bluuebunny/crossref_metadata_2025 huggingface dataset:
A small animal Raman instrument for rapid, wide-area, spectroscopic imaging
Authors: Bohndiek Sarah E. , Wagadarikar Ashwin , Zavaleta Cristina L. , Van de Sompel Dominique , Garai Ellis , Jokerst Jesse V. , Yazdanfar Siavash , Gambhir Sanjiv S.
Published: July 2013

Raman spectroscopy, amplified by surface enhanced Raman scattering (SERS) nanoparticles, is a molecular imaging modality with ultra-high sensitivity and the unique ability to multiplex readouts from different molecular targets using a single wavelength of excitation. This approach holds exciting prospects for a range of applications in medicine, including identification and characterization of malignancy during endoscopy and intraoperative image guidance of surgical resection. The development of Raman molecular imaging with SERS nanoparticles is presently limited by long acquisition times, poor spatial resolution, small field of view, and difficulty in animal handling with existing Raman spectroscopy instruments. Our goal is to overcome these limitations by designing a bespoke instrument for Raman molecular imaging in small animals. Here, we present a unique and dedicated small-animal Raman imaging instrument that enables rapid, high-spatial resolution, spectroscopic imaging over a wide field of view (> 6 cm
2
), with simplified animal handling. Imaging of SERS nanoparticles in small animals demonstrated that this small animal Raman imaging system can detect multiplexed SERS signals in both superficial and deep tissue locations at least an order of magnitude faster than existing systems without compromising sensitivity.
Photochemistry of aqueous pyruvic acid
Authors: Griffith Elizabeth C. , Carpenter Barry K. , Shoemaker Richard K. , Vaida Veronica
Published: July 2013

The study of organic chemistry in atmospheric aerosols and cloud formation is of interest in predictions of air quality and climate change. It is now known that aqueous phase chemistry is important in the formation of secondary organic aerosols. Here, the photoreactivity of pyruvic acid (PA; CH
3
COCOOH) is investigated in aqueous environments characteristic of atmospheric aerosols. PA is currently used as a proxy for α-dicarbonyls in atmospheric models and is abundant in both the gas phase and the aqueous phase (atmospheric aerosols, fog, and clouds) in the atmosphere. The photoreactivity of PA in these phases, however, is very different, thus prompting the need for a mechanistic understanding of its reactivity in different environments. Although the decarboxylation of aqueous phase PA through UV excitation has been studied for many years, its mechanism and products remain controversial. In this work, photolysis of aqueous PA is shown to produce acetoin (CH
3
CHOHCOCH
3
), lactic acid (CH
3
CHOHCOOH), acetic acid (CH
3
COOH), and oligomers, illustrating the progression from a three-carbon molecule to four-carbon and even six-carbon molecules through direct photolysis. These products are detected using vibrational and electronic spectroscopy, NMR, and MS, and a reaction mechanism is presented accounting for all products detected. The relevance of sunlight-initiated PA chemistry in aqueous environments is then discussed in the context of processes occurring on atmospheric aerosols.
Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ
Authors: Pertsinidis Alexandros , Mukherjee Konark , Sharma Manu , Pang Zhiping P. , Park Sang Ryul , Zhang Yunxiang , Brunger Axel T. , Südhof Thomas C. , Chu Steven
Published: July 2013

Significance

Synaptic vesicle fusion is catalyzed by multiprotein complexes that bring two lipid bilayers into close opposition. Several assembly mechanisms have been proposed for the synaptic vesicle fusion machinery, but exactly how these proteins interact in vivo remains unclear. We developed two-color fluorescence nanoscopy to directly visualize molecular interactions in situ and discovered that syntaxin-1, SNAP-25, and Munc18-1 (mammalian uncoordinated-18), three essential components for neurotransmission, closely colocalize on the plasma membrane, suggesting possible pathways for SNARE-mediated membrane fusion. Our superresolution method provides a framework for delineating the molecular underpinnings of the synaptic vesicle fusion machinery.
Detecting surface bundles in finite covers of hyperbolic closed 3-manifolds
Authors: Renard Claire
Published: July 2013

The main theorem of this article provides sufficient conditions for a degree

d
d

finite cover

M
′

M’

of a hyperbolic 3-manifold

M
M

to be a surface bundle. Let

F
F

be an embedded, closed and orientable surface of genus

g
g

, close to a minimal surface in the cover

M
′

M’

, splitting

M
′

M’

into a disjoint union of

q
q

handlebodies and compression bodies. We show that there exists a fiber in the complement of

F
F

provided that

d
d

,

q
q

and

g
g

satisfy some inequality involving an explicit constant

k
k

depending only on the volume and the injectivity radius of

M
M

. In particular, this theorem applies to a Heegaard splitting of a finite covering

M
′

M’

, giving an explicit lower bound for the genus of a strongly irreducible Heegaard splitting of

M
′

M’

. Applying the main theorem to the setting of a circular decomposition associated to a non-trivial homology class of

M
M

gives sufficient conditions for this homology class to correspond to a fibration over the circle. Similar methods also lead to a sufficient condition for an incompressible embedded surface in

M
M

to be a fiber.
Role of Fission Yeast Primase Catalytic Subunit in the Replication Checkpoint
Authors: Griffiths Dominic J. F. , Liu Vivian F. , Nurse Paul , Wang Teresa S.-F.
Published: July 2013

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe,spp1+and spp2+(S. pombe primase 1 and 2).spp1+encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polα to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1+gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1+exhibit an allele-specific abnormal mitotic phenotype. Mutations ofspp1+reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polα–primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polα has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polα. Together, these have led us to propose that suboptimal cellular levels of polα–primase complex due to the allele-specific mutations of Spp1 might not allow Polα to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.
Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast Microscopy
Authors: Patki Varsha , Buxton Joanne , Chawla Anil , Lifshitz Larry , Fogarty Kevin , Carrington Walter , Tuft Richard , Corvera Silvia
Published: July 2013

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.
Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells
Authors: Shoeman Robert L. , Hüttermann Claudia , Hartig Roland , Traub Peter
Published: July 2013

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin+] cells, whereas no changes were observed in SW 13 [vimentin−] cells after microinjection of protease. Treatment of SW 13 [vimentin−] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin−] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.
ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion
Authors: Jahraus Andrea , Egeberg Morten , Hinner Bernhard , Habermann Anja , Sackman Erich , Pralle Arnd , Faulstich Heinz , Rybin Vladimir , Defacque Hélène , Griffiths Gareth
Published: July 2013

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol ( Jahraus et al., 1998 ). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP ( Defacque et al., 2000a ), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.
Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways
Authors: Yahara Natsuko , Ueda Takashi , Sato Ken , Nakano Akihiko
Published: July 2013

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.
Transforming Growth Factor-β1 Mediates Epithelial to Mesenchymal Transdifferentiation through a RhoA-dependent Mechanism
Authors: Bhowmick Neil A. , Ghiassi Mayshan , Bakin Andrei , Aakre Mary , Lundquist Christopher A. , Engel Michael E. , Arteaga Carlos L. , Moses Harold L.
Published: July 2013

Transforming growth factor-β1 (TGF-β) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-β are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-β–induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-β do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-β rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160ROCK, by the expression of dominant-negative mutants, inhibited TGF-β–mediated EMT. The data suggest that TGF-β rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.
Regulation of Cell Cycle Progression by Swe1p and Hog1p Following Hypertonic Stress
Authors: Alexander Matthew R. , Tyers Mike , Perret Mireille , Craig B. Maureen , Fang Karen S. , Gustin Michael C.
Published: July 2013

Exposure of yeast cells to an increase in external osmolarity induces a temporary growth arrest. Recovery from this stress is mediated by the accumulation of intracellular glycerol and the transcription of several stress response genes. Increased external osmolarity causes a transient accumulation of 1N and 2N cells and a concomitant depletion of S phase cells. Hypertonic stress triggers a cell cycle delay in G2 phase cells that appears distinct from the morphogenesis checkpoint, which operates in early S phase cells. Hypertonic stress causes a decrease in CLB2 mRNA, phosphorylation of Cdc28p, and inhibition of Clb2p-Cdc28p kinase activity, whereas Clb2 protein levels are unaffected. Like the morphogenesis checkpoint, the osmotic stress-induced G2 delay is dependent upon the kinase Swe1p, but is not tightly correlated with inhibition of Clb2p-Cdc28p kinase activity. Thus, deletion ofSWE1 does not prevent the hypertonic stress-induced inhibition of Clb2p-Cdc28p kinase activity. Mutation of the Swe1p phosphorylation site on Cdc28p (Y19) does not fully eliminate the Swe1p-dependent cell cycle delay, suggesting that Swe1p may have functions independent of Cdc28p phosphorylation. Conversely, deletion of the mitogen-activated protein kinase HOG1 does prevent Clb2p-Cdc28p inhibition by hypertonic stress, but does not block Cdc28p phosphorylation or alleviate the cell cycle delay. However, Hog1p does contribute to proper nuclear segregation after hypertonic stress in cells that lack Swe1p. These results suggest a hypertonic stress-induced cell cycle delay in G2 phase that is mediated in a novel way by Swe1p in cooperation with Hog1p.
Structure and Function of a Vimentin-associated Matrix Adhesion in Endothelial Cells
Authors: Gonzales Meredith , Weksler Babette , Tsuruta Daisuke , Goldman Robert D. , Yoon Kristine J. , Hopkinson Susan B. , Flitney Frederick W. , Jones Jonathan C. R.
Published: July 2013

The α4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the α4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by αv and β3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an αvβ3-integrin–dependent manner. The αvβ3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the αvβ3 integrin-positive focal contacts. We have investigated the function of α4-laminin and αvβ3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.
Urokinase Receptors Promote β1 Integrin Function through Interactions with Integrin α3β1
Authors: Wei Ying , Eble Johannes A. , Wang Zemin , Kreidberg Jordan A. , Chapman Harold A.
Published: July 2013

The urokinase receptor (uPAR) is linked to cellular migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate cell signaling in response to urokinase (uPA) binding. The mechanisms for these activities remain incompletely defined, although uPAR was recently identified as acis-acting ligand for the β2 integrin CD11b/CD18 (Mac-1). Here we show that a major β1 integrin partner for uPAR/uPA signaling is α3. In uPAR-transfected 293 cells uPAR complexed (>90%) with α3β1 and antibodies to α3 blocked uPAR-dependent vitronectin (Vn) adhesion. Soluble uPAR bound to recombinant α3β1 in a uPA-dependent manner (Kd< 20 nM) and binding was blocked by a 17-mer α3β1 integrin peptide (α325) homologous to the CD11b uPAR-binding site. uPAR colocalized with α3β1 in MDA-MB-231 cells and uPA (1 nM) enhanced spreading and focal adhesion kinase phosphorylation on fibronectin (Fn) or collagen type I (Col) in a pertussis toxin- and α325-sensitive manner. A critical role of α3β1 in uPA signaling was verified by studies of epithelial cells from α3-deficient mice. Thus, uPAR preferentially complexes with α3β1, promoting direct (Vn) and indirect (Fn, Col) pathways of cell adhesion, the latter a heterotrimeric G protein-dependent mechanism of signaling between α3β1 and other β1 integrins.
Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis
Authors: Bello Valérie , Goding James W. , Greengrass Vicki , Sali Adnan , Dubljevic Valentina , Lenoir Christelle , Trugnan Germain , Maurice Michèle
Published: July 2013

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.
Zyxin Is not Colocalized with Vasodilator-stimulated Phosphoprotein (VASP) at Lamellipodial Tips and Exhibits Different Dynamics to Vinculin, Paxillin, and VASP in Focal Adhesions
Authors: Rottner Klemens , Krause Matthias , Gimona Mario , Small J. Victor , Wehland Jürgen
Published: July 2013

Actin polymerization is accompanied by the formation of protein complexes that link extracellular signals to sites of actin assembly such as membrane ruffles and focal adhesions. One candidate recently implicated in these processes is the LIM domain protein zyxin, which can bind both Ena/vasodilator-stimulated phosphoprotein (VASP) proteins and the actin filament cross-linking protein α-actinin. To characterize the localization and dynamics of zyxin in detail, we generated both monoclonal antibodies and a green fluorescent protein (GFP)-fusion construct. The antibodies colocalized with ectopically expressed GFP-VASP at focal adhesions and along stress fibers, but failed to label lamellipodial and filopodial tips, which also recruit Ena/VASP proteins. Likewise, neither microinjected, fluorescently labeled zyxin antibodies nor ectopically expressed GFP-zyxin were recruited to these latter sites in live cells, whereas both probes incorporated into focal adhesions and stress fibers. Comparing the dynamics of zyxin with that of the focal adhesion protein vinculin revealed that both proteins incorporated simultaneously into newly formed adhesions. However, during spontaneous or induced focal adhesion disassembly, zyxin delocalization preceded that of either vinculin or paxillin. Together, these data identify zyxin as an early target for signals leading to adhesion disassembly, but exclude its role in recruiting Ena/VASP proteins to the tips of lamellipodia and filopodia.
Cross Talk between β1and αVIntegrins: β1Affects β3mRNA Stability
Authors: Retta Saverio Francesco , Cassarà Georgia , D'Amato Monica , Alessandro Riccardo , Pellegrino Maurizio , Degani Simona , De Leo Giacomo , Silengo Lorenzo , Tarone Guido
Published: July 2013

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of β1-null GD25 cells ectopically expressing the β1A integrin subunit, we provide evidence for the existence of a cross talk between β1and αVintegrins that affects the ratio of αVβ3and αVβ5integrin cell surface levels. In particular, we demonstrate that a down-regulation of αVβ3and an up-regulation of αVβ5occur as a consequence of β1A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms β1B and β1D, as well as two β1cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (β1TR) or only its “variable” region (β1COM), we show that the effects of β1over αVintegrins take place irrespective of the type of β1isoform, but require the presence of the “common” region of the β1cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby β1integrins exert theirtrans-acting functions, we have found that the down-regulation of αVβ3is due to a decreased β3subunit mRNA stability, whereas the up-regulation of αVβ5is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.
Lumenal Endosomal and Golgi-Retrieval Determinants Involved in pH-sensitive Targeting of an Early Golgi Protein
Authors: Bachert Collin , Lee Tina H. , Linstedt Adam D.
Published: July 2013

Despite the potential importance of retrieval-based targeting, few Golgi cisternae-localized proteins have been demonstrated to be targeted by retrieval, and the putative retrieval signals remain unknown. Golgi phosphoprotein of 130 kDa (GPP130) is acis-Golgi protein that allows assay of retrieval-based targeting because it redistributes to endosomes upon treatment with agents that disrupt lumenal pH, and it undergoes endosome-to-Golgi retrieval upon drug removal. Analysis of chimeric molecules containing domains from GPP130 and the plasma membrane protein dipeptidylpeptidase IV indicated that GPP130 targeting information is contained entirely within its lumenal domain. Dissection of the lumenal domain indicated that a predicted coiled-coil stem domain adjacent to the transmembrane domain was both required and sufficient for pH-sensitive Golgi localization and endosome-to-Golgi retrieval. Further dissection of this stem domain revealed two noncontiguous stretches that each conferred Golgi localization separated by a stretch that conferred endosomal targeting. Importantly, in the absence of the endosomal determinant the Golgi targeting of constructs containing either or both of the Golgi determinants became insensitive to pH disruption by monensin. Because monensin blocks endosome-to-Golgi transport, the finding that the endosomal determinant confers monensin sensitivity suggests that the endosomal determinant causes GPP130 to traffic to endosomes from which it is normally retrieved. Thus, our observations identify Golgi and endosomal targeting determinants within a lumenal predicted coiled-coil domain that appear to act coordinately to mediate retrieval-based targeting of GPP130.
Gγ inDictyostelium: Its Role in Localization of Gβγ to the Membrane Is Required for Chemotaxis in Shallow Gradients
Authors: Zhang Ning , Long Yu , Devreotes Peter N.
Published: July 2013

G-protein–mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organismDictyostelium discoideum. Database searches have yielded 11 Gα-subunits, a single Gβ-subunit, but no Gγ-subunits. We report here the purification, cDNA isolation, and functional analysis of a Gγ-subunit. Like Gβ, the Gγ appears to be unique and hybridization studies show that Gγ and Gβ are expressed in parallel during development. Species-wide sequence comparisons of Gγ-subunits and γ-like domains of RGS proteins reveal short stretches of highly conserved residues as well as the common CXXL motif at the COOH-terminal of Gγs that target Gβγs to plasma membrane. Overexpression of a CSVL-deleted Gγ (GγΔ) in wild-type cells shifts Gβγ to the cytosol and selectively impairs certain G-protein–mediated signal transduction pathways. These cells are able to respond to increments in the stimulus, but are unable to sense chemoattractant gradients. They neither move directionally nor recruit PH-domains to their leading edge. Thus, a full complement of membrane-tethered Gβγ is required for sensing shallow gradients, but is not essential for responses to increments in extracellular stimuli.
The Cytoplasmic Domain of the Integrin α9 Subunit Requires the Adaptor Protein Paxillin to Inhibit Cell Spreading but Promotes Cell Migration in a Paxillin-independent Manner
Authors: Young Bradford A. , Taooka Yasuyuki , Liu Shouchun , Askins Karen J. , Yokosaki Yasuyuki , Thomas Sheila M. , Sheppard Dean
Published: July 2013

The integrin α9 subunit forms a single heterodimer, α9β1. The α9 subunit is most closely related to the α4 subunit, and like α4 integrins, α9β1 plays an important role in leukocyte migration. The α4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the α9 cytoplasmic domain has similar effects, a series of chimeric and deleted α9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an α9β1-specific ligand. Like α4, the α9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the α9 cytoplasmic domain and to a similar level as α4. In paxillin−/−cells, α9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the α9-paxillin interaction prevented α9 from inhibiting cell spreading but had no effect on α9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin α subunits enhance migration and inhibit cell spreading are distinct and that the α9 and α4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.
Vps26p, a Component of Retromer, Directs the Interactions of Vps35p in Endosome-to-Golgi Retrieval
Authors: Reddy Jonathan V. , Seaman Matthew N.J.
Published: July 2013

Endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p is mediated by a recently discovered membrane coat complex termed retromer. Retromer comprises five conserved proteins: Vps35p, Vps29p, Vps5p, Vps17p, and Vps26p. Vps35p recognizes cargo molecules such as Vps10p and interacts strongly with Vps29p. Vps5p forms a subcomplex with Vps17p and has been proposed to play a structural role by self-assembling into large multimeric structures. The function of Vps26p is currently unknown. We have investigated the role that Vps26p plays in retromer-mediated endosome-to-Golgi transport by analyzing dominant negative alleles of Vps26p. These mutants have identified a crucial region of Vps26p that plays an important role in its function. Functional domains of Vps26p have been investigated by the creation of yeast-mouse hybrid molecules in which domains of Vps26p have been replaced by the similar domain in the protein encoded by the mouse VPS26 gene, Hβ58. These domain swap experiments have shown that Vps26p promotes the interactions between the cargo-selective component Vps35p and the structural components Vps5p/Vps17p.
Glucocorticoid Receptor Activation of the IκBα Promoter within Chromatin
Authors: Deroo Bonnie J. , Archer Trevor K.
Published: July 2013

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive IκBα gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFκB). Glucocorticoids inhibit NFκB activity in some tissues by elevating the levels of IκBα. We found that glucocorticoids activated the IκBα promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the IκBα promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the IκBα promoter is assembled as phased nucleosomes that adopt an “open” chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.
In Vivo Association of Ku with Mammalian Origins of DNA Replication
Authors: Novac Olivia , Matheos Diamanto , Araujo Felipe D. , Price Gerald B. , Zannis-Hadjopoulos Maria
Published: July 2013

Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association withors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80+/+and Ku80−/−mouse embryonic fibroblasts. A decrease of ∼70% in in vitro DNA replication was observed when the Ku80−/−extracts were used, compared with the Ku80+/+extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.
pkl1+andklp2+: Two Kinesins of the Kar3 Subfamily in Fission Yeast Perform Different Functions in Both Mitosis and Meiosis
Authors: Troxell Cynthia L. , Sweezy Mark A. , West Robert R. , Reed Karen D. , Carson Bryan D. , Pidoux Alison L. , Cande W. Zacheus , McIntosh J. Richard
Published: July 2013

We have identified Klp2p, a new kinesin-like protein (KLP) of the KAR3 subfamily in fission yeast. The motor domain of this protein is 61% identical and 71% similar to Pkl1p, another fission yeast KAR3 protein, yet the two enzymes are different in behavior and function. Pkl1p is nuclear throughout the cell cycle, whereas Klp2p is cytoplasmic during interphase. During mitosis Klp2p enters the nucleus where it forms about six chromatin-associated dots. In metaphase-arrested cells these migrate back and forth across the nucleus. During early anaphase they segregate with the chromosomes into two sets of about three, fade, and are replaced by other dots that form on the spindle interzone. Neitherklp2+norpkl1+is essential, and the double deletion is also wild type for both vegetative and sexual reproduction. Each deletion rescues different alleles ofcut7ts, a KLP that contributes to spindle formation and elongation. When either or both deletions are combined with a dynein deletion, vegetative growth is normal, but sexual reproduction fails: klp2Δ,dhc1-d1 in karyogamy, pkl1Δ,dhc1-d1 in multiple phases of meiosis, and the triple deletion in both. Deletion of Klp2p elongates a metaphase-arrested spindle, but pkl1Δshortens it. The anaphase spindle of klp2Δ becomes longer than the cell, leading it to curl around the cell's ends. Apparently, Klp2p promotes spindle disassembly and contributes to the behavior of mitotic chromosomes.
MCM2–7 Proteins Are Essential Components of Prereplicative Complexes that Accumulate Cooperatively in the Nucleus during G1-phase and Are Required to Establish, But Not Maintain, the S-phase Checkpoint
Authors: Labib Karim , Kearsey Stephen E. , Diffley John F.X.
Published: July 2013

A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2–7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2–7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.
Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association
Authors: van Eenennaam Hans , van der Heijden Annemarie , Janssen Rolf J. R. J. , van Venrooij Walther J. , Pruijn Ger J. M.
Published: July 2013

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.
Na,K-ATPase Activity Is Required for Formation of Tight Junctions, Desmosomes, and Induction of Polarity in Epithelial Cells
Authors: Rajasekaran Sigrid A. , Palmer Lawrence G. , Moon Sun Y. , Peralta Soler Alejandro , Apodaca Gerard L. , Harper Jeffrey F. , Zheng Yi , Rajasekaran Ayyappan K.
Published: July 2013

Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na+homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.
Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit
Authors: Reilly Barbara A. , Kraynack Bryan A. , VanRheenen Susan M. , Waters M. Gerard
Published: July 2013

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show thatdsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of ∼80 and ∼55 kDa. The ∼80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is ∼50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the δ-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.
Dynamics of Ankyrin-containing Complexes in Chicken Embryonic Erythroid Cells: Role of Phosphorylation
Authors: Ghosh Sourav , Cox John V.
Published: July 2013

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.
G2/M Arrest Caused by Actin Disruption Is a Manifestation of the Cell Size Checkpoint in Fission Yeast
Authors: Rupes̆ Ivan , Webb Bradley A. , Mak Alan , Young Paul G.
Published: July 2013

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.
A Novel Karyoskeletal Protein: Characterization of Protein NO145, the Major Component of Nucleolar Cortical Skeleton inXenopusOocytes
Authors: Kneissel Sandra , Franke Werner W. , Gall Joseph G. , Heid Hans , Reidenbach Sonja , Schnölzer Martina , Spring Herbert , Zentgraf Hanswalter , Schmidt-Zachmann Marion S.
Published: July 2013

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to Mr145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from differentXenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli ofXenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.