bluuebunny/crossref_metadata_2025 huggingface dataset:
Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations.
Authors: Letai A , Coulombe P A , Fuchs E
Published: May 2004

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.
Antigen-receptor complex stimulation triggers protein kinase C-dependent CD11a/CD18-cytoskeleton association in T lymphocytes.
Authors: Pardi R , Inverardi L , Rugarli C , Bender J R
Published: May 2004

Although it is well accepted that intercellular adhesion involving the CD11a/CD18 (LFA-1) complex is critical in a wide array of T cell-dependent processes, recent demonstrations of an LFA-1 high avidity state, induced by triggering the T cell receptor (TCR) complex, has raised questions about the intracellular signals generated and molecular events leading to effective cell coupling, as well as their orderly sequence. In this study, we assessed the effects of T cell activation on the actin-based cytoskeleton, and LFA-1, as well as their interaction. Crosslinking the TCR complex with anti-CD3 mAb resulted in actin polymerization and colocalization with LFA-1, as detected by fluorescence microscopy. This association was confirmed by immunoprecipitating LFA-1 from the detergent insoluble, cytoskeletal-associated membrane fraction after TCR crosslinking. These consequences were inhibited by the protein kinase C (PKC) inhibitor staurosporine or by PKC desensitization, as was a transient CD11a hyperphosphorylation, induced by monoclonal anti-CD3. Furthermore, a small percentage of beta 2-deficient T cells maintained the ability to rearrange the cytoskeleton in response to TCR complex activation, with F-actin-VLA4 colocalization. These results provide evidence that the important consequences of TCR-induced signal transduction include a PKC-dependent cytoskeletal rearrangement, involving an association between leukocyte integrins and F-actin. We discuss the implications of these findings with respect to effective T cell functions.
Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect.
Authors: Levin D E , Bartlett-Heubusch E
Published: May 2004

The PKC1 gene of Saccharomyces cerevisiae encodes a homologue of the Ca(2+)-dependent isozymes of mammalian protein kinase C (Levin, D.cE., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990. Cell. 62:213-224). Cells depleted of the PKC1 gene product display a uniform phenotype, a behavior indicating a defect in the cell division cycle (cdc). These cells arrest division after DNA replication, but before mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrest growth with small buds. We created conditional alleles of PKC1 to explore the nature of this unusual cdc defect. In contrast to PKC1-depleted cells, all of the conditional pkc1 mutants isolated were suppressed by the addition of CaCl2 to the medium, suggesting that the mutant enzymes could be activated by Ca2+. Arrest of growth and cell division in the conditional mutants was accompanied by cessation of protein synthesis, rapid loss of viability, and release of cellular material into the medium, suggesting cell lysis. This conclusion was supported by the observation that a pkc1 deletion mutant was capable of proliferation in osmotically stabilized medium, but underwent rapid cell lysis when shifted to hypo-osmotic medium. We have incorporated these observations into a model to explain the cdc-specific arrest of pkc1 mutants.
The plus ends of stable microtubules are the exclusive nucleating structures for microtubules in the axon.
Authors: Baas P W , Ahmad F J
Published: May 2004

Microtubules (MTs) in the axon have a uniform polarity orientation that is recapitulated during recovery from episodes of MT depolymerization (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu. 1984. J. Cell Biol. 99:1289-1295). This tight regulation of their organization indicates that axonal MTs are spatially regulated by discrete nucleating structures comparable in function to the centrosome. Several authors have proposed that an especially stable class of MTs in the axon may serve as these nucleating structures. In a previous report (Baas, P. W., and M. M. Black. 1990. J. Cell Biol. 111:495-509), we determined that the axons of cultured sympathetic neurons contain two classes of MT polymer, stable and labile, that differ in their sensitivity to nocodazole by roughly 35-fold. The stable and labile polymer represent long-lived and recently assembled polymer, respectively. We also determined that these two classes of polymer can be visually distinguished at the immunoelectron microscopic level based on their content of tyrosinated alpha-tubulin: the labile polymer stains densely, while the stable polymer does not stain. In the present study, we have taken advantage of these observations to directly identify MT nucleating structures in the axon. Neuron cultures were treated with nocodazole for 6 h to completely depolymerize the labile polymer in the axon, and substantially shorten the stable polymer. The cultures were then rinsed free of the drug, permitted to reassemble polymer for various periods of time, and prepared for immunoelectron microscopic localization of tyrosinated alpha-tubulin. Serial reconstruction of consecutive thin sections was undertaken to determine the spatial relationship between the stable MTs and the newly assembled polymer. All of the new polymer assembled in direct continuity with the plus ends of stable MTs, indicating that these ends are assembly competent, and hence capable of acting as nucleating structures. Our results further indicate that no self-assembly of MTs occurs in the axon, nor do any MT nucleating structures exist in the axon other than the plus ends of stable MTs. Thus the plus ends of stable MTs are the exclusive nucleating structures for MTs in the axon.
MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites.
Authors: Cusella-De Angelis M G , Lyons G , Sonnino C , De Angelis L , Vivarelli E , Farmer K , Wright W E , Molinaro M , Bouchè M , Buckingham M
Published: May 2004

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.
Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans.
Authors: Shieh M T , WuDunn D , Montgomery R I , Esko J D , Spear P G
Published: May 2004

The role of cell surface heparan sulfate in herpes simplex virus (HSV) infection was investigated using CHO cell mutants defective in various aspects of glycosaminoglycan synthesis. Binding of radiolabeled virus to the cells and infection were assessed in mutant and wild-type cells. Virus bound efficiently to wild-type cells and initiated an abortive infection in which immediate-early or alpha viral genes were expressed, despite limited production of late viral proteins and progeny virus. Binding of virus to heparan sulfate-deficient mutant cells was severely impaired and mutant cells were resistant to HSV infection. Intermediate levels of binding and infection were observed for a CHO cell mutant that produced undersulfated heparan sulfate. These results show that heparan sulfate moieties of cell surface proteoglycans serve as receptors for HSV.
Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.
Authors: West C M , Erdos G W
Published: May 2004

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.
NuMA: an unusually long coiled-coil related protein in the mammalian nucleus.
Authors: Yang C H , Lambie E J , Snyder M
Published: May 2004

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.
Interferon action: nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster.
Authors: Choubey D , Lengyel P
Published: May 2004

The interferons are cytokines with antiviral, cell growth regulatory, and immunomodulatory activities. These activities are mediated by the proteins induced by the interferons. Earlier we described a gene cluster (the 200 cluster) consisting of at least six adjacent, interferon-activatable genes located next to the erythroid alpha-spectrin locus on murine chromosome 1. The genes of the cluster arose by repeated gene duplication and they specify proteins with pronounced sequence similarity. We have now raised polyclonal antibodies against a segment from one of these proteins (the 204 protein of 72 kD). Using these, we established that the 204 protein is a phosphoprotein whose level in cells from various murine lines can be increased up to 75-fold upon treatment with alpha interferon. Experiments involving fractionation of cell lysates and indirect immunofluorescence microscopy of control and interferon-treated cells revealed that the 204 protein is nucleolar and nucleoplasmic. This conclusion was confirmed by co-localization with B23, a known nucleolar protein. The 204 protein is the first interferon-induced protein found to be located in the nucleoli, the subcellular organelles of ribosomal RNA production and ribosome assembly. It remains to be seen whether the 204 protein affects any of these processes. Studies on 204 protein function should be facilitated by the availability of complete cDNA clones and the finding of cell lines in which the expression of this protein is impaired.
Evidence for the regulation of exocytic transport by protein phosphorylation.
Authors: Davidson H W , McGowan C H , Balch W E
Published: May 2004

We investigated the effects of the protein phosphatase inhibitors okadaic acid and microcystin-LR upon transport of newly synthesized proteins through the exocytic pathway. Treatment of CHO cells with 1 microM okadaic acid rapidly inhibited movement of a marker protein (vesicular stomatitis virus G protein) from the endoplasmic reticulum to the Golgi compartment. Both okadaic acid and microcystin-LR also inhibited transport in an in vitro assay reconstituting movement to the Golgi compartment, at concentrations equivalent to those required to inhibit phosphorylase phosphatase activity. Inhibition both in vivo and in vitro could be antagonized by protein kinase inhibitors, suggesting that protein phosphorylation was directly responsible for this effect. An early stage in the transport reaction associated with vesicle formation or targeting was inhibited by protein phosphorylation, which could be reversed by fractions enriched in protein phosphatase 2A. Protein kinase antagonists did not inhibit transport between sequential compartments of the exocytic pathway in vitro, suggesting that protein phosphorylation is not itself required for vesicular transport. During mitosis, vesicular transport is inhibited simultaneous to the activation of maturation-promoting factor. It is proposed that the inhibition caused by okadaic acid and microcystin-LR involves a similar mechanism to that responsible for the mitotic arrest of vesicular transport.
Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.
Authors: Tan A , Bolscher J , Feltkamp C , Ploegh H
Published: May 2004

The involvement of GTP-binding proteins in the intracellular transport of the secretory glycoprotein alpha 1-antitrypsin was investigated in streptolysin O-permeabilized HepG2 cells. This permeabilization procedure allows ready access to the intracellular milieu of the membrane-impermeant, nonhydrolyzable GTP analog GTP gamma S. In streptolysin O-permeabilized HepG2 cells, the constitutive secretory pathway remains functional and is sensitive to GTP gamma S. Exposure of HepG2 cells to brefeldin A resulted in redistribution of Golgi-resident glycosyltransferases (including both alpha 2----3 and alpha 2----6 sialyltransferases) to the ER. This redistribution was sensitive to GTP gamma S. Our results suggest that GTP-binding proteins are involved in the regulation not only of the anterograde, but also of the retrograde, pathway.
Primary structure of NuMA, an intranuclear protein that defines a novel pathway for segregation of proteins at mitosis.
Authors: Compton D A , Szilak I , Cleveland D W
Published: May 2004

From a collection of monoclonal antibodies that specifically bind to various parts of the mitotic apparatus in human cells (1991. J. Cell Biol. 112: 1083-1097), two (1F1 and 1H1) recognize a greater than 200-kD intranuclear protein that associates with the spindle immediately upon nuclear envelope breakdown and progresses down the spindle microtubules to concentrate ultimately at the pericentrosomal region. At the completion of anaphase this protein dissociates from the spindle microtubules and is imported into the regenerating nuclei through the nuclear pores. Overlapping cDNA clones that span the entire length of the corresponding 7.2-kb mRNA reveal an encoded polypeptide of 236,278 D that is predicted to contain two globular domains separated by a discontinuous alpha-helix with characteristics for adopting a coiled-coil structure. The corresponding gene is highly conserved but neither the DNA sequence nor the predicted amino acid sequence shows significant homology to any previously reported. Since the cDNA also encodes the epitopes recognized by antibodies specific for two previously described proteins, NuMA and centrophilin, and all three show similar molecular weights and localization during the cell cycle, NuMA, centrophilin, and the 1F1/1H1 antigen represent either the same protein or a family of proteins, for which the original name, NuMA, seems most appropriate. While the function of NuMA remains uncertain, its unusual pattern of segregation at mitosis defines a novel pathway for the segregation of nuclear proteins during cell division.
Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to-pole motion.
Authors: Shelden E , Wadsworth P
Published: May 2004

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.
Protein phosphatase type 1 in mammalian cell mitosis: chromosomal localization and involvement in mitotic exit.
Authors: Fernandez A , Brautigan D L , Lamb N J
Published: May 2004

We have examined the role of protein phosphatase type 1 (PP-1) in mammalian cell mitosis. Immunofluorescence using anti-PP-1 antibodies revealed that PP-1, which is mainly localized in the cytoplasm of G1 and S phase cells, accumulates in the nucleus during G2 phase and intensely colocalizes with individual chromosomes at mitosis. This increase in nuclear PP-1 in G2/M cells was confirmed by immunoblotting on subcellular fractions. Microinjection of neutralizing anti-PP-1 antibodies before division blocked cells at metaphase, whereas injection of PP-1 in one pole of an anaphase B cell accelerated cytokinesis and the reflattening of the injected cell. These results reveal a specific cell cycle-dependent redistribution of PP-1 and its involvement in reversing p34cdc2-induced effects after mid-mitosis in mammalian cells.
Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.
Authors: Buendia B , Draetta G , Karsenti E
Published: May 2004

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.
GAP-43 is expressed by nonmyelin-forming Schwann cells of the peripheral nervous system.
Authors: Curtis R , Stewart H J , Hall S M , Wilkin G P , Mirsky R , Jessen K R
Published: May 2004

Recently it has been demonstrated that the growth-associated protein GAP-43 is not confined to neurons but is also expressed by certain central nervous system glial cells in tissue culture and in vivo. This study has extended these observations to the major class of glial cells in the peripheral nervous system, Schwann cells. Using immunohistochemical techniques, we show that GAP-43 immunoreactivity is present in Schwann cell precursors and in mature non-myelin-forming Schwann cells both in vitro and in vivo. This immunoreactivity is shown by Western blotting to be a membrane-associated protein that comigrates with purified central nervous system GAP-43. Furthermore, metabolic labeling experiments demonstrate definitively that Schwann cells in culture can synthesize GAP-43. Mature myelin-forming Schwann cells do not express GAP-43 but when Schwann cells are removed from axonal contact in vivo by nerve transection GAP-43 expression is upregulated in nearly all Schwann cells of the distal stump by 4 wk after denervation. In contrast, in cultured Schwann cells GAP-43 is not rapidly upregulated in cells that have been making myelin in vivo. Thus the regulation of GAP-43 appears to be complex and different from that of other proteins associated with nonmyelin-forming Schwann cells such as N-CAM, glial fibrillary acidic protein, A5E3, and nerve growth factor receptor, which are rapidly upregulated in myelin-forming cells after loss of axonal contact. These observations suggest that GAP-43 may play a more general role in the nervous system than previously supposed.
Intercellular space is affected by the polysialic acid content of NCAM.
Authors: Yang P , Yin X , Rutishauser U
Published: May 2004

We have previously proposed that polysialic acid (PSA), which is attached to NCAM on the cell surface, can serve to regulate a variety of cell-cell interactions. The present study provides evidence that hydrated PSA influences a sufficiently large volume at the cell surface to exert broad steric effects, and that the removal of PSA in fact causes a detectable change in intercellular space. Using F11 neuron/neuroblastoma hybrid cells as a model system, the measured density and size of PSA suggests that a substantial fraction of the space between two apposed cell surface membranes could be sterically influenced by the presence of PSA. Specific enzymatic removal of PSA, which is similar in magnitude to changes that occur in many tissues during normal development, caused about a 25% decrease in the distance between two apposed cells. By contrast, removal of both heparan sulfate and chondroitin sulfate from the cells had no effect on this parameter. It is proposed that such changes in membrane-membrane distance could serve to alter selectively the efficiency of encounter between complementary receptors on apposing cells, and explain at least in part the broad biological influences of PSA.
Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons
Authors: Okabe S , Hirokawa N
Published: May 2004

To characterize the behavior of axonal microtubules in vivo, we analyzed the movement of tubulin labeled with caged fluorescein after activation to be fluorescent by irradiation of 365-nm light. When mouse sensory neurons were microinjected with caged fluorescein-labeled tubulin and then a narrow region of the axon was illuminated with a 365-nm microbeam, photoactivated tubulin was stationary regardless of the position of photoactivation. We next introduced caged fluorescein-labeled tubulin into Xenopus embryos and nerve cells isolated from injected embryos were analyzed by photoactivation. In this case, movement of the photoactivated zone toward the axon tip was frequently observed. The photoactivated microtubule segments in the Xenopus axon moved out from their initial position without significant spreading, suggesting that fluorescent microtubules are not sliding as individual filaments, but rather translocating en bloc. Since these observations raised the possibility that the mechanism of nerve growth might differ between two types of neurons, we further characterized the movement of another component of the axon structure, the plasma membrane. Analysis of the position of polystyrene beads adhering to the neurites of Xenopus neurons revealed anterograde movement of the beads at the rate similar to the rate of microtubule movement. In contrast, no movement of the beads relative to the cell body was observed in mouse sensory neurons. These results suggest that the mode of translocation of cytoskeletal polymers and some components of the axon surface differ between two neuron types and that most microtubules are stationary within the axon of mammalian neurons where the surface-related motility of the axon is not observed.
Tissue and cellular distribution of the extended family of protein kinase C isoenzymes
Authors: Wetsel WC , Khan WA , Merchenthaler I , Rivera H , Halpern AE , Phung HM , Negro-Vilar A , Hannun YA
Published: May 2004

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.
The Caenorhabditis elegans unc-93 gene encodes a putative transmembrane protein that regulates muscle contraction
Authors: Levin JZ , Horvitz HR
Published: May 2004

unc-93 is one of a set of five interacting genes involved in the regulation or coordination of muscle contraction in Caenorhabditis elegans. Rare altered-function alleles of unc-93 result in sluggish movement and a characteristic "rubber band" uncoordinated phenotype. By contrast, null alleles cause no visibly abnormal phenotype, presumably as a consequence of the functional redundancy of unc-93. To understand better the role of unc-93 in regulating muscle contraction, we have cloned and molecularly characterized this gene. We isolated transposon-insertion alleles and used them to identify the region of DNA encoding the unc-93 protein. Two unc-93 proteins differing at their NH2 termini are potentially encoded by transcripts that differ at their 5' ends. The putative unc-93 proteins are 700 and 705 amino acids in length and have two distinct regions: the NH2 terminal portion of 240 or 245 amino acids is extremely hydrophilic, whereas the rest of the protein has multiple potential membrane-spanning domains. The unc-93 transcripts are low in abundance and the unc-93 gene displays weak codon usage bias, suggesting that the unc-93 protein is relatively rare. The unc-93 protein has no sequence similarity to proteins listed in current data-bases. Thus, unc-93 is likely to encode a novel membrane-associated muscle protein. We discuss possible roles for the unc-93 protein either as a component of an ion transport system involved in excitation-contraction coupling in muscle or in coordinating muscle contraction between muscle cells by affecting the functioning of gap junctions.
The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation
Authors: Migliaccio G , Nicchitta CV , Blobel G
Published: May 2004

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.
Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5-trisphosphate-dependent mechanism
Authors: Sandberg K , Ji H , Iida T , Catt KJ
Published: May 2004

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.
Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan
Authors: Carey DJ , Evans DM , Stahl RC , Asundi VK , Conner KJ , Garbes P , Cizmeci-Smith G
Published: May 2004

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.
Kinetics of binding, endocytosis, and recycling of EGF receptor mutants
Authors: Felder S , LaVin J , Ullrich A , Schlessinger J
Published: May 2004

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.
Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13)
Authors: Snipes GJ , Suter U , Welcher AA , Shooter EM
Published: May 2004

We have recently described a novel cDNA, SR13 (Welcher, A. A., U. Suter, M. De Leon, G. J. Snipes, and E. M. Shooter. 1991. Proc. Natl. Acad. Sci. USA. 88:7195-7199), that is repressed after sciatic nerve crush injury and shows homology to both the growth arrest-specific mRNA, gas3 (Manfioletti, G., M. E. Ruaro, G. Del Sal, L. Philipson, and C. Schneider, 1990. Mol. Cell Biol. 10:2924-2930), and to the myelin protein, PASII (Kitamura, K., M. Suzuki, and K. Uyemura. 1976. Biochim. Biophys. Acta. 455:806-816). In this report, we show that the 22-kD SR13 protein is expressed in the compact portion of essentially all myelinated fibers in the peripheral nervous system. Although SR13 mRNA was found in the central nervous system, no corresponding SR13 protein could be detected by either immunoblot analysis or by immunohistochemistry. Northern and immunoblot analysis of SR13 mRNA and protein expression during development of the peripheral nervous system reveal a pattern similar to other myelin proteins. Furthermore, we demonstrate by in situ mRNA hybridization on tissue sections and on individual nerve fibers that SR13 mRNA is produced predominantly by Schwann cells. We conclude that the SR13 protein is apparently exclusively expressed in the peripheral nervous system where it is a major component of myelin. Thus, we propose the name Peripheral Myelin Protein-22 (PMP-22) for the proteins and cDNA previously designated PASII, SR13, and gas3.
Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells
Authors: Green SA , Kelly RB
Published: May 2004

Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.