bluuebunny/crossref_metadata_2025 huggingface dataset:
Perineurial cells coexpress genes encoding interstitial collagens and basement membrane zone components.
Authors: Jaakkola S , Peltonen J , Uitto J J
Published: May 2004

Perineurial cell cultures were established from the sciatic nerves of adult Wistar rats. Highly enriched cultures were studied with respect to the production of extracellular matrix components under conditions free from the influence of Schwann cells, axons, or the extracellular matrix of peripheral nerves. Indirect immunofluorescence staining revealed the presence of collagen type IV epitopes, and electron microscopy demonstrated patches of basement membrane on the perineurial cell surfaces. Collagenous fibrils with a diameter of 15-20 nm were also observed in the intracellular space. SDS-PAGE of radiolabeled medium proteins showed a pattern of bands suggesting the synthesis and secretion of fibronectin, and type I and IV collagens. Northern hybridizations revealed characteristic polymorphic mRNA transcripts corresponding to fibronectin, laminin B2 chain, as well as to the alpha-chain subunits of type I, III, and IV collagens. Furthermore, in situ hybridizations suggested expression of these genes by cultured perineurial cells without apparent heterogeneity within the cell populations. In situ hybridizations of sciatic nerve tissue from 2-wk-old rats also suggested that perineurial cells express alpha 1(I) and alpha 2(IV) collagen, as well as laminin B2 chain genes in vivo. This profile of matrix gene expression is different from that of Schwann cells, which do not synthesize fibronectin, or that of fibroblastic cells, which do not form a cell surface basement membrane. The capability of perineurial cells to express genes for the basement membrane zone and for interstitial collagens further adds to our understanding of the functional role of perineurial cells in developing and healing peripheral nerve, as well as in certain neoplastic lesions of neural origin, such as von Recklinghausen's neurofibromas.
Autonomous splicing and complementation of in vivo-assembled spliceosomes.
Authors: Zeitlin S , Wilson R C , Efstratiadis A
Published: May 2004

We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.
The XLR gene product defines a novel set of proteins stabilized in the nucleus by zinc ions.
Authors: Garchon H J , Davis M M
Published: May 2004

The major product of the XLR (X-chromosomal, lymphocyte-regulated) locus is found to be a 30-kD nuclear protein with a relatively short (t1/2 approximately equal to 2 h) half-life. Together with its stage- and tissue-specific pattern of expression, this suggests a role for this protein in the regulation of differentiation in T and B lymphocytes. Interestingly, the XLR protein almost completely leaches out of the nucleus after lysis of cells in low salt buffer, but is stabilized in that location by metal cations, particularly Zn++. This stabilization is reversible by chelating agents (o-phenanthroline, EDTA) which also release a number of other polypeptides in addition to XLR. These results suggest that XLR represents a novel class of nuclear proteins, and that cations such as zinc may play a role in the localization of these proteins in the nucleus.
Nascent secretory chain binding and translocation are distinct processes: differentiation by chemical alkylation.
Authors: Nicchitta C V , Blobel G
Published: May 2004

We have investigated the effects of chemical alkylation of microsomal membranes on nascent chain binding and translocation. Assays were conducted using either full-length or truncated preprolactin transcripts in combination with a reconstituted membrane system consisting of proteolyzed rough microsomes and the cytoplasmic domain of the signal recognition particle receptor. Treatment of rough microsomes with N-ethylmaleimide was observed to inhibit preprolactin processing at a site other than the signal recognition particle or the signal recognition particle receptor. As formation of a translocation competent junction between the ribosome/nascent chain complex and the membrane has recently been demonstrated to require GTP (Connolly, T., and R. Gilmore. J. Cell Biol. 1986. 103:2253-2261), the effects of membrane alkylation on this parameter were assessed. N-ethylmaleimide treatment did not inhibit nascent chain targeting or GTP-dependent signal sequence insertion. Translocation of the targeted and inserted nascent chain was, however, blocked. These data indicate (a) that the process of nascent chain translocation is distinct from targeting and signal sequence insertion, and (b) translocation of the peptide chain across the membrane is mediated by an N-ethylmaleimide-sensitive membrane protein component(s). To further substantiate the observation that nascent chain targeting and signal sequence insertion can be distinguished from translocation, the temperature dependencies of the two phenomena were compared. Signal sequence insertion occurred at low temperatures (4 degrees C) and was maximal between 10 and 15 degrees C. Translocation was only observed at higher temperatures and was maximal between 25 and 30 degrees C.
Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor.
Authors: Andrews D W , Lauffer L , Walter P , Lingappa V R
Published: May 2004

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.
Selective retention of monoglucosylated high mannose oligosaccharides by a class of mutant vesicular stomatitis virus G proteins.
Authors: Suh K , Bergmann J E , Gabel C A
Published: May 2004

Cells infected with a temperature-sensitive mutant of vesicular stomatitis virus, ts045, or transfected with the plasmid vector pdTM12 produce mutant forms of the G protein that remain within the ER. The mutant G proteins were isolated by immunoprecipitation from cells metabolically labeled with [2-3H]mannose to facilitate analysis of the protein-linked oligosaccharides. The 3H-labeled glycopeptides recovered from the immunoprecipitated G proteins contained high mannose-type oligosaccharides. Structural analysis, however, indicated that 60-78% of the 3H-mannose-labeled oligosaccharides contained a single glucose residue and no fewer than eight mannose residues. The 3H-labeled ts045 oligosaccharides were deglucosylated and processed to complex-type units after the infected cells were returned to the permissive temperature. When shifted to the permissive temperature in the presence of a proton ionophore, the G protein oligosaccharides were deglucosylated but remained as high mannose-type units. The glucosylated state was observed, therefore, when the G protein existed in an altered conformation. The ts045 G protein oligosaccharides were deglucosylated in vitro by glucosidase II at both the permissive and nonpermissive temperatures. G protein isolated from ts045-infected cells labeled with [6-3H]galactose in the presence of cycloheximide contained 3H-glucose-labeled monoglucosylated oligosaccharides, indicating that the high mannose oligosaccharides were glucosylated in a posttranslational process. These results suggest that aberrant G proteins are selectively modified by resident ER enzymes to retain monoglucosylated oligosaccharides.
Cloning of cDNAs encoding two related 100-kD coated vesicle proteins (alpha-adaptins).
Authors: Robinson M S
Published: May 2004

Coat proteins of approximately 100-kD (adaptins) are components of the adaptor complexes which link clathrin to receptors in coated vesicles. The alpha-adaptins, which are found exclusively in endocytic coated vesicles, separate into two bands on SDS gels, designated A and C (Robinson, M. S., 1987. J. Cell Biol. 104:887-895). Two distinct cDNAs (sequences 1 and 2) encoding the two alpha-adaptins were cloned from a mouse brain cDNA library. Southern blotting indicates that there is one copy of each of the two alpha-adaptin genes, and that there are no additional closely related genes. Based on the size of the predicted protein products of the two genes (108 and 104 kD), the relative abundance of the two messages in brain and liver, and the reactivity of a sequence 1 fusion protein with different antibodies, it was possible to conclude that sequence 1 codes for A and sequence 2 for C. The two protein sequences are strikingly homologous to each other (84% identical amino acids), the major difference being an additional stretch of 41 amino acids, rich in prolines and acidic residues, inserted into the COOH-terminal half of A. In situ hybridization carried out on mouse brain sections indicates that the same cell type may express both transcripts, but that their relative expressions vary. Antipeptide antibodies are now being raised to find out whether the proteins are localized in functionally distinct populations of endocytic coated vesicles.
Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH.
Authors: Heuser J
Published: May 2004

Lysosomes labeled by uptake of extracellular horseradish peroxidase display remarkable changes in shape and cellular distribution when cytoplasmic pH is experimentally altered. Normally, lysosomes in macrophages and fibroblasts cluster around the cell center. However, when the cytoplasmic pH is lowered to approximately pH 6.5 by applying acetate or by various other means, lysosomes promptly move outward and accumulate in tight clusters at the very edge of the cell, particularly in regions that are actively ruffling before acidification but become quiescent. This movement follows the distribution of microtubules in these cells, and does not occur if microtubules are depolymerized with nocodazole before acidification. Subsequent removal of acetate or the other stimuli to acidification results in prompt resumption of ruffling activity and return of lysosomes into a tight cluster at the cell center. This is correlated with a rebound alkalinization of the cytoplasm. Correspondingly, direct application of weak bases also causes hyperruffling and unusually complete withdrawal of lysosomes to the cell center. Thus, lysosomes appear to be acted upon by microtubule-based motors of both the anterograde (kinesin) type as well as the retrograde (dynein) type, or else they possess bidirectional motors that are reversed by changes in cytoplasmic pH. During the outward movements induced by acidification, lysosomes also appear to be smaller and more predominantly vesicular than normal, while during inward movements they appear to be more confluent and elongated than normal, often becoming even more tubular than in phorbol-treated macrophages (Phaire-Washington, L., S. C. Silverstein, and E. Wang. 1980. J. Cell Biol. 86:641-655). These size and shape changes suggest that cytoplasmic pH also affects the fusion/fission properties of lysosomes. Combined with pH effects on their movement, the net result during recovery from acidification is a stretching of lysosomes into tubular forms along microtubules.
Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens.
Authors: Ludwig M , Gibbs S P
Published: May 2004

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.
Microtubule dynamics investigated by microinjection of Paramecium axonemal tubulin: lack of nucleation but proximal assembly of microtubules at the kinetochore during prometaphase.
Authors: Geuens G , Hill A M , Levilliers N , Adoutte A , DeBrabander M
Published: May 2004

Microtubule (MT) dynamics in PtK2 cells have been investigated using in vivo injection of unmodified Paramecium ciliary tubulin and time-lapse fixation. The sites of incorporation of the axonemal tubulin were localized using a specific antibody which does not react with vertebrate cytoplasmic tubulin (Adoutte, A., M. Claisse, R. Maunoury, and J. Beisson. 1985. J. Mol. Evol. 22:220-229), followed by immunogold labeling, Nanovid microscopy, and ultrastructural observation of the same cells. We confirm data from microinjection of labeled tubulins in other cell types (Soltys, B. J., and G. G. Borisy. 1985. J. Cell Biol. 100:1682-1689; Mitchison, T., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527; Schulze, E., and M. Kirschner. 1986. J. Cell Biol. 102:1020-1031). In agreement with the dynamic instability model (Mitchison, T., and M. Kirschner. 1984. Nature (Lond.). 312:237-242), during interphase, fast (2.6 microns/min) distal growth of MTs occurs, together with new centrosomal nucleation. Most of the cytoplasmic MT complex is replaced within 15-30 min. During mitosis, astral MTs display the same pattern of renewal, but the turnover of the MT system is much faster (approximately 6 min). We have concentrated on the construction of the kinetochore fibers during prometaphase and observe that (a) incorporation of tubulin in the vicinity of the kinetochores is not seen during prophase and early prometaphase as long as the kinetochores are not yet connected to a pole by MTs; (b) proximal time-dependent incorporation occurs only into preexisting kinetochore MTs emanating from centrosomes. Consequently, in undisturbed prometaphase cells, the kinetochores probably do not act as independent nucleation sites. This confirms a model in which, at prometaphase, fast probing centrosomal MTs are grabbed by the kinetochores, where tubulin incorporation then takes place.
The isolation and in situ location of adligin: the microtubule cross-linking protein from Caenorhabditis elegans.
Authors: Aamodt E , Holmgren R , Culotti J
Published: May 2004

Microtubules isolated from the nematode Caenorhabditis elegans contain long stretches of periodic cross-links formed by microtubule-associated proteins (MAPs). These cross-links are 5.7 nm long, 3 nm wide, and occur at one tubulin dimer (8-nm) intervals along the walls of microtubules (Aamodt, E., and J. Culotti, 1986. J. Cell Biol. 103:23-31). The structural protein of the cross-links was isolated from the MAPs by centrifugation and exclusion chromatography. The cross-links were formed exclusively from the most prevalent MAP, a 32,000 mol wt protein. We suggest the name adligin for this MAP. Adligin eluted from the exclusion column at 33,000 mol wt indicating that it was a monomer in solution. Antibodies were made against the purified adligin and affinity purified. The affinity-purified antibodies were used to locate adligin in situ and to determine its distribution relative to that of tubulin by the use of double label immunofluorescence. The anti-adligin antibodies labeled a fibrous network in the cytoplasm of most cells of C. elegans. Neurons were labeled especially well. This labeling pattern was similar to the labeling pattern obtained with antitubulin, but anti-adligin labeled some granules in the gut that were not labeled with antitubulin. These results suggest that adligin may be part of the interphase microtubule network in C. elegans.
Differential axonal transport of isotubulins in the motor axons of the rat sciatic nerve.
Authors: Denoulet P , Filliatreau G , de Néchaud B , Gros F , Di Giamberardino L
Published: May 2004

The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.
Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients.
Authors: Fisher P R , Merkl R , Gerisch G
Published: May 2004

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.
A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane.
Authors: Haeuptle M T , Flint N , Gough N M , Dobberstein B
Published: May 2004

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.
Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.
Authors: Sporn L A , Marder V J , Wagner D D
Published: May 2004

von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160-168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185-190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10-min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule-depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane.
Endocytosis from coated pits of Shiga toxin: a glycolipid-binding protein from Shigella dysenteriae 1.
Authors: Sandvig K , Olsnes S , Brown J E , Petersen O W , van Deurs B
Published: May 2004

Evidence is presented that endocytosis is involved in the transport to the cytosol of the cytotoxin from Shigella dysenteriae 1, Shiga toxin, which acts by removal of a single adenine residue in 28-S ribosomal RNA. Inhibition of endocytosis by ATP depletion of the cells prevented toxin uptake. Exposure of HeLa S3 and Vero cells to toxin at low extracellular pH, where translocation to the cytosol, but not endocytosis is inhibited, allowed the toxin to accumulate in a compartment where it was protected against antibodies to the toxin. Upon transfer of the cells to normal medium endocytosed toxin entered the cytosol. Electron microscopical studies of cells exposed at 0 degrees C to a toxin-horseradish peroxidase (HRP) conjugate, or to unconjugated toxin followed by horse antitoxin antibodies and then protein G-gold, revealed that the Shiga toxin binding sites were randomly distributed on the cell surface, without any preference to, for example, coated pits. In contrast, when cells were exposed to toxin at 37 degrees C, the binding sites were preferentially localized in coated pits. The Shiga-HRP conjugate was also seen in endosomes, lysosomes, and in the Golgi region. Endocytosis by the coated pit/coated vesicle pathway was selectively inhibited by acidification of the cytosol. Under these conditions, both the uptake of toxin-HRP conjugates and intoxication of the cells were inhibited. Evidence from the literature as well as our own results suggest that Shiga toxin binding sites are glycolipids. Thus, Shiga toxin appears to be the first example of a lipid-binding ligand that is endocytosed from coated pits.
Erythrocyte morphology reflects the transbilayer distribution of incorporated phospholipids.
Authors: Daleke D L , Huestis W H
Published: May 2004

The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.
Monoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells.
Authors: Pfister K K , Wagner M C , Stenoien D L , Brady S T , Bloom G S
Published: May 2004

Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.
Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments.
Authors: Albers K , Fuchs E
Published: May 2004

We have deleted cDNA sequences encoding portions of the amino- and carboxy-terminal end of a human type I epidermal keratin K14, and examined the molecular consequences of forcing the expression of these mutants in simple epithelial and squamous cell carcinoma lines. To follow the expression of our mutant products in transfected cells, we have tagged the 3' end of the K14 coding sequence with a sequence encoding an antigenic domain of the neuropeptide substance P. Using DNA transfection and immunohistochemistry (with an antibody against substance P), we have defined the limits of K14 sequence necessary to incorporate into a keratin filament network in vivo without disrupting its architecture. We have also uncovered major differences in the behavior of carboxy- and amino-terminal alpha-helical mutants which do perturb the cytoskeletal network of IFs: whereas carboxy terminal mutants give rise to aggregates of keratin in the cytoplasm, amino-terminal mutants tend to produce aggregates of keratins which seem to localize at the nuclear surface. An examination of the phenotypes generated by the carboxy and amino-terminal mutants and the behavior of cells at late times after transfection suggests a model whereby initiation of filament assembly occurs at discrete sites on the nuclear envelope and filaments grow from the nucleus toward the cytoplasm.
Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures.
Authors: Angelides K J , Smith K E , Takeda M
Published: May 2004

We have explored the dynamics of intermediate filament assembly and subunit exchange using fluorescently labeled neurofilament proteins and a fluorescence resonance energy transfer assay. Neurofilaments (NFs) are assembled from three highly phosphorylated proteins with molecular masses of 180 (NF-H), 130 (NF-M), and 66 kD (NF-L) of which NF-L forms the structural core. The core component, NF-L, was stoichiometrically labeled at cysteine 321 with fluorescein, coumarin, or biotin-maleimide to produce assembly-competent fluorescent or biotinylated derivatives, respectively. Using coumarin-labeled NF-L as fluorescence donor and fluorescein-labeled NF-L as the fluorescence acceptor, assembly of NF filaments was induced by rapidly raising the NaCl concentration to 170 mM, and the kinetics was followed by the decrease in the donor fluorescence. Assembly of NF-L subunits into filaments does not require nucleotide binding or hydrolysis but is strongly dependent on ionic strength, pH, and temperature. The critical concentration of NF-L, that concentration that remains unassembled at equilibrium with fully formed filaments, is 38 micrograms/ml or 0.6 microM. Under physiological salt conditions NF-L filaments also undergo extensive subunit exchange. Kinetic analysis and evaluation of several possible mechanisms indicate that subunit exchange is preceded by dissociation of subunits from the filament and generation of a kinetically active pool of soluble subunits. Given the concentration of NF-L found in nerve cells and the possibility of regulating this pool, these results provide the first information that intermediate filaments are dynamic structures and that NF-L within the NF complex is in dynamic equilibrium with a small but kinetically active pool of unassembled NF-L units.
Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters.
Authors: Tanner L I , Lienhard G E
Published: May 2004

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.
The hyaluronate receptor is preferentially expressed on proliferating epithelial cells.
Authors: Alho A M , Underhill C B
Published: May 2004

In the present study, we have examined the distribution of the hyaluronate receptor as well as hyaluronate itself in a variety of adult tissues. The hyaluronate receptor was localized with a monoclonal antibody, termed K-3, while hyaluronate was localized using proteolytic fragments of cartilage proteoglycan. Staining with the K-3 monoclonal antibody revealed that the hyaluronate receptor was present in a variety of epithelia including the skin, cheek, tongue, esophagus, vagina, intestines, oviduct, and bladder. However, it was notably absent from epithelial cells of the cornea and stomach as well as from endothelial cells of blood vessels. When present, the hyaluronate receptor was preferentially located in regions of active cell growth, such as in the basal layers of stratified epithelium and at the base of the crypts of Lieberkuhn in intestinal epithelium. A similar phenomenon was observed in cultured 3T3 cells. Cultures of 3T3 cells that were actively proliferating were found to have greater amounts of the receptor than their nonproliferating counterparts. When the various tissues were examined for hyaluronate, it was found to have a widespread distribution, being present in most of the basement membranes and between the cells in stratified epithelium. Indeed, in many cases, the distribution of hyaluronate closely paralleled that of the hyaluronate receptor. These results suggest that the interaction between hyaluronate and its receptor is involved in cell-to-substratum adhesion.
Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.
Authors: Weidman P J , Melançon P , Block M R , Rothman J E
Published: May 2004

An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.
Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis.
Authors: Croze E , Ivanov I E , Kreibich G , Adesnik M , Sabatini D D , Rosenfeld M G
Published: May 2004

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.
The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts.
Authors: Liscum L , Ruggiero R M , Faust J R
Published: May 2004

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.
In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.
Authors: Ahrem B , Hoffschulte H K , Müller M
Published: May 2004

In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated. To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system. In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization. However, if inverted membrane vesicles from E. coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membrane-containing subfraction (enriched in leader [signal] peptidase activity). This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides. Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme. Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles. Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force.
The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.
Authors: Stoller T J , Shields D
Published: May 2004

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min. Globin degradation was insensitive to chloroquine, a drug which inhibits lysosomal proteases, but was inhibited at 16 degrees C suggesting proteolysis occurred during transport to the cis-Golgi apparatus. In contrast to the control globin, approximately 30% of the somatostatin propeptide-globin fusion protein was transported to the distal elements of the Golgi apparatus where it was endoproteolytically processed. Processing of the chimera occurred in an acidic intracellular compartment since cleavage was inhibited by 25 microM chloroquine. 60% of the transported chimera was cleaved at the Arg-Lys processing site in native prosomatostatin yielding "mature" alpha-globin. Most significantly, approximately 50% of processed alpha-globin was sorted to the regulated pathway and secreted in response to 8-Br-cAMP. We conclude that the somatostatin propeptide mediated transport of alpha-globin from the endoplasmic reticulum to the trans-Golgi network by protecting molecules from degradation and in addition, facilitated packaging of alpha-globin into vesicles whose secretion was stimulated by cAMP.