bluuebunny/crossref_metadata_2025 huggingface dataset:
Gα COOH-Terminal Minigene Vectors Dissect Heterotrimeric G Protein Signaling
Authors: Gilchrist Annette , Li Anli , Hamm Heidi E
Published: May 2003

The COOH-termini of heterotrimeric guanine nucleotide-binding protein (G protein) α subunits (Gα) are critical for both binding to their cognate G protein-coupled receptors (GPCRs) and determining specificity. Additionally, synthetic peptides corresponding to the COOH-terminus can serve as competitive inhibitors of receptor-G protein interactions, presumably by blocking the site on the GPCR that normally binds the G protein. To selectively antagonize G protein signal transduction events, we have generated minigene vectors that encode 14 unique COOH-terminal sequence for the 16 Gα subunits. Minigene vectors expressing Gα COOH-terminal peptides, or the control minigene vector, which expresses the inhibitory Gα subunit (G
i
) peptide in random order, can be systematically introduced into cells by transfection and used to determine which G protein underlies a given GPCR-mediated response. Because Gα COOH-terminal minigene vectors selectively block signal transduction through a given G protein, they are a powerful tool for dissecting out which G protein mediates a given biochemical or physiological function. This also provides a novel strategy for exploring the coupling mechanisms of receptors that interact with multiple G proteins, as well as for teasing out the downstream responses mediated by a specific G protein.
Mechanotransduction: All Signals Point to Cytoskeleton, Matrix, and Integrins
Authors: Alenghat Francis J. , Ingber Donald E.
Published: May 2003

Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.
ECM Remodeling Regulates Angiogenesis: Endothelial Integrins Look for New Ligands
Authors: Stupack Dwayne G. , Cheresh David A.
Published: May 2003

The process of angiogenesis is a dynamic one. Vascular endothelial cells are changing at the same time the extracellular matrix is being remodeled. Stupack and Cheresh discuss how remodeling of the extracellular matrix (ECM) and changes in the endothelial cell protein production and integrin expression contribute to the complex process of new blood vessel growth from an existing vascular bed.
Knowing How to Navigate: Mechanisms of Semaphorin Signaling in the Nervous System
Authors: He Zhigang , Wang Kevin C. , Koprivica Vuk , Ming Guoli , Song Hong-Jun
Published: May 2003

Neuronal connections are made during embryonic development with astonishing precision to ultimately form the physical basis for the central nervous system's main capacity: information processing. Over the past few decades, much has been learned about the general principles of axon guidance. A key finding to emerge is that extracellular cues play decisive roles in establishing the connections. One family of such cues, the semaphorin proteins, was first identified as repellents for navigating axons during brain wiring. Recent studies have implicated these molecules in many other processes of neuronal development, including axonal fasciculation, target selection, neuronal migration, and dendritic guidance, as well as in the remodeling and repair of the adult nervous system. It appears that responding neuronal processes sense these semaphorin signals by a family of transmembrane molecules, namely the plexins, even though neuropilins were also found to be required for mediating the interaction between plexins and class 3 semaphorins. Our understanding of the intracellular signaling machinery linking the receptors to the cytoskeleton machinery is still incomplete, but several molecules have been implicated in mediating or modulating semaphorin-induced responses. Adding to the complexity of semaphorin biology, new findings implicate semaphorins in functioning not only as signaling ligands, but also as signal-transducing receptors. Thus, semaphorins may serve as important probes for exploring the mechanisms of intercellular communication during the development and function of the nervous system.
The SAP Family: A New Class of Adaptor-Like Molecules That Regulates Immune Cell Functions
Authors: Veillette André
Published: May 2003

EAT-2 is a member of a newly described adaptor-like protein family consisting, at the moment, of EAT-2 and the signaling lymphocytic-activation molecule (SLAM)-associated protein (SAP). Both proteins are expressed in immune cells and can bind to members of the SLAM family of immune receptors. However, differences between the two proteins exist. There appears to be little overlap in the types of immune cells that express SAP and EAT-2, and the Src homology 2 (SH2) domain of SAP can bind to unphosphorylated tyrosines, whereas the SH2 domain of EAT-2 cannot. Veillette discusses new findings on the functions of EAT-2 and SAP and how they might regulate signals emanating from the SLAM family of receptors.
Infrared-Guided Laser Stimulation of Neurons in Brain Slices
Authors: Dodt Hans-Ulrich , Eder Matthias , Schierloh Anja , Zieglgänsberger Walter
Published: May 2003

Infrared-guided laser stimulation is a new technique that allows precise and rapid stimulation of visualized neurons in brain slices. Infrared imaging of neurons with a new contrast system is combined with the photolytic release of caged neurotransmitters by an ultraviolet (UV) laser. Addition of caged neurotransmitters to the superfusion medium of neurons in brain slices allows local excitation in the micrometer range with a focused spot of UV light. In this way, the distribution of glutamate or γ-aminobutyric acid (GABA) receptors on neuronal dendrites can be mapped. Furthermore, this technique can be used to map the connectivity of neuronal networks through the controlled stimulation of neighboring neurons. Because the laser stimulation can be performed much faster than can paired recording, it is also possible to search for synaptic connections between distant neurons that have a low probability of connectivity.
The Dawn of the SPPARMs?
Authors: Rangwala Shamina M. , Lazar Mitchell A.
Published: May 2003

Thiazolidinediones (TZDs) are used as antidiabetic agents in the treatment of type II diabetes. These compounds are ligands for the nuclear hormone receptor PPARγ, which is highly expressed in adipose tissue. PPARγ acts as a molecular switch in the process of fat cell development. The quest for the ideal antidiabetic agent is challenged by the need to develop PPARγ ligands that improve insulin sensitivity, but do not promote fat cell formation. A newly described PPARγ ligand may represent an initial step in this direction and could lead to improved agents for treating insulin resistance in type II diabetes.
How and Why Does the Immunological Synapse Form? Physical Chemistry Meets Cell Biology
Authors: Chakraborty Arup K.
Published: May 2003

During T lymphocyte (T cell) recognition of an antigen, a highly organized and specific pattern of membrane proteins forms in the junction between the T cell and the antigen-presenting cell (APC). This specialized cell-cell junction is called the immunological synapse. It is several micrometers large and forms over many minutes. A plethora of experiments are being performed to study the mechanisms that underlie synapse formation and the way in which information transfer occurs across the synapse. The wealth of experimental data that is beginning to emerge must be understood within a mechanistic framework if it is to prove useful in developing modalities to control the immune response. Quantitative models can complement experiments in the quest for such a mechanistic understanding by suggesting experimentally testable hypotheses. Here, a quantitative synapse assembly model is described. The model uses concepts developed in physical chemistry and cell biology and is able to predict the spatiotemporal evolution of cell shape and receptor protein patterns observed during synapse formation. Attention is directed to how the juxtaposition of model predictions and experimental data has led to intriguing hypotheses regarding the role of null and self peptides during synapse assembly, as well as correlations between T cell effector functions and the robustness of synapse assembly. We remark on some ways in which synergistic experiments and modeling studies can improve current models, and we take steps toward a better understanding of information transfer across the T cell-APC junction.
Software for Signaling Networks, Electronic and Cellular
Authors: Lok Larry
Published: May 2003

Cellular signaling networks have many similarities with electronic circuits. Therefore, the tools used to test and analyze electronic circuits can be adapted to analyze cellular signaling processes. Lok describes some of these signaling network analysis tools, their underlying principles, and their limitations.
Ordered Just So: Lipid Rafts and Lymphocyte Function
Authors: Sedwick Caitlin E. , Altman Amnon
Published: May 2003

Immunologists have long been occupied with the description of cellular activation signaling events that originate with the stimulation of multichain immunoreceptors at the cell surface. These signals are transmitted by a protein-partner-signaling cascade through the cytoplasm to the nucleus, where they culminate in changes in gene expression, metabolic state, and entry into cell cycle. For T cells and B cells, these signaling cascades start with the ligation of the T cell receptor (TCR) and B cell receptor (BCR), respectively, and result in the recruitment and activation of related families of signaling molecules at the cell surface. Until recently, this gathering of signaling proteins was thought to occur within the featureless plasma membrane, a cellular organ that was envisioned as a boundary between the inner and outer components of the cell, but which contributed little to the signaling process. However, the past few years have seen the gradual realization that activation of signaling in lymphocytes takes place in and around specialized membrane subdomains called lipid rafts (also known as DIGs and GEMs). Here, we provide a brief overview of the analogous structures and compositions of lipid raft-associated signaling complexes in T cells and B cells, and the ways in which lymphocytes--and their pathogen adversaries--use lipid rafts to their benefit.
Molecular BKology: The Study of Splicing and Dicing
Authors: Fury Matthew , Marx Steven O. , Marks Andrew R.
Published: May 2003

The activity of ion channels is regulated by several different mechanisms. Studies have been undertaken to determine how the large-conductance Ca
2+
-sensitive K
+
channel (the BK channel) is regulated. Fury
et al
. discuss how the presence or absence of alternatively spliced regions in the BK channel α subunit can act as a molecular switch by which different kinases activate the BK channel.
Argosomes: Intracellular Transport Vehicles for Intercellular Signals?
Authors: Christian Jan L.
Published: May 2003

Cell clusters in the immature tissues of developing organisms create morphogen gradients that guide cellular differentiation into specific cell fates. Although the process of simple diffusion in gradient establishment has been well studied, there are other mechanisms by which cells establish morphogen gradients. Christian discusses the recent findings that morphogens may establish gradients through the use of plasma membrane-containing exovesicles, termed "argosomes."
Dancing with Multiple Partners
Authors: Woodside Darren G.
Published: May 2003

Transmembrane proteins, such as G protein-coupled receptors (GPCRs) and integrins, activate intracellular signaling pathways through interactions with downstream binding partners. Woodside discusses two examples in which GPCRs and integrins interact in a noncompeting manner with more than one partner. The specific GPCR described is the thrombin receptor, in experiments where G protein peptides selectively block signaling through a particular G protein that does not appear to inhibit coupling of the receptor to other G proteins. The second system described is the αIIbβ3 integrin and its activation of the nonreceptor tyrosine kinase Syk. Syk appeared capable of interacting with both the integrin and intracellular domains of immune response receptors, because binding of Syk to the integrin was not inhibited by peptides based on the Syk binding site in immune response receptors. Thus, multiple, noncompeting binding partners add to the complexity of signal transduction outputs from a single receptor complex.
A Herd of Cats, but No Talking DogsReview and commentary on
Methods in Cellular Imaging
, edited by Ammasi Periasamy, Ph.D., Oxford University Press, Oxford, 2001, 423 pp. $99.50, ISBN: 0-19-513936-4
Authors: Edidin Michael , McCaffery J. Michael
Published: May 2003

For biologists wanting to see their cells,
Methods in Cellular Imaging
is just the right resource. Edidin and McCaffery point out the strengths and weaknesses of the book. With coverage of both the classic technologies and emerging techniques, the book serves as a useful reference tool and resource for novices as well as experts.
Visualizing Cellular Phosphoinositide Pools with GFP-Fused Protein-Modules
Authors: Balla Tamas , Várnai Péter
Published: May 2003

Inositol phospholipids are well known for their pivotal role in calcium signaling as precursors of important second messengers generated in response to various stimuli. However, over the last 10 years, inositides have also emerged as universal signaling components present in virtually every membrane of eukaryotic cells. These lipids are locally produced and degraded by the numerous inositide kinase and phosphatase enzymes, to control the recruitment and activity of protein signaling complexes in specific membrane compartments. The spatial and temporal constraints imposed on changes in cellular inositides pose new challenges in finding experimental techniques through which such changes can be examined. Taking advantage of the protein domains selected by evolution to recognize cellular phosphoinositides, we have created fluorescent molecules by fusing these domains to the improved version of green fluorescent protein (EGFP); the distribution of these fusion proteins can be followed within live cells, thereby reporting on changes in phosphoinositides. Although this technique is one of the few that provide information on phosphoinositide dynamics in live cells with subcellular resolution and has rapidly gained popularity, it also has limitations that need to be taken into account when interpreting the data. Here, we summarize our experience in designing and using these constructs and review our position concerning the interpretation of the data obtained by this technique.
Confocal Imaging of Subcellular Ca
2+
Concentrations Using a Dual-Excitation Ratiometric Indicator Based on Green Fluorescent Protein
Authors: Shimozono Satoshi , Fukano Takashi , Nagai Takeharu , Kirino Yutaka , Mizuno Hideaki , Miyawaki Atsushi
Published: May 2003

Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca
2+
indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca
2+
imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca
2+
dynamics or Ca
2+
changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca
2+
using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca
2+
waves from the cytosol to the nucleus and changes in Ca
2+
concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.
The Message and the Messenger: Delivering RNA in Neurons
Authors: Kosik Kenneth S. , Krichevsky Anna M.
Published: May 2003

Synaptic plasticity results in enduring changes in synaptic function. Localized protein synthesis is part of this process. Kosik and Krichevsky describe how a dynamic macromolecular structure, the RNA granule, may be a key element contributing to changes in protein production leading to synaptic plasticity.
Calcium Release and Influx in Yeast: TRPC and VGCC Rule Another Kingdom
Authors: Bonilla Myriam , Cunningham Kyle W.
Published: May 2003

Calcium is one of the most ubiquitous second messengers, in addition to being a fundamentally important cofactor for many proteins' functions. Thus, cells from fungi to plants to animals have evolved mechanisms for carefully controlling calcium concentrations in their organelles and cytosol. This perspective discusses the similarities and differences between yeast and animals in cation channels of two families: the transient receptor potential channels (TRPCs) and voltage-gated calcium channels (VGCCs). One of the key differences highlighted is in the channels implicated in the response of yeast and animal cells to depletion of calcium from intracellular stores, and the differences in localization of these channels between yeast and animal cells. Understanding the logic of fungal Ca
2+
channels, therefore, may provide new insights into the organization and regulation of cellular calcium signaling networks in animals.
ADAP-ting TCR Signaling to Integrins
Authors: Griffiths Emily K. , Penninger Josef M.
Published: May 2003

Adaptor proteins are essential components of T cell receptor (TCR) signaling cascades regulating gene transcription and cytoskeletal reorganization. The molecular adaptor adhesion- and degranulation-promoting adaptor protein (ADAP), also known as Fyn binding protein (FYB) or Slp-76-associated protein of 130 kilodaltons (SLAP-130), interacts with a number of signaling intermediates including Slp-76, the Src family tyrosine kinase Fyn, vasodilator-stimulated phosphoprotein (VASP), and the actin-nucleating protein WASP. Recently ADAP was shown genetically to positively regulate T cell activation, TCR-induced integrin clustering, and T cell adhesion. The mechanism by which ADAP couples TCR stimulation to integrin clustering remains unclear; however, studies of ADAP, the exchange factor Vav1, and WASP suggest that TCR and integrin clustering may be controlled by distinct signaling pathways.
CTLA-4 Negative Signaling via Lipid Rafts: A New Perspective
Authors: Rudd Christopher E. , Martín Margarita , Schneider Helga
Published: May 2003

Proper function of the immune system requires that activation of T cells is precisely regulated. Responses to the T cell receptor are modulated by signals from other receptors. CTLA-4 (cytotoxic T lymphocyte antigen-4, also called CD152), for example, inhibits cytokine production and proliferation of T cells. Activation of T cells is associated with the accumulation of signaling proteins in lipd rafts--microdomains of the plasma membrane enriched in cholesterol and glycosphingolipds. Rudd
et al.
discuss evidence that CTLA-4 might inhibit cytokine production and T cell proliferation by limiting the assembly of lipid rafts, which are critical to the formation of a functional immunological synapse between antigen-presenting cells and T cells.
Flow Cytometric Analysis of T Cell Receptor Signal Transduction
Authors: Zell Traci , Jenkins Marc K.
Published: May 2003

Intracellular assays of signal transduction in T cells stimulated by antigen in vivo have been limited by requirements for large numbers of highly purified cells to perform conventional biochemical techniques. Here, we describe a technique using flow cytometry to study signaling events in T cells after antigen recognition in the body.
Protein Lipid Overlay Assay
Authors: Dowler Simon , Kular Gursant , Alessi Dario R.
Published: May 2003

The Protein Lipid Overlay (PLO) assay enables the identification of the lipid ligands with which lipid binding proteins interact. This assay also provides qualitative information on the relative affinity with which a protein binds to a lipid. In the PLO assay, serial dilutions of different lipids are spotted onto a nitrocellulose membrane to which they attach. These membranes are then incubated with a lipid binding protein possessing an epitope tag. The membranes are washed and the protein, still bound to the membrane by virtue of its interaction with lipid(s), is detected by immunoblotting with an antibody recognizing the epitope tag. This procedure requires only a few micrograms of protein and is quicker and cheaper to perform than other methods that have been developed to assess protein-lipid interactions. The reagents required for the PLO assay are readily available from commercial sources and the assay can be performed in any laboratory, even by those with no prior expertise in this area.
Endocannabinoids and Neuroprotection
Authors: Mechoulam R. , Spatz M. , Shohami E.
Published: May 2003

Traumatic brain injury (TBI) releases harmful mediators that lead to secondary damage. On the other hand, neuroprotective mediators are also released, and the balance between these classes of mediators determines the final outcome after injury. Recently, it was shown that the endogenous brain cannabinoids anandamide and 2-Arachidonoyl glycerol (2-AG) are also formed after TBI in rat and mouse respectively, and when administered after TBI, they reduce brain damage. In the case of 2-AG, better results are seen when it is administered together with related fatty acid glycerol esters. Significant reduction of brain edema, better clinical recovery, and reduced infarct volume and hippocampal cell death are noted. This new neuroprotective mechanism may involve inhibition of transmitter release and of inflammatory response. 2-AG is also a potent modulator of vascular tone, and counteracts the endothelin (ET-1)-induced vasoconstriction that aggravates brain damage; it may thus help to restore blood supply to the injured brain.
Do Defects in Ion Channel Glycosylation Set the Stage for Lethal Cardiac Arrhythmias?
Authors: Fozzard Harry A. , Kyle John W.
Published: May 2003

Many ion channels are modified by the addition of carbohydrate residues. Fozzard and Kyle discuss evidence that sialic acid residues on glycosylated cardiac sodium and potassim channels may be important for preventing early after-depolarizations that can result in cardiac arrhythmias.
Opening Pandora's Box: Clinical Data and the Study of Complex Diseases
Authors: Liebman Michael N.
Published: May 2003

Complex diseases have complex phenotypes, and proper diagnosis requires that the analysis take into account the patient's history and exposure to environmental factors, as well as genetic information. Signaling information is one aspect of a grander "biomedical informatics" approach advocated for a better understanding of a patient's medically relevant disease phenotype.
Lethal Weight Loss: The Focus Shifts to Signal Transduction
Authors: Tracey Kevin J.
Published: May 2003

A hallmark of life-threatening disease in vertebrates is cachexia, a syndrome of weight loss with progressive erosion of body protein. Tumor necrosis factor (TNF) and other endogenously derived factors are sufficient to mediate the pathophysiology of cachexia in vivo, but the downstream signaling pathways have remained a mystery until recently. Tracey describes the involvement of the stress-activated protein kinase p38 and the transcriptional regulators nuclear factor kappa B and peroxisome proliferator-activated receptor gamma coactivator-1 in causing alterations in myocytes and skeletal muscle physiology. Furthermore, soluble factors including TNF and proteolysis-inducing factor may enhance protein degradation through the ubiquitin-proteosome pathway.
Harvesting Cells Under Anchorage-Independent Cell Transformation Conditions for Biochemical Analyses
Authors: Dong Zigang , Cmarik Joan L.
Published: May 2003

Most molecular biology and biochemical analyses use cultured cells grown in anchorage-dependent monolayer conditions. The standard oncogenic transformation assay for cell lines is usually performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. However, cells suspended in soft agar cannot be readily recovered for studying inducible biochemical and molecular events. We developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent conditions.
Death Receptor Signaling Giving Life to Ectodermal Organs
Authors: Thesleff Irma , Mikkola Marja L.
Published: May 2003

A new tumor necrosis factor (TNF) pathway has been identified that has an important function in the regulation of embryonic development. Three key components of this pathway are previously unknown proteins: the TNF ligand ectodysplasin (also known as EDA), its death domain-containing receptor EDAR, and the death domain adapter molecule EDARADD. This pathway was discovered and delineated through the cloning of genes that cause human hypohidrotic ectodermal dysplasia (HED) syndromes and by analysis of the corresponding mouse mutants (
Tabby
,
downless
, and
crinkled
) showing defects in hair, teeth, and several exocrine glands. EDAR signaling is mediated by the activation of nuclear factor kappa B, but other downstream targets are not known. Ectodysplasin-EDAR signaling mediates cell interactions within the ectoderm and regulates the initiation and morphogenesis of hair and teeth. It is also necessary for the development of fish scales, indicating that this pathway and its function have been conserved during the evolution of ectodermal organs.
Sticky Caveats in an Otherwise Glowing Report: Oligomerizing Fluorescent Proteins and Their Use in Cell Biology
Authors: Zacharias David A.
Published: May 2003

Fluorescent proteins from sea creatures have revolutionized the study of cell biology and signal transduction in many ways. Zacharias discusses some of the technical caveats to working with these proteins when they are fused to cellular proteins to track protein localization and interactions. Special attention is paid to problems arising from oligomerization of these fluorescent proteins and how that impacts protein interactions detected by fluorescence resonance energy transfer (FRET).
Amplification of Signaling Events in Bacteria
Authors: Dahlquist Frederick W.
Published: May 2003

Bacteria respond to extremely shallow chemical gradients by modifying their motility in a process called chemotaxis. This chemotactic response is characterized by high sensitivity to small concentration differences, which extends over a large range of concentrations. This combination of high signal gain and large dynamic range results from both a memory of past events and the ability to amplify small differences in signal between the memory and the current environment. Dahlquist describes the signaling mechanism used by bacteria to regulate the flagellar motor and the places in this pathway where signal amplification may occur.
Information Processing in Bacterial Chemotaxis
Authors: Stock Jeffry B. , Levit Mikhail N. , Wolanin Peter M.
Published: May 2003

Motile bacteria respond to attractants and repellents in their environment by changing their movement. Stock
et al
. describe the similarities of the bacterial chemotaxis signaling system to eukaryotic signaling cascades. Also included is a discussion of how the ordered signaling complex of the receptor, the kinase CheA, and the kinase regulator CheW can be thought of as a primitive "probrain" to allow the integration of signals to produce the optimal cellular response.
An Attractive Surface: Gram-Negative Bacterial Biofilms
Authors: Schembri Mark A. , Givskov Michael , Klemm Per
Published: May 2003

In nature, most bacteria live in close association with surfaces as complex communities referred to as biofilms. Community members within these compact microbial consortia show extraordinary resistance to conventional antibiotics, biocides, and hydrodynamic shear forces when compared to their planktonic counterparts. The buildup of these surface-associated bacterial communities is a highly organized and complex process that requires many signal transduction mechanisms to orchestrate the different stages of development. In this review, we describe several types of signal transduction that Gram-negative bacteria employ during the adhesion and expansion stages of biofilm formation, as well as discuss quorum-sensing in relation to the production of virulence factors.
RNA Editing of Neurotransmitter Receptors in the Mammalian Brain
Authors: Schmauss Claudia , Howe James R.
Published: May 2003

RNA editing refers to various posttranscriptional mechanisms that alter the nucleotide sequence of RNA. In the mammalian brain, RNA editing results in significant changes in the functional properties of receptors for the important neurotransmitters glutamate and serotonin. These changes result from site-specific deamination of single adenosines in the pre-messenger RNA encoding these receptors. Here, we review what is known about the mechanisms underlying this editing, the consequences of RNA editing for glutamate and serotonin receptor function, and recent studies on transgenic mice and human post-mortem tissue that have begun to elucidate the role of RNA editing in the intact mammalian brain.
TRAF1: Lord Without A RING
Authors: Zapata Juan M. , Reed John C.
Published: May 2003

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) constitute a family of adaptor proteins that associate with the cytosolic tail of particular TNF-family receptors (TNFR) and regulate cytokine signaling by linking TNFRs with downstream protein kinases, ubiquitin ligases, and other effector proteins. A total of six members of this family (TRAF1-6) have been identified in mammals. TRAF1 is unique among TRAFs because it lacks a RING finger domain present in TRAF2-6 that has been shown to be required for TRAF2- and TRAF6-mediated activities. TRAF1 also has the most restricted expression among TRAFs, and is found almost exclusively in activated lymphocytes, dendritic cells, and certain epithelia. Recent evidence obtained from
TRAF1
-/-
mice shows that TRAF1-deficient T cells are hyper-responsive to TNF-α, having increased T cell receptor (TCR)-dependent T cell proliferation rates in vitro. Also, these
TRAF1
-/-
mice had increased sensitivity to TNF-α-induced skin necrosis in vivo. These results support a role for TRAF1 as a negative regulator of signaling by certain TNF-family receptors. This review summarizes current knowledge about TRAF1, focusing on the new information provided by these
TRAF1
-deficient mice. Also, the pros and cons of TRAFs as potential targets for drug discovery are discussed.
Paradigms of Growth Control: Relation to Cdk Activation
Authors: Olashaw Nancy , Pledger W. J.
Published: May 2003

The cyclin-dependent kinases (CDKs) play a key role in cell cycle control, and in this review, we focus on the events that regulate their activities. Emphasis is placed on the CDKs that function during the G
1
phase of the cell cycle and on the CDK inhibitor p27
Kip1
. We discuss how CDK activation relates to two basic concepts of cell cycle regulation: (i) the need for multiple mitogens for the proliferation of nontransformed cells and (ii) the inhibitory effect of high culture density on proliferative capacity. We also describe how Cdk2 modulates the expression of the α subunit of the interleukin-2 receptor in T cells, and address the question of whether p27
Kip1
functions as an activator or inhibitor of the CDKs associated with the D cyclins.
KSR--A Regulator and Scaffold Protein of the MAPK Pathway
Authors: Raabe Thomas , Rapp Ulf R.
Published: May 2003

Raabe and Rapp discuss how the scaffolding proteins may spatially localize mitogen-activated protein kinase (MAPK) signaling cassettes to allow signaling specificity. They focus on the adaptor kinase suppressor of Ras (KSR) and describe how the interactions between KSR and other proteins and the subcellular localization of KSR are regulated by phosphorylation.
The Slice Overlay Assay: A Versatile Tool to Study the Influence of Extracellular Signals on Neuronal Development
Authors: Polleux Franck , Ghosh Anirvan
Published: May 2003

We have developed a technique that allows characterization and identification of extracellular signals that regulate various aspects of neuronal differentiation. In this in vitro assay, dissociated cells isolated from the developing cerebral wall are labeled and cultured over organotypic cortical slices. We have used this slice overlay assay to identify some of the extracellular cues that regulate differentiation and patterning of axons and dendrites in the cerebral cortex. This assay can be easily adapted to identify factors that control other aspects of neuronal development, such as proliferation and survival, and can also be used to evaluate the role of extracellular signals in the development of nonneural tissues.
Analysis of Glutamate Receptor Surface Expression in Acute Hippocampal Slices
Authors: Grosshans D. R. , Clayton D. A. , Coultrap S. J. , Browning M. D.
Published: May 2003

Trafficking of receptors to and from the cell surface is a powerful mechanism for regulating neuronal excitability. To date, the majority of studies concerning glutamate receptor trafficking have been performed in neuronal cultures in which surface expression can be readily assayed by immunofluorescence techniques. Results from such studies have had important implications in the field of synaptic plasticity. However, cultured neurons are, by necessity, prepared from very young animals. Moreover, although an enhancement of excitatory neurotransmission can be induced in such systems, classic long-term potentiation (LTP) can be produced only in acute slices or in vivo. To study trafficking in adult tissues, we have adapted two biochemical techniques, proteolysis and cross-linking. These techniques help define surface-expressed and intracellular pools of native receptors in acute hippocampal slices.
Homer as Both a Scaffold and Transduction Molecule
Authors: Fagni Laurent , Worley Paul F. , Ango Fabrice
Published: May 2003

Increasing evidence shows that scaffold proteins not only control membrane assembly of receptors and channels, but also modulate intracellular signaling by assembled receptors. The Homer family of proteins act as scaffolds to bind clusters of proteins and glutamate receptors at postsynaptic sites. We review results of cloning and gene expression of this protein family, and summarize roles in glutamate receptor function and intracellular signaling in neurons. Homer proteins trigger the localization of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) to the postsynaptic plasma membrane. They can also alter the kinetics and peak amplitude of the intracellular Ca
2+
responses of mGlu1 and mGlu5 receptors. Homer proteins can either prevent or promote spontaneous activation of these receptors, depending on the type of Homer protein isoform expressed.
Estrogen Actions in the Brain
Authors: Wang Ling , Andersson Sandra , Warner Margaret , Gustafsson Jan-Åke
Published: May 2003

Understanding of the mechanisms of estrogen action in the brain has always been poor. Neurons in several brain regions do not harbor estrogen receptor α (ERα) and yet are estrogen responsive. It was formerly thought that these responses represented indirect actions of estrogen. It is now evident that these neurons express ERβ and that estrogen receptors have diverse actions in the central nervous system. By clear delineation of the cellular expression and function of the two estrogen receptors, it is likely that, in the future, selective ERα and ERβ ligands will be developed and used for treatment of depression and behavioral disorders and may be useful in preventing degenerative diseases, such as Alzheimer's and Parkinson's disease.
Rapid Actions of Steroid Receptors in Cellular Signaling Pathways
Authors: Cato Andrew C. B. , Nestl Andrea , Mink Sigrun
Published: May 2003

Steroid hormones regulate cellular processes by binding to intracellular receptors that, in turn, interact with discrete nucleotide sequences to alter gene expression. Because most steroid receptors in target cells are located in the cytoplasm, they need to get into the nucleus to alter gene expression. This process typically takes at least 30 to 60 minutes. In contrast, other regulatory actions of steroid hormones are manifested within seconds to a few minutes. These time periods are far too rapid to be due to changes at the genomic level and are therefore termed nongenomic or rapid actions, to distinguish them from the classical steroid hormone action of regulation of gene expression. The rapid effects of steroid hormones are manifold, ranging from activation of mitogen-activated protein kinases (MAPKs), adenylyl cyclase (AC), protein kinase C (PKC), and heterotrimeric guanosine triphosphate-binding proteins (G proteins). In some cases, these rapid actions of steroids are mediated through the classical steroid receptor that can also function as a ligand-activated transcription factor, whereas in other instances the evidence suggests that these rapid actions do not involve the classical steroid receptors. One candidate target for the nonclassical receptor-mediated effects are G protein-coupled receptors (GPCRs), which activate several signal transduction pathways. One characteristic of responses that are not mediated by the classical steroid receptors is insensitivity to steroid antagonists, which has contributed to the notion that a new class of steroid receptors may be responsible for part of the rapid action of steroids. Evidence suggests that the classical steroid receptors can be localized at the plasma membrane, where they may trigger a chain of reactions previously attributed only to growth factors. Identification of interaction domains on the classical steroid receptors involved in the rapid effects, and separation of this function from the genomic action of these receptors, should pave the way to a better understanding of the rapid action of steroid hormones.
Cytoskeletal Proteins and Gene Regulation: Form, Function, and Signal Transduction in the Nucleus
Authors: de Lanerolle Primal , Cole Anthony B.
Published: May 2003

The cytoskeleton is important for controlling cell shape and cell motility and for organizing signaling complexes. Additionally, connections are being found between cytoskeletal proteins and the regulation of gene expression in the nucleus. De Lanerolle and Cole discuss new insights from plants that show that a plant member of the COOH-terminal binding protein (CtBP) and the brefeldin A adenosine diphosphate (ADP)-ribosylated subtrates (BARS) proteins, ANGUSTIFOLIA, may be a regulator of both the microtubule-based cytoskeleton and a transcriptional regulator. Additionally, they describe how actin and myosins may play similar roles in regulating gene expression in the nucleus of mammalian cells.
Microtubule-Actin Cross-talk at Focal Adhesions
Authors: Palazzo Alexander F. , Gundersen Gregg G.
Published: May 2003

Focal adhesions are dynamic structures in which traction forces are exerted against the substratum during cell migration and are sites for the organization of signaling complexes. Palazzo and Gundersen discuss how focal adhesions may also be the site of cross-talk between the actin-based and microtubule-based cytoskeletons. Microtubules appear to deliver factors that can regulate the formation and dissolution of focal adhesions, whereas focal adhesions contribute to microtubule localization and stability.
Screening for Gene Regulation Mutants by Bioluminescence Imaging
Authors: Chinnusamy Viswanathan , Stevenson Becky , Lee Byeong-ha , Zhu Jian-Kang
Published: May 2003

Because plants cannot move, they have evolved complex sensing and response systems to cope with the physical environment. Adverse environmental conditions, such as those causing abiotic stress, often cause significant losses in crop productivity and quality. Because of a paucity of well-defined visible phenotypes, conventional genetic screens have not been very successful in isolating abiotic stress signal transduction mutants of plants. Here, we describe a reporter gene-based strategy to screen for mutants affected in abiotic stress-regulated gene transcription. Our genetic screen uses the firefly luciferase reporter gene driven by the cold, drought, salt, and abscisic acid (ABA)-responsive
RD29A
promoter (
RD29A::LUC
).
Arabidopsis
plants transformed with the
RD29A::LUC
reporter emit bioluminescence in response to cold, drought, salt, or ABA treatment. After mutagenesis of these plants with ethyl methanesulfonate (EMS), mutants can be screened from the M2 population by monitoring the level of stress-inducible bioluminescence with a high-throughput, low-light imaging system. This protocol describes in detail the procedures for this luciferase reporter-based genetic screen for
Arabidopsis
mutants defective in abiotic stress signaling.
Cross-Talk in Abscisic Acid Signaling
Authors: Fedoroff Nina V.
Published: May 2003

"Cross-talk" in hormone signaling reflects an organism's ability to integrate different inputs and respond appropriately, a crucial function at the heart of signaling network operation. Abscisic acid (ABA) is a plant hormone involved in bud and seed dormancy, growth regulation, leaf senescence and abscission, stomatal opening, and a variety of plant stress responses. This review summarizes what is known about ABA signaling in the control of stomatal opening and seed dormancy and provides an overview of emerging knowledge about connections between ABA, ethylene, sugar, and auxin synthesis and signaling.
Multivesicular Bodies and Multivesicular Endosomes: The "Ins and Outs" of Endosomal Traffic
Authors: Stahl Philip D. , Barbieri M. Alejandro
Published: May 2003

Multivesicular endosomes (MVEs) are complex intracellular organelles that function in endocytosis. A major function of the endocytic pathway is to sort internalized macromolecules and membrane proteins. Appropriately sorted proteins, such as epidermal growth factor (EGF) receptor (EGFR), are incorporated into MVEs before transport to the lysosomal compartment, where degradation occurs. Thus, MVEs operate in the endosome-to-lysosome portion of the pathway. In yeast cells, where MVE formation has been extensively studied, the pathway terminates in the yeast vacuole, which is equivalent to the vertebrate lysosome. MVEs arise by invagination of the limiting membrane of an endosomal vesicle such that many small internal vesicles are formed, hence the term "multivesicular endosome." In part, the internalization and targeting of membrane proteins to the MVE involves ubiquitin, a small protein associated with protein degradation. In reticulocytes and certain antigen-presenting cells, MVEs are routed to the plasma membrane rather than the lysosome, releasing small vesicles called "exosomes" back into the extracellular space.
Analysis of Phosphorylation-Dependent Protein-Protein Interactions Using a Bacterial Two-Hybrid System
Authors: Shaywitz Adam J. , Dove Simon L. , Greenberg Michael E. , Hochschild Ann
Published: May 2003

Phosphorylation-dependent protein-protein interactions provide the foundation for a multitude of intracellular signal transduction pathways. One of the goals of signal transduction research is to more precisely understand the nature of these phosphorylation-dependent interactions. Here, we describe a bacterial two-hybrid assay that allows for the rapid, efficient analysis of phosphorylation-dependent protein-protein interactions. In this system, the interacting protein domains are provided as fusion proteins in
Escherichia coli
. cells that contain a eukaryotic kinase. Specific phosphorylation of one of the fused protein domains results in a protein-protein interaction that can be detected as a change in the expression of a reporter gene. We also describe how this system can be modified to permit the use of cDNA libraries to identify either novel binding partners for a phosphorylated substrate or novel kinases that can induce a specific protein-protein interaction.
Binary and Graded Responses in Gene Networks
Authors: Louis Matthieu , Becskei Attila
Published: May 2003

Although gene expression can be regulated in a graded or a binary fashion, the majority of eukaryotic genes are either fully activated or not expressed at all in individual cells. This binary response might be an inherent property of many eukaryotic promoters. Analysis of transcription under the control of yeast
GAL1
promoter suggests, however, that graded and binary modes of transcription are not mutually exclusive, but that both can occur at the same promoter when the activity of different signaling pathways is varied. In view of that, it can be expected that forthcoming experimental studies on the combinatorial effects of signaling and transcriptional mechanisms will reveal new strategies for generating graded or binary responses.
Dubble or Nothing? Is HAUSP Deubiquitylating Enzyme the Final Arbiter of p53 Levels?
Authors: Wood Stephen A.
Published: May 2003

Signal transduction processes can be regulated by biochemical modifications that affect protein activity or localization and by protein stability. Proteins implicated in cancer, such as β-catenin and p53, are regulated by a combination of posttranslational modifications and protein degradation by the ubiquitin-proteasome pathway. Wood explores how ubiquitylation of these proteins may not be as unidirectional as previously thought. With the identification of substrate-specific deubiquitylating enzymes, ubiquitylation may not always lead to protein destruction, but may provide another finely tunable step for controlling protein activity.
CD28 Costimulation: A Source of Vav-1 for TCR Signaling with the Help of SLP-76?
Authors: Michel Frédérique , Acuto Oreste
Published: May 2003

T cells require dual stimulation to become activated. When T cells encounter antigen-presenting cells, both the T cell receptor (TCR) and the CD28 coreceptor are ligated and activated. Michel and Acuto discuss how the adaptor SLP-76, which is recruited to the activated TCR complex, and the Rho family guanosine triphosphatase exchanger Vav-1, which is recruited by the CD28 receptor and TCR, may form a macromolecular complex that results in T cells activation. Vav-1 may serve as a central integrator between CD28 signaling and TCR signaling through its indirect effects on phosphoinositide 3-kinase-dependent signaling.