bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of M-phase chromatin on the anisotropy of microtubule asters.
Authors: Dogterom M , Félix M A , Guet C C , Leibler S
Published: May 2004

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" by kinetochores, may be stabilized by chromatin in two different ways: short-range stabilization effects may affect microtubules in close contact with the chromatin, while long-range stabilization effects may "guide" microtubule growth towards the chromatin (e.g., by introducing a diffusive gradient of an enzymatic activity that affects microtubule assembly). Here, we use both meiotic and mitotic extracts from Xenopus laevis eggs to study microtubule aster formation and microtubule dynamics in the presence of chromatin. In "low-speed" meiotic extracts, in the presence of salmon sperm chromatin, we find that short-range stabilization effects lead to a strong anisotropy of the microtubule asters. Analysis of the dynamic parameters of microtubule growth show that this anisotropy arises from a decrease in the catastrophe frequency, an increase in the rescue frequency and a decrease in the growth velocity. In this system we also find evidence for long-range "guidance" effects, which lead to a weak anisotropy of the asters. Statistically relevant results on these long-range effects are obtained in "high-speed" mitotic extracts in the presence of artificially constructed chromatin stripes. We find that aster anisotropy is biased in the direction of the chromatin and that the catastrophe frequency is reduced in its vicinity. In this system we also find a surprising dependence of the catastrophe and the rescue frequencies on the length of microtubules nucleated from centrosomes: the catastrophe frequency increase and the rescue frequency decreases with microtubule length.
Microtubule transport and assembly during axon growth.
Authors: Yu W , Schwei M J , Baas P W
Published: May 2004

There is controversy concerning the mechanisms by which the axonal microtubule (MT) array is elaborated, with some models focusing on MT assembly and other models focusing on MT transport. We have proposed a composite model in which MT assembly and transport are both important (Joshi, H.C., and P.W. Baas. 1993. J. Cell Biol. 121:1191-1196). In the present study, we have taken a novel approach to evaluate the merits of this proposal. Biotinylated tubulin was microinjected into cultured neurons that had already grown short axons. The axons were then permitted to grow longer, after which the cells were prepared for immunoelectron microscopic analyses. We reasoned that any polymer that assembled or turned over subunits after the introduction of the probe should label for biotin, while any polymer that was already assembled but did not turnover should not label. Therefore, the presence in the newly grown region of the axon of any unlabeled MT polymer is indicative of MT transport. In sampled regions, the majority of the polymer was labeled, indicating that MT assembly events are active during axon growth. Varying amounts of unlabeled polymer were also present in the newly grown regions, indicating that MT transport also occurs. Together these findings demonstrate that MT assembly and transport both contribute to the elaboration of the axonal MT array.
Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells.
Authors: Saada A , Reichert F , Rotshenker S
Published: May 2004

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.
Partial laminin alpha2 chain restoration in alpha2 chain-deficient dy/dy mouse by primary muscle cell culture transplantation.
Authors: Vilquin J T , Kinoshita I , Roy B , Goulet M , Engvall E , Tomé F , Fardeau M , Tremblay J P
Published: May 2004

Laminin-2 is a component of skeletal and cardiac basal lamina expressed in normal mouse and human. Laminin alpha2 chain (LAMA2), however, is absent from muscles of some congenital muscular dystrophy patients and the dystrophia muscularis (dy/dy) mouse model. LAMA2 restoration was investigated following cell transplantation in vivo in dy/dy mouse. Allogeneic primary muscle cell cultures expressing the beta-galactosidase transgene under control of a muscular promoter, or histocompatible primary muscle cell cultures, were transplanted into dy/dy mouse muscles. FK506 immunosuppression was used in noncompatible models. All transplanted animals expressed LAMA2 in these immunologically-controlled models, and the degrees of LAMA2 restoration were shown to depend on the age of the animal at transplantation, on muscle pretreatment, and on duration time after transplantation in some cases. LAMA2 did not always colocalize with new or hybrid muscle fibers formed by the fusion of donor myoblasts. LAMA2 deposition around muscle fibers was often segmental and seemed to radiate from the center to the periphery of the injection site. Allogeneic conditionally immortalized pure myogenic cells expressing the beta-galactosidase transgene were characterized in vitro and in vivo. When injected into FK506-immunosuppressed dy/dy mice, these cells formed new or hybrid muscle fibers but essentially did not express LAMA2 in vivo. These data show that partial LAMA2 restoration is achieved in LAMA2-deficient dy/dy mouse by primary muscle cell culture transplantation. However, not all myoblasts, or myoblasts alone, or the muscle fibers they form are capable of LAMA2 secretion and deposition in vivo.
Recognition of two classes of oligoproline sequences in profilin-mediated acceleration of actin-based Shigella motility.
Authors: Zeile W L , Purich D L , Southwick F S
Published: May 2004

The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.
Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast.
Authors: Wells W A , Murray A W
Published: May 2004

The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis.
Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s).
Authors: Yamamoto A , Guacci V , Koshland D
Published: May 2004

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA-damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.
Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells.
Authors: Yoshimori T , Keller P , Roth M G , Simons K
Published: May 2004

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.
The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.
Authors: Dyer J M , McNew J A , Goodman J M
Published: May 2004

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.
Induction of DNA synthesis and apoptosis in cardiac myocytes by E1A oncoprotein.
Authors: Liu Y , Kitsis R N
Published: May 2004

Beginning during the second half of gestation, increasing numbers of cardiac myocytes withdraw from the cell cycle such that DNA synthesis is no longer detectable in these cells by neonatal day 17 in vivo. The mechanisms that exclude these and other terminally differentiated cells from the cell division cycle are poorly understood. To begin to explore the molecular basis of the barrier to G1/S progression in cardiac myocytes, we used adenoviruses to express wild-type and mutant E1A proteins in primary cultures from embryonic day 20 rats. While most of these cardiac myocytes are ordinarily refractory to DNA synthesis, even in the presence of serum growth factors, expression of wild-type E1A stimulates DNA synthesis in up to 94% or almost all successfully transduced cells. Rather than complete the cell cycle, however, these cells undergo apoptosis. Apoptosis is limited to those cells that engage in DNA synthesis, and the kinetics of the two processes suggest that DNA synthesis precedes apoptosis. Mutations in E1A that disable it from binding Rb and related pocket proteins have little effect on its ability to stimulate DNA synthesis in cardiac myocytes. In contrast, mutants that are defective in binding the cellular protein p300 stimulate DNA synthesis 2.4-4.1-fold less efficiently, even in the context of retained E1A pocket protein binding. In the absence of ElA pocket protein binding, the usual situation in the cell, loss of p300 binding severely decreases the ability of ElA to stimulate DNA synthesis. These results suggest that the barrier to G1/S progression in cardiac myocytes is mediated. at least in part, by the same molecules that gate the G1/S transition in actively cycling cells, and that p300 or related family members play an important role in this process.
Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.
Authors: French L E , Hahne M , Viard I , Radlgruber G , Zanone R , Becker K , Müller C , Tschopp J
Published: May 2004

The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.
Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein.
Authors: Zhu X , Ohtsubo M , Böhmer R M , Roberts J M , Assoian R K
Published: May 2004

Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1- and E-dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.
Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation.
Authors: De Arcangelis A , Neuville P , Boukamel R , Lefebvre O , Kedinger M , Simon-Assmann P
Published: May 2004

The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation.
A novel role for the integrin-binding III-10 module in fibronectin matrix assembly.
Authors: Hocking D C , Smith R K , McKeown-Longo P J
Published: May 2004

Fibronectin matrix assembly is a cell-dependent process which is upregulated in tissues at various times during development and wound repair to support the functions of cell adhesion, migration, and differentiation. Previous studies have demonstrated that the alpha 5 beta 1 integrin and fibronectin's amino terminus and III-1 module are important in fibronectin polymerization. We have recently shown that fibronectin's III-1 module contains a conformationally sensitive binding site for fibronectin's amino terminus (Hocking, D.C., J. Sottile, and P.J. McKeown-Longo. 1994. J. Biol. Chem. 269: 19183-19191). The present study was undertaken to define the relationship between the alpha 5 beta 1 integrin and fibronectin polymerization. Solid phase binding assays using recombinant III-10 and III-1 modules of human plasma fibronectin indicated that the III-10 module contains a conformation-dependent binding site for the III-1 module of fibronectin. Unfolded III-10 could support the formation of a ternary complex containing both III-1 and the amino-terminal 70-kD fragment, suggesting that the III-1 module can support the simultaneous binding of III-10 and 70 kD. Both unfolded III-10 and unfolded III-1 could support fibronectin binding, but only III-10 could promote the formation of disulfide-bonded multimers of fibronectin in the absence of cells. III-10-dependent multimer formation was inhibited by both the anti-III-1 monoclonal antibody, 9D2, and amino-terminal fragments of fibronectin. A fragment of III-10, termed III-10/A, was able to block matrix assembly in fibroblast monolayers. Similar results were obtained using the III-10A/RGE fragment, in which the RGD site had been mutated to RGE, indicating that III-I0/A was blocking matrix assembly by a mechanism distinct from disruption of integrin binding. Texas red-conjugated recombinant III-1,2 localized to beta 1-containing sites of focal adhesions on cells plated on fibronectin or the III-9,10 modules of fibronectin. Monoclonal antibodies against the III-1 or the III-9,10 modules of fibronectin blocked binding of III-1,2 to cells without disrupting focal adhesions. These data suggest that a role of the alpha 5 beta 1 integrin in matrix assembly is to regulate a series of sequential self-interactions which result in the polymerization of fibronectin.
Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen.
Authors: Satoh M , Hirayoshi K , Yokota S , Hosokawa N , Nagata K
Published: May 2004

Heat shock protein 47 (HSP47), a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER. We previously demonstrated that HSP47 transiently associated with newly synthesized procollagen in the ER (Nakai, A., M. Satoh, K. Hirayoshi, and K. Nagata. 1992. J. Cell Biol. 117:903-914). In the present work, we examined the location where HSP47 binds to and dissociates from newly synthesized procollagen within the cells, and whether HSP47 associates with nascent single procollagen polypeptide chains and/or with mature triple-helix procollagen. This was accomplished by biochemical coprecipitation with anti-HSP47 and anticollagen antibodies, combined with pulse-label and chase experiments in the presence or absence of various inhibitors for protein secretion, as well as by confocal laser microscopic observation of the cells double stained with both antibodies. We further examined whether the RDEL (Arg-Asp-Glu-Leu) sequence at the COOH terminus of HSP47 can act as an ER-retention signal, as the KDEL sequence does. When the secretion of procollagen was inhibited by the presence of alpha, alpha'-dipyridyl, an iron chelator that inhibits procollagen triple-helix formation, or by the presence of brefeldin A. which inhibits protein transport between the ER and the Golgi apparatus, procollagen was found to be bound to HSP47 during the chase period in the intermediate compartment. In contrast, the dissociation of procollagen chains from HSP47 was not inhibited when procollagen secretion was inhibited by monensin or bafilomycin A1, both of which are known to be inhibitors of post-cis-Golgi transport. These findings suggest that HSP47 and procollagen dissociated between the post-ER and the cis-Golgi compartments. HSP47 was shown to bind to nascent, single-polypeptide chains of newly synthesized procollagen, as well as to the mature triple-helix form of procollagen. HSP47 with the RDEL sequence deleted was secreted out of the cells, which suggests that the RDEL sequence actually acts as an ER-retention signal, as the KDEL sequence does. This secreted HSP47 did not acquire endoglycosidase H resistance. The biological significance of the interaction between HSP47 and procollagen in the central secretory pathway, as well as possible mechanisms for this pathway, will be discussed.
RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus.
Authors: Palacios I , Weis K , Klebe C , Mattaj I W , Dingwall C
Published: May 2004

Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have different defects; Q69L (glutamine 69 changed to leucine) is defective in GTP hydrolysis while T24N (threonine 24 changed to asparagine) is defective in binding GTP. Both protein and snRNP import are sensitive either to the presence of the two mutant Ran proteins, which act as dominant negative inhibitors of nuclear import, or to incubation with nonhydrolyzable GTP analogues. This demonstrates that there is a requirement for a GTPase activity for the import of U snRNPs, as well as proteins, into the nucleus. The dominant negative effects of the two mutant Ran proteins indicate that the pathways of protein and snRNP import share at lease one common component.
Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases.
Authors: Cooper A A , Stevens T H
Published: May 2004

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.
Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells.
Authors: Müsch A , Xu H , Shields D , Rodriguez-Boulan E
Published: May 2004

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.
Three-dimensional structure of the Z band in a normal mammalian skeletal muscle.
Authors: Schroeter J P , Bretaudiere J P , Sass R L , Goldstein M A
Published: May 2004

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three-dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z-filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.
A purified Drosophila septin complex forms filaments and exhibits GTPase activity.
Authors: Field C M , al-Awar O , Rosenblatt J , Wong M L , Alberts B , Mitchison T J
Published: May 2004

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.
Cell type-specific roles for Cdc42, Rac, and RhoL in Drosophila oogenesis.
Authors: Murphy A M , Montell D J
Published: May 2004

The Rho subfamily of GTPases has been shown to regulate cellular morphology. We report the discovery of a new member of the Rho family, named RhoL, which is equally similar to Rac, Rho, and Cdc42. Expression of a dominant-negative RhoL transgene in the Drosophila ovary caused nurse cells to collapse and fuse together. Mutant forms of Cdc42 mimicked this effect. Expression of constitutively active RhoL led to nurse cell subcortical actin breakdown and disruption of nurse cell-follicle cell contacts, followed by germ cell apoptosis. In contrast, Rac activity was specifically required for migration of a subset of follicle cells called border cells. All three activities were necessary for normal transfer of nurse cell cytoplasm to the oocyte. These results suggest that Rho protein activities have cell type-specific effects on morphogenesis.
Neurotrophins promote the survival and development of neurons in the cerebellum of hypothyroid rats in vivo.
Authors: Neveu I , Arenas E
Published: May 2004

The development of cerebellar cortex is strongly impaired by thyroid hormone (T3) deficiency, leading to altered migration, differentiation, synaptogenesis, and survival of neurons. To determine whether alteration in the expression of neurotrophins and/or their receptors may contribute to these impairments, we first analyzed their expression using a sensitive RNAse protection assay and in situ hybridization; second, we administered the deficient neurotrophins to hypothyroid animals. We found that early hypothyroidism disrupted the developmental pattern of expression of the four neurotrophins, leading to relatively higher levels of NGF and neurotrophin 4/5 mRNAs and to a severe deficit in NT-3 and brain-derived neurotrophic factor (BDNF) mRNA expression, without alteration in the levels of the full-length tyrosine kinase (trk) B and trkC receptor mRNAs. Grafting of P3 hypothyroid rats with cell lines expressing high levels of neurotrophin 3 (NT-3) or BDNF prevented hypothyroidism-induced cell death in neurons of the internal granule cell layer at P15. In addition, we found that NT-3, but not BDNF, induced the differentiation and/or migration of neurons in the external granule cell layer, stimulated the elaboration of the dendritic tree by Purkinje cells, and promoted the formation of the mature pattern of synaptic afferents to Purkinje cell somas. Thus, our results indicate that both granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo and suggest that T3 may regulate the levels of neurotrophins to promote the development of cerebellum.
Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites.
Authors: Dubreuil R R , MacVicar G , Dissanayake S , Liu C , Homer D , Hortsch M
Published: May 2004

The protein ankyrin links integral membrane proteins to the spectrin-based membrane skeleton. Ankyrin is often concentrated within restricted membrane domains of polarized epithelia and neurons, but the mechanisms responsible for membrane targeting and its segregation within a continuous lipid bilayer remain unexplained. We provide evidence that neuroglian, a cell adhesion molecule related to L1 and neurofascin, can transmit positional information directly to ankyrin and thereby polarize its distribution in Drosophila S2 tissue culture cells. Ankyrin was not normally associated with the plasma membrane of these cells. Upon expression of an inducible neuroglian minigene, however, cells aggregated into large clusters and ankyrin became concentrated at sites of cell-cell contact. Spectrin was also recruited to sites of cell contact in response to neuroglian expression. The accumulation of ankyrin at cell contacts required the presence of the cytoplasmic domain of neuroglian since a glycosyl phosphatidylinositol-linked form of neuroglian failed to recruit ankyrin to sites of cell-cell contact. Double-labeling experiments revealed that, whereas ankyrin was strictly associated with sites of cell-cell contact, neuroglian was more broadly distributed over the cell surface. A direct interaction between neuroglian and ankyrin was demonstrated using yeast two-hybrid analysis. Thus, neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of cell contact and not with other regions of the plasma membrane.
Heterophilic interactions of DM-GRASP: GRASP-NgCAM interactions involved in neurite extension.
Authors: DeBernardo A P , Chang S
Published: May 2004

DM-GRASP is an immunoglobulin superfamily cell adhesion molecule that is expressed in both the developing nervous and immune system. Specific populations of neurons respond to DM-GRASP substrates appears to require homophilic interactions between DM-GRASP molecules. We were interested in determining whether DM-GRASP interacts heterophilically with other ligands as well. We have found that eleven proteins from embryonic chick brain membranes consistently bind to and elute from a DM-GRASP-Sepharose affinity column. One of these proteins is DM-GRASP itself, consistent with its known homophilic binding. Another protein, at 130 kD, is immunoreactive with monoclonal antibodies to NgCAM. Other neural cell adhesion molecules were not detected in the eluate. The DM-GRASP-Sepharose eluate also contains a potent neurite stimulating activity, which cannot be accounted for by either DM-GRASP or NgCAM. To investigate the interaction of DM-GRASP and NgCAM, antibodies against DM-GRASP were added to neuronal cultures extending neurites on an NgCAM substrate. The presence of antibodies to DM-GRASP decreased neurite extension on laminin, suggesting that the antibody is not toxic or generally inhibiting motility. We present two possible models for the DM-GRASP-NgCAM association and a hypothesis for neural cell adhesion function that features the dimerization of cell adhesion molecules.
Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis.
Authors: Kanai Y , Kanai-Azuma M , Noce T , Saido T C , Shiroishi T , Hayashi Y , Yazaki K
Published: May 2004

The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells.
Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2.
Authors: Schwall R H , Chang L Y , Godowski P J , Kahn D W , Hillan K J , Bauer K D , Zioncheck T F
Published: May 2004

Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.
Intron-dependent recruitment of pre-mRNA splicing factors to sites of transcription.
Authors: Huang S , Spector D L
Published: May 2004

We have examined the nuclear localization of transiently and stably expressed nascent RNA transcripts containing or lacking introns in order to determine if the spatial association of RNA transcripts and pre-mRNA splicing factors in nuclei is random or functionally significant. Our findings show that the association between nascent RNA and splicing factors in the nucleus is intron-dependent when the RNAs are either transiently or stably expressed. Furthermore, our data indicate that splicing factors are recruited to the transcription sites. The presence of both pre-and mRNA at these locations suggest that pre-mRNA splicing occurs at these sites of transcription. In addition, electron microscopic examination of the highly active transcription sites has revealed a granular appearance which closely resembles, but is functionally different from, interchromatin granule clusters. Our findings demonstrate that the nucleus is highly organized and dynamic with regard to the functions of the transcription and pre-mRNA splicing.