bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts.
Authors: Piñol-Roma S , Swanson M S , Gall J G , Dreyfuss G
Published: May 2004

Immediately after the initiation of transcription in eukaryotes, nascent RNA polymerase II transcripts are bound by nuclear proteins resulting in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. hnRNP complexes from HeLa cell nuclei contain greater than 20 major proteins in the molecular mass range of 34,000-120,000 D. Among these are the previously described A, B, and C groups of proteins (34,000-43,000 D) and several larger, and as yet uncharacterized, proteins. Here we describe the isolation and characterization of a novel hnRNP protein termed the L protein (64-68 kD by mobility in SDS-polyacrylamide gels). Although L is a bona fide component of hnRNP complexes, it also appears to be a different type of hnRNP protein from those previously characterized. A considerable amount of L is found outside hnRNP complexes, and monoclonal antibodies to the L protein also strongly stain unidentified discrete nonnucleolar structures, in addition to nucleoplasm, in HeLa cell nuclei. Interestingly, the same antibodies stain the majority of nonnucleolar nascent transcripts from the loops of lampbrush chromosomes in the newt, but the most intense staining is localized to the landmark giant loops. The L protein is the first protein of giant loops identified so far, and antibodies to it thus provide a useful tool with which to study these unique RNAs. In addition, isolation and sequencing of cDNA clones for the L protein from human cells predicts a glycine- and proline-rich protein of 60,187 D, which contains two 80 amino acid segments only distantly related to the RNP consensus sequence-type RNA-binding domain. The L protein, therefore, is a new type of hnRNP protein.
Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.
Authors: Kindle K L , Schnell R A , Fernández E , Lefebvre P A
Published: May 2004

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.
Protein import into mitochondria: ATP-dependent protein translocation activity in a submitochondrial fraction enriched in membrane contact sites and specific proteins.
Authors: Pon L , Moll T , Vestweber D , Marshallsay B , Schatz G
Published: May 2004

To identify the membrane regions through which yeast mitochondria import proteins from the cytoplasm, we have tagged these regions with two different partly translocated precursor proteins. One of these was bound to the mitochondrial surface of ATP-depleted mitochondria and could subsequently be chased into mitochondria upon addition of ATP. The other intermediate was irreversibly stuck across both mitochondrial membranes at protein import sites. Upon subfraction of the mitochondria, both intermediates cofractionated with membrane vesicles whose buoyant density was between that of inner and outer membranes. When these vesicles were prepared from mitochondria containing the chaseable intermediate, they internalized it upon addition of ATP. A non-hydrolyzable ATP analogue was inactive. This vesicle fraction contained closed, right-side-out inner membrane vesicles attached to leaky outer membrane vesicles. The vesicles contained the mitochondrial binding sites for cytoplasmic ribosomes and contained several mitochondrial proteins that were enriched relative to markers of inner or outer membranes. By immunoelectron microscopy, two of these proteins were concentrated at sites where mitochondrial inner and outer membranes are closely apposed. We conclude that these vesicles contain contact sites between the two mitochondrial membranes, that these sites are the entry point for proteins into mitochondria, and that the isolated vesicles are still translocation competent.
Identification of a human protein that interacts with nuclear localization signals.
Authors: Li R H , Thomas J O
Published: May 2004

Through a series of label transfer experiments, we have identified a HeLa cell nuclear protein that interacts with nuclear localization signals (NLSs). The protein has a molecular weight of 66,000 and an isoelectric point of approximately 6. It associates with a synthetic peptide that contains the SV-40 T antigen NLS peptide but not with an analogous peptide in which an asparagine is substituted for an essential lysine (un-NLS peptide). In addition to these peptides, several proteins have been tested as label donors. With the proteins, there is a correlation between nuclear localization (assayed with lysolecithin-permeabilized cells) and label transfer to the 66-kD protein. The NLS peptide (but not the un-NLS peptide) competes with the proteins in label transfer experiments, but neither wheat germ agglutinin nor ATP has an effect. These results suggest that the 66-kD protein functions as an NLS receptor in the first step of nuclear localization. In the course of this work, we have observed that the Staphylococcus aureus protein A is a strongly karyophilic protein. Its dramatic nuclear localization properties suggest that it may have multiple copies of an NLS.
SEC62 encodes a putative membrane protein required for protein translocation into the yeast endoplasmic reticulum.
Authors: Deshaies R J , Schekman R
Published: May 2004

Yeast sec62 mutant cells are defective in the translocation of several secretory precursor proteins into the lumen of the endoplasmic reticulum (Rothblatt et al., 1989). The deficiency, which is most restrictive for alpha-factor precursor (pp alpha F) and preprocarboxypeptidase Y, has been reproduced in vitro. Membranes isolated from mutant cells display low and labile translocation activity with pp alpha F translated in a wild-type cytosol fraction. The defect is unique to the membrane fraction because cytosol from mutant cells supports translocation into membranes from wild-type yeast. Invertase assembly is only partly affected by the sec62 mutation in vivo and is nearly normal with mutant membranes in vitro. A potential membrane location for the SEC62 gene product is supported by evaluation of the molecular clone. DNA sequence analysis reveals a 32-kD protein with no obvious NH2-terminal signal sequence but with two domains of sufficient length and hydrophobicity to span a lipid bilayer. Sec62p is predicted to display significant NH2- and COOH-terminal hydrophilic domains on the cytoplasmic surface of the ER membrane. The last 30 amino acids of the COOH terminus may form an alpha-helix with 14 lysine and arginine residues arranged uniformly about the helix. This domain may allow Sec62p to interact with other proteins of the putative translocation complex.
A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein.
Authors: Sadler I , Chiang A , Kurihara T , Rothblatt J , Way J , Silver P
Published: May 2004

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.
A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus.
Authors: Nakano A , Muramatsu M
Published: May 2004

SAR1, a gene that has been isolated as a multicopy suppressor of the yeast ER-Golgi transport mutant sec12, encodes a novel GTP-binding protein. Its nucleotide sequence predicts a 21-kD polypeptide that contains amino acid sequences highly homologous to GTP-binding domains of many ras-related proteins. Gene disruption experiments show that SAR1 is essential for cell growth. To test its function further, SAR1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SAR1 copy disrupted. This mutant grows normally in galactose medium but arrests growth 12-15 h after transfer to glucose medium. At the same time, mutant cells accumulate ER precursor forms of a secretory pheromone, alpha-mating factor, and a vacuolar enzyme, carboxypeptidase Y. We propose that Sec12p and Sarlp collaborate in directing ER-Golgi protein transport.
Association of ligand-receptor complexes with actin filaments in human neutrophils: a possible regulatory role for a G-protein.
Authors: Särndahl E , Lindroth M , Bengtsson T , Fällman M , Gustavsson J , Stendahl O , Andersson T
Published: May 2004

Most ligand-receptor interactions result in an immediate generation of various second messengers and a subsequent association of the ligand-receptor complex to the cytoskeleton. Depending on the receptor involved, this linkage to the cytoskeleton has been suggested to play a role in the termination of second messenger generation and/or the endocytic process whereby the ligand-receptor complex is internalized. We have studied how the binding of chemotactic peptide-receptor complexes to the cytoskeleton of human neutrophils is accomplished. As much as 76% of the tritiated formylmethionyl-leucyl-phenylalanine (fMet-Leu-[3H]Phe) specifically bound to intact cells, obtained by a 30-s stimulation with 20 nM fMet-Leu-[3H]Phe, still remained after Triton X-100 extraction. Preincubating intact cells with dihydrocytochalasin B (dhCB) or washing the cytoskeletal preparation with a high concentration of potassium, reduced the binding of ligand-receptor complexes to the cytoskeleton by 46% or more. Inhibition of fMet-Leu-Phe-induced generation of second messengers by ADP-ribosylating the alpha-subunit of the receptor-coupled G-protein with pertussis toxin, did not reduce the binding of ligand-receptor complexes to the cytoskeleton. However, using guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) to prevent the dissociation of the fMet-Leu-Phe-associated G-protein within electrically permeabilized cells, led to a pronounced reduction (62%) of the binding between ligand-receptor complexes and the cytoskeleton. In summary, in human neutrophils the rapid association between chemotactic peptide-receptor complexes and the cytoskeleton is dependent on filamentous actin. This association is most likely regulated by the activation and dissociation of the fMet-Leu-Phe-associated G-protein.
Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family.
Authors: Garcia A , Coudrier E , Carboni J , Anderson J , Vandekerkhove J , Mooseker M , Louvard D , Arpin M
Published: May 2004

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625-14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.
Identification of a talin binding site in the cytoskeletal protein vinculin.
Authors: Jones P , Jackson P , Price G J , Patel B , Ohanion V , Lear A L , Critchley D R
Published: May 2004

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.
Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma-enteric isoform in differentiating striated myocytes.
Authors: Sawtell N M , Lessard J L
Published: May 2004

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha-vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.
Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function.
Authors: Cleves A E , Novick P J , Bankaitis V A
Published: May 2004

The budding mode of Saccharomyces cerevisiae cell growth demands that a high degree of secretory polarity be established and directed toward the emerging bud. We report here our demonstration that mutations in SAC1, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing sec14 lethalities associated with yeast Golgi defects. Moreover, these sac1 suppressor properties also extended to sec6 and sec9 secretory vesicle defects. The genetic data are consistent with the notion that SAC1p modulates both secretory pathway and actin cytoskeleton function. On this basis, we suggest that SAC1p may represent one aspect of the mechanism whereby secretory and cytoskeletal activities are coordinated, so that proper spatial regulation of secretion might be achieved.
Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.
Authors: Miller K G , Field C M , Alberts B M
Published: May 2004

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.
Identification of microtubule-associated proteins in the centrosome, spindle, and kinetochore of the early Drosophila embryo.
Authors: Kellogg D R , Field C M , Alberts B M
Published: May 2004

We have developed affinity chromatography methods for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts and have used them to analyze the cytoskeleton of the early Drosophila embryo. More than 50 Drosophila embryo proteins bind to microtubule affinity columns. To begin to characterize these proteins, we have generated individual mouse polyclonal antibodies that specifically recognize 24 of them. As judged by immunofluorescence, some of the antigens localize to the mitotic spindle in the early Drosophila embryo, while others are present in centrosomes, kinetochores, subsets of microtubules, or a combination of these structures. Since 20 of the 24 antibodies stain microtubule structures, it is likely that most of the proteins that bind to our columns are associated with microtubules in vivo. Very few MAPS seem to be identically localized in the cell, indicating that the microtubule cytoskeleton is remarkably complex.
Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity.
Authors: Driscoll M , Dean E , Reilly E , Bergholz E , Chalfie M
Published: May 2004

Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta-tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.
Gap junctional communication in the extraembryonic tissues of the gastrulating mouse embryo.
Authors: Kalimi G H , Lo C W
Published: May 2004

We characterized gap junctional communication in the extraembryonic tissues of the 7.5-d gastrulating mouse embryo. At this stage of development, the extraembryonic tissues form a large part of the conceptus, and link the embryo proper to the maternal tissue. Using Lucifer yellow injections, cells in most extraembryonic tissues were observed to be very well dye coupled, the only exception being the peripheral regions of the ectoplacental cone. Of particular interest was the fact that no dye coupling was detected between the three major extraembryonic tissues. Thus, the extraembryonic ectoderm (EEC), the extraembryonic endoderm (EEN), and the ectoplacental cone (EPC) corresponded to separate communication compartments, with the EPC being further subdivided into three compartments. Interestingly, the EEN was observed to exhibit a very low level of dye coupling with the adjacent visceral embryonic endoderm (EN), and consistent with the latter dye coupling results was the finding that the EEN was ionically coupled to the EN, but not with any other extraembryonic tissues. However, in the EPC, ionic coupling studies show that the central region was well coupled ionically to the EEC, but only weakly coupled to the peripheral EPC. These findings, in conjunction with our previous study (1988. J. Cell Biol. 107:241-255), demonstrate that the 7.5-d mouse conceptus is subdivided into at least nine major Lucifer yellow-delineated communication compartments, with ionic coupling across some of these compartments effectively unifying the embryo into two large domains corresponding to the embryo proper and the major extraembryonic tissues.
Junctional communication is induced in migrating capillary endothelial cells.
Authors: Pepper M S , Spray D C , Chanson M , Montesano R , Orci L , Meda P
Published: May 2004

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.
The cytomechanics of axonal elongation and retraction.
Authors: Dennerll T J , Lamoureux P , Buxbaum R E , Heidemann S R
Published: May 2004

Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.
Changes in microtubule polarity orientation during the development of hippocampal neurons in culture.
Authors: Baas P W , Black M M , Banker G A
Published: May 2004

Microtubules in the dendrites of cultured hippocampal neurons are of nonuniform polarity orientation. About half of the microtubules have their plus ends oriented distal to the cell body, and the other half have their minus ends distal; in contrast, microtubules in the axon are of uniform polarity orientation, all having their plus ends distal (Baas, P.W., J.S. Deitch, M. M. Black, and G. A. Banker. 1988. Proc. Natl. Acad. Sci. USA. 85:8335-8339). Here we describe the developmental changes that give rise to the distinct microtubule patterns of axons and dendrites. Cultured hippocampal neurons initially extend several short processes, any one of which can apparently become the axon (Dotti, C. G., and G. A. Banker. 1987. Nature [Lond.]. 330:477-479). A few days after the axon has begun its rapid growth, the remaining processes differentiate into dendrites (Dotti, C. G., C. A. Sullivan, and G. A. Banker. 1988. J. Neurosci. 8:1454-1468). The polarity orientation of the microtubules in all of the initial processes is uniform, with plus ends distal to the cell body, even through most of these processes will become dendrites. This uniform microtubule polarity orientation is maintained in the axon at all stages of its growth. The polarity orientation of the microtubules in the other processes remains uniform until they begin to grow and acquire the morphological characteristics of dendrites. It is during this period that microtubules with minus ends distal to the cell body first appear in these processes. The proportion of minus end-distal microtubules gradually increases until, by 7 d in culture, about equal numbers of dendritic microtubules are oriented in each direction. Thus, the establishment of regional differences in microtubule polarity orientation occurs after the initial polarization of the neuron and is temporally correlated with the differentiation of the dendrites.
Differential effects of heparin, fibronectin, and laminin on the phosphorylation of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A.
Authors: Feige J J , Bradley J D , Fryburg K , Farris J , Cousens L C , Barr P J , Baird A
Published: May 2004

Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP-dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.
Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.
Authors: Schreiner C L , Bauer J S , Danilov Y N , Hussein S , Sczekan M M , Juliano R L
Published: May 2004

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.
Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth.
Authors: Hann B C , Poritz M A , Walter P
Published: May 2004

We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Römisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Nature (Lond.). 340:478-482; Bernstein H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, P. Walter. 1989. Nature (Lond.). 340:482-486). To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes. The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively. Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain). In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain. SRP54sp contains a 480-nucleotide intron located 78 nucleotides from the 5' end of the open reading frame. Although the function of the yeast homologues is unknown, gene disruption experiments in S. cerevisiae show that the gene is essential for growth. The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.
Endocytosis in filter-grown Madin-Darby canine kidney cells.
Authors: Bomsel M , Prydz K , Parton R G , Gruenberg J , Simons K
Published: May 2004

In this paper, we have characterized the apical and basolateral endocytic pathways of epithelial MDCK cells grown on filters. The three-dimensional organization of the endocytic compartments was analyzed by confocal microscopy after internalization of a fluorescent fluid-phase marker from either side of the cell layer. After 5 min of internalization, distinct sets of apical and basolateral early endosomes were observed lining the plasma membrane domain from which internalization had occurred. At later time points, the apical and the basolateral endocytic pathways were shown to converge in the perinuclear region. Mixing of two different fluorescent markers could be detected after their simultaneous internalization from opposite sides of the cell layer. The extent of the meeting was quantitated by measuring the amount of complex formed intracellularly between avidin internalized from the apical side and biotinylated horseradish peroxidase (HRP) from the basolateral side. After 15 min, 14% of the avidin marker was complexed with the biotinylated HRP and this value increased to 50% during a subsequent chase of 60 min in avidin-free medium. We also determined the kinetics of fluid internalization, recycling, transcytosis, and intracellular retention using HRP as a marker. Fluid was internalized with the same rates from either surface domain (1.2 x 10(-4) microns 3/min per microns 2 of surface area). However, significant differences were observed for each pathway in the amounts and kinetics of marker recycled and transcytosed. The content of apical early endosomes was primarily recycled and transcytosed (45% along Bach route after 1 h internalization), whereas delivery to late endocytic compartments was favored from the basolateral early endosome (77% after 1 h). Our results demonstrate that early apical and basolateral endosomes are functionally and topologically distinct, but that the endocytic pathways converge at later stages in the perinuclear region of the cell.
Meeting of the apical and basolateral endocytic pathways of the Madin-Darby canine kidney cell in late endosomes.
Authors: Parton R G , Prydz K , Bomsel M , Simons K , Griffiths G
Published: May 2004

Electron microscopic approaches have been used to study the endocytic pathways from the apical and basolateral surface domains of the polarized epithelial cell, MDCK strain I, grown on polycarbonate filters. The cells were incubated at 37 degrees C in the presence of two distinguishable markers administered separately to the apical or the basolateral domain. Initially each marker was visualized within distinct apical or basolateral peripheral endosomes. However, after 15 min at 37 degrees C, both markers were observed within common perinuclear structures. The compartment in which meeting first occurred was shown to be a late endosome (prelysosome) that labeled extensively with antibodies against the cation-independent mannose-6-phosphate receptor (MPR) on cryosections. With increasing incubation times, markers passed from these MPR-positive structures into a common set of MPR-negative lysosomes that were mainly located in the apical half of the cell. A detailed quantitative analysis of the endocytic pathways was carried out using stereological techniques in conjunction with horseradish peroxidase and acid phosphatase cytochemistry. This enabled us to estimate the absolute volumes and membrane surface areas of the endocytic organelles involved in apical and basolateral endocytosis.
Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains.
Authors: Hunziker W , Mellman I
Published: May 2004

Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII-B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and -B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.
Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.
Authors: Picard A , Capony J P , Brautigan D L , Dorée M
Published: May 2004

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.
Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells.
Authors: Klatte D H , Kurpakus M A , Grelling K A , Jones J C
Published: May 2004

Treatment of bovine tongue mucosa with 1 M KCl induced a split in the lamina densa of the basement membrane zone (BMZ). The epithelium was then separated from the underlying connective tissue. Electron microscopic analysis of the stripped epithelium revealed that hemidesmosomes and their associated intermediate filaments (IF) remain along the basal surface of the epithelium. This surface was solubilized in an SDS/urea-containing buffer. Characterization of components of this protein mixture was undertaken using human autoantibodies from bullous pemphigoid (BP) patients that have been shown to recognize hemidesmosomal plaque elements (Mutasim, D. F., Y. Takahashi, R. S. Labib, G. J. Anhalt, H. P. Patel, and L. A. Diaz. 1985. J. Invest. Dermatol. 84:47-53) and by production of mAbs. Affinity-purified autoantibodies directed against 180- and 240-kD polypeptides present in the protein preparation generated strong immunofluorescence staining patterns along the BMZ of bovine tongue mucosa. Furthermore, immunogold localization revealed that these two polypeptides are associated with the hemidesmosomal plaque. A mAb preparation directed against a 125-kD polypeptide present in this same protein mixture lamina lucida side of the hemidesmosome. Autoantibodies in BP serum samples, affinity-purified 180-kD autoantibodies and the mAb preparation generated a punctate stain along the substratum attached surface of epithelial cells maintained on glass substrata for approximately 1 wk. The spots appeared to be associated with bundles of IF in cultured mouse keratinocytes. These monospecific antibody probes should prove invaluable for the study of hemidesmosome structure, assembly, and function.