bluuebunny/crossref_metadata_2025 huggingface dataset:
Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.
Authors: Matsuyoshi N , Hamaguchi M , Taniguchi S , Nagafuchi A , Tsukita S , Takeichi M
Published: May 2004

Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.
The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset.
Authors: Williams B C , Karr T L , Montgomery J M , Goldberg M L
Published: May 2004

Mutations in the gene l(1)zw10 disrupt the accuracy of chromosome segregation in a variety of cell types during the course of Drosophila development. Cytological analysis of mutant larval brain neuroblasts shows very high levels of aneuploid cells. Many anaphase figures are aberrant, the most frequent abnormality being the presence of lagging chromosomes that remain in the vicinity of the metaphase plate when the other chromosomes have migrated toward the spindle poles. Finally, the centromeric connection between sister chromatids in mutant neuroblasts treated with colchicine often appears to be broken, in contrast with similarly treated control neuroblasts. The 85-kD protein encoded by the l(1)zw10 locus displays a dynamic pattern of localization in the course of the embryonic cell cycle. It is excluded from the nuclei during interphase, but migrates into the nuclear zone during prometaphase. At metaphase, the zw10 antigen is found in a novel filamentous structure that may be specifically associated with kinetochore microtubules. Upon anaphase onset, there is an extremely rapid redistribution of the zw10 protein to a location at or near the kinetochores of the separating chromosomes.
The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment.
Authors: Hobman T C , Woodward L , Farquhar M G
Published: May 2004

Evidence is accumulating that a distinct compartment(s) exists in the secretory pathway interposed between the rough ER (RER) and the Golgi stack. In this study we have defined a novel post-RER, pre-Golgi compartment where unassembled subunits of rubella virus (RV) E1 glycoprotein accumulate. When RV E1 is expressed in CHO cells in the absence of E2 glycoprotein, transport of E1 to the Golgi complex is arrested. The compartment in which E1 accumulates consists of a tubular network of smooth membranes which is in continuity with the RER but has distinctive properties from either the RER, Golgi, or previously characterized intermediate compartments. It lacks RER and Golgi membrane proteins and is not disrupted by agents which disrupt either the RER (thapsigargin, ionomycin) or Golgi (nocodazole and brefeldin A). However, luminal ER proteins bearing the KDEL signal have access to this compartment. Kinetically the site of E1 arrest lies distal to or at the site where palmitylation occurs and proximal to the low temperature 15 degrees C block. Taken together the findings suggest that the site of E1 arrest corresponds to, or is located close to the exit site from the ER. This compartment could be identified morphologically because it is highly amplified in cells overexpressing unassembled E1 subunits, but it may have its counterpart among the transitional elements of non-transfected cells. We conclude that the site of E1 arrest may represent a new compartment or a differentiated proximal moiety of the intermediate compartment.
In AtT20 and HeLa cells brefeldin A induces the fusion of tubular endosomes and changes their distribution and some of their endocytic properties.
Authors: Tooze J , Hollinshead M
Published: May 2004

We have studied the effects of brefeldin A (BFA) on the tubular endosomes in AtT20 and HeLa cells (Tooze, J., and M. Hollinshead. 1991. J. Cell Biol. 115:635-653) by electron microscopy of cells labeled with three endocytic tracers, HRP, BSA-gold, and transferrin conjugated to HRP, and by immunofluorescence microscopy. For the latter we used antibodies specific for transferrin receptor, and, in the case of AtT20 cells, also antibodies specific for synaptophysin. In HeLa cells BFA at concentrations ranging from 1 micrograms to 10 micrograms/ml causes the dispersed patches of network of preexisting tubular early endosomes to be incorporated within 5 min into tubules approximately 50 nm in diameter but up to 40-50 microns long. These long, straight tubular endosomes are aligned along microtubules; they branch relatively infrequently to form an open network or reticulum extending from the cell periphery to the microtubule organizing center (MTOC). As the incubation with BFA is prolonged beyond 5 min, a steady state is reached in which many tubules are located in a dense network enclosing the centrioles, with branches extending in a more open network to the periphery. This effect of BFA, which is fully reversed within 15-30 min of washing out, is inhibited by pre-incubating the cells with sodium azide and 2-deoxy-D-glucose. In AtT20 cells BFA at 5 micrograms/ml or above causes the same sorts of changes, preexisting tubular endosomes are recruited into a more continuous endosomal network, and there is a massive accumulation of this network around the MTOC. Maintenance of the BFA-induced endosomal reticulum in both cell types is dependent upon the integrity of microtubules. In AtT20 cells BFA at 1 microgram/ml has no detectable effect on the early endosomal system but the Golgi stacks are converted to clusters of tubules and vesicles that remain in the region of the MTOC during prolonged incubations. Therefore, the Golgi apparatus in these cells is more sensitive to BFA than the early endosomes. The morphological evidence suggests that all the tubular early endosomes in BFA-treated HeLa and AtT20 cells are linked together in a single reticulum. Consistent with this, incubations as short as 1-3 min with 10 or 20 mg/ml HRP in the medium result in the entire endosomal reticulum in most of the BFA-treated cells being filled with HRP reaction product.(ABSTRACT TRUNCATED AT 400 WORDS)
2-Deoxyglucose and cytochalasin D modulate aldolase mobility in living 3T3 cells.
Authors: Pagliaro L , Taylor D L
Published: May 2004

Approximately 23% of the glycolytic enzyme aldolase in the perinuclear region of Swiss 3T3 cells is immobile as measured by FRAP. Previous studies suggest that the immobile fraction may be associated with the actin cytoskeleton (Pagliaro, L. and D. L. Taylor. 1988. J. Cell Biol. 107:981-991), and it has been proposed that the association of some glycolytic enzymes with the cytoskeleton could have functional significance, perhaps involving a fundamental relationship between glycolysis, cytoplasmic organization, and cell motility. We have tested the effect of a key glycolytic inhibitor and an actin cytoskeletal modulator on the mobility of aldolase in living cells directly, using fluorescent analog cytochemistry and FRAP. We report here that the competitive hexokinase inhibitor 2-deoxyglucose releases the bound fraction of aldolase in 3T3 cells within 10 min, and that this process is reversible upon washout of the inhibitor. A similar result is produced with the actin-binding agent, cytochalasin D. These results are consistent with models in which glycolytic enzymes are not exclusively diffusion-limited, soluble proteins, but may exist partially in the solid phase of cytoplasm. Such organization has significant implications for both the modulation of cytoplasmic structure and for cellular metabolism.
Cooperation between PDGF and FGF converts slowly dividing O-2Aadult progenitor cells to rapidly dividing cells with characteristics of O-2Aperinatal progenitor cells.
Authors: Wolswijk G , Noble M
Published: May 2004

We have shown previously that oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells isolated from adult rat optic nerves can be distinguished in vitro from their perinatal counterparts on the basis of their much slower rates of division, differentiation, and migration when grown in the presence of cortical astrocytes or PDGF. This behavior is consistent with in vivo observations that there is only a modest production of oligodendrocytes in the adult CNS. As such a behavior is inconsistent with the likely need for a rapid generation of oligodendrocytes following demyelinating damage to the mature CNS, we have been concerned with identifying in vitro conditions that allow O-2Aadult progenitor cells to generate rapidly large numbers of progeny cells. We now provide evidence that many slowly dividing O-2Aadult progenitor cells can be converted to rapidly dividing cells by exposing adult optic nerve cultures to both PDGF and bFGF. In addition, these O-2Aadult progenitor cells appear to acquire other properties of O-2Aperinatal progenitor cells, such as bipolar morphology and high rate of migration. Although many O-2Aadult progenitor cells in cultures exposed to bFGF alone also divide rapidly, these cells are multipolar and migrate little in vitro. Oligodendrocytic differentiation of O-2Aadult progenitor cells, which express receptors for bFGF in vitro, is almost completely inhibited in cultures exposed to bFGF or bFGF plus PDGF. As bFGF and PDGF appear to be upregulated and/or released after injury to the adult brain, this particular in vitro response of O-2Aadult progenitor cells to PDGF and bFGF may be of importance in the generation of large numbers of new oligodendrocytes in vivo following demyelination.
Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity.
Authors: Flaumenhaft R , Abe M , Mignatti P , Rifkin D B
Published: May 2004

Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries.
Distinct cellular expression pattern of annexins in Hydra vulgaris.
Authors: Schlaepfer D D , Bode H R , Haigler H T
Published: May 2004

The annexins are a structurally related family of Ca2+ and phospholipid binding proteins whose function has not been clearly defined. Further investigations of annexin function may be enhanced by studying simpler organisms that express fewer annexin gene products. We previously characterized annexin XII from the freshwater cnidarian Hydra vulgaris (Schlaepfer, D. D., D. A. Fisher, M. E. Brandt, H. R. Bode, J. Jones, and H. T. Haigler. 1992. J. Biol. Chem. 267:9529-9539). In this report, we detected one other hydra annexin (40 kD) by screening hydra cell extracts with antibodies raised against peptides from highly conserved regions of known annexins. The 40-kD protein was expressed at less than 1% of annexin XII levels. These biochemical studies indicate that hydra contain a very limited number of annexin gene products. The cellular hydra annexin distribution was analyzed by indirect immunofluorescence. Using affinity-purified antibodies to annexin XII, the epithelial battery cells were stained throughout the tentacle. A lower level of annexin XII staining was detected in peduncle region epithelial cells. No other cell types showed detectable annexin XII staining. The anti-peptide antibody that specifically detected the 40-kD hydra annexin, maximally stained the cytoplasm of nematocytes. The immunofluorescent results showed that annexin XII and the 40-kD annexin were not co-expressed in the same cells. Since the hydra annexins localized to specific subsets of the total hydra cell types, it is likely that these proteins perform specialized biological roles, and not general "housekeeping" functions which are part of the essential molecular machinery of all cells.
Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.
Authors: Giancotti F G , Stepp M A , Suzuki S , Engvall E , Ruoslahti E
Published: May 2004

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.
Type VI collagen microfibrils: evidence for a structural association with hyaluronan.
Authors: Kielty C M , Whittaker S P , Grant M E , Shuttleworth C A
Published: May 2004

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.
The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes.
Authors: Mathews P M , Martinie J B , Fambrough D M
Published: May 2004

A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.
Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis.
Authors: Bowser R , Müller H , Govindan B , Novick P
Published: May 2004

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.
Kinectin, a major kinesin-binding protein on ER.
Authors: Toyoshima I , Yu H , Steuer E R , Sheetz M P
Published: May 2004

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing. mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.
Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracellular targeting function.
Authors: Paschal B M , Mikami A , Pfister K K , Vallee R B
Published: May 2004

In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74-kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length cDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain cDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit. Comparison with known sequences revealed that the carboxyl-terminal half of the polypeptide is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70-kD intermediate chain of flagellar outer arm dynein. Immunoblot analysis with a monoclonal antibody to the 74-kD species indicated a widespread tissue distribution, as expected for a cytoplasmic dynein subunit. Nonetheless, the antibody recognized a 67-kD species in ram sperm flagella and pig tracheal cilia, supporting the existence of distinct but related cytoplasmic and axonemal polypeptides in mammals. In view of evidence for a role for the ODA6 gene product in anchoring flagellar dynein to the A subfiber microtubule in the axoneme, we predict an analogous role for the 74-kD polypeptide, perhaps in mediating the interaction of cytoplasmic dynein with membranous organelles and kinetochores.
Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas.
Authors: Mastronarde D N , O'Toole E T , McDonald K L , McIntosh J R , Porter M E
Published: May 2004

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.
Extragenic suppressors of paralyzed flagellar mutations in Chlamydomonas reinhardtii identify loci that alter the inner dynein arms.
Authors: Porter M E , Power J , Dutcher S K
Published: May 2004

We have analyzed extragenic suppressors of paralyzed flagella mutations in Chlamydomonas reinhardtii in an effort to identify new dynein mutations. A temperature-sensitive allele of the PF16 locus was mutagenized and then screened for revertants that could swim at the restrictive temperature (Dutcher et al. 1984. J. Cell Biol. 98:229-236). In backcrosses of one of the revertant strains to wild-type, we recovered both the original pf16 mutation and a second, unlinked suppressor mutation with its own flagellar phenotype. This mutation has been identified by both recombination and complementation tests as a new allele of the previously uncharacterized PF9 locus on linkage group XII/XIII. SDS-PAGE analysis of isolated flagellar axonemes and dynein extracts has demonstrated that the pf9 strains are missing four polypeptides that form the I1 inner arm dynein subunit. The primary effect of the loss of the I1 subunit is a decrease in the forward swimming velocity due to a change in the flagellar waveform. Both the flagellar beat frequency and the axonemal ATPase activity are nearly wild-type. Examination of axonemes by thin section electron microscopy and image averaging methods reveals that a specific domain of the inner arm complex is missing in the pf9 mutant strains (see accompanying paper by Mastronarde et al.). When combined with other flagellar defects, the loss of the I1 subunit has synergistic effects on both flagellar assembly and flagellar motility. These synthetic phenotypes provide a screen for new suppressor mutations in other loci. Using this approach, we have identified the first interactive suppressors of a dynein arm mutation and an unusual bypass suppressor mutation.
A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts.
Authors: Symons M H , Mitchison T J
Published: May 2004

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.
Nectadrin, the heat-stable antigen, is a cell adhesion molecule.
Authors: Kadmon G , Eckert M , Sammar M , Schachner M , Altevogt P
Published: May 2004

Nectadrin, the cell surface glycoprotein recognized by the novel mAb 79, was found to be immunologically identical to the heat-stable antigen (HSA). It is a glycoprotein with a polypeptide core of only 30 amino acids and a very high carbohydrate content (Wenger, R. H., M. Ayane, R. Bose, G. Köhler, and P. J. Nielsen. 1991. Eur. J. Immunol. 21:1039-1046). Immunocytological studies using cultured splenic B-lymphocytes, neuroblastoma cells, and cerebellar cells indicated that nectadrin is preferentially expressed at sites of cell-cell contact. Purified nectadrin and monoclonal nectadrin antibody 79, but not other monoclonal nectadrin antibodies, inhibited the aggregation of B-lymphocytes by 70%, suggesting that nectadrin may act as a cell adhesion molecule. Nectadrin was purified from a mouse lymphoma cell line in two forms of 40-60 and 23-30 kD. The lower molecular weight form appears to be generated from the higher molecular weight form by degradative removal of saccharide residues characteristic of complex type oligosaccharide side chains. Latex beads coated with purified nectadrin aggregated and the rate of their aggregation depended on the molecular form of nectadrin, with the larger form being more potent than the smaller one in mediating bead aggregation. Nectadrin thus appears to be a self-binding cell adhesion molecule of a structurally novel type in that its extensive glycan structures may be implicated in mediating cell adhesion.
Bravo/Nr-CAM is closely related to the cell adhesion molecules L1 and Ng-CAM and has a similar heterodimer structure.
Authors: Kayyem J F , Roman J M , de la Rosa E J , Schwarz U , Dreyer W J
Published: May 2004

Diverse cell-surface molecules of the nervous system play an important role in specifying cell interactions during development. Using a method designed to generate mAbs against neural surface molecules of defined molecular weight, we have previously reported on the surface protein, Bravo, found in the developing avian retinotectal system. Bravo is immunologically detected on developing optic fibers in the retina, but absent from distal regions of the same fibers in the tectum. We have isolated cDNA clones encompassing the entire coding region of Bravo, including clones containing five alternative sequences of cDNA. These putative alternatively spliced sequences encode stretches of polypeptide ranging in length from 10-93 amino acids and are predicted to be both extra- and intracellular. The deduced primary structure of Bravo reveals that, like the cell adhesion molecules (CAMs) chicken Ng-CAM and mouse L1, Bravo is composed of six Ig-like domains, five fibronectin type III repeats, a transmembrane domain, and a short cytoplasmic region. Recently, the cDNA sequence of a related molecule, Nr-CAM, was reported and its possible identity with Bravo discussed (Grumet, M., V. Mauro, M. P. Burgoon, G. E. Edelman, and B. A. Cunningham. 1991. J. Cell Biol. 113:1399-1412). Here we confirm this identity and moreover show that Bravo is found on Müller glial processes and end-feet in the developing retina. In contrast to the single polypeptide chain structure of Nr-CAM reported previously, we show that Bravo has a heterodimer structure composed of an alpha chain of M(r) 140/130 and a beta chain of 60-80 kD. As with L1 and Ng-CAM, the two chains of Bravo are generated from an intact polypeptide by cleavage at identical locations and conserved sites within all three molecules (Ser-Arg/Lys-Arg). The similar domain composition and heterodimer structure, as well as the 40% amino acid sequence identity of these molecules, defines them as an evolutionarily related subgroup of CAMs. The relationship of Bravo to molecules known to be involved in cell adhesion and process outgrowth, combined with its pattern of expression and numerous potential isoforms, suggests a complex role for this molecule in cell interactions during neural development.
In vitro assembly of prenucleolar bodies in Xenopus egg extract.
Authors: Bell P , Dabauvalle M C , Scheer U
Published: May 2004

Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.
Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.
Authors: Huovila A P , Eder A M , Fuller S D
Published: May 2004

Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 min) pulse, increased in concentration to peak at 60 min, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (t 1/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.
Cytoplasmic dynein participates in the centrosomal localization of the Golgi complex.
Authors: Corthésy-Theulaz I , Pauloin A , Pfeffer S R
Published: May 2004

The localization of the Golgi complex depends upon the integrity of the microtubule apparatus. At interphase, the Golgi has a restricted pericentriolar localization. During mitosis, it fragments into small vesicles that are dispersed throughout the cytoplasm until telophase, when they again coalesce near the centrosome. These observations have suggested that the Golgi complex utilizes a dynein-like motor to mediate its transport from the cell periphery towards the minus ends of microtubules, located at the centrosome. We utilized semi-intact cells to study the interaction of the Golgi complex with the microtubule apparatus. We show here that Golgi complexes can enter semi-intact cells and associate stably with cytoplasmic constituents. Stable association, termed here "Golgi capture," requires ATP hydrolysis and intact microtubules, and occurs maximally at physiological temperature in the presence of added cytosolic proteins. Once translocated into the semi-intact cell cytoplasm, exogenous Golgi complexes display a distribution similar to endogenous Golgi complexes, near the microtubule-organizing center. The process of Golgi capture requires cytoplasmic tubulin, and is abolished if cytoplasmic dynein is immunodepleted from the cytosol. Cytoplasmic dynein, prepared from CHO cell cytosol, restores Golgi capture activity to reactions carried out with dynein immuno-depleted cytosol. These results indicate that cytoplasmic dynein can interact with isolated Golgi complexes, and participate in their accumulation near the centrosomes of semi-intact, recipient cells. Thus, cytoplasmic dynein appears to play a role in determining the subcellular localization of the Golgi complex.
Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.
Authors: Ghinea N , Vu Hai M T , Groyer-Picard M T , Houllier A , Schoëvaërt D , Milgrom E
Published: May 2004

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.
The establishment of polarized membrane traffic in Xenopus laevis embryos.
Authors: Roberts S J , Leaf D S , Moore H P , Gerhart J C
Published: May 2004

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin was polarized. Oogenic prolactin was secreted only into the blastocoel (from the cleavage membrane), none could be detected in the external medium (from the original oocyte membrane). These results provide the first direct evidence that the oocyte synthesizes a cache of vesicles for specific recruitment to the embryonic cleavage membranes which are polarized beginning with the first cleavage division.
Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling.
Authors: Maycox P R , Link E , Reetz A , Morris S A , Jahn R
Published: May 2004

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.
Localization of dystrophin COOH-terminal domain by the fracture-label technique.
Authors: Squarzoni S , Sabatelli P , Maltarello M C , Cataldi A , di Primio R , Maraldi N M
Published: May 2004

The precise localization of dystrophin in the skeletal muscle cell should contribute to a better understanding of the yet unclear functional role of this protein, both in normal and in Duchenne muscular dystrophy. Immunocytochemical studies did not give conclusive results on the localization of dystrophin with respect to the sarcolemma and to the cytoskeletal components. To improve the reliability of the electron microscopic immunocytochemical localization of dystrophin, a mAb against the COOH-terminus of the molecule has been used in association with the fracture-label technique, which, causing a partition of the membrane in protoplasmic and exoplasmic halves, allows a more precise dystrophin localization. The results obtained indicate that dystrophin is associated with the protoplasmic half of the plasmalemma, and the observation that it does not randomly follow the partition of the membrane is consistent with a stable association with the cytoskeleton.