bluuebunny/crossref_metadata_2025 huggingface dataset:
42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor.
Authors: Coppard N J , Poulsen K , Madsen H O , Frydenberg J , Clark B F
Published: May 2004

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.
Formation and intracellular transport of a heterodimeric viral spike protein complex.
Authors: Persson R , Pettersson R F
Published: May 2004

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.
Extracellular ATP as a trigger for apoptosis or programmed cell death.
Authors: Zheng L M , Zychlinsky A , Liu C C , Ojcius D M , Young J D
Published: May 2004

Extracellular ATP is shown here to induce programmed cell death (or apoptosis) in thymocytes and certain tumor cell lines. EM studies indicate that the ATP-induced death of thymocytes and susceptible tumor cells follows morphological changes usually associated with glucocorticoid-induced apoptosis of thymocytes. These changes include condensation of chromatin, blebbing of the cell surface, and breakdown of the nucleus. Cytotoxicity assays using double-labeled cells show that ATP-mediated cell lysis is accompanied by fragmentation of the target cell DNA. DNA fragmentation can be set off by ATP but not the nonhydrolysable analogue ATP gamma S nor other nucleoside-5'-triphosphates. ATP-induced DNA fragmentation but not ATP-induced 51Cr release can be blocked in cells pretreated with inhibitors of protein or RNA synthesis or the endonuclease inhibitor, zinc; whereas pretreatment with calmidazolium, a potent calmodulin antagonist, blocks both DNA fragmentation and 51Cr release. The biochemical and morphological changes caused by ATP are preceded by a rapid increase in the cytoplasmic calcium of the susceptible cell. Calcium fluxes by themselves, however, are not sufficient to cause apoptosis, as the pore-forming protein, perforin, causes cell lysis without DNA fragmentation or the morphological changes associated with apoptosis. Taken together, these results indicate that ATP can cause cell death through two independent mechanisms, one of which, requiring an active participation on the part of the cell, takes place through apoptosis.
Purification and characterization of an 82-kD membrane protein as a neurite outgrowth factor binding protein: possible involvement of NOF binding protein in axonal outgrowth in developing retina.
Authors: Taniura H , Kuo C H , Hayashi Y , Miki N
Published: May 2004

Neurite outgrowth factor (NOF) is a glycoprotein isolated from an extract of gizzard that induces neurite outgrowth from cultured retinal or ciliary ganglionic (CG) neurons. We have reported that a glycoprotein of approximately 82 kD solubilized from gizzard muscles binds to NOF (ligand blotting) and inhibits the neurite promoting activity of NOF (inhibition assay). The 82-kD protein (NOF binding protein) was purified from gizzard muscle membranes as a doublet band on SDS-PAGE and a polyclonal antibody was raised against it. An NOF binding protein in developing retina exhibited the same physicochemical properties as that of the gizzard muscle. Quantitative decrease in NOF binding protein in embryonic retinas was observed after day 11 by the inhibition assay, ligand blotting, and immunoblotting, its decrease being parallel with reduction of NOF-induced neurite outgrowth of embryonic retinas. In an immunohistochemical study, the antibody stained only the optic fiber layers of the retinas of 8-d embryos, and this staining was no longer detectable in retinas of 18-d embryos. These results suggest that the 82-kD protein is a novel membrane protein that behaves as an NOF receptor and that the loss of neuritic response of the retinal neurons to NOF reflects a decrease in NOF receptor molecules.
Activation of LFA-1 through a Ca2(+)-dependent epitope stimulates lymphocyte adhesion.
Authors: van Kooyk Y , Weder P , Hogervorst F , Verhoeven A J , van Seventer G , te Velde A A , Borst J , Keizer G D , Figdor C G
Published: May 2004

The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-ligand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 alpha mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2(+)-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-1: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16+ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16+) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.
Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis.
Authors: Weller A , Beck S , Ekblom P
Published: May 2004

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).
The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.
Authors: Su B , Waneck G L , Flavell R A , Bothwell A L
Published: May 2004

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.
Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.
Authors: Lynch R M , Fogarty K E , Fay F S
Published: May 2004

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.
Monoclonal antibody mapping of structural and functional plectin epitopes.
Authors: Foisner R , Feldman B , Sander L , Wiche G
Published: May 2004

To map structural and functional epitopes of the cytomatrix protein plectin, a set of mAbs was prepared by immunization of mice. Using immunoblot analysis of plectin fragments obtained after limited digestion with various proteases, two groups of mAbs were distinguished. The epitopes of one group (1) were located on a 130-kD terminal segment of the plectin 300-kD polypeptide chain, whereas those of the other group (2) bound within a 40kD segment confined to a central domain of the polypeptide chain. Domains containing the epitopes of group 2 mAbs were shown to include in vitro phosphorylation sites for kinase A, whereas kinase C phosphorylation sites were found on the same terminal segment that contained group 1 mAb epitopes. Rotary shadowing EM of mAb (Fab fragment) -decorated plectin molecules at various states of aggregation, ranging from characteristic dumbbell-shaped single molecules to highly complex multimeric structures, revealed that the epitopes of group 1 as well as those of group 2 mAbs were located on plectin's roughly 200-nm long rod domain interlinking its two globular end domains. Epitopes of group 1 mAbs were localized within a region near the center of the rod, those of group 2 in more peripheral sections near the globular end domains. Solid-phase binding assays carried out in the presence of Fab fragments of mAbs demonstrated an interference of certain group 1 mAbs in the interactions of plectin with vimentin and lamin B. On the other hand, plectin's self-interaction was inhibited mainly by Fab fragments with epitopes in the peripheral rod domain (group 2 mAbs). Together, these results suggested that the molecular binding sites of plectin for vimentin and lamin B, as well as the phosphorylation sites for kinase C, were confined to a defined central section of plectin's rod domain. In addition, they suggest an involvement of peripheral rod sections in plectin self-association.
The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments.
Authors: Hartwig J H , DeSisto M
Published: May 2004

We used high-resolution EM and immunocytochemistry in combination with different specimen preparation techniques to resolve the ultrastructure of the resting platelet cytoskeleton. The periphery of the cytoskeleton, an electron-dense subplasmalemmal region in thin section electron micrographs, is a tightly woven planar sheet composed of a spectrin-rich network whose interstices contain GPIb/IX-actin-binding protein (ABP) complexes. This membrane skeleton connects to a system of curved actin filaments (F-actin) that emanate from a central oval core of F-actin cross-linked by ABP. The predominant interaction of the radial actin filaments with the membrane skeleton is along their sides, and the strongest connection between the membrane skeleton and F-actin is via ABP-GPIb ligands, although there is evidence for spectrin attaching to the ends of the radial actin filaments as well. Since a mechanical separation of the F-actin cores and radial F-actin-GPIb-ABP complexes from the underlying spectrin-rich skeleton leads to the latter's expansion, it follows that the spectrin-based skeleton of the resting cell may be held in a compressed form by interdigitating GPIb/IX complexes which are immobilized by radial F-actin-ABP anchors.
Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor.
Authors: Sorkin A , Westermark B , Heldin C H , Claesson-Welsh L
Published: May 2004

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.
A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.
Authors: Dessev G , Iovcheva-Dessev C , Bischoff J R , Beach D , Goldman R
Published: May 2004

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L. Rebhun, and R. Goldman. 1989. Dev. Biol. 131:469-504). To identify these factors, we have fractionated the oocyte extracts. The nuclear lamina disassembly (NLD) activity, together with a protein kinase activity specific for L67, appear as a single peak throughout a number of purification steps. This peak also contains p34cdc2, cyclin B, and histone H1-kinase activity, which are components of the M-phase promoting factor (MPF). The NLD/L67-kinase activity is depleted by exposure of this purified material to Sepharose conjugated to p13suc1, and is restored upon addition of a p34cdc2/p62 complex from HeLa cells. The latter complex phosphorylates L67 and induces NLD in the absence of other clam oocyte proteins. Our results suggest that a single protein kinase activity (p34cdc2-H1 kinase, identical with MPF) phosphorylates the lamin and is involved in the meiotic breakdown of the nuclear envelope in clam oocytes.
Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism.
Authors: Lippincott-Schwartz J , Glickman J , Donaldson J G , Robbins J , Kreis T E , Seamon K B , Sheetz M P , Klausner R D
Published: May 2004

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.
Immunolocalization and molecular properties of a high molecular weight microtubule-bundling protein (syncolin) from chicken erythrocytes.
Authors: Feick P , Foisner R , Wiche G
Published: May 2004

A protein of apparent molecular weight 280,000 (syncolin), which is immunoreactive with antibodies to hog brain microtubule-associated protein (MAP) 2, was purified from chicken erythrocytes. Immunofluorescence microscopy of bone marrow cells revealed the presence of syncolin in cells at all stages of erythrocyte differentiation. In early erythroblasts syncolin was diffusely distributed throughout the cytoplasm. At later stages it was found along microtubules of the marginal band, as confirmed by immunoelectron microscopy. The association of syncolin with the marginal band was dependent on the integrity of microtubules, as demonstrated by temperature-dependent de- and repolymerization or marginal band microtubules. Syncolin cosedimented in a saturable manner with microtubules assembled in vitro, and it was displaced from the polymer by salt. Brain as well as erythrocyte microtubules, reconstituted with taxol from MAP-free tubulin and purified syncolin, were aggregated into dense bundles containing up to 15 microtubules, as determined by electron microscopy. On the ultrastructural level, syncolin molecules were visualized as globular or ringlike structures, in contrast to the thin, threadlike appearance of filamentous MAPs, such as brain MAP 2. According to ultrastructural measurements and gel permeation chromatography, syncolin's molecular weight was approximately 1 x 10(6). It is suggested that syncolin's specific function is the cross-linking of microtubules in the marginal band and, by implication, the stabilization of this structure typical for nucleated (chicken) erythrocytes.
The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms.
Authors: Piperno G , Ramanis Z
Published: May 2004

A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.
Intracellular free calcium oscillates during cell division of Xenopus embryos.
Authors: Grandin N , Charbonneau M
Published: May 2004

In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.
Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry.
Authors: Fushimi K , Verkman A S
Published: May 2004

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.
The structure of plasmodesmata as revealed by plasmolysis, detergent extraction, and protease digestion.
Authors: Tilney L G , Cooke T J , Connelly P S , Tilney M S
Published: May 2004

Plasmodesmata or intercellular bridges that connect plant cells are cylindrical channels approximately 40 nm in diameter. Running through the center of each is a dense rod, the desmotubule, that is connected to the endoplasmic reticulum of adjacent cells. Fern, Onoclea sensibilis, gametophytes were cut in half and the cut surfaces exposed to the detergent, Triton X 100, then fixed. Although the plasma membrane limiting the plasmodesma is solubilized partially or completely, the desmotubule remains intact. Alternatively, if the cut surface is exposed to papain, then fixed, the desmotubule disappears, but the plasma membrane limiting the plasmodesmata remains intact albeit swollen and irregular in profile. Gametophytes were plasmolyzed, and then fixed. As the cells retract from their cell walls they leave behind the plasmodesmata still inserted in the cell wall. They can break cleanly when the cell proper retracts or can pull away portions of the plasma membrane of the cell with them. Where the desmotubule remains intact, the plasmodesma retains its shape. These images and the results with detergents and proteases indicate that the desmotubule provides a cytoskeletal element for each plasmodesma, an element that not only stabilizes the whole structure, but also limits its size and porosity. It is likely to be composed in large part of protein. Suggestions are made as to why this structure has been selected for in evolution.
Identification of a novel nuclear domain.
Authors: Ascoli C A , Maul G G
Published: May 2004

For most known nuclear domains (ND), specific functions have been identified. In this report we used murine mAbs and human autoantibodies to investigate precisely circumscribed structures 0.2-0.3 micron in diameter which appear as "nuclear dots" distributed throughout the nucleoplasm. Nuclear dots are metabolically stable and resistant to nuclease digestion and salt extraction. The localization of nuclear dots is separate from kinetochores, centromeres, sites of mRNA processing and tRNA synthesis, nuclear bodies, and chromosomes. The nuclear dots, therefore, represent a novel ND. Nuclear dots break down as cells enter metaphase and reassemble at telophase. In interphase cells, nuclear dots are frequently "paired," and some are visible as "doublets" when stained with one particular antiserum. The number of dot doublets increased when quiescent cells were stimulated with serum although the total number of dots did not change substantially. One of the antigens was identified as a protein with a molecular mass of approximately 55 kD showing three charge isomers in the pI range of 7.4 to 7.7. Autoantibodies affinity purified from this nuclear dot protein (NDP-55) show nuclear dots exclusively. Nuclear dot-negative rat liver parenchymal cells became positive after chemical hepatectomy, suggesting involvement of the NDP-55 in the proliferative state of cells.
A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.
Authors: Thrift R N , Andrews D W , Walter P , Johnson A E
Published: May 2004

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.
Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.
Authors: Goda Y , Pfeffer S R
Published: May 2004

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.
Constitutive and basal secretion from the endocrine cell line, AtT-20.
Authors: Matsuuchi L , Kelly R B
Published: May 2004

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.
Mutagenesis of the human transferrin receptor: two cytoplasmic phenylalanines are required for efficient internalization and a second-site mutation is capable of reverting an internalization-defective phenotype.
Authors: McGraw T E , Pytowski B , Arzt J , Ferrone C
Published: May 2004

Site-specific mutagenesis has been used to define the sequences required for efficient internalization of the human transferrin receptor. It has previously been shown that the sole cytoplasmic tyrosine, at position 20, is required for efficient internalization. When two other cytoplasmic aromatic residues, the phenylalanines at positions 13 and 23, are substituted with alanines internalization is also reduced. The phenylalanine 23 mutation decreases the internalization rate constant approximately threefold, and mutation of phenylalanine 13 decreases it by approximately twofold. The mutation at position 23 has as serious an effect on internalization as substitution with a nonaromatic amino acid for the single tyrosine. These results demonstrate the importance of several aromatic amino acids in maintaining efficient internalization of the transferrin receptor. Substitution of a tyrosine at a second site, for a serine at position 34, within the cytoplasmic domain of a transferrin receptor with a nonaromatic amino acid at position 20, results in a complete reversion of the internalization-defective phenotype. This reversion is completely dependent upon a tyrosine, as phenylalanine substituted at position 34 does not revert the internalization-defective phenotype. This result demonstrates that a tyrosine placed outside of its native context can still function in the internalization of the transferrin receptor, suggesting a flexibility in surrounding sequences required for efficient internalization.
Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.
Authors: Gundersen D , Orlowski J , Rodriguez-Boulan E
Published: May 2004

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.
Binding of actin to liver cell membranes: the state of membrane-bound actin.
Authors: Tranter M P , Sugrue S P , Schwartz M A
Published: May 2004

Previous work has shown that actin binds specifically and saturably to liver membranes stripped of endogenous actin (Tranter, M. P., S. P. Sugrue, and M. A. Schwartz. 1989. J. Cell Biol. 109:2833-2840). Scatchard plots of equilibrium binding data were linear, indicating that binding is not cooperative, as would be expected for F- or G-actin. To determine the state of membrane-bound actin, we have analyzed the binding of F- and G-actin to liver cell membranes. G-actin in low salt depolymerization buffer and EF-actin, a derivative that polymerizes very poorly in solution, bind to liver cell membranes as well as untreated actin in polymerization buffer. Phalloidin-stabilized F-actin binds, but to a lesser extent. The binding of F- and G-actins are mutually competitive and are inhibited by ATP, suggesting that both forms of actin bind to the same sites. For untreated actin in polymerization buffer, the time course of binding is biphasic, with an initial rapid component which is followed by a plateau phase, then a second, slower component. The binding kinetics of pure F-actin and pure G-actin are both monophasic and match the fast and slower components, respectively, of untreated actin. In the reconstituted system, membrane-bound actin does not stain with rhodamine-phalloidin, nor are actin filaments detected by EM. Distinct regions of amorphous material, however, are visible, which stain with an anti-actin antibody. The exact nature of this material has yet to be determined. A model of actin binding is presented.
Formation and contraction of a microfilamentous shell in saponin-permeabilized platelets.
Authors: Stark F , Golla R , Nachmias V T
Published: May 2004

To study the mechanism of granule centralization in platelets, we permeabilized with saponin in either EGTA (5 mM) or calcium (1 or 10 microM). Under all conditions, platelets retained 40-50% of their total actin and greater than 70% of their actin-binding protein (ABP) but lost greater than 80% of talin and myosin to the supernatant. Thin sections of platelets permeabilized in EGTA showed a microfilament network under the residual plasma membrane and throughout the cytoplasm. Platelets permeabilized in calcium contained a microfilament shell partly separated from the residual membrane. The shell stained brightly for F-actin. A less dense microfilament shell was also seen in sections of ADP-stimulated intact platelets subsequently permeabilized in EGTA. In the presence of 1 mM ATP gamma S and calcium, myosin was retained (70%) and was localized by indirect immunofluorescence in bright central spots that also stained intensely for F-actin. Electron micrographs showed centralized granules surrounded by a closely packed mass of microfilaments much like the structures seen in thrombin-stimulated intact platelets subsequently permeabilized in EGTA. Permeabilization in calcium, ATP, and okadaic acid, produced the same configuration of centralized granules and packed microfilaments; myosin was retained and the myosin regulatory light chain became phosphorylated. Microtubule coil disassembly before permeabilization did not inhibit granule centralization. These results suggest a possible mechanism for granule centralization in these models. The cytoskeletal network first separates from some of its connections to the plasma membrane by a calcium-dependent mechanism not involving ABP proteolysis. Phosphorylated myosin interacts with the microfilaments to contract the shell moving the granules to the platelet's center.
Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development.
Authors: Maher P A
Published: May 2004

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.
A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy.
Authors: Polans A S , Buczyłko J , Crabb J , Palczewski K
Published: May 2004

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.
Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle.
Authors: Bowser R , Novick P
Published: May 2004

SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast. Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane. The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl. Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane. Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p. However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes. Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable. During the course of these studies we have found that two activities associated with the yeast Golgi apparatus, Kex2 endopeptidase and GDPase, are in separable subcompartments.
Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor.
Authors: Dadabay C Y , Patton E , Cooper J A , Pike L J
Published: May 2004

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.