bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmonic Roche lobe in metal-dielectric-metal structure
Authors: Shiu Ruei-Cheng , Lan Yung-Chiang
Published: July 2013

This study investigates a plasmonic Roche lobe that is based on a metal-dielectric-metal (MDM) structure using finite-difference time-domain simulations and theoretical analyses. The effective refractive index of the MDM structure has two centers and is inversely proportional to the distance from the position of interest to the centers, in a manner that is analogous to the gravitational potential in a two-star system. The motion of surface plasmons (SPs) strongly depends on the ratio of permittivities at the two centers. The Lagrange point is an unstable equilibrium point for SPs that propagate in the system. After the SPs have passed through the Lagrange point, their spread drastically increases.
Projection-reduction method applied to deriving non-linear optical conductivity for an electron-impurity system
Authors: Kang Nam Lyong , Lee Sang-Seok , Choi Sang Don
Published: July 2013

The projection-reduction method introduced by the present authors is known to give a validated theory for optical transitions in the systems of electrons interacting with phonons. In this work, using this method, we derive the linear and first order nonlinear optical conductivites for an electron-impurity system and examine whether the expressions faithfully satisfy the quantum mechanical philosophy, in the same way as for the electron-phonon systems. The result shows that the Fermi distribution function for electrons, energy denominators, and electron-impurity coupling factors are contained properly in organized manners along with absorption of photons for each electron transition process in the final expressions. Furthermore, the result is shown to be represented properly by schematic diagrams, as in the formulation of electron-phonon interaction. Therefore, in conclusion, we claim that this method can be applied in modeling optical transitions of electrons interacting with both impurities and phonons.
The effect of essential oils of Zataria multiflora and Mentha spicata on the in vitro rumen fermentation, and growth and deaminative activity of amino acid-fermenting bacteria isolated from Mehraban sheep
Authors: Taghavi-Nezhad M. , Alipour D. , Flythe M. D. , Zamani P. , Khodakaramian G.
Published: July 2013

Gas (CO2 and CH4) and ammonia production in the rumen represent major sources of lost carbon and nitrogen, respectively. The essential oils of some plants have been shown to decrease gas and ammonia production by selectively inhibiting rumen microbes. Particularly, those of Zataria multiflora (ZEO; thymol 21%, carvacrol 32%) and Mentha spicata (SEO; carvone 55%) were evaluated in vitro as ruminant-feed additives. The experiments employed mixed rumen microbes and a hyper-ammonia-producing bacterium (HAP) isolated from the rumen of a Mehraban sheep. Both ZEO and SEO decreased in vitro fibre digestibility and also gas production by mixed rumen microbes that were fermenting a typical growing-lamb diet. ZEO decreased ammonia concentration in mixed culture of rumen microbes, but SEO exerted the opposite effect. A bacterial isolate (MT8) was obtained from the rumen of a Mehraban sheep, and the 16S rRNA gene sequence indicated that it was most closely related to Clostridium bifermentans. Isolate MT8 exhibited rapid ammonia production when peptides were the growth substrate, which indicated that MT8 was a HAP. Both oils inhibited the growth and ammonia production of isolate MT8. However, ZEO decreased ammonia production at lower doses, and to a greater degree, than did SEO. These results indicated that both essential oils could potentially be used to modulate rumen fermentation. The detrimental effects on fibre digestion could be problematic in high-forage diets, and this requires further investigation. Isolate MT8 is the first described HAP from the Mehraban sheep rumen. Results on ammonia production by isolate MT8 and mixed rumen microbes indicate differential mode of action of each oil on this parameter.
How psychological framing affects economic market prices in the lab and field
Authors: Sonnemann Ulrich , Camerer Colin F. , Fox Craig R. , Langer Thomas
Published: July 2013

A fundamental debate in social sciences concerns how individual judgments and choices, resulting from psychological mechanisms, are manifested in collective economic behavior. Economists emphasize the capacity of markets to aggregate information distributed among traders into rational equilibrium prices. However, psychologists have identified pervasive and systematic biases in individual judgment that they generally assume will affect collective behavior. In particular, recent studies have found that judged likelihoods of possible events vary systematically with the way the entire event space is partitioned, with probabilities of each of
N
partitioned events biased toward 1/
N
. Thus, combining events into a common partition lowers perceived probability, and unpacking events into separate partitions increases their perceived probability. We look for evidence of such bias in various prediction markets, in which prices can be interpreted as probabilities of upcoming events. In two highly controlled experimental studies, we find clear evidence of partition dependence in a 2-h laboratory experiment and a field experiment on National Basketball Association (NBA) and Federation Internationale de Football Association (FIFA World Cup) sports events spanning several weeks. We also find evidence consistent with partition dependence in nonexperimental field data from prediction markets for economic derivatives (guessing the values of important macroeconomic statistics) and horse races. Results in any one of the studies might be explained by a specialized alternative theory, but no alternative theories can explain the results of all four studies. We conclude that psychological biases in individual judgment can affect market prices, and understanding those effects requires combining a variety of methods from psychology and economics.
Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels
Authors: McCormack Ken , Santos Sonia , Chapman Mark L. , Krafte Douglas S. , Marron Brian E. , West Christopher W. , Krambis Michael J. , Antonio Brett M. , Zellmer Shannon G. , Printzenhoff David , Padilla Karen M. , Lin Zhixin , Wagoner P. Kay , Swain Nigel A. , Stupple Paul A. , de Groot Marcel , Butt Richard P. , Castle Neil A.
Published: July 2013

Significance

Voltage-gated sodium (Na
v
) channels contribute to physiological and pathophysiological electrical signaling in nerve and muscle cells. Because Na
v
channel isoforms exhibit tissue-specific expression, subtype selective modulation of this channel family provides important drug development opportunities. However, most available Na
v
channel modulators are unable to distinguish between Na
v
channel subtypes, which limits their therapeutic utility because of cardiac or nervous system toxicity. This study describes a new class of subtype selective Na
v
channel inhibitors that interact with a region of the channel that controls voltage sensitivity. This interaction site may enable development of selective therapeutic interventions with reduced potential for toxicity.
The mTOR pathway negatively controls ATM by up-regulating miRNAs
Authors: Shen Changxian , Houghton Peter J.
Published: July 2013

The ataxia telangiectasia mutated (ATM) checkpoint is the central surveillance system that maintains genome integrity. We found that in the context of childhood sarcoma, mammalian target of rapamycin (mTOR) signaling suppresses ATM by up-regulating miRNAs targeting
ATM
. Pharmacological inhibition or genetic down-regulation of the mTOR pathway resulted in increase of
ATM
mRNA and protein both in mouse sarcoma xenografts and cultured cells. mTOR Complex 1 (mTORC1) suppresses ATM via S6K1/2 signaling pathways. microRNA-18a and microRNA-421, both of which target ATM, are positively controlled by mTOR signaling. Our findings have identified a negative feedback loop for the signaling between ATM and mTOR pathways and suggest that oncogenic growth signals may promote tumorigenesis by dampening the ATM checkpoint.
MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS
Authors: Lee Jae Keun , Shin Jin Hee , Hwang Sang Gil , Gwag Byoung Joo , McKee Ann C. , Lee Junghee , Kowall Neil W. , Ryu Hoon , Lim Dae-Sik , Choi Eui-Ju
Published: July 2013

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species–dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.
LNK
genes integrate light and clock signaling networks at the core of the
Arabidopsis
oscillator
Authors: Rugnone Matias L. , Faigón Soverna Ana , Sanchez Sabrina E. , Schlaen Ruben Gustavo , Hernando Carlos Esteban , Seymour Danelle K. , Mancini Estefanía , Chernomoretz Ariel , Weigel Detlef , Más Paloma , Yanovsky Marcelo J.
Published: July 2013

Light signaling pathways and the circadian clock interact to help organisms synchronize physiological and developmental processes with periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Here we describe a family of night light–inducible and clock-regulated genes (
LNK
) that play a key role linking light regulation of gene expression to the control of daily and seasonal rhythms in
Arabidopsis thaliana
. A genomewide transcriptome analysis revealed that most light-induced genes respond more strongly to light during the subjective day, which is consistent with the diurnal nature of most physiological processes in plants. However, a handful of genes, including the homologous genes
LNK1
and
LNK2,
are more strongly induced by light in the middle of the night, when the clock is most responsive to this signal. Further analysis revealed that the morning phased
LNK1
and
LNK2
genes control circadian rhythms, photomorphogenic responses, and photoperiodic dependent flowering, most likely by regulating a subset of clock and flowering time genes in the afternoon.
LNK1
and
LNK2
themselves are directly repressed by members of the
TIMING OF CAB1 EXPRESSION
/
PSEUDO RESPONSE REGULATOR
family of core-clock genes in the afternoon and early night. Thus,
LNK1
and
LNK2
integrate early light signals with temporal information provided by core oscillator components to control the expression of afternoon genes, allowing plants to keep track of seasonal changes in day length.
Superconductivity in highly disordered dense carbon disulfide
Authors: Dias Ranga P. , Yoo Choong-Shik , Struzhkin Viktor V. , Kim Minseob , Muramatsu Takaki , Matsuoka Takahiro , Ohishi Yasuo , Sinogeikin Stanislav
Published: July 2013

High pressure plays an increasingly important role in both understanding superconductivity and the development of new superconducting materials. New superconductors were found in metallic and metal oxide systems at high pressure. However, because of the filled close-shell configuration, the superconductivity in molecular systems has been limited to charge-transferred salts and metal-doped carbon species with relatively low superconducting transition temperatures. Here, we report the low-temperature superconducting phase observed in diamagnetic carbon disulfide under high pressure. The superconductivity arises from a highly disordered extended state (
CS4
phase or phase III[
CS4
]) at ∼6.2 K over a broad pressure range from 50 to 172 GPa. Based on the X-ray scattering data, we suggest that the local structural change from a tetrahedral to an octahedral configuration is responsible for the observed superconductivity.
Transformation of odor selectivity from projection neurons to single mushroom body neurons mapped with dual-color calcium imaging
Authors: Li Hao , Li Yiming , Lei Zhengchang , Wang Kaiyu , Guo Aike
Published: July 2013

Although the response properties of most neurons are, to a large extent, determined by the presynaptic inputs that they receive, comprehensive functional characterization of the presynaptic inputs of a single neuron remains elusive. Toward this goal, we introduce a dual-color calcium imaging approach that simultaneously monitors the responses of a single postsynaptic neuron together with its presynaptic axon terminal inputs in vivo. As a model system, we applied the strategy to the feed-forward connections from the projection neurons (PNs) to the Kenyon cells (KCs) in the mushroom body of
Drosophila
and functionally mapped essentially all PN inputs for some of the KCs. We found that the output of single KCs could be well predicted by a linear summation of the PN input signals, indicating that excitatory PN inputs play the major role in generating odor-selective responses in KCs. When odors failed to activate KC output, local calcium transients restricted to individual postsynaptic sites could be observed in the KC dendrites. The response amplitudes of the local transients often correlated linearly with the presynaptic response amplitudes, allowing direct assay of the strength of single synaptic sites. Furthermore, we found a scaling relationship between the total number of PN terminals that a single KC received and the average synaptic strength of these PN-KC synapses. Our strategy provides a unique perspective on the process of information transmission and integration in a model neural circuit and may be broadly applicable for the study of the origin of neuronal response properties.
Dynamics of mechanosensing in the bacterial flagellar motor
Authors: Lele Pushkar P. , Hosu Basarab G. , Berg Howard C.
Published: July 2013

Mechanosensing by flagella is thought to trigger bacterial swarmer-cell differentiation, an important step in pathogenesis. How flagellar motors sense mechanical stimuli is not known. To study this problem, we suddenly increased the viscous drag on motors by a large factor, from very low loads experienced by motors driving hooks or hooks with short filament stubs, to high loads, experienced by motors driving tethered cells or 1-μm latex beads. From the initial speed (after the load change), we inferred that motors running at very low loads are driven by one or at most two force-generating units. Following the load change, motors gradually adapted by increasing their speeds in a stepwise manner (over a period of a few minutes). Motors initially spun exclusively counterclockwise, but then increased the fraction of time that they spun clockwise over a time span similar to that observed for adaptation in speed. Single-motor total internal reflection fluorescence imaging of YFP–MotB (part of a stator force-generating unit) confirmed that the response to sudden increments in load occurred by the addition of new force-generating units. We estimate that 6–11 force-generating units drive motors at high loads. Wild-type motors and motors locked in the clockwise or counterclockwise state behaved in a similar manner, as did motors in cells deleted for the motor protein gene
fliL
or for genes in the chemotaxis signaling pathway. Thus, it appears that stators themselves act as dynamic mechanosensors. They change their structure in response to changes in external load. How such changes might impact cellular functions other than motility remains an interesting question.
Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II
Authors: Liu Xin , Kapoor Tarun M. , Chen James K. , Huse Morgan
Published: July 2013

Centrosome reorientation to the immunological synapse maintains the specificity of T-cell effector function by facilitating the directional release of cytokines and cytolytic factors toward the antigen-presenting cell. This polarization response is driven by the localized accumulation of diacylglycerol, which recruits multiple protein kinase (PK)C isozymes to the synaptic membrane. Here, we used T-cell receptor (TCR) photoactivation and imaging methodology to demonstrate that PKCs control centrosome dynamics through the reciprocal localization of two motor complexes, dynein and nonmuscle myosin (NM)II. Dynein accumulated in the region of TCR stimulation, whereas NMII clustered in the back of the cell, behind the polarizing centrosome. PKC activity, which shaped both dynein and NMII accumulation within this framework, controlled NMII localization directly by phosphorylating inhibitory sites within the myosin regulatory light chain, thereby suppressing NMII clustering in the region of TCR stimulation. Concurrently, phosphorylation of distinct sites within myosin regulatory light chain by Rho kinase drove NMII clustering in areas behind the centrosome. These results reveal a role for NMII in T-cell polarity and demonstrate how it is regulated by upstream signals.
Kinetic response of a photoperturbed allosteric protein
Authors: Buchli Brigitte , Waldauer Steven A. , Walser Reto , Donten Mateusz L. , Pfister Rolf , Blöchliger Nicolas , Steiner Sandra , Caflisch Amedeo , Zerbe Oliver , Hamm Peter
Published: July 2013

By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery.
Real-time observation of fluctuations at the driven-dissipative Dicke phase transition
Authors: Brennecke Ferdinand , Mottl Rafael , Baumann Kristian , Landig Renate , Donner Tobias , Esslinger Tilman
Published: July 2013

We experimentally study the influence of dissipation on the driven Dicke quantum phase transition, realized by coupling external degrees of freedom of a Bose–Einstein condensate to the light field of a high-finesse optical cavity. The cavity provides a natural dissipation channel, which gives rise to vacuum-induced fluctuations and allows us to observe density fluctuations of the gas in real-time. We monitor the divergence of these fluctuations over two orders of magnitude while approaching the phase transition, and observe a behavior that deviates significantly from that expected for a closed system. A correlation analysis of the fluctuations reveals the diverging time scale of the atomic dynamics and allows us to extract a damping rate for the external degree of freedom of the atoms. We find good agreement with our theoretical model including dissipation via both the cavity field and the atomic field. Using a dissipation channel to nondestructively gain information about a quantum many-body system provides a unique path to study the physics of driven-dissipative systems.
BET proteins promote efficient murine leukemia virus integration at transcription start sites
Authors: Sharma Amit , Larue Ross C. , Plumb Matthew R. , Malani Nirav , Male Frances , Slaughter Alison , Kessl Jacques J. , Shkriabai Nikolozi , Coward Elizabeth , Aiyer Sriram S. , Green Patrick L. , Wu Li , Roth Monica J. , Bushman Frederic D. , Kvaratskhelia Mamuka
Published: July 2013

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.
Massively parallel in vivo enhancer assay reveals that highly local features determine thecis-regulatory function of ChIP-seq peaks
Authors: White Michael A. , Myers Connie A. , Corbo Joseph C. , Cohen Barak A.
Published: July 2013

Transcription factors (TFs) recognize short sequence motifs that are present in millions of copies in large eukaryotic genomes. TFsmust distinguish their target binding sites from a vast genomic excess of spurious motif occurrences; however, it is unclear whether functional sites are distinguished from nonfunctional motifs by local primary sequence features or by the larger genomic context in which motifs reside. We used a massively parallel enhancer assay in living mouse retinas to compare 1,300 sequences bound in the genome by the photoreceptor transcription factor Cone-rod homeobox (Crx), to 3,000 control sequences. We found that very short sequences bound in the genome by Crx activated transcription at high levels, whereas unbound genomic regions with equal numbers of Crx motifs did not activate above background levels, even when liberated from their larger genomic context. High local GC content strongly distinguishes bound motifs from unbound motifs across the entire genome. Our results show that thecis-regulatory potential of TF-bound DNA is determined largely by highly local sequence features and not by genomic context.
Antibodies that bind complex glycosaminoglycans accumulate in the Golgi
Authors: Radic Marko , Weigert Martin G. , Khan Salar N. , Han Jiong , Kalinina Olga , Luning Prak Eline T.
Published: July 2013

Light (L) chains that edit anti-DNA heavy (H) chains rescue B-cell development by suppressing DNA binding. However, exceptional editor L chains allow B cells to reach splenic compartments even though their B-cell receptors remain autoreactive. Such incompletely edited B cells express multireactive antibodies that accumulate in the Golgi and are released as insoluble, amyloid-like immune complexes. Here, we examine examples of incomplete editing from the analysis of variable to joining (VJ) gene junction of the variable (Vλx) editor L chain. When paired with the anti-DNA heavy chain, VH56R, the Vλx variants yield antibodies with differing specificities, including glycosaminoglycan reactivity. Our results implicate these specificities in the evasion of receptor editing through intracellular sequestration of IgM and the release of insoluble IgM complexes. Our findings can be extrapolated to human L chains and have implications for understanding a latent component of the Ig repertoire that could exert pathogenic and protective functions.
Efficient viral delivery system for unnatural amino acid mutagenesis in mammalian cells
Authors: Chatterjee Abhishek , Xiao Han , Bollong Michael , Ai Hui-Wang , Schultz Peter G.
Published: July 2013

Here we report the development of a baculovirus-based delivery system that enables the efficient incorporation of unnatural amino acids into proteins in mammalian cells. We have exploited the large cargo-capacity (>30 kb) and stability of the double-stranded DNA genome of baculovirus to deliver to a variety of cell types all of the components required to genetically incorporate novel amino acids. These include the engineered tRNA/aminoacyl-tRNA synthetase pair and the nonsense mutant of the target gene. Mammalian cell transduction efficiency of baculovirus was significantly improved by incorporating genetic elements from mammalian viruses. Two polyspecific tRNA/aminoacyl-tRNA synthetase pairs were inserted into this expression system, enabling the site-specific incorporation of a variety of unnatural amino acids with novel chemical and biological properties into proteins.
Importance of trivalency and the e
g
1
configuration in the photocatalytic oxidation of water by Mn and Co oxides
Authors: Maitra Urmimala , Naidu B. S. , Govindaraj A. , Rao C. N. R.
Published: July 2013

Prompted by the early results on the catalytic activity of LiMn
2
O
4
and related oxides in the photochemical oxidation of water, our detailed study of several manganese oxides has shown that trivalency of Mn is an important factor in determining the catalytic activity. Thus, Mn
2
O
3
, LaMnO
3
, and MgMn
2
O
4
are found to be very good catalysts with turnover frequencies of 5 × 10
−4
s
−1
, 4.8 × 10
−4
s
−1
, and 0.8 ×10
−4
s
−1
, respectively. Among the cobalt oxides, Li
2
Co
2
O
4
and LaCoO
3
—especially the latter—exhibit excellent catalytic activity, with the turnover frequencies being 9 × 10
−4
s
−1
and 1.4 × 10
−3
s
−1
, respectively. The common feature among the catalytic Mn and Co oxides is not only that Mn and Co are in the trivalent state, but Co
3+
in the Co oxides is in the intermediate t
2g
5
e
g
1
state whereas Mn
3+
is in the t
2g
3
e
g
1
state. The presence of the e
g
1
electron in these Mn and Co oxides is considered to play a crucial role in the photocatalytic properties of the oxides.
The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function
Authors: Jha Archana , Ahuja Malini , Maléth József , Moreno Claudia M. , Yuan Joseph P. , Kim Min Seuk , Muallem Shmuel
Published: July 2013

Ca2+ influx by store-operated Ca2+ channels (SOCs) mediates all Ca2+-dependent cell functions, but excess Ca2+ influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca2+ sensor STIM1. Slow Ca2+-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448–530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448–490) and STIM1(490–530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448–490) lobe restricted, whereas the STIM1(490–530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.
Augmin-dependent microtubule nucleation at microtubule walls in the spindle
Authors: Kamasaki Tomoko , O’Toole Eileen , Kita Shigeo , Osumi Masako , Usukura Jiro , McIntosh J. Richard , Goshima Gohta
Published: July 2013

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.
GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6
Authors: Wong Siew Heng , Xu Yue , Zhang Tao , Griffiths Gareth , Lowe Stephen Loucian , Subramaniam V. Nathan , Seow Kah Tong , Hong Wanjin
Published: July 2013

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.
Phototactic Migration ofDictyosteliumCells Is Linked to a New Type of Gelsolin-related Protein
Authors: Stocker Susanne , Hiery Mary , Marriott Gerard
Published: July 2013

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type inDictyostelium, as well as in heterologous cells from lower to higher eukaryotes.
Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells
Authors: Vilhardt Frederik , Nielsen Morten , Sandvig Kirsten , van Deurs Bo
Published: July 2013

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44Acells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.
Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network
Authors: Eng Francis J. , Varlamov Oleg , Fricker Lloyd D.
Published: July 2013

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.
Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions
Authors: Helfman David M. , Levy Esther T. , Berthier Christine , Shtutman Michael , Riveline Daniel , Grosheva Inna , Lachish-Zalait Aurelie , Elbaum Michael , Bershadsky Alexander D.
Published: July 2013

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.
Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts
Authors: Gallo Rita , Serafini Marco , Castellani Loriana , Falcone Germana , Alemà Stefano
Published: July 2013

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src–induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures.
Membrane-anchored Plakoglobins Have Multiple Mechanisms of Action in Wnt Signaling
Authors: Klymkowsky Michael W. , Williams Bart O. , Barish Grant D. , Varmus Harold E. , Vourgourakis Yanni E.
Published: July 2013

In Wnt signaling, β-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing geneSiamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic β-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize β-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of β-catenin turnover. Expression of cnxPg increases levels of cytosolic β-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize β-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with β-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both β-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm ofXenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on β-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.
Rab5 Induces Rac-independent Lamellipodia Formation and Cell Migration
Authors: Spaargaren Marcel , Bos Johannes L.
Published: July 2013

Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative Rab5 inhibits vesicle fusion. Here we show that introduction of active Rab5 in fibroblasts also induced reorganization of the actin cytoskeleton but not of microtubule filaments, resulting in prominent lamellipodia formation. The Rab5-induced lamellipodia formation did not require activation of PI3-K or the GTPases Ras, Rac, Cdc42, or Rho, which are all strongly implicated in cytoskeletal reorganization. Furthermore, lamellipodia formation by insulin, Ras, or Rac was not affected by expression of dominant negative Rab5. In addition, cells expressing active Rab5 displayed a dramatic stimulation of cell migration, with the lamellipodia serving as the leading edge. Both lamellipodia formation and cell migration were dependent on actin polymerization but not on microtubules. These results demonstrate that Rab5 induces lamellipodia formation and cell migration and that the Rab5-induced lamellipodia formation occurs by a novel mechanism independent of, and distinct from, PI3-K, Ras, or Rho-family GTPases. Thus, Rab5 can control not only endocytosis but also actin cytoskeleton reorganization and cell migration, which provides strong support for an intricate relationship between these processes.
Phosphorylation Controls Timing of Cdc6p Destruction: A Biochemical Analysis
Authors: Elsasser Suzanne , Chi Yong , Yang Ping , Campbell Judith L.
Published: July 2013

The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation. We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during theSaccharomyces cerevisiae cell cycle. Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed. First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase. However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p. SCF-Cdc4p– and N-terminal phosphorylation site–dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle. The stable cdc6 mutants show no apparent replication defects in wild-type strains. However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex.
Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos inXenopusOocytes in Response to Progesterone
Authors: Frank-Vaillant Marie , Jessus Catherine , Ozon René , Maller James L. , Haccard Olivier
Published: July 2013

Progesterone-induced meiotic maturation of Xenopusoocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immatureXenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.