).
[PubMed]
Phosphorylation Isn’t Everything
Authors: Adler Elizabeth M.
Published: December 2007

Interferon-α (IFN-α) binds to its receptors (IFNαR1 and IFNαR2), stimulating their heterodimerization and the activation of receptor-associated tyrosine kinases. IFN-α receptor phosphorylation enables the recruitment and activation of signal transducer and activator of transcription 2 (STAT2) and the consequent recruitment and activation of STAT1. STAT1 and STAT2 heterodimerize with interferon regulatory

factor 9 (IRF9) to form the interferon-stimulated gene factor 3 (ISGF3) complex, which translocates to the nucleus to activate target genes. Noting that IFN-α stimulates the interaction of STAT2 with the histone acetyltransferase transcriptional coactivator CREB-binding protein (CBP), Tang
et al
. investigated the role of acetylation in IFN-α signaling. IFN-α treatment led to an increase in cytoplasmic CBP in HeLa and other cells, concurrent with a decrease in nuclear CBP, and promoted the coimmunoprecipitation of CBP with IFNαR2. Mutational analysis indicated that CBP bound to a region between IFNαR2 Gly
335
and Ser
392
and that phosphorylation of two serines within this region stabilized the interaction. IFN-α elicited IFNαR2 acetylation, which was abolished by siRNA knockdown of CBP, and a combination of mutational and mass spectrometry analysis indicated that acetylation took place on IFNαR2 Lys
399
. IRF9 associated with IFNαR2 through Lys
399
and was itself acetylated by CBP, as were STAT2 and STAT1, and CBP overexpression stimulated ISGF3-dependent transcriptional activity, whereas siRNA directed against CBP inhibited it. Moreover, CBP-mediated acetylation of IFNαR2, IRF9, and STAT2 were required for IFN-α-dependent association of ISGF3 with chromatin and antiviral activity. Thus, the authors conclude that acetylation--as well as phosphorylation--plays a key role in type I interferon receptor signaling.

X. Tang, J.-S. Gao, Y.-j. Guan, K. E. McLane, Z.-L. Yuan, B. Ramratnam, Y. E. Chin, Acetylation-dependent signal transduction for type I interferon receptor.
Cell
131
, 93-105 (2007).
[PubMed]
Fight, Flight, or Inflame
Authors: Foley John F.
Published: December 2007

The catecholamines epinephrine and norepinephrine, components of the adrenergic nervous system, modulate lymphocyte proliferation and differentiation; however, their role in inflammation is unclear. Flierl
et al
. investigated whether macrophages and neutrophils (together called phagocytes) could serve as cellular sources of these hormones and how this might affect inflammation. The authors used enzyme-linked immunosorbant assays to show that exposure of mouse phagocytes to lipopolysaccharide (LPS) in vitro resulted in the rapid secretion of both epinephrine and norepinephrine. Real-time reverse transcription polymerase chain reaction and Western blotting assays showed that the abundance of mRNA and protein for known catecholamine-generating enzymes was increased in LPS-treated cells compared with untreated cells. The authors then studied the role of catecholamines in two animal models of lung inflammatory injury, one of which was caused by the administration of LPS. Leakage of albumin into the lung, an indicator of injury, was higher in LPS-treated mice than in control mice. Treatment of mice with an antagonist of the α
2
-adrenoreceptor reduced the severity of LPS-induced lung injury, as assessed by both measurement of albumin leakage and histological analysis of lung sections, whereas treatment with an α
2
-adrenoreceptor agonist exacerbated the damage. Blocking other adrenoreceptors had no effect on LPS-induced lung inflammation. Pharmacological blockade of sympathetic neurons indicated that they were not the source of the catecholamines; T cells were also not the source, because T cell-depleted mice still showed lung inflammation after treatment with LPS, and α
2
-adrenoreceptor blockade still reduced LPS-induced injury in these mice. This study identifies phagocytes as a significant source of catecholamines and raises the possibility of blocking adrenoreceptors in the treatment of inflammation.

M. A. Flierl, D. Rittirsch, B. A. Nadeau, A. J. Chen, J. V. Sarma, F. S. Zetoune, S. R. McGuire, R. P. List, D. E. Day, L. M. Hoesel, H. Gao, N. Van Rooijen, M. S. Huber-Lang, R. R. Neubig, P. A. Ward, Phagocyte-derived catecholamines enhance acute inflammatory injury.
Nature
449
, 721-725 (2007).
[PubMed]
Almost in a Day’s Work
Authors: Hines Pamela J.
Published: December 2007

Many plants coordinate their annual time of flowering with particular seasons of the year, and one indicator that they use to determine the change of seasons is day length. Sawa
et al
. (see the Perspective by Rubio and Deng) provide some insights into the molecular interactions that translate day length into flowering initiation. The day needs to be long enough for the blue-light stimulated expression of the proteins FKF1 (flavin-binding, kelch repeat, F-box1) and GIGANTIA (GI) to coordinate. The daily rise in expression of GI lags far enough behind the daily rise in expression of FKF1 that, in the shorter days of the year, daylight has waned by the time there is enough GI to form complexes with FKF1. With the longer days of spring and summer, there is enough time to form complexes and signal to the
CONSTANS
gene to trigger the flowering pathway.

M. Sawa, D. A. Nusinow, S. A. Kay, T. Imaizumi, FKF1 and GIGANTEA complex formation is required for day-length measurement in
Arabidopsis
.
Science
318
, 261-265 (2007).
[Abstract]
[Full Text]

V. Rubio, X. W. Deng, Standing on the shoulders of GIGANTEA.
Science
318
, 206-207 (2007).
[Summary]
[Full Text]
VHL: Ligase and Kinase Adaptor
Authors: Gough Nancy R.
Published: December 2007

Elevated activity of the transcription factor nuclear factor κB (NF-κB) contributes to tumorigenesis by decreasing apoptosis caused by cytokines, such as tumor necrosis factor-α (TNF-α). Clear cell renal carcinoma, a common kidney cancer, is frequently associated with mutations in or decreased expression of the gene encoding von Hippel Lindau (VHL), a protein well known for its role as an E3 ubiquitin ligase substrate recognition subunit mediating ubiquitination of the hypoxia-responsive transcription factor HIF-1α. However, several lines of evidence suggest that the role of VHL in these tumors is not related to its regulation of HIF-1α. Cells deficient in VHL activity also often exhibit elevated NF-κB signaling. Yang
et al
. affinity purified proteins that interacted with VHL and identified the adaptor protein CARD9, which is involved in activation of NF-κB. The interaction between CARD9 and VHL was confirmed using multiple assays--in vitro with recombinant proteins, in transfected cells, and with endogenous proteins. CARD9 consistently appeared as a doublet when assayed by electrophoresis, the top band was absent in CARD9 isolated from VHL-deficient cells, and both molecular forms collapsed into a single faster-migrating form when the samples were treated with phosphatase. VHL interacts with casein kinase 2 (CK2), and CARD9 has two CK2 consensus phosphorylation sites in the C-terminal region. CARD9 was a substrate for CK2 in vitro. Overexpression of VHL increased the interaction between CK2 and CARD9, and purified dephosphorylated CARD9 was phosphorylated by incubation with a VHL-containing cell extract. Inhibition of CK2 activity (by siRNA or with pharmacological agents) decreased the phosphorylation of CARD9. Phosphorylation of CARD9 by CK2 appeared to inhibit the ability of CARD9 to inhibit NF-κB, because cells expressing CARD9 mutants lacking the C-terminal domain or with mutated phosphorylation sites or cells in which CK2 was inhibited pharmacologically exhibited enhanced NF-κB activation. In
VHL
−/−
renal carcinoma cells, knockdown of CARD9 with siRNA attenuated the NF-κB activity, increased apoptosis in response to TNF-α, and decreased tumor growth in a mouse xenograft assay. Thus, VHL appears to have a second important function with relevance for cancer--serving as a kinase adaptor to regulate NF-κB activity.

H. Yang, Y. A. Minamishima, Q. Yan, S. Schlisio, B. L. Ebert, X. Zhang, L. Zhang, W. Y. Kim, A. F. Olumi, W. G. Kaelin Jr., pVHL acts as an adaptor to promote the inhibitor phosphorylation of the NF-κB agonist Card9 by CK2.
Mol. Cell
28
, 15-27 (2007).
[PubMed]
Unraveling Ras Interactions
Authors: Ray L. Bryan
Published: December 2007

Activated signaling through the small guanosine triphosphatase Ras is often associated with abnormal proliferation of cancer cells. Stites
et al.
used a combination of mathematical modeling and experiments to gain new insights into the regulatory properties of the network of factors that influence Ras (or are regulated by it) and how these factors are altered in cancer cells. Their results offer insight into why particular activating Ras mutants, but not others, are found in cancer cells, and suggest a pharmacological strategy that may preferentially impede excessive signaling in networks containing an oncogenically mutated form of Ras while sparing normal cells.

E. C. Stites, P. C. Trampont, Z. Ma, K. S. Ravichandran, Network analysis of oncogenic Ras activation in cancer.
Science
318
, 463-467 (2007).
[Abstract]
[Full Text]
Lock and Load
Authors: Foley John F.
Published: December 2007

Helicobacter pylori
is a Gram-negative bacterium associated with stomach ulcers and gastric cancer. As Hauck discusses in commentary,
H. pylori
injects its virulence factors, which include the oncoprotein cytotoxin-associated gene A (CagA), into gastric epithelial cells using a multisubunit complex called the type-IV secretion pilus. CagA is then tyrosine-phosphorylated by Src, leading to restructuring of the actin cytoskeleton and the elongation of infected cells. To identify the host receptor targeted by
H. pylori
, Kwok
et al
. used confocal microscopy to determine that phosphorylated CagA was predominantly localized to focal adhesions in infected human gastric adenocarcinoma (AGS) cells. Focal adhesions are sites where clusters of integrins, heterodimeric adhesion receptors, anchor the cell to the extracellular matrix. The addition of blocking antibodies against the integrins α
5
and β
1
inhibited
H. pylori
-induced phosphorylation of CagA in AGS cells. The authors identified CagL as the only protein associated with the type-IV secretion system that contained the arginine-glycine-aspartate (RGD) motif that is required to bind to integrins. Microscopic analysis showed that CagL was localized to the bacterial pilus during infection of AGS cells.
H. pylori
that expressed CagL proteins with mutated RGD motifs that could not bind to integrins failed to translocate CagA into AGS cells. Western blotting showed that the tyrosine kinases focal adhesion kinase (FAK) and Src were phosphorylated in AGS cells infected with
H. pylori
that expressed wild-type CagL, but not with
H. pylori
that expressed a CagL with a mutated RGD motif. These data uncover a new role for integrins in microbial pathogenesis and suggest CagL as a potential drug target in the treatment of
H. pylori
infections.

T. Kwok, D. Zabler, S. Urman, M. Rohde, R. Hartig, S. Wessler, R. Misselwitz, J. Berger, N. Sewald, W. König, S. Backert,
Helicobacter
exploits integrin for type IV secretion and kinase activation.
Nature
449
, 862-866 (2007).
[PubMed]

C. R. Hauck, Preparing the shot.
Nature
449
, 798-799 (2007).
[PubMed]
The Best Offense Is Someone Else’s Defense
Authors: Hines Pamela J.
Published: December 2007

Part of the strategy that
Agrobacterium
uses to attack its host plant cells includes transferring part of its genome into the plant cell’s genome. This DNA transfer event has been effectively used as a tool for plant genetic transformation in the lab. In the natural interaction,
Agrobacterium
uses a complex of proteins, including both its own and the host’s, to effect the transfer of its DNA into the plant cell nucleus. One of the plant’s proteins, VIP1, was known to function for
Agrobacterium
DNA transfer, but its use to the host plant was unknown. Djamei
et al.
have now discovered that the plant’s VIP1 is supposed to function in a pathogen defense cascade. Thus,
Agrobacterium
derails the plant’s defense response by using one of the plant’s own effectors to carry the bacterial DNA into the plant nucleus.

A. Djamei, A. Pitzschke, H. Nakagami, I. Rajh, H. Hirt, Trojan horse strategy in
Agrobacterium
transformation: Abusing MAPK defense signaling.
Science
318
, 453-456 (2007).
[Abstract]
[Full Text]
Roundabout the Dendritic Tree?
Authors: Adler Elizabeth M.
Published: December 2007

The secreted protein Slit and its receptor Robo stimulate growth cone collapse and repel axons from the midline during development. Noting that Slit signaling can promote neurite branching in vitro, and that expression of Slit and Robo persists beyond the developmental stage at which axonal repulsion occurs, Furrer
et al
. investigated their role in vivo in dendritic development of a
Drosophila
neuron, the anterior corner cell (aCC) motoneuron. Visualization of individual neurons filled with a fluorescent dye revealed that, in
Drosophila
embryos lacking Robo, aCCs had stunted collateral dendrites. Similar results were seen when RNAi constructs directed against Robo were specifically expressed in aCCs, whereas expression of wild-type Robo in aCCs of flies lacking Robo substantially rescued dendrogenesis. Immunocytochemical analysis of extracellular Slit during embryonic development of the
Drosophila
central nervous system (CNS) revealed that Slit had begun to accumulate in the region at which aCC dendrites develop (as well as was found at the midline) just as their dendrogenesis began. In developing flies lacking Robo (which would be expected to capture Slit at the cell surface), this secondary site of Slit accumulation was shifted medially, as were the small aCC dendrites. In flies lacking functional Slit, dendrites failed to develop. Ectopic expression of Slit in muscles contacted by motoneurons, however, did not stimulate collateral dendrogenesis; therefore, the effect of Slit may be limited to the CNS environment. Thus, the authors conclude that, in contrast to its effects on axons, in the developing
Drosophila
CNS Slit signaling through Robo regulates dendrogenesis.

M.-P. Furrer, I. Vasenkova, D. Kamiyama, Y. Rosado, A. Chiba, Slit and Robo control the development of dendrites in
Drosophila
CNS.
Development
134
, 3795-3804 (2007).
[Abstract]
[Full Text]
Focus Issue: Decisions, Great and Small
Authors: Adler Elizabeth M. , Gough Nancy R.
Published: December 2007

This Focus Issue of
Science
’s STKE concerns cellular "decision-making" in the nervous system, with Perspectives on how diet affects the "decision" of a neuron to fire and on the "decision" of progenitor cells to become neurons or glia.
Ras Versus Fas
Authors: Foley John F.
Published: December 2007

Cancer cells in which
Ras
oncogenes are activated often exhibit epigenetic silencing of
Fas
, leading to the prevention of Fas-ligand-mediated apoptosis. Inactivation of
Fas
is usually due to hypermethylation at its promoter, but how this silencing is directed is unknown. Gazin
et al
. transduced
K-ras-
transformed NIH 3T3 cells that did not express Fas on the cell surface with a library of retroviruses containing small hairpin RNAs (shRNAs) and used immunomagnetic beads to isolate cells that regained surface expression of Fas. The shRNAs that promoted expression of Fas on the cell surface were sequenced, thus identifying 28 target genes, which encoded signaling molecules, regulators of gene expression, and proteins not previously associated with Ras. The abundance of these genes was higher in
K-ras
-transformed NIH 3T3 cells than in NIH 3T3 cells, as measured by real-time reverse transcription polymerase chain reaction assays. Methylated DNA immunoprecipitation assays showed that although the
Fas
promoter was hypermethylated in
K-ras
-transformed NIH 3T3 cells, it was not hypermethylated in any of the 28 cell lines, each of which expressed a single shRNA targeting one of the identified genes. Chromatin immunoprecipitation assays established that the DNA methyltransferase DNMT1 was associated with the
Fas
promoter in
K-ras
-transformed NIH-3T3 cells but that this association was decreased in all 28 shRNA-containing cell lines. At least 21 of the 28 genes identified by the screen were also required for the hypermethylation of 5 other genes that are known to be silenced in
Ras
-transformed cells. Tumors in mice resulting from injection of any of 9 of the
K-ras
-transformed NIH 3T3 shRNA-containing cell lines were smaller than those in mice injected with
K-ras
-transformed NIH 3T3 cells. These results suggest that oncogenic Ras does indeed direct epigenetic silencing of
Fas
(and other genes) through a number of effectors, which might serve as targets for new therapies.

C. Gazin, N. Wajapeyee, S. Gobeil, C.-M. Virbasius, M. R. Green, An elaborate pathway required for Ras-mediated epigenetic silencing.
Nature
449
, 1073-1077 (2007).
[PubMed]
A "Rsk"y Shortcut in Dendritic Cells
Authors: Ray L. Bryan
Published: December 2007

Dendritic cells have a key role in protecting organisms from infections. These cells detect antigens derived from invading bacteria or viruses and present them to T cells. Zaru
et al.
explored the roles of protein kinases activated in response to mitogen-activated protein kinases (MAPKs) and not only demonstrate the importance of various MAPK-activated protein kinases (MKs)--the MKs known as MK2 and MK3 and the p90 ribosomal S6 kinases (Rsks)--but also reveal a previously unrecognized regulatory network for control of downstream kinases in a MAPK system. The authors monitored Toll-like receptor-activated endocytosis by detecting uptake of fluorescently labeled dextran and observed that dendritic cells lacking both MK2 and MK3 had a reduced response. They also observed that pharmacological inhibition of Rsk family members with two structurally distinct inhibitors inhibited Toll-like receptor-induced endocytosis. The surprise was that inhibition of ERK1 and ERK2, the MAPKs known to activate Rsks, could not block receptor-induced Rsk activation in this system. Inhibition of ERKs only blocked Rsk activation in cells lacking MK2 and MK3, kinases that are activated by the MAPK p38 rather then ERKs. Rsks have two kinase domains, and the C-terminal kinase domain of Rsk proteins is phosphorylated by ERKs and autophosphorylates a serine residue located between the two kinase domains [thus recruiting yet another kinase (PDK1) to complete the activation]. Zaru
et al
. realized, however, that this critical serine residue in Rsk fits the consensus for recognition by MK2 and MK3 and showed that MK2 could phosphorylate Rsk2 at this site in vitro. Thus, the p38 MAPK provides an alternative mechanism for activating Rsks that bypasses the mechanism used for their activation by the ERK MAPKs, a mechanism that appears to be restricted to dendritic cells. The authors and Karin (in associated commentary) speculate on ways in which this modified regulatory scheme might be advantageous in these particular cells.

R. Zaru, N. Ronkina, M. Gaestel, J. S. C. Arthur, C. Watts, The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways.
Nat. Immunol.
8
, 1227-1235 (2007).
[PubMed]

M. Karin, Rsk Tolls the bell for endocytosis in DCs.
Nat. Immunol.
8
, 1197-1199 (2007).
[PubMed]
ADP for ATP Exchange Mechanism
Authors: Gough Nancy R.
Published: December 2007

The signal transduction ATPases with numerous domains (STAND) family of proteins can be subdivided, according to the characteristics of the nucleotide-binding oligomerization domain (NOD), into three groups: the AP subfamily that includes APAF-1, an apoptotic mediator; the NACHT subfamily members that are involved in mammalian innate immunity; and the MalT subfamily of bacterial transcription factors. Although the presence of ATP is critical for the activation of these proteins, which are generally monomeric in the inactive state and oligomerize upon activation, it has been unclear what role ATP plays in this process and whether ATP hydrolysis was involved. Marquenet and Richet determined that the
Escherichia coli
MalT transcription factor cycles between an ADP-bound inactive form and an ATP-bound active form and that intrinsic ATP hydrolysis was required to return the protein to the inactive state. This is quite similar to the mechanism by which heterotrimeric guanine nucleotide-binding proteins (G proteins) and small guanosine triphosphatases (GTPases) function, except that the latter bind guanine nucleotides instead of adenosine nucleotides. Endogenous inhibitors of MalT--MalY, Aes, and MalK--bind and stabilize the monomeric form and, in the presence of ATP, maltotriose stimulates the oligomerization and formation of active MalT. The authors compared the activities of mutant MalT proteins that bound, but could not hydrolyze ATP, with the activity of wild-type MalT in vivo. MalT-D129A was most extensively studied. In vivo, MalT-D129A was hyperactive, stimulating transcription of a reporter to higher levels than the wild-type protein, and the activity of the ATP hydrolysis mutant was also not inhibited by MalK or MalY. Gel filtration assays showed that MalT-D129A did not bind effectively to MalY and that MalT-D129A existed in an oligomeric state with no detectable monomers in the presence or absence of added maltotriose or ATP. The wild-type protein bound MalY and existed as only monomers in the absence of maltotriose and ATP and as both oligomeric and monomeric forms in the presence of maltotriose and ATP. Assays to detect the bound nucleotide showed that MalT-D129A was bound to ATP in a 1:1 stoichometry, suggesting that it purified in an ATP-bound state. In contrast, wild-type MalT in the absence of ATP and maltotriose, either alone or when bound to MalY, was bound to ADP. Maltotriose stimulated the exchange of ADP for ATP on wild-type MalT, which was detected either using nonhydrolyzable analogs of ATP or with radiolabeled ATP. Thus, maltotriose appears to switch MalT from an inactive ADP-bound form to an activated ATP-bound form, and hydrolysis of ATP is necessary for MalT cycling and regulation. It will be interesting to see if other NOD proteins show similar adenine nucleotide exchange and hydrolysis as part of their signaling mechanism.

E. Marquenet, E. Richet, How integration pf positive and negative regulatory signals by a STAND signaling protein depends on ATP hydrolysis.
Mol. Cell
28
, 187-199 (2007).
[PubMed]
Extracellular Death Factor
Authors: Ash Caroline
Published: December 2007

Programmed cell death (PCD) has traditionally been considered to be restricted to eukaryotic multicellular organisms; however, several genetic modules in prokaryotes are known to mediate a type of programmed cell death. In
Escherichia coli
,
mazF
encodes a stable toxin, and
mazE
encodes a labile antitoxin, that prevents the lethal effect of MazF. Kolodkin-Gal
et al.

(see the Perspective by Kolter) now show that
E. coli
mazEF
-mediated cell death is a population phenomenon that requires a quorum-sensing signal molecule, extracellular death factor (EDF). EDF is a symmetric, linear pentapeptide whose amino acid sequence is Asn-Asn-Trp-Asn-Asn. In synthetic peptides, the symmetrical arrangement of each of the five amino acids of EDF was important for
mazEF
-mediated killing activity.

I. Kolodkin-Gal, R. Hazan, A. Gaathon, S. Carmeli, H. Engelberg-Kulka, A linear pentapeptide is a quorum-sensing factor required for
mazEF
-mediated cell death in
Escherichia coli
.
Science
318
, 652-655 (2007).
[Abstract]
[Full Text]

R. Kolter, Deadly priming.
Science
318
, 578-579 (2007).
[Summary]
[Full Text]
The Makings of Self-Pollination
Authors: Hines Pamela J.
Published: December 2007

Plants that produce both male and female gametes use various strategies to determine whether they will predominantly self-fertilize or predominantly use pollen from other flowers. Although compatibility of cell surface receptors is part of the story, the geometry of flower parts also affects what pollen lands on the stigma. In the flower of the domesticated tomato, the stigma is buried within the anthers, which makes self-fertilization more likely. In wild relatives of the tomato, however, the stigma projects beyond the anthers and favors cross-fertilization. Chen
et al
. now identify the gene that regulates the length of the style--and thus regulates the relative geometry of stigma and anthers in the tomato.

K.-Y. Chen, B. Cong, R. Wing, J. Vrebalov, S. D. Tanksley, Changes in regulation of a transcription factor lead to autogamy in cultivated tomatoes.
Science
318
, 643-645 (2007).
[Abstract]
[Full Text]