bluuebunny/crossref_metadata_2025 huggingface dataset:
An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3-mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation.
Authors: Massia S P , Hubbell J A
Published: May 2004

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.
Evolutionary conservation of a microbody targeting signal that targets proteins to peroxisomes, glyoxysomes, and glycosomes.
Authors: Keller G A , Krisans S , Gould S J , Sommer J M , Wang C C , Schliebs W , Kunau W , Brody S , Subramani S
Published: May 2004

Peroxisomes, glyoxysomes, glycosomes, and hydrogenosomes have each been classified as microbodies, i.e., subcellular organelles with an electron-dense matrix that is bound by a single membrane. We investigated whether these organelles might share a common evolutionary origin by asking if targeting signals used for translocation of proteins into these microbodies are related. A peroxisomal targeting signal (PTS) consisting of the COOH-terminal tripeptide serine-lysine-leucine-COOH has been identified in a number of peroxisomal proteins (Gould, S.J., G.-A. Keller, N. Hosken, J. Wilkinson, and S. Subramani. 1989. J. Cell Biol. 108:1657-1664). Antibodies raised to a peptide ending in this sequence (SKL-COOH) recognize a number of peroxisomal proteins. Immunocryoelectron microscopy experiments using this anti-SKL antibody revealed the presence of proteins containing the PTS within glyoxysomes of cells from Pichia pastoris, germinating castor bean seeds, and Neurospora crassa, as well as within the glycosomes of Trypanosoma brucei. Western blot analysis of purified organelle fractions revealed the presence of many proteins containing this PTS in both glyoxysomes and glycosomes. These results indicate that at least one of the signals, and therefore the mechanism, for protein translocation into peroxisomes, glyoxysomes, and glycosomes has been conserved, lending support to a common evolutionary origin for these microbodies. Hydrogenosomes, the fourth type of microbody, did not contain proteins that cross-reacted with the anti-PTS antibody, suggesting that this organelle is unrelated to microbodies.
Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii.
Authors: Baldan B , Girard-Bascou J , Wollman F A , Olive J
Published: May 2004

To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments.
Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization.
Authors: Terasaki M , Jaffe L A
Published: May 2004

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.
Desmin/vimentin intermediate filaments are dispensable for many aspects of myogenesis.
Authors: Schultheiss T , Lin Z X , Ishikawa H , Zamir I , Stoeckert C J , Holtzer H
Published: May 2004

An expression vector was prepared containing a cDNA coding for a truncated version of the intermediate filament (IF) protein desmin. The encoded truncated desmin protein lacks a portion of the highly conserved alpha-helical rod region as well as the entire nonhelical carboxy-terminal domain. When transiently expressed in primary fibroblasts, or in differentiating postmitotic myoblasts and multinucleated myotubes, the truncated protein induces the complete dismantling of the preexisting vimentin or desmin/vimentin IF networks, respectively. Instead, in both cell types vimentin and desmin are packaged into hybrid spheroid bodies scattered throughout the cytoplasm. Despite the complete lack of intact IFs, myoblasts and myotubes expressing truncated desmin assemble and laterally align normal striated myofibrils and contract spontaneously in a manner indistinguishable from that of control myogenic cells. In older cultures the spheroid bodies shift from a longitudinal to a predominantly transverse orientation and loosely align along the I-Z-I-regions of striated myofibrils (Bennett, G.S., S. Fellini, Y. Toyama, and H. Holtzer. 1979. J. Cell Biol. 82:577-584), analogous to the translocation of intact desmin/vimentin IFs in control muscle. These results suggest the need for a critical reexamination of currently held concepts regarding the functions of desmin IFs during myogenesis.
Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes.
Authors: Cerneus D P , van der Ende A
Published: May 2004

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.
PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis.
Authors: Höhfeld J , Veenhuis M , Kunau W H
Published: May 2004

Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3-mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E. coli. Pas3p was identified as a 48 kD peroxisomal integral membrane protein. It is shown that a lack of this protein causes the peroxisome-deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes. Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule is exposed to the cytosol. These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery.
Biophysical properties and microfilament assembly in neutrophils: modulation by cyclic AMP.
Authors: Downey G P , Elson E L , Schwab B , Erzurum S C , Young S K , Worthen G S
Published: May 2004

The microfilament lattice, composed primarily of filamentous (F)-actin, determines in large part the mechanical (deformability) properties of neutrophils, and thus may regulate the ability of neutrophils to transit a microvascular bed. Circulating factors may stimulate the neutrophil to become rigid and therefore be retained in the capillaries. We hypothesized that cell stiffening might be attenuated by an increase in intracellular cAMP. A combination of cell filtration and cell poking (mechanical indentation) was used to measure cell deformability. Neutrophils pretreated with dibutyryl cAMP (db-cAMP) or the combination of prostaglandin E2 (PGE2, a stimulator of adenylate cyclase) and isobutylmethylxanthine (IBMX, an inhibitor of phosphodiesterase) demonstrated significant inhibition of the n-formyl-methionyl-leucyl-phenylalanine (fMLP)-inducing stiffening. The inhibition of cell stiffening was associated with an increase in intracellular cAMP as measured by enzyme-linked immunoassay (EIA) and an increase in the activity of the cAMP-dependent kinase (A-kinase). Treatment with PGE2 and IBMX also resulted in a decrease in the F-actin content of stimulated neutrophils as assayed by NBD-phallacidin staining and flow cytometry or by changes in right angle light scattering. Direct addition of cAMP to electropermeabilized neutrophils resulted in attenuation of fMLP-induced actin assembly. Neutrophils stimulated with fMLP demonstrated a rapid redistribution of F-actin from a diffuse cortical location to a peripheral ring as assessed by conventional and scanning confocal fluorescence microscopy. Pretreatment of neutrophils with the combination of IBMX and PGE2 resulted in incomplete development and fragmentation of the cortical ring. We conclude that assembly and redistribution of F-actin may be responsible for cell stiffening after exposure to stimulants and that this response was attenuated by agents that increase intracellular cAMP, by altering the amount and spatial organization of the microfilament component of the cytoskeleton.
Immunocytochemical localization of casein kinase II during interphase and mitosis.
Authors: Yu I J , Spector D L , Bae Y S , Marshak D R
Published: May 2004

We have developed specific antibodies to synthetic peptide antigens that react with the individual subunits of casein kinase II (CKII). Using these antibodies, we studied the localization of CKII in asynchronous HeLa cells by immunofluorescence and immunoelectron microscopy. Further studies were done on HeLa cells arrested at the G1/S transition by hydroxyurea treatment. Our results indicate that the CKII alpha and beta subunits are localized in the cytoplasm during interphase and are distributed throughout the cell during mitosis. Further electron microscopic investigation revealed that CKII alpha subunit is associated with spindle fibers during metaphase and anaphase. In contrast, the CKII alpha' subunit is localized in the nucleus during G1 and in the cytoplasm during S. Taken together, our results suggest that CKII may play significant roles in cell division control by shifting its localization between the cytoplasm and nucleus.
Purkinje cell degeneration associated with erythroid ankyrin deficiency in nb/nb mice.
Authors: Peters L L , Birkenmeier C S , Bronson R T , White R A , Lux S E , Otto E , Bennett V , Higgins A , Barker J E
Published: May 2004

Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.
Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.
Authors: Brunner G , Gabrilove J , Rifkin D B , Wilson E L
Published: May 2004

Basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow stromal cells and stimulates haematopoiesis in vitro. We report here that primary human bone marrow cultures contain bFGF and express heparin-like bFGF binding sites on the cell surface and in the extracellular matrix (ECM). bFGF bound predominantly to a 200-kD cell surface heparan sulfate proteoglycan (HSPG), which was also found in conditioned medium. bFGF was released from bone marrow cultures by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) and, less efficiently, by plasmin. Solubilized bFGF was found as a complex with the 200-kD HSPG. The complex was biologically active as shown by its ability to stimulate plasminogen activator production in bovine aortic endothelial cells. bFGF-HSPG complexes of bovine endothelial cells, however, were not released by PI-PLC. While only trace amounts of the bFGF-binding 200-kD HSPG were released spontaneously from bone marrow cultures, incubation with PI-PLC solubilized almost all of the 200-kD HSPG. The HSPG could be metabolically labeled with ethanolamine or palmitate, which was partially removed by treatment with PI-PLC. These findings indicate linkage of the HSPG to the cell surface via a phosphatidylinositol anchor. Plasmin released the 200-kD HSPG less efficiently than PI-PLC. We conclude that HSPGs of human bone marrow serve as a reservoir for bFGF, from which it can be released in a biologically active form via a dual mechanism; one involving a putative endogenous phospholipase, the other involving the proteolytic cascade of plasminogen activation.
Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.
Authors: Nagai T , Yamakawa N , Aota S , Yamada S S , Akiyama S K , Olden K , Yamada K M
Published: May 2004

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.
Phosphoinositide kinase, diacylglycerol kinase, and phospholipase C activities associated to the cytoskeleton: effect of epidermal growth factor.
Authors: Payrastre B , van Bergen en Henegouwen P M , Breton M , den Hartigh J C , Plantavid M , Verkleij A J , Boonstra J
Published: May 2004

In this paper we demonstrate that cytoskeletons isolated from A431 cells have associated with them high activities of several kinases involved in inositol lipid metabolism, such as phosphatidylinositol kinase, phosphatidylinositol phosphate kinase, and diacylglycerol kinase. In addition also phospholipase C activity was detected on isolated cytoskeletons. Controlled extraction of the cytoskeletons followed by in vitro polymerization of actin demonstrated an association of the kinases to the actin filament system consisting of actin and a number of actin-binding proteins. The cytoskeleton-associated lipid kinase activities were significantly increased upon treatment of intact cells with EGF. These data suggest that the association of the phosphoinositide kinases, diacylglycerol kinase, phospholipase C, and also the EGF receptor to the cytoskeleton may play a role in the efficient signal transduction induced by EGF, by providing a matrix for the various components involved in signal transduction.
Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.
Authors: Cameron P L , Südhof T C , Jahn R , De Camilli P
Published: May 2004

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.
Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts.
Authors: Hartman D S , Millar N S , Claudio T
Published: May 2004

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR.
Coordinate developmental regulation of purine catabolic enzyme expression in gastrointestinal and postimplantation reproductive tracts.
Authors: Witte D P , Wiginton D A , Hutton J J , Aronow B J
Published: May 2004

Using histochemical detection, we have visualized in situ the complete metabolic pathway for the degradation of purine nucleotides. From the tongue to the ileum, diverse epithelial cell types lining the lumen of the mouse gastrointestinal (GI) tract strongly coexpress each of the five key purine catabolic enzymes. Dramatic increases in the expression of each enzyme occurred during postnatal maturation of the GI tract. Using in situ hybridization, an intense accumulation of adenosine deaminase (ADA) mRNA was detected only within GI epithelial cells undergoing postmitotic differentiation. In a similar manner, at the developing maternal-fetal interface, high level expression of the purine catabolic pathway also occurred in a unique subset of maternal decidual cells previously known to express high levels of alkaline phosphatase and ADA. This induction occurred almost immediately after implantation in the periembryonic maternal decidual cells, shortly thereafter in antimesometrial decidual cells, and later in cells of the placental decidua basalis: all of which contain cell types thought to be undergoing programmed cell death. The expression of the pathway at the site of embryo implantation appears to be critical because its pharmacologic inhibition during pregnancy has been found to be embryolethal or teratogenic. Purine destruction at these nutritional interfaces (placenta and gastrointestinal tract) seem to override any potential economy of purine salvage, and may represent biochemical adaptation to nucleic acid breakdown occurring in the context of dietary digestion or extensive programmed cell death.
A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein.
Authors: Swift A M , Machamer C E
Published: May 2004

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.
Paxillin is a major phosphotyrosine-containing protein during embryonic development.
Authors: Turner C E
Published: May 2004

Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development.
Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen.
Authors: Takada Y , Murphy E , Pil P , Chen C , Ginsberg M H , Hemler M E
Published: May 2004

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3-specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.
Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium.
Authors: Kinosita K , Itoh H , Ishiwata S , Hirano K , Nishizaka T , Hayakawa T
Published: May 2004

A new microscope technique, termed "W" (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized.
Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.
Authors: Zhang F , Crise B , Su B , Hou Y , Rose J K , Bothwell A , Jacobson K
Published: May 2004

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.
The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae.
Authors: McCaffrey M , Johnson J S , Goud B , Myers A M , Rossier J , Popoff M R , Madaule P , Boquet P
Published: May 2004

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.
Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.
Authors: Ettinger W F , Theg S M
Published: May 2004

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.
A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment.
Authors: Moran P , Caras I W
Published: May 2004

The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, does not become GPI anchored. We now show that this null sequence for GPI attachment can be converted to a strong GPI signal by mutating a pair of residues (valine-glutamate) in the LDLR sequence at a position corresponding to the normal cleavage/attachment site, to serine-glycine, as found in the DAF sequence. A single mutation (converting valine at the anchor addition site to serine, the normal acceptor for GPI addition in DAF) was insufficient to produce GPI anchoring, as was mutation of the valine-glutamate pair to serine-phenylalanine (a bulky residue). These results suggest that a pair of small residues (presumably flanking the cleavage point) is required for GPI attachment. By introducing the sequence serine-glycine (comprising a cleavage-attachment site for GPI addition) at different positions in the LDLR sequence of the fusion protein, HLD, we show that optimal GPI attachment requires a processing site positioned 10-12 residues NH2-terminal to the hydrophobic domain, the efficiency anchor attachment dropping off sharply as the cleavage site is moved beyond these limits. These data suggest that the GPI signal consists solely of a hydrophobic domain combined with a processing site composed of a pair of small residues, positioned 10-12 residues NH2-terminal to the hydrophobic domain. No other structural motifs appear necessary.
Filensin: a new vimentin-binding, polymerization-competent, and membrane-associated protein of the lens fiber cell.
Authors: Merdes A , Brunkener M , Horstmann H , Georgatos S D
Published: May 2004

We have studied the molecular properties of a 100-kD protein, termed filensin, which we have isolated from porcine lens membranes. Filensin represents a membrane-associated element, resistant to salt and nonionic detergent treatment, and extractable only by alkali or high concentrations of urea. By indirect immunofluorescence and immunoelectron microscopy, this protein can be localized at the periphery of the lens fiber cells. Immunochemical analysis suggests that filensin originates from a larger 110-kD component which is abundantly expressed in lens but not in other tissues. Purified filensin polymerizes in a salt-dependent fashion and forms irregular fibrils (integral of 10 nm in diameter) when reconstituted into buffers of physiological ionic strength and neutral pH. Radiolabeled filensin binds specifically to lens vimentin under isotonic conditions, as demonstrated by affinity chromatography and ligand-blotting assays. By the latter approach, filensin also reacts with a 47-kD peripheral membrane protein of the lens cells. Purified filensin binds to PI, a synthetic peptide modelled after a segment of the COOH-terminal domain of peripherin (a type III intermediate filament protein highly homologous to vimentin), but not to various other peptides including the NH2-terminal headpiece of vimentin and derivatives of its middle (rod) domain. The filensin-PI binding is inhibited by purified lamin B, which is known to interact in vitro with PI (Djabali, K., M.-M. Portier, F. Gros, G. Blobel, and S. D. Georgatos. 1991. Cell. 64:109-121). Finally, limited proteolysis indicates that the filensin-vimentin interaction involves a 30-kD segment of the filensin molecule. Based on these observations, we postulate that the lens fiber cells express a polymerization-competent protein which is tightly associated with the plasma membrane and has the potential to serve as an anchorage site for vimentin intermediate filaments.
Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein.
Authors: Bloch R J , Resneck W G , O'Neill A , Strong J , Pumplin D W
Published: May 2004

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.
Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity.
Authors: Batistatou A , Greene L A
Published: May 2004

Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the neuronal cell death which occurs after neurotrophic factor deprivation. In this experimental paradigm, nerve growth factor (NGF) rescues the cells from death. It is reported here that serum-deprived PC12 cells manifest an endonuclease activity that leads to internucleosomal cleavage of their cellular DNA. This activity is detected within 3 h of serum withdrawal and several hours before any morphological sign of cell degeneration or death. NGF and serum, which promote survival of the cells, inhibit the DNA fragmentation. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases in vitro, suppresses the endonuclease activity and promotes long-term survival of PC12 cells in serum-free cultures. This effect appears to be independent of macromolecular synthesis. In addition, ATA promotes long-term survival of cultured sympathetic neurons after NGF withdrawal. ATA neither promotes nor maintains neurite outgrowth. It is hypothesized that the activation of an endogenous endonuclease could be responsible for neuronal cell death after neurotrophic factor deprivation and that growth factors could promote survival by leading to inhibition of constitutively present endonucleases.