bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrafast dynamics of orbital-order-induced polarization
Authors: Ogawa Naoki , Ogimoto Yasushi , Miyano Kenjiro
Published: June 2013

Ultrafast photoinduced modulation is demonstrated in the orbital-order-induced polarization in manganite thin films. The evolution of the polarization modulation and the lattice temperature rise after photoexcitation are tracked separately by monitoring the relevant nonlinear optical susceptibility tensor elements. Substantial portion of the polarization disappears within the excitation pulse width (∼120 fs), indicating the purely electronic origin triggered by an interband excitation. We propose oxide heterostructure devices incorporating polar gating layers capable of ultrafast optical control of electronic phases.
99.996 % 12C films isotopically enriched and deposited in situ
Authors: Dwyer K. J. , Pomeroy J. M. , Simons D. S.
Published: June 2013

Ionizing natural abundance carbon dioxide gas, we extract and mass select the ions, depositing thin films isotopically enriched to 99.9961(4) % 12C as measured by secondary ion mass spectrometry (SIMS). In solid state quantum information, coherence times of nitrogen-vacancy (NV) centers in 12C enriched diamond exceeding milliseconds demonstrate the viability of NV centers as qubits, motivating improved isotopic enrichment. NV centers in diamond are particularly attractive qubit candidates due to the optical accessibility of the spin states. We present SIMS analysis and cross-sectional scanning electron microscopy of 12C enriched thin film samples grown with this method.
Improvement of efficiency and electrical properties using intentionally formed V-shaped pits in InGaN/GaN multiple quantum well light-emitting diodes
Authors: Han Sang-Heon , Lee Dong-Yul , Shim Hyun-Wook , Wook Lee Jeong , Kim Dong-Joon , Yoon Sukho , Sun Kim Young , Kim Sung-Tae
Published: June 2013

We demonstrate a high efficiency and an improvement of the electrical properties in InGaN/GaN multiple quantum well light-emitting diodes (LEDs) using intentionally formed V-shaped pits. Efficiency droop behaviors are measured and LEDs with V-shaped pits act like LEDs with a low dislocation density. The reverse voltage at −10 μA of LEDs with V-shaped pits shows −120 V, which is comparable to p-i-n rectifiers grown on a free-standing GaN, and reverse leakage current is decreased indicating electrical passivation of dislocation. A calculated diode ideality factor shows that electron tunneling at low forward voltage is suppressed in LEDs with V-shaped pits.
Terahertz emission from a GaAs/AlAs coupled multilayer cavity with nonlinear optical susceptibility inversion
Authors: Kitada Takahiro , Katoh Sho , Takimoto Toshikazu , Nakagawa Yoshinori , Morita Ken , Isu Toshiro
Published: June 2013

Terahertz wave generation was demonstrated by the difference frequency generation of two cavity modes in a polarization-controlled GaAs/AlAs coupled multilayer cavity. Inversion of the second-order nonlinear optical susceptibility was achieved by face-to-face bonding of the two halves of a coupled cavity structure grown on a (113)B-oriented GaAs substrate. Signal enhancement due to this inversion was observed through the comparison of inverted and normal coupled cavity samples in both simulated and experimentally observed terahertz waveforms produced by simultaneous excitation of two cavity modes using femtosecond laser pulses.
Theory of mass-independent fractionation of isotopes, phase space accessibility, and a role of isotopic symmetry
Authors: Marcus Rudolph A.
Published: June 2013

Key experimental and theoretical features of mass-independent fractionation (MIF) of isotopes, also known as the η-effect, are summarized, including its difference from the exit channel zero-point energy difference effect. The latter exactly cancels in the MIF. One key experimental result is that the MIF for O
3
formation is a low-pressure phenomenon and, moreover, that it decreases with increasing pressure of third bodies at pressures far below the “Lindemann fall-off” pressur
e
s for three-body recombination of O and O
2
. A possible origin of the MIF is discussed in terms of a role for isotopologue symmetry in intramolecular energy sharing. An explanation is suggested for the large difference in the fall-off pressure for recombination and the pressure for a large decrease in MIF, in terms of a difference between deactivating collisions and what we term here “symmetry-changing collisions”. It is noted that the theory of the MIF involves four recombination rate constants and an equilibrium constant, for each trace isotope, seven rate constants in all and two equilibrium constants. A conceptual shortcut is noted. Experimental and computational information that may provide added insight into the MIF mechanism and tests is described.
Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue
Authors: Gerdes Michael J. , Sevinsky Christopher J. , Sood Anup , Adak Sudeshna , Bello Musodiq O. , Bordwell Alexander , Can Ali , Corwin Alex , Dinn Sean , Filkins Robert J. , Hollman Denise , Kamath Vidya , Kaanumalle Sireesha , Kenny Kevin , Larsen Melinda , Lazare Michael , Li Qing , Lowes Christina , McCulloch Colin C. , McDonough Elizabeth , Montalto Michael C. , Pang Zhengyu , Rittscher Jens , Santamaria-Pang Alberto , Sarachan Brion D. , Seel Maximilian L. , Seppo Antti , Shaikh Kashan , Sui Yunxia , Zhang Jingyu , Ginty Fiona
Published: July 2013

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.
Earliest floral grave lining from 13,700–11,700-y-old Natufian burials at Raqefet Cave, Mt. Carmel, Israel
Authors: Nadel Dani , Danin Avinoam , Power Robert C. , Rosen Arlene M. , Bocquentin Fanny , Tsatskin Alexander , Rosenberg Danny , Yeshurun Reuven , Weissbrod Lior , Rebollo Noemi R. , Barzilai Omry , Boaretto Elisabetta
Published: July 2013

Flowering plants possess mechanisms that stimulate positive emotional and social responses in humans. It is difficult to establish when people started to use flowers in public and ceremonial events because of the scarcity of relevant evidence in the archaeological record. We report on uniquely preserved 13,700–11,700-y-old grave linings made of flowers, suggesting that such use began much earlier than previously thought. The only potentially older instance is the questionable use of flowers in the Shanidar IV Neanderthal grave. The earliest cemeteries (
ca
. 15,000–11,500 y ago) in the Levant are known from Natufian sites in northern Israel, where dozens of burials reflect a wide range of inhumation practices. The newly discovered flower linings were found in four Natufian graves at the burial site of Raqefet Cave, Mt. Carmel, Israel. Large identified plant impressions in the graves include stems of sage and other
Lamiaceae
(
Labiatae
; mint family) or
Scrophulariaceae
(figwort family) species; accompanied by a plethora of phytoliths, they provide the earliest direct evidence now known for such preparation and decoration of graves. Some of the plant species attest to spring burials with a strong emphasis on colorful and aromatic flowers. Cave floor chiseling to accommodate the desired grave location and depth is also evident at the site. Thus, grave preparation was a sophisticated planned process, embedded with social and spiritual meanings reflecting a complex preagricultural society undergoing profound changes at the end of the Pleistocene.
Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo
Authors: Beans Elizabeth J. , Fournogerakis Dennis , Gauntlett Carolyn , Heumann Lars V. , Kramer Rainer , Marsden Matthew D. , Murray Danielle , Chun Tae-Wook , Zack Jerome A. , Wender Paul A.
Published: June 2013

Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4
+
T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4
+
T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4
+
T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication.
Context-dependent signaling defines roles of BMP9 and BMP10 in embryonic and postnatal development
Authors: Chen Hao , Brady Ridgway John , Sai Tao , Lai Joyce , Warming Søren , Chen Hanying , Roose-Girma Merone , Zhang Gu , Shou Weinian , Yan Minhong
Published: June 2013

Many important signaling pathways rely on multiple ligands. It is unclear if this is a mechanism of safeguard via redundancy or if it serves other functional purposes. In this study, we report unique insight into this question by studying the activin receptor-like kinase 1 (ALK1) pathway. Despite its functional importance in vascular development, the physiological ligand or ligands for ALK1 remain to be determined. Using conventional knockout and specific antibodies against bone morphogenetic protein 9 (BMP9) or BMP10, we showed that BMP9 and BMP10 are the physiological, functionally equivalent ligands of ALK1 in vascular development. Timing of expression dictates the in vivo requisite role of each ligand, and concurrent expression results in redundancy. We generated mice (
Bmp10

9/9

) in which the coding sequence of
Bmp9
replaces that of
Bmp10
. Surprisingly, analysis of
Bmp10

9/9

mice demonstrated that BMP10 has an exclusive function in cardiac development, which cannot be substituted by BMP9. Our study reveals context-dependent significance in having multiple ligands in a signaling pathway.
High-resolution view of bacteriophage lambda gene expression by ribosome profiling
Authors: Liu Xiaoqiu , Jiang Huifeng , Gu Zhenglong , Roberts Jeffrey W.
Published: June 2013

Bacteriophage lambda is one of the most extensively studied organisms and has been a primary model for understanding basic modes of genetic regulation. Here, we examine the progress of lambda gene expression during phage development by ribosome profiling and, thereby, provide a very-high-resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. However, many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function.
Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts
Authors: Tanabe Koji , Nakamura Michiko , Narita Megumi , Takahashi Kazutoshi , Yamanaka Shinya
Published: June 2013

Pluripotency can be induced in somatic cells by forced expression of POU domain, class 5, transcription factor 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), myelocytomatosis oncogene (c-MYC) (OSKM). However, factor-mediated direct reprogramming is generally regarded as an inefficient and stochastic event. Contrary to this notion, we herein demonstrate that most human adult dermal fibroblasts initiated the reprogramming process on receiving the OSKM transgenes. Within 7 d, ∼20% of these transduced cells became positive for the TRA-1-60 antigen, one of the most specific markers of human pluripotent stem cells. However, only a small portion (∼1%) of these nascent reprogrammed cells resulted in colonies of induced pluripotent stem cells after replating. We found that many of the TRA-1-60–positive cells turned back to be negative again during the subsequent culture. Among the factors that have previously been reported to enhance direct reprogramming, LIN28, but not Nanog homeobox (NANOG), Cyclin D1, or p53 shRNA, significantly inhibited the reversion of reprogramming. These data demonstrate that maturation, and not initiation, is the limiting step during the direct reprogramming of human fibroblasts toward pluripotency and that each proreprogramming factor has a different mode of action.
Spatial interactions among ecosystem services in an urbanizing agricultural watershed
Authors: Qiu Jiangxiao , Turner Monica G.
Published: July 2013

Understanding spatial distributions, synergies, and tradeoffs of multiple ecosystem services (benefits people derive from ecosystems) remains challenging. We analyzed the supply of 10 ecosystem services for 2006 across a large urbanizing agricultural watershed in the Upper Midwest of the United States, and asked the following: (i) Where are areas of high and low supply of individual ecosystem services, and are these areas spatially concordant across services? (ii) Where on the landscape are the strongest tradeoffs and synergies among ecosystem services located? (iii) For ecosystem service pairs that experience tradeoffs, what distinguishes locations that are “win–win” exceptions from other locations? Spatial patterns of high supply for multiple ecosystem services often were not coincident; locations where six or more services were produced at high levels (upper 20th percentile) occupied only 3.3% of the landscape. Most relationships among ecosystem services were synergies, but tradeoffs occurred between crop production and water quality. Ecosystem services related to water quality and quantity separated into three different groups, indicating that management to sustain freshwater services along with other ecosystem services will not be simple. Despite overall tradeoffs between crop production and water quality, some locations were positive for both, suggesting that tradeoffs are not inevitable everywhere and might be ameliorated in some locations. Overall, we found that different areas of the landscape supplied different suites of ecosystem services, and their lack of spatial concordance suggests the importance of managing over large areas to sustain multiple ecosystem services.
The physical environment and child development: An international review
Authors: Ferguson Kim T. , Cassells Rochelle C. , MacAllister Jack W. , Evans Gary W.
Published: June 2013

A growing body of research in the United States and Western Europe documents significant effects of the physical environment (toxins, pollutants, noise, crowding, chaos, and housing, school and neighborhood quality) on children and adolescents' cognitive and socioemotional development. Much less is known about these relations in other contexts, particularly the global South. We thus briefly review the evidence for relations between child development and the physical environment in Western contexts, and discuss some of the known mechanisms behind these relations. We then provide a more extensive review of the research to date outside of Western contexts, with a specific emphasis on research in the global South. Where the research is limited, we highlight relevant data documenting the physical environment conditions experienced by children, and make recommendations for future work. In these recommendations, we highlight the limitations of employing research methodologies developed in Western contexts (Ferguson & Lee, 2013). Finally, we propose a holistic, multidisciplinary, and multilevel approach based on Bronfenbrenner's (1979) bioecological model to better understand and reduce the aversive effects of multiple environmental risk factors on the cognitive and socioemotional development of children across the globe.
USP18 inhibits NF-κB and NFAT activation during Th17 differentiation by deubiquitinating the TAK1–TAB1 complex
Authors: Liu Xikui , Li Hongxiu , Zhong Bo , Blonska Marzenna , Gorjestani Sara , Yan Ming , Tian Qiang , Zhang Dong-Er , Lin Xin , Dong Chen
Published: July 2013

Reversible ubiquitin modification of cell signaling molecules has emerged as a critical mechanism by which cells respond to extracellular stimuli. Although ubiquitination of TGF-β–activated kinase 1 (TAK1) is critical for NF-κB activation in T cells, the regulation of its deubiquitination is unclear. We show that USP18, which was previously reported to be important in regulating type I interferon signaling in innate immunity, regulates T cell activation and T helper 17 (Th17) cell differentiation by deubiquitinating the TAK1–TAB1 complex. USP18-deficient T cells are defective in Th17 differentiation and Usp18−/− mice are resistant to experimental autoimmune encephalomyelitis (EAE). In response to T cell receptor engagement, USP18-deficient T cells exhibit hyperactivation of NF-κB and NFAT and produce increased levels of IL-2 compared with the wild-type controls. Importantly, USP18 is associated with and deubiquitinates the TAK1–TAB1 complex, thereby restricting expression of IL-2. Our findings thus demonstrate a previously uncharacterized negative regulation of TAK1 activity during Th17 differentiation, suggesting that USP18 may be targeted to treat autoimmune diseases.
Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection
Authors: Cmarko Dusan , Verschure Pernette J. , Martin Terence E. , Dahmus Michael E. , Krause Sabine , Fu Xiang-Dong , van Driel Roel , Fakan Stanislav
Published: July 2013

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.
Association of Rab25 and Rab11a with the Apical Recycling System of Polarized Madin–Darby Canine Kidney Cells
Authors: Casanova James E. , Wang Xiaoye , Kumar Ravindra , Bhartur Sheela G. , Navarre Jennifer , Woodrum Julie E. , Altschuler Yoram , Ray Greg S. , Goldenring James R.
Published: July 2013

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin–Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.
Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria
Authors: Fünfschilling Ursula , Rospert Sabine
Published: July 2013

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.
A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling
Authors: Suto Kimihiko , Nagata Akihisa , Murakami Hiroshi , Okayama Hiroto
Published: July 2013

Fission yeast rad22+, a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22+, designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolatedrad22+and its novel homologuerti1+as multicopy suppressors of this mutant. rti1+suppresses all the defects of cells lacking rad22+. Mating type switch-inactive heterothallic cells lacking eitherrad22+or rti1+are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture ofrad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However,rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation.rad22+genetically interacts withrad11+, which encodes the large subunit of replication protein A. Deletion ofrad22+/rti1+or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4–6°C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.
TheSaccharomyces cerevisiaeHomologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex
Authors: Madania Ammar , Dumoulin Pascal , Grava Sandrine , Kitamoto Hiroko , Schärer-Brodbeck Claudia , Soulard Alexandre , Moreau Violaine , Winsor Barbara
Published: July 2013

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least oneARP3 ts mutant are synthetically lethal withlas17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δcells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.
Cell Cycle–regulated Transcription in Fission Yeast: Cdc10–Res Protein Interactions during the Cell Cycle and Domains Required for Regulated Transcription
Authors: Whitehall Simon , Stacey Peter , Dawson Keren , Jones Nic
Published: July 2013

In Schizosaccharomyces pombe the MBF (DSC1) complex mediates transcriptional activation at Start and is composed of a common subunit called Cdc10 in combination with two alternative DNA-binding partners, Res1 and Res2. It has been suggested that a high-activity MBF complex (at G1/S) is switched to a low-activity complex (in G2) by the incorporation of the negative regulatory subunit Res2. We have analyzed MBF protein–protein interactions and find that both Res proteins are associated with Cdc10 throughout the cell cycle, arguing against this model. Furthermore we demonstrate that Res2 is capable of interacting with a mutant form of Cdc10 that has high transcriptional activity. It has been shown previously that both Res proteins are required for periodic cell cycle–regulated transcription. Therefore a series of Res1–Res2 hybrid molecules was used to determine the domains that are specifically required to regulate periodic transcription. In Res2 the nature of the C-terminal region is critical, and in both Res1 and Res2, a domain overlapping the N-terminal ankyrin repeat and a recently identified activation domain is important for mediating cell cycle–regulated transcription.
Keratocytes Generate Traction Forces in Two Phases
Authors: Burton Kevin , Park Jung H. , Taylor D. Lansing
Published: July 2013

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.
Mammalian Transcription Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress
Authors: Haze Kyosuke , Yoshida Hiderou , Yanagi Hideki , Yura Takashi , Mori Kazutoshi
Published: July 2013

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.
Sequential Actions of Phospholipase D and Phosphatidic Acid Phosphohydrolase 2b Generate Diglyceride in Mammalian Cells
Authors: Sciorra Vicki A. , Morris Andrew J.
Published: July 2013

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1–enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.
Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells
Authors: Blagoveshchenskaya Anastasiya D. , Hewitt Eric W. , Cutler Daniel F.
Published: July 2013

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.
Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response
Authors: de Virgilio Maddalena , Kitzmüller Claudia , Schwaiger Eva , Klein Michael , Kreibich Gert , Ivessa N. Erwin
Published: July 2013

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.
A Modulatory Role for Clathrin Light Chain Phosphorylation in Golgi Membrane Protein Localization during Vegetative Growth and during the Mating Response ofSaccharomyces cerevisiae
Authors: Chu Diana S. , Pishvaee Babak , Payne Gregory S.
Published: July 2013

The role of clathrin light chain phosphorylation in regulating clathrin function has been examined in Saccharomyces cerevisiae. The phosphorylation state of yeast clathrin light chain (Clc1p) in vivo was monitored by [32P]phosphate labeling and immunoprecipitation. Clc1p was phosphorylated in growing cells and also hyperphosphorylated upon activation of the mating response signal transduction pathway. Mating pheromone-stimulated hyperphosphorylation of Clc1p was dependent on the mating response signal transduction pathway MAP kinase Fus3p. Both basal and stimulated phosphorylation occurred exclusively on serines. Mutagenesis of Clc1p was used to map major phosphorylation sites to serines 52 and 112, but conversion of all 14 serines in Clc1p to alanines [S(all)A] was necessary to eliminate phosphorylation. Cells expressing the S(all)A mutant Clc1p displayed no defects in Clc1p binding to clathrin heavy chain, clathrin trimer stability, sorting of a soluble vacuolar protein, or receptor-mediated endocytosis of mating pheromone. However, the trans-Golgi network membrane protein Kex2p was not optimally localized in mutant cells. Furthermore, pheromone treatment exacerbated the Kex2p localization defect and caused a corresponding defect in Kex2p-mediated maturation of the α-factor precursor. The results reveal a novel requirement for clathrin during the mating response and suggest that phosphorylation of the light chain subunit modulates the activity of clathrin at the trans-Golgi network.
Elucidation of a PTS–Carbohydrate Chemotactic Signal Pathway inEscherichia coliUsing a Time-resolved Behavioral Assay
Authors: Lux Renate , Munasinghe V. Ranjit N. , Castellano Fred , Lengeler Joseph W. , Corrie John E. T. , Khan Shahid
Published: July 2013

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)–carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates d-glucose and its nonmetabolizable analog methyl α-d-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 ± 3.1 s−1. The response threshold was <10 nM for glucose. Responses to methyl α-d-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP–CheW–CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.
TheDrosophila melanogasterHomologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes
Authors: Reynaud Enrique , Lomelı́ Hilda , Vázquez Martha , Zurita Mario
Published: July 2013

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.
Hypoxic Regulation of Vascular Endothelial Growth Factor mRNA Stability Requires the Cooperation of Multiple RNA Elements
Authors: Dibbens J. A. , Miller D. L. , Damert A. , Risau W. , Vadas M. A. , Goodall G. J.
Published: July 2013

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3′ untranslated region (3′UTR), but also contains destabilizing elements in the 5′UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5′UTR, coding region, and 3′UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.
Active Nucleocytoplasmic Shuttling Required for Function and Regulation of Stress-Activated Kinase Spc1/StyI in Fission Yeast
Authors: Gaits Frédérique , Russell Paul
Published: July 2013

Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeastSchizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1–Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.