bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Insulin Receptor Substrate Proteins with Bcl-2 and Their Effects on Its Phosphorylation and Antiapoptotic Function
Authors: Ueno Hiroo , Kondo Eisaku , Yamamoto-Honda Ritsuko , Tobe Kazuyuki , Nakamoto Tetsuya , Sasaki Ko , Mitani Kinuko , Furusaka Akihiro , Tanaka Teruji , Tsujimoto Yoshihide , Kadowaki Takashi , Hirai Hisamaru
Published: July 2013

Insulin receptor substrate (IRS) proteins are docking proteins that couple growth factor receptors to various effector molecules, including phosphoinositide-3 kinase, Grb-2, Syp, and Nck. Here we show that IRS-1 associates with the loop domain of Bcl-2 and synergistically up-regulates antiapoptotic function of Bcl-2. IRS-2 but not IRS-3 binds to Bcl-2, and IRS-1 associates with Bcl-XL but not with Bax or Bik. Overexpression of IRS-1 suppresses phosphorylation of Bcl-2 induced by stimulation with insulin, and the hypophosphorylation may lead to its enhanced antiapoptotic activity. The binding site for Bcl-2 is located on the carboxyl half-domain of IRS-1. IRS-3, which lacks the corresponding region, dominant-negatively abrogates the survival effects of IRS-1 and Bcl-2. For the antiapoptotic activity of IRS-1, binding to Bcl-2 is more critical than activating phosphoinositide-3 kinase. Our results indicate that IRS proteins transmit signals from the insulin receptor to Bcl-2, thus regulating cell survival probably through regulating phosphorylation of Bcl-2.
Identification of Novel, Evolutionarily Conserved Cdc42p-interacting Proteins and of Redundant Pathways Linking Cdc24p and Cdc42p to Actin Polarization in Yeast
Authors: Bi Erfei , Chiavetta John B. , Chen Herman , Chen Guang-Chao , Chan Clarence S. M. , Pringle John R.
Published: July 2013

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p–Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination withMSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation.MSB3 has a close homologue (designatedMSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues inSchizosaccharomyces pombe, Drosophila(pollux), and humans (the oncogenetre17). Deletion of either MSB3 orMSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, andGIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, andMSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.
Discontinuous Actin Hexagon, a Protein Essential for Cortical Furrow Formation inDrosophila, Is Membrane Associated and Hyperphosphorylated
Authors: Zhang Claire X. , Rothwell Wendy F. , Sullivan William , Hsieh Tao-shih
Published: July 2013

discontinuous actin hexagon (dah) is a maternal-effect gene essential for the formation of cortical furrows during Drosophila embryogenesis, and DAH protein colocalizes with actin in these furrows. Biochemical fractionation experiments presented here demonstrate that DAH is highly enriched in the membrane fraction and that its membrane association is resistant to high-salt and alkaline washes. Furthermore, it partitions into the detergent phase of the Triton X-114 solution, indicating its tight binding to the membranes. DAH can also interact with the actin cytoskeleton, because a fraction of DAH remains insoluble to nonionic detergent along with actin. These biochemical characterizations suggest that DAH may play a role in the linkage of the actin cytoskeleton to membranes. Using phosphatase inhibitors, we detected multiple phosphorylated forms of DAH in embryonic extracts. The DAH phosphorylation peaks during cellularization, a stage at which DAH function is critical. A kinase activity is coimmunoprecipitated with the DAH complex and hyperphosphorylates DAH in vitro. Purified casein kinase I can also hyperphosphorylate DAH in the immune complex. Both DAH localization and phosphorylation are disrupted in another maternal-effect mutant, nuclear-fallout. It is possible that nuclear-fallout collaborates withdah and directs DAH protein localization to the cortical furrows.
Transforming Growth Factor β1Selectively Inhibits the Cyclic AMP-dependent Proliferation of Primary Thyroid Epithelial Cells by Preventing the Association of Cyclin D3–cdk4 with Nuclear p27kip1
Authors: Depoortere Fabienne , Pirson Isabelle , Bartek Jiri , Dumont Jacques E. , Roger Pierre P.
Published: July 2013

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27kip1but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor β1(TGFβ1) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFβ receptor signaling, because they did not affect the TGFβ-induced nuclear translocation of Smad 2 and 3. TGFβ inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFβ did not inhibit c-myc expression. In TSH-stimulated cells, TGFβ did not affect the expression of cyclin D3, cdk4, and p27kip1, nor the induced formation of cyclin D3–cdk4 complexes, but it prevented the TSH-induced relocalization of p27kip1from cdk2 to cyclin D3–cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3–cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27kip1away from cdk2. This study dissociates the assembly of cyclin D3–cdk4 complexes from their nuclear localization and association with p27kip1. It provides a new mechanism of regulation of proliferation by TGFβ, which points out the subcellular location of cyclin d–cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.
Activation of Wee1 by p42 MAPK In Vitro and in CyclingXenopusEgg Extracts
Authors: Walter Sarah A. , Guadagno Sarah N. , Ferrell James E.
Published: July 2013

Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. In the present study, we have investigated the mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest, usingXenopus egg extracts as a model system. We found that Wee1-depleted extracts were unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restored their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK was found to phosphorylate recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK resulted in a modest (∼2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, we present evidence that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts resulted in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully.
Functional Interactions between the p35 Subunit of the Arp2/3 Complex and Calmodulin in Yeast
Authors: Schaerer-Brodbeck Claudia , Riezman Howard
Published: July 2013

The end9-1 (arc35-1) mutant was identified as an endocytosis mutant and is a mutant allele ofARC35 that encodes a subunit of the Arp2/3 complex. As for other mutants in the Arp2/3 complex, arc35-1 is defective for endocytosis and organization of the actin cytoskeleton. Both defects can be suppressed by overexpression of calmodulin. Analysis of a collection of temperature-sensitive cmd1mutants for their ability to suppress either the endocytic defect and/or the actin defect indicates that the two defects are tightly coupled. We demonstrate that Arc35p and Cmd1p interact and that Arc35p is required for cortical localization of calmodulin. This is the first report linking Arp2/3 complex function with calmodulin through which it exercises at least one of its endocytic functions.
Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase
Authors: Nguyen Aaron Ngocky , Lee Albert , Place Warren , Shiozaki Kazuhiro
Published: July 2013

In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeastSchizosaccharomyces pombe. The fission yeastmpr1+gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative stress is severely impaired in the Δmpr1 mutant as well as in thempr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine. In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1. Unexpectedly, when exposed to hydrogen peroxide, Δmpr1 cells can induce the catalase gene ctt1+, one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation. We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1+expression upon oxidative stress independently of the Spc1 stress-activated protein kinase. These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression.
Mitotic Chromosome Condensation Requires Brn1p, the Yeast Homologue of Barren
Authors: Lavoie Brigitte D. , Tuffo K. Michelle , Oh Scott , Koshland Doug , Holm Connie
Published: July 2013

In vitro studies suggest that the Barren protein may function as an activator of DNA topoisomerase II and/or as a component of theXenopus condensin complex. To better understand the role of Barren in vivo, we generated conditional alleles of the structural gene for Barren (BRN1) in Saccharomyces cerevisiae. We show that Barren is an essential protein required for chromosome condensation in vivo and that it is likely to function as an intrinsic component of the yeast condensation machinery. Consistent with this view, we show that Barren performs an essential function during a period of the cell cycle when chromosome condensation is established and maintained. In contrast, Barren does not serve as an essential activator of DNA topoisomerase II in vivo. Finally,brn1 mutants display additional phenotypes such as stretched chromosomes, aberrant anaphase spindles, and the accumulation of cells with >2C DNA content, suggesting that Barren function influences multiple aspects of chromosome transmission and dynamics.
Chromosome Condensation Factor Brn1p Is Required for Chromatid Separation in Mitosis
Authors: Ouspenski Ilia I. , Cabello Olga A. , Brinkley B. R.
Published: July 2013

This work describes BRN1, the budding yeast homologue of Drosophila Barren andXenopus condensin subunit XCAP-H. TheDrosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Somebrn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations ofBRN1 can be suppressed by overexpression of a novel geneYCG1, which is homologous to anotherXenopus condensin subunit, XCAP-G. Overexpression ofSMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.
gdt1, a New Signal Transduction Component for Negative Regulation of the Growth–Differentiation Transition inDictyostelium discoideum
Authors: Zeng Changjiang , Anjard Christophe , Riemann Karsten , Konzok Angelika , Nellen Wolfgang
Published: July 2013

Discoidin I expression was used as a marker to screen for mutants affected in the growth–differentiation transition (GDT) ofDictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function.gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1−phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein α2 display a loss of discoidin expression and do not aggregate.gdt1−/Gα2−double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Gα2.
Truncation Mutants of the Tight Junction Protein ZO-1 Disrupt Corneal Epithelial Cell Morphology
Authors: Ryeom Sandra W. , Paul David , Goodenough Daniel A.
Published: July 2013

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.
The p21 Rho-activating Toxin Cytotoxic Necrotizing Factor 1 Is Endocytosed by a Clathrin-independent Mechanism and Enters the Cytosol by an Acidic-dependent Membrane Translocation Step
Authors: Contamin Stephanette , Galmiche Antoine , Doye Anne , Flatau Gilles , Benmerah Alexandre , Boquet Patrice
Published: July 2013

Cytotoxic necrotizing factor 1 (CNF1), a protein produced by pathogenic strains of Escherichia coli, activates the p21 Rho-GTP-binding protein, inducing a profound reorganization of the actin cytoskeleton. CNF1 binds to its cell surface receptor on HEp-2 cells with high affinity (Kd= 20 pM). In HEp-2 cells the action of CNF1 is not blocked in the presence of filipin, a drug described to reduce cholera toxin internalization by the caveolae-like mechanism. Moreover, HEp-2 cells, which express a dominant negative form of proteins that impair the formation of clathrin coated-vesicles and internalization of transferrin (Eps15, dynamin or intersectin-Src homology 3), are still sensitive to CNF1. In this respect, the endocytosis of CNF1 is similar to the plant toxin ricin. However, unlike ricin toxin, CNF1 does not cross the Golgi apparatus and requires an acidic cell compartment to transfer its enzymatic activity into the cytosol in a manner similar to that required by diphtheria toxin. As shown for diphtheria toxin, the pH-dependent membrane translocation step of CNF1 could be mimicked at the level of the plasma membrane by a brief exposure to a pH of ≤5.2. CNF1 is the first bacterial toxin described that uses both a clathrin-independent endocytic mechanism and an acidic-dependent membrane translocation step in its delivery of the catalytic domain to the cell cytosol.
Stage-Specific Expression and Targeting of Cyst Wall Protein–Green Fluorescent Protein Chimeras inGiardia
Authors: Hehl Adrian B. , Marti Matthias , Köhler Peter
Published: July 2013

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)–green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5′ flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3′ flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1–GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.
Structure–Function Relationships in Yeast Tubulins
Authors: Richards Kristy L. , Anders Kirk R. , Nogales Eva , Schwartz Katja , Downing Kenneth H. , Botstein David
Published: July 2013

A comprehensive set of clustered charged-to-alanine mutations was generated that systematically alter TUB1, the major α-tubulin gene of Saccharomyces cerevisiae. A variety of phenotypes were observed, including supersensitivity and resistance to the microtubule-destabilizing drug benomyl, lethality, and cold- and temperature-sensitive lethality. Many of the most benomyl-sensitivetub1 alleles were synthetically lethal in combination with tub3Δ, supporting the idea that benomyl supersensitivity is a rough measure of microtubule instability and/or insufficiency in the amount of α-tubulin. The systematictub1 mutations were placed, along with the comparable set of tub2 mutations previously described, onto a model of the yeast α–β-tubulin dimer based on the three-dimensional structure of bovine tubulin. The modeling revealed a potential site for binding of benomyl in the core of β-tubulin. Residues whose mutation causes cold sensitivity were concentrated at the lateral and longitudinal interfaces between adjacent subunits. Residues that affect binding of the microtubule-binding protein Bim1p form a large patch across the exterior-facing surface of α-tubulin in the model. Finally, the positions of the mutations suggest that proximity to the α–β interface may account for the finding of synthetic lethality of five viable tub1 alleles with the benomyl-resistant but otherwise entirely viable tub2-201allele.
Inactivation oflmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants
Authors: Temesvari Lesly , Zhang Linyi , Fodera Brent , Janssen Klaus-Peter , Schleicher Michael , Cardelli James A.
Published: July 2013

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.
Sorting of Membrane and Fluid at the Apical Pole of Polarized Madin-Darby Canine Kidney Cells
Authors: Leung Som-Ming , Ruiz Wily G. , Apodaca Gerard
Published: July 2013

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (≤2.5 min at 37°C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor–bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome.
Accumulation of rab4GTP in the Cytoplasm and Association with the Peptidyl-Prolyl Isomerase Pin1 during Mitosis
Authors: Gerez Lisya , Mohrmann Karin , van Raak Marcel , Jongeneelen Mandy , Zhou Xiao Zhen , Lu Kun Ping , van der Sluijs Peter
Published: July 2013

Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34cdc2kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.
AtVPS45 Complex Formation at thetrans-Golgi Network
Authors: Bassham Diane C. , Sanderfoot Anton A. , Kovaleva Valentina , Zheng Haiyan , Raikhel Natasha V.
Published: July 2013

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide–sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.
Progesterone Synthesized by Schwann Cells during Myelin Formation Regulates Neuronal Gene Expression
Authors: Chan Jonah R. , Rodriguez-Waitkus Paul M. , Ng Benjamin K. , Liang Peng , Glaser Michael
Published: July 2013

Previously, progesterone was found to regulate the initiation and biosynthetic rate of myelin synthesis in Schwann cell/neuronal cocultures. The mRNA for cytochrome P450scc (converts cholesterol to pregnenolone), 3β-hydroxysteroid dehydrogenase (3β-HSD, converts pregnenolone to progesterone), and the progesterone receptor were found to be markedly induced during active myelin synthesis. However, the cells in the cocultures responsible for these changes were not identified. In this study, in situ hybridization was used to determine the localization of the enzymes responsible for steroid biosynthesis. The mRNA for cytochrome P450scc and 3β-HSD were detected only in actively myelinating cocultures and were localized exclusively in the Schwann cells. Using immunocytochemistry, with minimal staining of the Schwann cells, we found the progesterone receptor in the dorsal root ganglia (DRG) neurons. The progesterone receptor in the neurons translocated into the nuclei of these cells when progesterone was added to neuronal cultures or during myelin synthesis in the cocultures. Additionally, a marked induction of the progesterone receptor was found in neuronal cultures after the addition of progesterone. The induction of various genes in the neurons was also investigated using mRNA differential display PCR in an attempt to elucidate the mechanism of steroid action on myelin synthesis. Two novel genes were induced in neuronal cultures by progesterone. These genes, along with the progesterone receptor, were also induced in cocultures during myelin synthesis, and their induction was blocked by RU-486 (a progesterone receptor antagonist). These genes were not induced in Schwann cells cultured alone after the addition of progesterone. These results suggest that progesterone is synthesized in Schwann cells and that it can indirectly regulate myelin formation by activating transcription via the classical steroid receptor in the DRG neurons.
The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination
Authors: Leverson Joel D. , Joazeiro Claudio A.P. , Page Andrew M. , Huang Han-kuei , Hieter Philip , Hunter Tony
Published: July 2013

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.
Syntaxin 7 and VAMP-7 are SolubleN-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages
Authors: Ward Diane McVey , Pevsner Jonathan , Scullion Matthew A. , Vaughn Michael , Kaplan Jerry
Published: July 2013

Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.
Luv1p/Rki1p/Tcs3p/Vps54p, a Yeast Protein That Localizes to the Late Golgi and Early Endosome, Is Required for Normal Vacuolar Morphology.
Authors: Conboy Michael J. , Cyert Martha S.
Published: July 2013

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. Theluv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth.luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H+-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn2+, Mn2+, and Cd2+) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 × g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein–Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.
Entry of the Two Infectious Forms of Vaccinia Virus at the Plasma Membane Is Signaling-Dependent for the IMV but Not the EEV
Authors: Locker Jacomine Krijnse , Kuehn Annett , Schleich Sibylle , Rutter Gaby , Hohenberg Heinrich , Wepf Roger , Griffiths Gareth
Published: July 2013

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.
EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts
Authors: Wilson Jean M. , de Hoop Meltsje , Zorzi Natasha , Toh Ban-Hock , Dotti Carlos G. , Parton Robert G.
Published: July 2013

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of “basolateral-type” endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.
Specific Sequence Motif of 8-Cys Repeats of TGF-β Binding Proteins, LTBPs, Creates a Hydrophobic Interaction Surface for Binding of Small Latent TGF-β
Authors: Saharinen Juha , Keski-Oja Jorma
Published: July 2013

Transforming growth factor (TGF)-βs are secreted in large latent complexes consisting of TGF-β, its N-terminal latency-associated peptide (LAP) propeptide, and latent TGF-β binding protein (LTBP). LTBPs are required for secretion and subsequent deposition of TGF-β into the extracellular matrix. TGF-β1 associates with the 3rd8-Cys repeat of LTBP-1 by LAP. All LTBPs, as well as fibrillins, contain multiple 8-Cys repeats. We analyzed the abilities of fibrillins and LTBPs to bind latent TGF-β by their 8-Cys repeats. 8-Cys repeat was found to interact with TGF-β1•LAP by direct cysteine bridging. LTBP-1 and LTBP-3 bound efficiently all TGF-β isoforms, LTBP-4 had a much weaker binding capacity, whereas LTBP-2 as well as fibrillins -1 and -2 were negative. A short, specific TGF-β binding motif was identified in the TGF-β binding 8-Cys repeats. Deletion of this motif in the 3rd8-Cys repeat of LTBP-1 resulted in loss of TGF-β•LAP binding ability, while its inclusion in non-TGF-β binding 3rd8-Cys repeat of LTBP-2 resulted in TGF-β binding. Molecular modeling of the 8-Cys repeats revealed a hydrophobic interaction surface and lack of three stabilizing hydrogen bonds introduced by the TGF-β binding motif necessary for the formation of the TGF-β•LAP - 8-Cys repeat complex inside the cells.
Nuclear Dynamics inArabidopsis thaliana
Authors: Chytilova Eva , Macas Jiri , Sliwinska Elwira , Rafelski Susanne M. , Lambert Georgina M. , Galbraith David W.
Published: July 2013

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm.  This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.
Analysis of mid1p, a Protein Required for Placement of the Cell Division Site, Reveals a Link between the Nucleus and the Cell Surface in Fission Yeast
Authors: Paoletti Anne , Chang Fred
Published: July 2013

mid1 is required for the proper placement of the contractile actin ring for cytokinesis at a medial site overlying the nucleus. Here we find that mid1 protein (mid1p) shuttles between the nucleus and a cortical medial broad band during interphase and early mitosis. The position of this broad band, which overlies the nucleus, is linked to nuclear position even in cells with displaced or multiple nuclei. We identified and created mutations in an NLS and in two crm1-dependent NES sequences in mid1p. NES mutations caused mid1p accumulation in the nucleus and loss of function. An NLS mutations greatly reduced nuclear localization but did not perturb cytoplasmic localization or function. mid1p localization to the medial broad band was also not dependent on mid1p PH domain or microtubule and actin cytoskeletons. Overexpression of mid1p produced ectopic cell growth at this band during interphase and abnormal karmellae-like nuclear membrane structures. In plo1–1, mid1p formed a medial broad band but did not incorporate into a tight ring, suggesting that polo kinase plo1p is required for activation of mid1p function. Thus, the mid1p broad band defines a compartment at the medial cell surface, whose localization is linked to the position of the nucleus, and whose function may be to position the plane of cell division.
Death Effector Domain Protein PEA-15 Potentiates Ras Activation of Extracellular Signal Receptor-activated Kinase by an Adhesion-independent Mechanism
Authors: Ramos Joe W. , Hughes Paul E. , Renshaw Mark W. , Schwartz Martin A. , Formstecher Etienne , Chneiweiss Hervé , Ginsberg Mark H.
Published: July 2013

PEA-15 is a small, death effector-domain (DED)–containing protein that was recently demonstrated to inhibit tumor necrosis factor-α–induced apoptosis and to reverse the inhibition of integrin activation due to H-Ras. This led us to investigate the involvement of PEA-15 in Ras signaling. Surprisingly, PEA-15 activates the extracellular signal receptor-activated kinase (ERK) mitogen-activated protein kinase pathway in a Ras-dependent manner. PEA-15 expression in Chinese hamster ovary cells resulted in an increased mitogen-activated protein kinase kinase and ERK activity. Furthermore, PEA-15 expression leads to an increase in Ras guanosine 5′-triphosphate loading. PEA-15 bypasses the anchorage dependence of ERK activation. Finally, the effects of PEA-15 on integrin signaling are separate from those on ERK activation. Heretofore, all known DEDs functioned in the regulation of apoptosis. In contrast, the DED of PEA-15 is essential for its capacity to activate ERK. The ability of PEA-15 to simultaneously inhibit apoptosis and potentiate Ras-to-Erk signaling may be of importance for oncogenic processes.
Cell Contact–dependent Activation of α3β1 Integrin Modulates Endothelial Cell Responses to Thrombospondin-1
Authors: Chandrasekaran Lakshmi , He Chao-Zhen , Al-Barazi Hebah , Krutzsch Henry C. , Iruela-Arispe M. Luisa , Roberts David D.
Published: July 2013

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified α3β1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an α3β1 integrin–binding peptide from TSP1 by normal endothelial cells is induced after loss of cell–cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, α3β1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell–cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the α3β1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the α3β1 integrin–binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by α3β1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.
Dynamics of Transitional Endoplasmic Reticulum Sites in Vertebrate Cells
Authors: Hammond Adam T. , Glick Benjamin S.
Published: July 2013

A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER–Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER–Golgi system is organized by mutual feedback between these two compartments.