bluuebunny/crossref_metadata_2025 huggingface dataset:
DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing
Authors: Shareef Mohammed Momin , King Chadwick , Damaj Mona , Badagu RamaKrishna , Huang Da Wei , Kellum Rebecca
Published: July 2013

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.
Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6
Authors: Wendler Franz , Page Lesley , Urbé Sylvie , Tooze Sharon A.
Published: July 2013

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t–t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.
A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum
Authors: Kamhi-Nesher Shiri , Shenkman Marina , Tolchinsky Sandra , Fromm Sharon Vigodman , Ehrlich Rachel , Lederkremer Gerardo Z.
Published: July 2013

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”
Distribution and Transport of Cholesterol inCaenorhabditis elegans
Authors: Matyash Vitali , Geier Christian , Henske Annemarie , Mukherjee Sushmita , Hirsh David , Thiele Christoph , Grant Barth , Maxfield Frederick R. , Kurzchalia Teymuras V.
Published: July 2013

Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1–L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol byC. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.
Identification of aSaccharomyces cerevisiaeGene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*
Authors: Alic Nazif , Higgins Vincent J. , Dawes Ian W.
Published: July 2013

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, theYNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival,OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification ofOCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.
Modulation of Cell-Substrate Adhesion by Arachidonic Acid: Lipoxygenase Regulates Cell Spreading and ERK1/2-inducible Cyclooxygenase Regulates Cell Migration in NIH-3T3 Fibroblasts
Authors: Stockton Rebecca A. , Jacobson Bruce S.
Published: July 2013

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.
Topological and Mutational Analysis ofSaccharomyces cerevisiaeSte14p, Founding Member of the Isoprenylcysteine Carboxyl Methyltransferase Family
Authors: Romano Julia D. , Michaelis Susan
Published: July 2013

Eukaryotic proteins that terminate in a CaaX motif undergo three processing events: isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. In Saccharomyces cerevisiae, the latter step is mediated by Ste14p, an integral endoplasmic reticulum membrane protein. Ste14p is the founding member of the isoprenylcysteine carboxyl methyltransferase (ICMT) family, whose members share significant sequence homology. Because the physiological substrates of Ste14p, such as Ras and the yeast a-factor precursor, are isoprenylated and reside on the cytosolic side of membranes, the Ste14p residues involved in enzymatic activity are predicted to be cytosolically disposed. In this study, we have investigated the topology of Ste14p by analyzing the protease protection of epitope-tagged versions of Ste14p and the glycosylation status of Ste14p-Suc2p fusions. Our data lead to a topology model in which Ste14p contains six membrane spans, two of which form a helical hairpin. According to this model most of the Ste14p hydrophilic regions are located in the cytosol. We have also generated ste14mutants by random and site-directed mutagenesis to identify residues of Ste14p that are important for activity. Notably, four of the five loss-of-function mutations arising from random mutagenesis alter residues that are highly conserved among the ICMT family. Finally, we have identified a novel tripartite consensus motif in the C-terminal region of Ste14p. This region is similar among all ICMT family members, two phospholipid methyltransferases, several ergosterol biosynthetic enzymes, and a group of bacterial open reading frames of unknown function. Site-directed and random mutations demonstrate that residues in this region play a critical role in the function of Ste14p.
Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin
Authors: Kee Sun-Ho , Steinert Peter M.
Published: July 2013

The association of the cytoskeleton with the cadherin–catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the E-cadherin–catenin–actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non- or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.
Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei
Authors: Tolonen Nina , Doglio Laura , Schleich Sibylle , Locker Jacomine Krijnse
Published: July 2013

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ∼45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [3H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.
AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes
Authors: Huizing Marjan , Sarangarajan Rangaprasad , Strovel Erin , Zhao Yang , Gahl William A. , Boissy Raymond E.
Published: July 2013

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the β3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the β3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3–deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.
Tubulin Sorting during Dimerization In Vivo
Authors: Hoyle Henry D. , Turner F. Rudolf , Brunick Linda , Raff Elizabeth C.
Published: July 2013

We demonstrate sorting of β-tubulins during dimerization in theDrosophila male germ line. Different β-tubulin isoforms exhibit distinct affinities for α-tubulin during dimerization. Our data suggest that differences in dimerization properties are important in determining isoform-specific microtubule functions. The differential use of β-tubulin during dimerization reveals structural parameters of the tubulin heterodimer not discernible in the resolved three-dimensional structure. We show that the variable β-tubulin carboxyl terminus, a surface feature in the heterodimer and in microtubules, and which is disordered in the crystallographic structure, is of key importance in forming a stable α-β heterodimer. If the availability of α-tubulin is limiting, α-β dimers preferentially incorporate intact β-tubulins rather than a β-tubulin missing the carboxyl terminus (β2ΔC). When α-tubulin is not limiting, β2ΔC forms stable α-β heterodimers. Once dimers are formed, no further sorting occurs during microtubule assembly: α-β2ΔC dimers are incorporated into axonemes in proportion to their contribution to the total dimer pool. Co-incorporation of β2ΔC and wild-type β2-tubulin results in nonmotile axonemes because of a disruption of the periodicity of nontubulin axonemal elements. Our data show that the β-tubulin carboxyl terminus has two distinct roles: 1) forming the α-β heterodimer, important for all microtubules and 2) providing contacts for nontubulin components required for specific microtubule structures, such as axonemes.
Differential Control of Transcription by DNA-bound Cyclins
Authors: Kim Tae-You , Kaelin William G.
Published: July 2013

Different cyclins mediate different cell-cycle transitions. Some cyclins, such as cyclin A and cyclin E, form stable complexes with proteins that bind directly or indirectly to DNA and thus might be recruited to certain regions of the genome at specific times in the cell cycle. Furthermore, cyclins contain structural motifs that are also present in known transcriptional modulators. We found that cyclin A is a potent transcriptional repressor and cyclin E is a potent transcriptional activator when bound to DNA via a heterologous DNA binding domain. The former activity was linked to the integrity of the cyclin A cyclin fold, whereas the latter activity related to the ability of cyclin E to activate cdk2 and recognize substrates. Furthermore, we found that cyclin E, but not cyclin A, activated transcription in a cell-cycle–dependent manner when present in physiological concentrations as an unfused protein. These results suggest that cyclin A and cyclin E intrinsically differ with respect to their ability to modulate transcription when tethered to DNA.
Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex
Authors: Lippé Roger , Miaczynska Marta , Rybin Vladimir , Runge Anja , Zerial Marino
Published: July 2013

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.
Biogenesis of Golgi Stacks in Imaginal Discs ofDrosophila melanogaster
Authors: Kondylis Vangelis , Goulding Sarah E. , Dunne Jonathan C. , Rabouille Catherine
Published: July 2013

We provide a detailed description of Golgi stack biogenesis that takes place in vivo during one of the morphogenetic events in the lifespan of Drosophila melanogaster. In early third-instar larvae, small clusters consisting mostly of vesicles and tubules were present in epithelial imaginal disk cells. As larvae progressed through mid- and late-third instar, these larval clusters became larger but also increasingly formed cisternae, some of which were stacked. In white pupae, the typical Golgi stack was observed. We show that larval clusters are Golgi stack precursors by 1) localizing various Golgi-specific markers to the larval clusters by electron and immunofluorescence confocal microscopy, 2) driving this conversion in wild-type larvae incubated at 37°C for 2 h, and 3) showing that this conversion does not take place in an NSF1 mutant (comt 17). The biological significance of this conversion became clear when we found that the steroid hormone 20-hydroxyecdysone (ecdysone) is critically involved in this conversion. In its absence, Golgi stack biogenesis did not occur and the larval clusters remained unaltered. We showed that dGM130 and sec23p expression increases approximately three- and fivefold, respectively, when discs are exposed to ecdysone in vivo and in vitro. Taken together, these results suggest that we have developed an in vivo system to study the ecdysone-triggered Golgi stack biogenesis.
Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein Interactions
Authors: Shav-Tal Yaron , Cohen Michal , Lapter Smadar , Dye Billy , Patton James G. , Vandekerckhove Joel , Zipori Dov
Published: July 2013

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1–70K and SR proteins and therefore may acquire new functions.
Mutant rab8 Impairs Docking and Fusion of Rhodopsin-bearing Post-Golgi Membranes and Causes Cell Death of TransgenicXenopusRods
Authors: Moritz Orson L. , Tam Beatrice M. , Hurd Larry L. , Peränen Johan , Deretic Dusanka , Papermaster David S.
Published: July 2013

Rab8 is a GTPase involved in membrane trafficking. In photoreceptor cells, rab8 is proposed to participate in the late stages of delivery of rhodopsin-containing post-Golgi membranes to the plasma membrane near the base of the connecting cilium. To test the function of rab8 in vivo, we generated transgenic Xenopus laevis expressing wild-type, constitutively active (Q67L), and dominant negative (T22N) forms of canine rab8 in their rod photoreceptors as green fluorescent protein (GFP) fusion proteins. Wild-type and constitutively active GFP-rab8 proteins were primarily associated with Golgi and post-Golgi membranes, whereas the dominant negative protein was primarily cytoplasmic. Expression of wild-type GFP-rab8 had minimal effects on cell survival and intracellular structures. In contrast, GFP-rab8T22N caused rapid retinal degeneration. In surviving peripheral rods, tubulo-vesicular structures accumulated at the base of the connecting cilium. Expression of GFP-rab8Q67L induced a slower retinal degeneration in some tadpoles. Transgene effects were transmitted to F1 offspring. Expression of the GFP-rab8 fusion proteins appears to decrease the levels of endogenous rab8 protein. Our results demonstrate a role for rab8 in docking of post-Golgi membranes in rods, and constitute the first report of a transgenic X. laevismodel of retinal degenerative disease.
Dominant Negativec-junInhibits Activation of the Cyclin D1 and Cyclin E Kinase Complexes
Authors: Hennigan Robert F. , Stambrook Peter J.
Published: July 2013

The AP-1 transcription factor is activated by oncogenic signal transduction cascades and its function is critical for both mitogenesis and carcinogenesis. To define the role of AP-1 in the context of a human fibrosarcoma cell line, HT1080, we expressed a dominant negativec-jun mutant fused to the green fluorescent protein in an ecdysone-inducible system. We demonstrated that high levels of this mutant, GFP-TAM67, inhibit AP-1 activity and arrest cells predominately in the G1 phase of the cell cycle. This arrest is reversible and occurs only above a threshold concentration; low to moderate levels of GFP-TAM67 are insufficient for growth arrest. Contrary to expectations based on the literature, GFP-TAM67 does not inhibit expression of cyclin D1, cyclin E, or their respective cyclin-dependent kinases. However, pRB is hypophosphorylated in GFP-TAM67-arrested cells and the activity of both the cyclin D1:cdk and the cyclin E:cdk complexes are impaired. Both of these complexes show an increased association with p21CIP1/WAF1, concomitantly with induction of the p21 mRNA by GFP-TAM67. These results suggest a novel function of AP-1 in the activation of the G1 cyclin:cdk complexes in human tumor cells by regulating the expression of the p21CIP1/WAF1gene.
Regulated Degradation of an Endoplasmic Reticulum Membrane Protein in a Tubular Lysosome inLeishmania mexicana
Authors: Mullin Kylie A. , Foth Bernardo J. , Ilgoutz Steven C. , Callaghan Judy M. , Zawadzki Jody L. , McFadden Geoffrey I. , McConville Malcolm J.
Published: July 2013

The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.
Sac1 Lipid Phosphatase and Stt4 Phosphatidylinositol 4-Kinase Regulate a Pool of Phosphatidylinositol 4-Phosphate That Functions in the Control of the Actin Cytoskeleton and Vacuole Morphology
Authors: Foti Michelangelo , Audhya Anjon , Emr Scott D.
Published: July 2013

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1tsallele and one of two temperature-sensitive PtdIns 4-kinase genes,stt4tsor pik1ts, we have demonstrated that the bulk of PtdIns(4)P that accumulates insac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated withstt4tsbut notpik1tsmutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1tsmutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.
Bud8p and Bud9p, Proteins That May Mark the Sites for Bipolar Budding in Yeast
Authors: Harkins Heidi A. , Pagé Nicolas , Schenkman Laura R. , De Virgilio Claudio , Shaw Sidney , Bussey Howard , Pringle John R.
Published: July 2013

The bipolar budding pattern of a /α Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identifiedBUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 orBUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- andO-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in abni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.
Cnm67p Is a Spacer Protein of theSaccharomyces cerevisiaeSpindle Pole Body Outer Plaque
Authors: Schaerer Florian , Morgan Garry , Winey Mark , Philippsen Peter
Published: July 2013

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted forCNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.
ASH1mRNA Localization in Three Acts
Authors: Beach Dale L. , Bloom Kerry
Published: July 2013

Novel green fluorescent protein (GFP) labeling techniques targeting specific mRNA transcripts reveal discrete phases of mRNA localization in yeast: packaging, transport, and docking. In budding yeast,ASH1 mRNA is translocated via actin and myosin to the tip of growing cells. A GFP-decorated reporter transcript containing the ASH1 3′ untranslated region gRNAASH1forms spots of fluorescence localized to a cortical domain at the bud tip, relocates to the mother-bud neck before cell separation, and finally migrates to the incipient bud site before the next budding cycle. The correct positioning of the mRNA requires at least six proteins: She1p-5p and Bud6p/Aip3p. gRNAASH1localization in mutant strains identified three functional categories for the She proteins: mRNA particle formation (She2p and She4p), mRNA transport into the bud (She1p/Myo4p and She3p), and mRNA tethering at the bud tip (She5p/Bni1p and Bud6p/Aip3p). Because localization of the mRNA within the bud does not a priori restrict the translated protein, we examine the distribution of a mother-specific protein (Yta6p) translated from a mRNA directed into the bud. Yta6p remains associated with the mother cortex despite localization of the mRNA to the bud. This video essay traces the life history of a localized mRNA transcript, describes the roles of proteins required to polarize and anchor the mRNA, and demonstrates at least one instance where mRNA localization does not effect protein localization.
Expression Patterns of Cell-type–specific Genes inDictyostelium
Authors: Iranfar Negin , Fuller Danny , Sasik Roman , Hwa Terence , Laub Michael , Loomis William F.
Published: July 2013

Cell-type specific genes were recognized by interrogating microarrays carrying Dictyostelium gene fragments with probes prepared from fractions enriched in prestalk and prespore cells. Cell-type specific accumulation of mRNA from 17 newly identified genes was confirmed by Northern analyses. DNA microarrays carrying 690 targets were used to determine expression profiles during development. The profiles were fit to a biologically based kinetic equation to extract the times of transcription onset and cessation. Although the majority of the genes that were cell-type enriched at the slug stage were first expressed as the prespore and prestalk cells sorted out in aggregates, some were found to be expressed earlier before the cells had even aggregated. These early genes may have been initially expressed in all cells and then preferentially turned over in one or the other cell type. Alternatively, cell type divergence may start soon after the initiation of development.
Aux1p/Swa2p Is Required for Cortical Endoplasmic Reticulum Inheritance inSaccharomyces cerevisiae
Authors: Du Yunrui , Pypaert Marc , Novick Peter , Ferro-Novick Susan
Published: July 2013

In the yeast Saccharomyces cerevisiae, the endoplasmic reticulum (ER) is found at the periphery of the cell and around the nucleus. The segregation of ER through the mother-bud neck may occur by more than one mechanism because perinuclear, but not peripheral ER, requires microtubules for this event. To identify genes whose products are required for cortical ER inheritance, we have used a Tn3-based transposon library to mutagenize cells expressing a green fluorescent protein-tagged ER marker protein (Hmg1p). This approach has revealed that AUX1/SWA2plays a role in ER inheritance. The COOH terminus of Aux1p/Swa2p contains a J-domain that is highly related to the J-domain of auxilin, which stimulates the uncoating of clathrin-coated vesicles. Deletion of the J-domain of Aux1p/Swa2p leads to vacuole fragmentation and membrane accumulation but does not affect the migration of peripheral ER into daughter cells. These findings suggest that Aux1p/Swa2p may be a bifunctional protein with roles in membrane traffic and cortical ER inheritance. In support of this hypothesis, we find that Aux1p/Swa2p localizes to ER membranes.
Deoxycholic Acid (DCA) Causes Ligand-independent Activation of Epidermal Growth Factor Receptor (EGFR) and FAS Receptor in Primary Hepatocytes: Inhibition of EGFR/Mitogen-activated Protein Kinase-Signaling Module Enhances DCA-induced Apoptosis
Authors: Qiao Liang , Studer Elaine , Leach Kevin , McKinstry Robert , Gupta Seema , Decker Roy , Kukreja Rakesh , Valerie Kristoffer , Nagarkatti Prakash , Deiry Wafik El , Molkentin Jeffrey , Schmidt-Ullrich Rupert , Fisher Paul B. , Grant Steven , Hylemon Philip B. , Dent Paul
Published: July 2013

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 μM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI3kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused ∼25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome cfrom mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-Sand c-FLIP-Lthat were reduced by inhibition of MAPK or PI3kinase. Constitutive overexpression of c-FLIP-sabolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.
Single Site α-Tubulin Mutation Affects Astral Microtubules and Nuclear Positioning during Anaphase inSaccharomyces cerevisiae: Possible Role for Palmitoylation of α-Tubulin
Authors: Caron Joan M. , Vega Leticia R. , Fleming James , Bishop Robert , Solomon Frank
Published: July 2013

We generated a strain of Saccharomyces cerevisiae in which the sole source of α-tubulin protein has a cys-to-ser mutation at cys-377, and then we examined microtubule morphology and nuclear positioning through the cell cycle. During G1 of the cell cycle, microtubules in the C377S α-tubulin (C377S tub1) mutant were indistinguishable from those in the control (TUB1) strain. However, mitotic C377Stub1 cells displayed astral microtubules that often appeared excessive in number, abnormally long, and/or misoriented compared with TUB1 cells. Although mitotic spindles were always correctly aligned along the mother-bud axis, translocation of spindles through the bud neck was affected. In late anaphase, spindles were often not laterally centered but instead appeared to rest along the sides of cells. When the doubling time was increased by growing cells at a lower temperature (15°C), we often found abnormally long mitotic spindles. No increase in the number of anucleate or multinucleate C377S mutant cells was found at any temperature, suggesting that, despite the microtubule abnormalities, mitosis proceeded normally. Because cys-377 is a presumptive site of palmitoylation in α-tubulin in S. cerevisiae, we next compared in vivo palmitoylation of wild-type and C377S mutant forms of the protein. We detected palmitoylated α-tubulin inTUB1 cells, but the cys-377 mutation resulted in approximately a 60% decrease in the level of palmitoylated α-tubulin in C377S tub1 cells. Our results suggest that cys-377 of α-tubulin, and possibly palmitoylation of this amino acid, plays a role in a subset of astral microtubule functions during nuclear migration in M phase of the cell cycle.
CENP-E Is Essential for Reliable Bioriented Spindle Attachment, but Chromosome Alignment Can Be Achieved via Redundant Mechanisms in Mammalian Cells
Authors: McEwen Bruce F. , Chan Gordon K.T. , Zubrowski Beata , Savoian Matthew S. , Sauer Matthew T. , Yen Tim J.
Published: July 2013

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with a mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, and electron microscopy to demonstrate that depletion of CENP-E from kinetochores via antibody microinjection reduces kinetochore microtubule binding by 23% at aligned chromosomes, and severely reduces microtubule binding at unaligned chromosomes. Disruption of CENP-E function also reduces tension across the centromere, increases the incidence of spindle pole fragmentation, and results in monooriented chromosomes approaching abnormally close to the spindle pole. Nevertheless, chromosomes show typical patterns of congression, fast poleward motion, and oscillatory motions. Furthermore, kinetochores of aligned and unaligned chromosomes exhibit normal patterns of checkpoint protein localization. These data are explained by a model in which redundant mechanisms enable kinetochore microtubule binding and checkpoint monitoring in the absence of CENP-E at kinetochores, but where reduced microtubule-binding efficiency, exacerbated by poor positioning at the spindle poles, results in chronically monooriented chromosomes and mitotic arrest. Chromosome position within the spindle appears to be a critical determinant of CENP-E function at kinetochores.