bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of dendritic microtubules establishes their nonuniform polarity orientation.
Authors: Sharp D J , Yu W , Baas P W
Published: May 2004

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus-ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.
Sequence and characterization of cytoplasmic nuclear protein import factor p97.
Authors: Chi N C , Adam E J , Adam S A
Published: May 2004

Nuclear location sequence-mediated binding of karyophilic proteins to the nuclear pore complexes is one of the earliest steps in nuclear protein import. We previously identified two cytosolic proteins that reconstitute this step in a permeabilized cell assay: the 54/56-kD NLS receptor and p97. A monoclonal antibody to p97 localizes the protein to the cytoplasm and the nuclear envelope. p97 is extracted from nuclear envelopes under the same conditions as the O-glycosylated nucleoporins indicating a tight association with the pore complex. The antibody inhibits import in a permeabilized cell assay but does not affect binding of karyophiles to the nuclear pore complex. Immunodepletion of p97 renders the cytosol inactive for import and identifies at least three other cytosolic proteins that interact with p97. cDNA cloning of p97 shows that it is a unique protein containing 23 cysteine residues. Recombinant p97 binds zinc and a bound metal ion is required for the nuclear envelope binding activity of the protein.
trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention.
Authors: Wahlberg J M , Geffen I , Reymond F , Simmen T , Spiess M
Published: May 2004

Unlike the wild-type asialoglycoprotein receptor subunit H1 which is transported to the cell surface, endocytosed and recycled, a mutant lacking residues 4-33 of the 40-amino acid cytoplasmic domain was found to be retained intracellularly upon expression in different cell lines. The mutant protein accumulated in the trans-Golgi, as judged from the acquisition of trans-Golgi-specific modifications of the protein and from the immunofluorescence staining pattern. It was localized to juxtanuclear, tubular structures that were also stained by antibodies against galactosyltransferase and gamma-adaptin. The results of further mutagenesis in the cytoplasmic domain indicated that the size rather than the specific sequence of the cytoplasmic domain determines whether H1 is retained in the trans-Golgi or transported to the cell surface. Truncation to less than 17 residues resulted in retention, and extension of a truncated tail by an unrelated sequence restored surface transport. The transmembrane segment of H1 was not sufficient for retention of a reporter molecule and it could be replaced by an artificial apolar sequence without affecting Golgi localization. The cytoplasmic domain thus appears to inhibit interaction(s) of the exoplasmic portion of H1 with trans-Golgi component(s) for example by steric hindrance or by changing the positioning of the protein in the membrane. This mechanism may also be functional in other proteins.
Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae.
Authors: TerBush D R , Novick P
Published: May 2004

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.
c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.
Authors: Chang J H , Gill S , Settleman J , Parsons S J
Published: May 2004

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-Src variants to potentiate or inhibit EGF-dependent DNA synthesis is the phosphotyrosine content of multiple cellular proteins, including p190-RhoGAP, a protein thought to regulate growth factor-induced actin cytoskeleton remodeling by modulating the activity of the small GTP binding protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whether tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dependent actin remodeling, we used conventional and confocal immunofluorescence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo control cells and cells that stably overexpress wild-type (K+) or kinase-defective (K-) c-Src. We found that in all cell lines, EGF induced a rapid and transient condensation of p190 and RasGAP into cytoplasmic, arclike structures. However, in K+ cells the rate of appearance and number of cells exhibiting arcs increased when compared with control cells. Conversely, K- cells exhibited delayed arc formation and a reduction in number of cells forming arcs. EGF-induced actin stress fiber disassembly and reassembly occurred with the same kinetics and frequency as did p190 and RasGAP rearrangements in all three cell lines. These results, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, suggest that c-Src regulates EGF-dependent actin cytoskeleton reorganization through phosphorylation of p190.
p120, a p120-related protein (p100), and the cadherin/catenin complex.
Authors: Staddon J M , Smales C , Schulze C , Esch F S , Rubin L L
Published: May 2004

Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, beta and gamma, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60src. In this paper, we show that endothelial and epithelial cells express p120 and p100, a 100-kD, p120-related protein. Peptide sequencing of p100 establishes it as highly related to p120. p120 and p100 both appear associated with the cadherin/catenin complex, but independent p120/catenin and p100/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/p100 antibody, and of p120/p100 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and beta-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and beta-catenin is similar in MDCK cells, but only approximately 20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.
Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells.
Authors: Sullivan R , Lo C W
Published: May 2004

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.
In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure.
Authors: Pittler S J , Fliesler S J , Fisher P L , Keller P K , Rapp L M
Published: May 2004

Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from those induced with lovastatin. These results implicate a defect in protein prenylation in the lovastatin-induced retinal degeneration, and suggest the presence of a dynamic pathway in the retina that requires isoprenylated proteins to maintain retinal cytoarchitecture.
Characterization of functional domains of the tenascin-R (restrictin) polypeptide: cell attachment site, binding with F11, and enhancement of F11-mediated neurite outgrowth by tenascin-R.
Authors: Nörenberg U , Hubert M , Brümmendorf T , Tárnok A , Rathjen F G
Published: May 2004

The extracellular matrix glycoprotein tenascin-R (TN-R) is a multidomain protein implicated in neural cell adhesion. To analyze the structure-function relationship of the different domains of TN-R, several recombinant TN-R fragments were expressed in bacterial cells. Two distinct binding regions were localized on the TN-R polypeptide: a region binding the axon-associated immunoglobulin (Ig)-like F11 protein and a cell attachment site. The binding region of the glycosylphosphatidylinositol (GPI)-anchored F11 was allocated to the second and third fibronectin type III (FNIII)-like domain within TN-R. By using a mutant polypeptide of F11 containing only Ig-like domains, a direct interaction between the Ig-like domains of F11 and FNIII-like domains 2-3 of TN-R was demonstrated. The interaction of TN-R with F11 in in vitro cultures enhanced F11-mediated neurite outgrowth, suggesting that the combined action of F11 and TN-R might be of regulatory influence on axon extension. A cell attachment region was identified in the FNIII-like domain eight of TN-R by domain-specific antibodies and fusion constructs. This site is distinct from the F11 binding site within TN-R.
DNM1, a dynamin-related gene, participates in endosomal trafficking in yeast.
Authors: Gammie A E , Kurihara L J , Vallee R B , Rose M D
Published: May 2004

We identified DNM1, a novel dynamin-related gene in Saccharomyces cerevisiae. Molecular and genetic mapping showed that DNM1 is the most proximal gene to the right of centromere 12, and is predicted to encode a protein of 85 kD, designated Dnm1p. The protein exhibits 41% overall identity with full-length dynamin I and 55% identity with the most highly conserved 400-amino acid GTPase region. Our findings show that like mammalian dynamin, Dnm1p participates in endocytosis; however, it is unlikely to be a cognate homologue. Cells with a disruption in the DNM1 gene showed mating response defects consistent with a delay in receptor-mediated endocytosis. The half-life of the Ste3p pheromone receptor was increased two- to threefold in the dnm1 mutant, demonstrating that Dnm1p participates in the constitutive turnover of the receptor. To define the step in the endocytic pathway at which Dnm1p acts, we analyzed mutant strains at both early and late steps of the process. Initial internalization of epitope-tagged pheromone receptor or of labeled pheromone proceeded with wild-type kinetics. However, delivery of the internalized receptor to the vacuole was greatly impeded during ligand-induced endocytosis. These data suggest that during receptor-mediated endocytosis, Dnm1p acts after internalization, but before fusion with the vacuole. The dnm1 mutant was not defective for sorting of vacuolar proteins, indicating that Dnm1p is not required for transport from the late endosome to the vacuole. Therefore, we suggest that Dnm1p participates at a novel step before fusion with the late endosome.
Expression of light meromyosin in Dictyostelium blocks normal myosin II function.
Authors: Burns C G , Reedy M , Heuser J , De Lozanne A
Published: May 2004

The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.
F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling.
Authors: Tilney L G , Tilney M S , Guild G M
Published: May 2004

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).
Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.
Authors: Olson K R , McIntosh J R , Olmsted J B
Published: May 2004

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.
Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis.
Authors: McCollum D , Balasubramanian M K , Pelcher L E , Hemmingsen S M , Gould K L
Published: May 2004

Schizosaccharomyces pombe cells divide by medial fission. One class of cell division mutants (cdc), the late septation mutants, defines four genes: cdc3, cdc4, cdc8, and cdc12 (Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Mol. & Gen. Genet. 146:167-178). We have cloned and characterized the cdc4 gene and show that the predicted gene product. Cdc4p, is a 141-amino acid polypeptide that is similar in sequence to EF-hand proteins including myosin light chains, calmodulin, and troponin C. Two temperature-sensitive lethal alleles, cdc4-8 and cdc4-31, accumulate multiple nuclei and multiple improper F-actin rings and septa but fail to complete cytokinesis. Deletion of cdc4 also results in a lethal terminal phenotype characterized by multinucleate, elongated cells that fail to complete cytokinesis. Sequence comparisons suggest that Cdc4p may be a member of a new class of EF-hand proteins. Cdc4p localizes to a ringlike structure in the medial region of cells undergoing cytokinesis. Thus, Cdc4p appears to be an essential component of the F-actin contractile ring. We find that Cdc4 protein forms a complex with a 200-kD protein which can be cross-linked to UTP, a property common to myosin heavy chains. Together these results suggest that Cdc4p may be a novel myosin light chain.
Members of the NAP/SET family of proteins interact specifically with B-type cyclins.
Authors: Kellogg D R , Kikuchi A , Fujii-Nakata T , Turck C W , Murray A W
Published: May 2004

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).
NAP1 acts with Clb1 to perform mitotic functions and to suppress polar bud growth in budding yeast.
Authors: Kellogg D R , Murray A W
Published: May 2004

NAP1 is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. We have examined the ability of the yeast mitotic cyclin Clb2 to function in cells that lack NAP1. Our results demonstrate that Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1 is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. Our experiments also suggest that NAP1 is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1 plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1 is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle.
A phosphatidylinositol 4,5-bisphosphate-sensitive casein kinase I alpha associates with synaptic vesicles and phosphorylates a subset of vesicle proteins.
Authors: Gross S D , Hoffman D P , Fisette P L , Baas P , Anderson R A
Published: May 2004

In interphase cells, alpha-casein kinase I (alpha-CKI) is found associated with cytosolic vesicular structures, the centrosome, and within the nucleus. To identify the specific vesicular structures with which alpha-CKI is associated, established cell lines and primary rat neurons were immunofluorescently labeled with an antibody raised to alpha-CKI. In nonneuronal cells, alpha-CKI colocalizes with vesicular structures which align with microtubules and are partially coincident with both Golgi and endoplasmic reticulum markers. In neurons, alpha-CKI colocalizes with synaptic vesicle markers. When synaptic vesicles were purified from rat brain, they were highly enriched in a CKI, based on activity and immunoreactivity. The synaptic vesicle-associated CKI is an extrinsic kinase and was eluted from synaptic vesicles and purified. This purified CKI has properties most similar to alpha-CKI. When the activities of casein kinase I or II were specifically inhibited on isolated synaptic vesicles, CKI was shown to phosphorylate a specific subset of vesicle proteins, one of which was identified as the synaptic vesicle-specific protein SV2. As with alpha-CKI, the synaptic vesicle CKI is inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). However, synthesis of PIP2 was detected only in plasma membrane-containing fractions. Therefore, PIP2 may spatially regulate CKI. Since PIP2 synthesis is required for secretion, this inhibition of CKI may be important for the regulation of secretion.
The chemotactic response to PDGF-BB: evidence of a role for Ras.
Authors: Kundra V , Anand-Apte B , Feig L A , Zetter B R
Published: May 2004

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility.
Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein.
Authors: Blystone S D , Lindberg F P , LaFlamme S E , Brown E J
Published: May 2004

Using a K562 cell transfection model, we have previously described a novel relationship between the integrins alpha v beta 3 and alpha 5 beta 1. alpha v beta 3 ligation was able to inhibit alpha 5 beta 1-mediated phagocytosis without effect on alpha 5 beta 1-mediated adhesion. The alpha v beta 3-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the beta 3 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of alpha 5 beta 1 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in alpha v beta 3 signal transduction, mimics the effects of alpha v beta 3 ligation only when the beta 3 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of alpha 5 beta 1, ligation of alpha v beta 3/IAP does not prevent acquisition of this high affinity state. We conclude that alpha v beta 3/IAP ligation initates a signal transduction cascade, dependent upon the beta 3 cytoplasmic tail, which inhibits the phagocytic function of alpha 5 beta 1 at a step subsequent to modulation of integrin affinity.
Large and small splice variants of collagen XII: differential expression and ligand binding.
Authors: Koch M , Bohrmann B , Matthison M , Hagios C , Trueb B , Chiquet M
Published: May 2004

Collagen XII has a short collagenous tail and a very large, three-armed NC3 domains consisting primarily of fibronectin type III repeats. Differential splicing within this domain gives rise to a large (320 kD) and a small (220 kD) subunit; the large but not the small can carry glycosaminoglycan. To investigate whether collagen XII variants have distinct expression patterns and functions, we generated antibody and cDNA probes specific for the alternatively spliced domain. We report here that the large variant has a more restricted expression in embryonic tissue than the small. For example, whereas the small variant is widespread in the dermis, the large is limited to the base of feather buds. Distinct proportions of mRNA for the two variants were detected depending on the tissue. Monoclonal antibodies allowed us to separate collagen XII variants, and to show that homo- and heterotrimers exist. Collagen XII variants differ in ligand binding. Small subunits interact weakly with heparin via their COOH-terminal domain. Large subunits have additional, stronger heparin-binding site(s) in their NH2-terminal extra domain. In vivo, both large and small collagen XII are associated with interstitial collagen. Here we show biochemically and ultrastructurally that collagen XII can be incorporated into collagen I fibrils when it is present during, but not after, fibril formation. Removal of the collagenous domain of collagen XII reduces its coprecipitation with collagen I. Our results indicate that collagen XII is specifically associated with fibrillar collagen, and that the large variant has binding sites for extracellular ligands not present in the small variant.
Early events in DNA replication require cyclin E and are blocked by p21CIP1.
Authors: Jackson P K , Chevalier S , Philippe M , Kirschner M W
Published: May 2004

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.
Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.
Authors: Labriola C , Cazzulo J J , Parodi A J
Published: May 2004

It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.
Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.
Authors: Brown W J , DeWald D B , Emr S D , Plutner H , Balch W E
Published: May 2004

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.
Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes.
Authors: Davidson H W
Published: May 2004

At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells. Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor-ligand complexes from the TGN towards lysosomes.
Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75.
Authors: Vijayasaradhi S , Xu Y , Bouchard B , Houghton A N
Published: May 2004

The structural and functional integrity of cytoplasmic organelles is maintained by intracellular mechanisms that sort and target newly synthesized proteins to their appropriate cellular locations. In melanocytic cells, melanin pigment is synthesized in specialized organelles, melanosomes. A family of melanocyte-specific proteins, known as tyrosinase-related proteins that regulate melanin pigment synthesis, is localized to the melanosomal membrane. The human brown locus protein, tyrosinase-related protein-1 or gp75, is the most abundant glycoprotein in melanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sorting of glycoprotein (gp)75, we constructed protein chimeras containing the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fibroblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelles. Signals in the gp75 cytoplasmic tail alone, were sufficient for intracellular retention and targeting of the chimeric proteins to the endosomal/lysosomal compartment. Analysis of subcellular localization of carboxy-terminal deletion mutants of gp75 and the CD8/gp75 chimeras showed that deletion of up amino acids from the gp75 carboxyl terminus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acids were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conserved among other melanosomal proteins. Further evidence showed that this hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanosomal proteins along the endosomal/lysosomal pathway lies within this sequence. These data provide evidence for common signals for intracellular sorting of melanosomal and lysosomal proteins, and support the notion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.
Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.
Authors: van Weert A W , Dunn K W , Gueze H J , Maxfield F R , Stoorvogel W
Published: May 2004

Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'-diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR-labeled late endosomes, but nearly completely abrogated transport to cathepsin D-labeled lysosomes. From these results, we conclude that trafficking through early and late endosomes, but not to lysosomes, continued upon inactivation of the vacuolar proton pump.
A truncated form of the Pho80 cyclin redirects the Pho85 kinase to disrupt vacuole inheritance in S. cerevisiae.
Authors: Nicolson T A , Weisman L S , Payne G S , Wickner W T
Published: May 2004

Partitioning of the vacuole during cell division in Saccharomyces cerevisiae begins during early S phase and ends in late G2 phase before the yeast nucleus migrates into the bud neck. We have isolated and characterized a new mutant, vac5-1, which is defective in vacuole segregation. Cells with the vac5-1 mutation can form large buds without vacuoles. The VAC5 gene was cloned and is identical to PHO80. PHO80 encodes a cyclin which acts in a complex with a cdc-like kinase, PHO85, as a negative regulator of two transcription factors (PHO2 and PHO4) that govern the expression of metabolic phosphatases. The vacuole inheritance defect in vac5-1 cells is dependent on the presence of the Pho85 kinase and its targets Pho4p and Pho2p. As with other alleles of PHO80, phosphatase levels are elevated in vac5-1 mutants. A suppressor, the COOH-terminal half of the Gal11 transcription factor, rescues the vac5-1 phenotype of defective vacuole inheritance without altering the vac5-1 phenotype of elevated phosphatase levels. In addition, neither maximal nor minimal levels of expression of the inducible "PHO" system phosphatases causes a vacuole inheritance defect. Though vac5-1 is recessive, pho80 delta or pho85 delta strains do not show a defect in vacuole inheritance, suggesting that vac5-1 is not a complete loss-of-function allele. Sequence analysis shows that the vac5-1 allele encodes a truncated form of the Pho80 cyclin and overexpression of vac5-1 in pho80 delta cells causes a vacuole inheritance defect. We conclude that the vac5-1 allele directs the Pho85 kinase to regulate, via transcription factors Pho4 and Pho2, genes that affect vacuole inheritance but which are not known to be under normal PHO pathway control.