bluuebunny/crossref_metadata_2025 huggingface dataset:
TwoDrosophilaInnexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels
Authors: Stebbings Lucy A. , Todman Martin G. , Phelan Pauline , Bacon Jonathan P. , Davies Jane A.
Published: July 2013

Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes,Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In pairedXenopus oocytes, the injection of Dm-inx2mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression ofDm-inx2 in vivo has limited effects on the viability ofDrosophila, and animals ectopically expressingDm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels.
Feedback Interactions between Cell–Cell Adherens Junctions and Cytoskeletal Dynamics in Newt Lung Epithelial Cells
Authors: Waterman-Storer Clare M. , Salmon Wendy C. , Salmon E.D.
Published: July 2013

To test how cell–cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell–cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell–cell contacts, cells were treated with nocodazole to inhibit MTs. After 1–2 h in either 10 μM or 100 nM nocodazole, breakage of cell–cell contacts occurred, indicating that MT growth is required for maintenance of cell–cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins α- and β-catenin were lost from adherens junctions as cell–cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell–cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.
Integrin α5/β1 Mediates Fibronectin-dependent Epithelial Cell Proliferation through Epidermal Growth Factor Receptor Activation
Authors: Kuwada Scott K. , Li Xiufen
Published: July 2013

Human integrin α5 was transfected into the integrin α5/β1–negative intestinal epithelial cell line Caco-2 to study EGF receptor (EGFR) and integrin α5/β1 signaling interactions involved in epithelial cell proliferation. On uncoated or fibronectin-coated plastic, the integrin α5 and control (vector only) transfectants grew at similar rates. In the presence of the EGFR antagonistic mAb 225, the integrin α5 transfectants and controls were significantly growth inhibited on plastic. However, when cultured on fibronectin, the integrin α5 transfectants were not growth inhibited by mAb 225. The reversal of mAb 225–mediated growth inhibition on fibronectin for the integrin α5 transfectants correlated with activation of the EGFR, activation of MAPK, and expression of proliferating cell nuclear antigen. EGFR kinase activity was necessary for both MAPK activation and integrin α5/β1–mediated cell proliferation. Although EGFR activation occurred when either the integrin α5–transfected or control cells were cultured on fibronectin, coprecipitation of the EGFR with SHC could be demonstrated only in the integrin α5–transfected cells. These results suggest that integrin α5/β1 mediates fibronectin-induced epithelial cell proliferation through activation of the EGFR.
Role of p97 and Syntaxin 5 in the Assembly of Transitional Endoplasmic Reticulum
Authors: Roy Line , Bergeron John J.M. , Lavoie Christine , Hendriks Rob , Gushue Jennifer , Fazel Ali , Pelletier Amélie , Morré D. James , Subramaniam V. Nathan , Hong Wanjin , Paiement Jacques
Published: July 2013

Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc−1) isolated from rat liver homogenates reconstitute tER by Mg2+GTP- and Mg2+ATP-hydrolysis–dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
Involvement of an SHP-2-Rho Small G Protein Pathway in Hepatocyte Growth Factor/Scatter Factor–induced Cell Scattering
Authors: Kodama Atsuko , Matozaki Takashi , Fukuhara Atsunori , Kikyo Mitsuhiro , Ichihashi Masamitsu , Takai Yoshimi
Published: July 2013

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase–type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.
Potential Role for Protein Kinases in Regulation of Bidirectional Endoplasmic Reticulum-to-Golgi Transport Revealed by Protein Kinase Inhibitor H89
Authors: Lee Tina H. , Linstedt Adam D.
Published: July 2013

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of βCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but βCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5′-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.
The AP-3 Complex Required for Endosomal Synaptic Vesicle Biogenesis is Associated with a Casein Kinase Ια-Like Isoform
Authors: Faundez Victor V. , Kelly Regis B.
Published: July 2013

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes ( Faundez et al., 1998 ). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15°C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the β3 subunit of the complex by a kinase similar to casein kinase 1α. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the β3A and β3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.
The YeastTEL1Gene Partially Substitutes for HumanATMin Suppressing Hyperrecombination, Radiation-Induced Apoptosis and Telomere Shortening in A-T Cells
Authors: Fritz Eberhard , Friedl Anna A. , Zwacka Ralf M. , Eckardt-Schupp Friederike , Meyn M. Stephen
Published: July 2013

Homozygous mutations in the human ATM gene lead to a pleiotropic clinical phenotype of ataxia-telangiectasia (A-T) patients and correlating cellular deficiencies in cells derived from A-T donors. Saccharomyces cerevisiae tel1 mutants lacking Tel1p, which is the closest sequence homologue to the ATM protein, share some of the cellular defects with A-T. Through genetic complementation of A-T cells with the yeast TEL1 gene, we provide evidence that Tel1p can partially compensate for ATM in suppressing hyperrecombination, radiation-induced apoptosis, and telomere shortening. Complementation appears to be independent of p53 activation. The data provided suggest that TEL1 is a functional homologue of human ATM in yeast, and they help to elucidate different cellular and biochemical pathways in human cells regulated by the ATM protein.
Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain
Authors: Zaliauskiene Lolita , Kang Sunghyun , Brouillette Christie G. , Lebowitz Jacob , Arani Ramin B. , Collawn James F.
Published: July 2013

How recycling receptors are segregated from down-regulated receptors in the endosome is unknown. In previous studies, we demonstrated that substitutions in the transferrin receptor (TR) transmembrane domain (TM) convert the protein from an efficiently recycling receptor to one that is rapidly down regulated. In this study, we demonstrate that the “signal” within the TM necessary and sufficient for down-regulation is Thr11Gln17Thr19(numbering in TM). Transplantation of these polar residues into the wild-type TR promotes receptor down-regulation that can be demonstrated by changes in protein half-life and in receptor recycling. Surprisingly, this modification dramatically increases the TR internalization rate as well (∼79% increase). Sucrose gradient centrifugation and cross-linking studies reveal that propensity of the receptors to self-associate correlates with down-regulation. Interestingly, a number of cell surface proteins that contain TM polar residues are known to be efficiently down-regulated, whereas recycling receptors for low-density lipoprotein and transferrin conspicuously lack these residues. Our data, therefore, suggest a simple model in which specific residues within the TM sequences dramatically influence the fate of membrane proteins after endocytosis, providing an alternative signal for down-regulation of receptor complexes to the well-characterized cytoplasmic tail targeting signals.
Syntaxin 17 Is Abundant in Steroidogenic Cells and Implicated in Smooth Endoplasmic Reticulum Membrane Dynamics
Authors: Steegmaier Martin , Oorschot Viola , Klumperman Judith , Scheller Richard H.
Published: July 2013

The endoplasmic reticulum (ER) consists of subcompartments that have distinct protein constituents, morphological appearances, and functions. To understand the mechanisms that regulate the intricate and dynamic organization of the endoplasmic reticulum, it is important to identify and characterize the molecular machinery involved in the assembly and maintenance of the different subcompartments. Here we report that syntaxin 17 is abundantly expressed in steroidogenic cell types and specifically localizes to smooth membranes of the ER. By immunoprecipitation analyses, syntaxin 17 exists in complexes with a syntaxin regulatory protein, rsly1, and/or two intermediate compartment SNARE proteins, rsec22b and rbet1. Furthermore, we found that syntaxin 17 is anchored to the smooth endoplasmic reticulum through an unusual mechanism, requiring two adjacent hydrophobic domains near its carboxyl terminus. Converging lines of evidence indicate that syntaxin 17 functions in a vesicle-trafficking step to the smooth-surfaced tubular ER membranes that are abundant in steroidogenic cells.
A Pcl-like Cyclin Activates the Res2p-Cdc10p Cell Cycle “Start” Transcriptional Factor Complex in Fission Yeast
Authors: Tanaka Koichi , Okayama Hiroto
Published: July 2013

In the fission yeast Schizosaccharomyces pombe, the “start” of the cell cycle is controlled by the two functionally redundant transcriptional regulator complexes, Res1p-Cdc10p and Res2p-Cdc10p, that activate genes essential for the onset and progression of S phase. The activity of the Res2p-Cdc10p complex is regulated at least by the availability of the Rep2trans-activator subunit in the mitotic cell cycle. We have recently isolated the pas1+gene as a multicopy suppressor of the res1 null mutant. This gene encodes a novel cyclin that shares homology with the Pho85 kinase–associated cyclins of the budding yeast Saccharomyces cerevisiae. Genetic analysis reveals that Pas1 cyclin is unrelated to phosphate metabolism and stimulates the G1-S transition by specifically activating the Res2p-Cdc10p complex independently of Rep2p. Pas1 cyclin also controls mating pheromone signaling. Cells lacking pas1+are highly sensitive to mating pheromone, responding with facilitated G1arrest and premature commitment to conjugation. Pas1 cyclin associates in vivo with both Cdc2 and Pef1 kinases, the latter of which is a fission yeast counterpart of the budding yeast Pho85 kinase, but genetic analysis indicates that the Pef1p-associated Pas1p is responsible for the activation of Res2p-Cdc10p during the G1-S transition.
Exposure at the Cell Surface Is Required for Gas3/PMP22 To Regulate Both Cell Death and Cell Spreading: Implication for the Charcot–Marie–Tooth Type 1A and Dejerine–Sottas Diseases
Authors: Brancolini Claudio , Edomi Paolo , Marzinotto Stefania , Schneider Claudio
Published: July 2013

Gas3/PMP22 is a tetraspan membrane protein highly expressed in myelinating Schwann cells. Point mutations in thegas3/PMP22 gene account for the dominant inherited peripheral neuropathies Charcot–Marie–Tooth type 1A disease (CMT1A) and Dejerine–Sottas syndrome (DSS). Gas3/PMP22 can regulate apoptosis and cell spreading in cultured cells.Gas3/PMP22 point mutations, which are responsible for these diseases, are defective in this respect. In this report, we demonstrate that Gas3/PMP22-WT is exposed at the cell surface, while its point-mutated derivatives are intracellularly retained, colocalizing mainly with the endoplasmic reticulum (ER). The putative retrieval motif present in the carboxyl terminus of Gas3/PMP22 is not sufficient for the intracellular sequestration of its point-mutated forms. On the contrary, the introduction of a retrieval signal at the carboxyl terminus of Gas3/PMP22-WT leads to its intracellular accumulation, which is accompanied by a failure to trigger cell death as well as by changes in cell spreading. In addition, by substituting the Asn at position 41 required for N-glycosylation, we provide evidence that N-glycosylation is required for the full effect on cell spreading, but it is not necessary for triggering cell death. In conclusion, we suggest that the DSS and the CMT1A neuropathies derived from point mutations ofGas3/PMP22 might arise, at the molecular level, from a reduced exposure of Gas3/PMP22 at the cell surface, which is required to exert its biological functions.
PECAM-1/CD31 Trans-homophilic Binding at the Intercellular Junctions Is Independent of Its Cytoplasmic Domain; Evidence for Heterophilic Interaction with Integrin αvβ3 in Cis
Authors: Wong Cindy W.Y. , Wiedle Guido , Ballestrem Christoph , Wehrle-Haller Bernhard , Etteldorf Susanne , Bruckner Monika , Engelhardt Britta , Gisler Roland H. , Imhof Beat A.
Published: July 2013

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin αvβ3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin αvβ3 associates with PECAM-1 on the same cell surface as in a cis manner.
Evidence for Functional Differentiation amongDrosophilaSeptins in Cytokinesis and Cellularization
Authors: Adam Jennifer C. , Pringle John R. , Peifer Mark
Published: July 2013

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. InDrosophila melanogaster, null mutations in thepnut septin gene are recessive lethal, but homozygouspnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue ofpnut homozygous mutant flies by apnut+transgene under control of thehsp70 promoter. Remarkably, the pnutgermline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both.
RGS4 and RGS2 Bind Coatomer and Inhibit COPI Association with Golgi Membranes and Intracellular Transport
Authors: Sullivan Brandon M. , Harrison-Lavoie Kimberly J. , Marshansky Vladimir , Lin Herbert Y. , Kehrl John H. , Ausiello Dennis A. , Brown Dennis , Druey Kirk M.
Published: July 2013

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.
Neuronal Cell Shape and Neurite Initiation Are Regulated by the Ndr Kinase SAX-1, a Member of the Orb6/COT-1/Warts Serine/Threonine Kinase Family
Authors: Zallen Jennifer A. , Peckol Erin L. , Tobin David M. , Bargmann Cornelia I.
Published: July 2013

The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those ofsax-1 mutants, and genetic interactions betweenrhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading.
Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells
Authors: Takeda Tetsuro , Go William Y. , Orlando Robert A. , Farquhar Marilyn Gist
Published: July 2013

Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor–expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation (∼60–80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafacienssialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.
The N-terminal Domain of the t-SNARE Vam3p Coordinates Priming and Docking in Yeast Vacuole Fusion
Authors: Laage Rico , Ungermann Christian
Published: July 2013

Homotypic fusion of yeast vacuoles requires a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with thecis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3ΔN) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons: Assembly, as well as disassembly of thecis-SNARE complex is more efficient on Vam3ΔN vacuoles; however, the HOPS complex is not associated well with the Vam3ΔN cis-complex. Thus, primed SNAREs from Vam3ΔN vacuoles cannot participate efficiently in the reaction becausetrans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion.
Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus
Authors: Carvalho Célia , Pereira Henrique M. , Ferreira João , Pina Cristina , Mendonça Denise , Rosa Agostinho C. , Carmo-Fonseca Maria
Published: July 2013

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric α-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or throughtrans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric α-satellite DNA in human lymphoid cells synchronized at G0/G1is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric α-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that “chromosomal environment” plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.
Tetraploid State Induces p53-dependent Arrest of Nontransformed Mammalian Cells in G1
Authors: Andreassen Paul R. , Lohez Olivier D. , Lacroix Françoise B. , Margolis Robert L.
Published: July 2013

A “spindle assembly” checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21WAF1 and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity.
The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions inSaccharomyces cerevisiae
Authors: Manning Brendan D. , Padmanabha Ramesh , Snyder Michael
Published: July 2013

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors ofcik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C.rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing eitherROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ andkar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore,rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.
A Novel Yeast Screen for Mitotic Arrest Mutants IdentifiesDOC1, a New Gene Involved in Cyclin Proteolysis
Authors: Hwang Lena H. , Murray Andrew W.
Published: July 2013

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, andCDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.
14–3-3 Inhibits theDictyosteliumMyosin II Heavy-Chain-specific Protein Kinase C Activity by a Direct Interaction: Identification of the 14–3-3 Binding Domain
Authors: Matto-Yelin Meirav , Aitken Alastair , Ravid Shoshana
Published: July 2013

Myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC), isolated from Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cyclic AMP. Immunoprecipitation of MHC-PKC revealed that it resides as a complex with several proteins. We show herein that one of these proteins is a homologue of the 14–3-3 protein (Dd14–3-3). This protein has recently been implicated in the regulation of intracellular signaling pathways via its interaction with several signaling proteins, such as PKC and Raf-1 kinase. We demonstrate that the mammalian 14–3-3 ζ isoform inhibits the MHC-PKC activity in vitro and that this inhibition is carried out by a direct interaction between the two proteins. Furthermore, we found that the cytosolic MHC-PKC, which is inactive, formed a complex with Dd14–3-3 in the cytosol in a cyclic AMP-dependent manner, whereas the membrane-bound active MHC-PKC was not found in a complex with Dd14–3-3. This suggests that Dd14–3-3 inhibits the MHC-PKC in vivo. We further show that MHC-PKC binds Dd14–3-3 as well as 14–3-3ζ through its C1 domain, and the interaction between these two proteins does not involve a peptide containing phosphoserine as was found for Raf-1 kinase. Our experiments thus show an in vivo function for a member of the 14–3-3 family and demonstrate that MHC-PKC interacts directly with Dd14–3-3 and 14–3-3ζ through its C1 domain both in vitro and in vivo, resulting in the inhibition of the kinase.
Interaction of Coatomer with Aminoglycoside Antibiotics: Evidence That Coatomer Has at Least Two Dilysine Binding Sites
Authors: Hudson Robert Tod , Draper Rockford K.
Published: July 2013

Coatomer is the soluble precursor of the COPI coat (coat protein I) involved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipitation first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by divalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more binding sites for neomycin. A variety of other aminoglycoside antibiotics precipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is known to interact with a motif (KKXX) containing adjacent lysine residues at the carboxyl terminus of the cytoplasmic domains of some membrane proteins resident in the endoplasmic reticulum. All of the antibiotics that interacted with coatomer contain at least two close amino groups, suggesting that the antibiotics might be interacting with the di-lysine binding site of coatomer. Consistent with this idea, di-lysine itself blocked the interaction of antibiotics with coatomer. Moreover, di-lysine and antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact with di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.
Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities
Authors: Taylor Randall S. , Jones Steven M. , Dahl Rolf H. , Nordeen Mark H. , Howell Kathryn E.
Published: July 2013

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.
Quality Control in the Secretory Pathway: The Role of Calreticulin, Calnexin and BiP in the Retention of Glycoproteins with C-Terminal Truncations
Authors: Zhang Jian-Xin , Braakman Ineke , Matlack Kent E.S. , Helenius Ari
Published: July 2013

Unlike properly folded and assembled proteins, most misfolded and incompletely assembled proteins are retained in the endoplasmic reticulum of mammalian cells and degraded without transport to the Golgi complex. To analyze the mechanisms underlying this unique sorting process and its fidelity, the fate of C-terminally truncated fragments of influenza hemagglutinin was determined. An assortment of different fragments was generated by adding puromycin at low concentrations to influenza virus-infected tissue culture cells. Of the fragments generated, <2% was secreted, indicating that the system for detecting defects in newly synthesized proteins is quite stringent. The majority of secreted species corresponded to folding domains within the viral spike glycoprotein. The retained fragments acquired a partially folded structure with intrachain disulfide bonds and conformation-dependent antigenic epitopes. They associated with two lectin-like endoplasmic reticulum chaperones (calnexin and calreticulin) but not BiP/GRP78. Inhibition of the association with calnexin and calreticulin by the addition of castanospermine significantly increased fragment secretion. However, it also caused association with BiP/GRP78. These results indicated that the association with calnexin and calreticulin was involved in retaining the fragments. They also suggested that BiP/GRP78 could serve as a backup for calnexin and calreticulin in retaining the fragments. In summary, the results showed that the quality control system in the secretory pathway was efficient and sensitive to folding defects, and that it involved multiple interactions with endoplasmic reticulum chaperones.
The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport
Authors: Pu Robert T. , Dasso Mary
Published: July 2013

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 fromXenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.
Nuclear Pore Complex Number and Distribution throughout theSaccharomyces cerevisiaeCell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes
Authors: Winey Mark , Yarar Defne , Giddings Thomas H. , Mastronarde David N.
Published: July 2013

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.
Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis
Authors: Fan Xinqing , Price Carolyn Mary
Published: July 2013

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis.Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA polα and polδ, our results suggest that this coordinate length regulation is mediated by DNA polymerase.