bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells.
Authors: Benavente R , Rose K M , Reimer G , Hügle-Dörr B , Scheer U
Published: May 2004

The formation of daughter nuclei and the reformation of nucleolar structures was studied after microinjection of antibodies to RNA polymerase I into dividing cultured cells (PtK2). The fate of several nucleolar proteins representing the three main structural subcomponents of the nucleolus was examined by immunofluorescence and electron microscopy. The results show that the RNA polymerase I antibodies do not interfere with normal mitotic progression or the early steps of nucleologenesis, i.e., the aggregation of nucleolar material into prenucleolar bodies. However, they inhibit the telophasic coalescence of the prenucleolar bodies into the chromosomal nucleolar organizer regions, thus preventing the formation of new nucleoli. These prenucleolar bodies show a fibrillar organization that also compositionally resembles the dense fibrillar component of interphase nucleoli. We conclude that during normal nucleologenesis the dense fibrillar component forms from preformed entities around nucleolar organizer regions, and that this association seems to be dependent on the presence of an active form of RNA polymerase I.
The subnuclear localization of tRNA ligase in yeast.
Authors: Clark M W , Abelson J
Published: May 2004

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.
Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle.
Authors: Weisman L S , Bacallao R , Wickner W
Published: May 2004

The vacuole of the yeast Saccharomyces cerevisiae was visualized with three unrelated fluorescent dyes: FITC-dextran, quinacrine, and an endogenous fluorophore produced in ade2 yeast. FITC-dextran, which enters cells by endocytosis, had been previously developed as a vital stain for yeast vacuoles. Quinacrine, which diffuses across membranes and accumulates in acidic compartments in mammalian cells, can also be used as a marker for yeast vacuoles. ade2 yeast accumulate an endogenous fluorophore in their vacuoles. Using these stains, yeast were examined for vacuole morphology throughout the cell division cycle. In both the parent cell and the bud, a single vacuole was the most common morphology at every stage. Two or more vacuoles could also be found in the mother cell or in the bud; however, this morphology was not correlated with any stage of the cell division cycle. Even small buds (in early S phase) often contained a small vacuole. By the time the bud was half the diameter of the mother cell, it almost always bore a vacuole. This picture of vacuole division and segregation differs from what is seen with synchronized cultures. In ade2 yeast, the bud usually inherits a substantial portion of its vacuole contents from the mother cell. We propose that vacuolar segregation is accomplished by vesicular traffic between the parent cell and the bud.
Dithiothreitol and the translocation of preprolactin across mammalian endoplasmic reticulum.
Authors: Ibrahimi I
Published: May 2004

The translocation mode of preprolactin (pPL) across mammalian endoplasmic reticulum was reinvestigated in light of recent findings that nascent secretory polypeptides synthesized in the presence of a highly reducing environment could be translocated posttranslationally and independently of their attachment to the ribosome (Maher, P. A., and S. J. Singer, 1986, Proc. Natl. Acad. Sci. USA, 83:9001-9005). The effects of the reducing agent dithiothreitol (DTT) on pPL synthesis and translocation were studied in this respect. The translocation of pPL was shown to take place only cotranslationally. The apparent posttranslational translocation was due to ongoing chain synthesis irrespective of the presence of high concentrations of DTT. When synthesis was completely blocked, no translocation was observed in the presence or absence of DTT. The synthesis of pPL was retarded by DTT, while its percent translocation was enhanced. The retardation in synthesis was reflected in reduced rates of initiation and elongation. As a consequence of this retardation, which increases the ratio of microsomes to nascent chains, and of possible effects on the conformation of nascent pPL and components of the translocation apparatus, DTT may expand the time and chain length windows for nascent chain translocation competence.
Lumenal location of the microsomal beta-glucuronidase-egasyn complex.
Authors: Brown J , Novak E K , Takeuchi K , Moore K , Medda S , Swank R T
Published: May 2004

Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.
Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary.
Authors: Hashimoto S , Fumagalli G , Zanini A , Meldolesi J
Published: May 2004

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.
Interactions of thrombospondin with endothelial cells: receptor-mediated binding and degradation.
Authors: Murphy-Ullrich J E , Mosher D F
Published: May 2004

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.
Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.
Authors: van Meer G , Stelzer E H , Wijnaendts-van-Resandt R W , Simons K
Published: May 2004

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.
Structure of the novel membrane-coating material in proton-secreting epithelial cells and identification as an H+ATPase.
Authors: Brown D , Gluck S , Hartwig J
Published: May 2004

Specialized proton-secreting cells known collectively as mitochondria-rich cells are found in a variety of transporting epithelia, including the kidney collecting duct (intercalated cells) and toad and turtle urinary bladders. These cells contain a population of characteristic tubulovesicles that are believed to be involved in the shuttling of proton pumps (H+ATPase) to and from the plasma membrane. These transporting vesicles have a dense, studlike material coating the cytoplasmic face of their limiting membranes and similar studs are also found beneath parts of the plasma membrane. We have recently shown that this membrane coat does not contain clathrin. The present study was performed to determine the structure of this coat in rapidly frozen and freeze-dried tissue, and to determine whether the coat contains a major membrane protein transported by these vesicles, a proton pumping H+ATPase. The structure of the coat was examined in proton-secreting, mitochondria-rich cells from toad urinary bladder epithelium by rapidly freezing portions of apical membrane and associated cytoplasm that were sheared away from the remainder of the cell using polylysine-coated coverslips. Regions of the underside of these apical membranes as large as 0.2 micron2 were decorated by studlike projections that were arranged into regular hexagonal arrays. Individual studs had a diameter of 9.5 nm and appeared to be composed of multiple subunits arranged around a central depression, possibly representing a channel. The studs had a density of approximately 16,800 per micron2 of membrane. Similar arrays of studs were also found on vesicles trapped in the residual band of cytoplasm that remained attached to the underside of the plasma membrane, but none were seen in adjacent granular cells. To determine whether these arrays of studs contained H+ATPase molecules, we examined a preparation of affinity-purified bovine medullary H+ATPase, using the same technique, after incorporation of the protein eluted from a monoclonal antibody affinity column into phospholipid liposomes. The affinity-purified protein was shown to be capable of ATP-dependent acidification. In such preparations, large paracrystalline arrays of studs identical in appearance to those seen in situ were found. The dimensions of the studs as well as the number per square micrometer of membrane were identical to those of toad bladder mitochondria-rich cells: 9.5 nm in diameter, 16,770 per micron2 of membrane.(ABSTRACT TRUNCATED AT 400 WORDS)
Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction.
Authors: Lopez L C , Shur B D
Published: May 2004

Gamete recognition in the mouse is mediated by galactosyltransferase (GalTase) on the sperm surface, which binds to its appropriate glycoside substrate in the egg zona pellucida (Lopez, L. C., E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, 1985, J. Cell Biol., 101:1501-1510). GalTase has been localized by indirect immunofluorescence to the dorsal surface of the anterior sperm head overlying the intact acrosome. Sperm binding to the zona pellucida triggers induction of the acrosome reaction, an exocytotic event that results in vesiculation and release of the outer acrosomal and overlying plasma membranes. Consequently, we examined the fate of sperm surface GalTase after the acrosome reaction. Contrary to our expectations, surface GalTase is not lost during the acrosome reaction despite the loss of its membrane domain. Rather, double-label indirect immunofluorescence assays show that GalTase is redistributed to the lateral surface of the sperm, coincident with the acrosome reaction. This apparent redistribution of GalTase was confirmed by direct enzymatic assays, which show that 90% of sperm GalTase activity is retained during the acrosome reaction. No GalTase activity is detectable on plasma membrane vesicles released during the acrosome reaction. In contrast, removal of plasma membranes by nitrogen cavitation releases GalTase activity from the sperm surface, showing that GalTase redistribution requires a physiological acrosome reaction. The selective redistribution of GalTase to a new membrane domain from one that is lost during the acrosome reaction suggests that GalTase is repositioned for some additional function after initial sperm-zona binding.
Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.
Authors: Dunia I , Manenti S , Rousselet A , Benedetti E L
Published: May 2004

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.
On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force.
Authors: Spurck T P , Pickett-Heaps J D
Published: May 2004

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.
Visualization of myosin in living cells.
Authors: Mittal B , Sanger J M , Sanger J W
Published: May 2004

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.
Abnormal expression and processing of keratins in pupoid fetus (pf/pf) and repeated epilation (Er/Er) mutant mice.
Authors: Fisher C , Jones A , Roop D R
Published: May 2004

The pupoid fetus (pf) and repeated epilation (Er) mutations of mice result in a failure of epidermal differentiation in homozygotes. Expression of the epidermal keratins has been followed in pf/pf and Er/Er mice by two-dimensional gel electrophoresis, and by immunohistochemistry and Western blotting using polyclonal antibodies that are monospecific for individual keratin polypeptides. Our results show that expression of the differentiation-specific keratins (K1 and K10) is delayed in both the pf/pf and Er/Er mutants and that, when these keratins do appear later in development, they are localized in the deeper layers of the thickened mutant epidermis. Conversely, K6 and K16, two keratins found in low abundance in normal epidermis, are abundant in mutant epidermis. In newborn mutant epidermis, K6 and K16 are found to be most abundant in the outermost epidermal cells, a distribution opposite to that of K1 and K10. These findings suggest that the expression of these hyperplastic keratins in mutant mice may occur to the exclusion of the differentiation-specific keratins both during development and in newborn animals. Differentiation, and an apparently normal pattern of keratin expression, occur when whole pf/pf or Er/Er skin is grafted to normal mice. These results suggest that the pf and Er genes may be expressed systemically and that transfer of the mutant skin to a "normal" environment results in the recovery of a normal phenotype.
Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues.
Authors: Couchman J R
Published: May 2004

A heparan sulfate proteoglycan (HSPG) synthesized by murine parietal yolk sac (PYS-2) cells has been characterized and purified from culture supernatants. A monospecific polyclonal antiserum was raised against it which showed activity against the HSPG core protein and basement membrane specificity in immunohistochemical studies on frozen tissue sections from many rat organs. However, there was no reactivity with some basement membranes, notably those of several smooth muscle types and cardiac muscle. In addition, it was found that pancreatic acinar basement membranes also lacked the HSPG type recognized by this antiserum. Those basement membranes that lacked the HSPG strongly stained with antisera against laminin and type IV collagen. The striking distribution pattern is possibly indicative of multiple species of basement membrane HSPGs of which one type is recognized by this antiserum. Further evidence for multiple HSPGs was derived from the finding that skeletal neuromuscular junction and liver epithelia also did not contain this type of HSPG, though previous reports have indicated the presence of HSPGs at these sites. The PYS-2 HSPG was shown to be antigenically related to the large, low buoyant density HSPG from the murine Engelbreth-Holm swarm tumor. It was, however, confirmed that only a single population of antibodies was present in the serum. Despite the presence of similar epitopes on these two proteoglycans of different hydrodynamic properties, it was apparent that the PYS-2 HSPG represents a basement membrane proteoglycan of distinct properties reflected in its restricted distribution in vivo.
Characterization of concentration gradients of a morphogenetically active retinoid in the chick limb bud.
Authors: Eichele G , Thaller C
Published: May 2004

It has long been suggested that the generation of biological patterns depends in part on gradients of diffusible substances. In an attempt to bridge the gap between this largely theoretical concept and experimental embryology, we have examined the physiology of diffusion gradients in an actual embryonic field. In particular, we have generated in the chick wing bud concentration gradients of the morphogenetically active retinoid TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl] benzoic acid, a synthetic vitamin A compound. Upon local application of TTNPB the normal 234 digit pattern is duplicated in a way that correlates with the geometry of the underlying TTNPB gradient; low doses of TTNPB lead to a shallow gradient and an additional digit 2, whereas higher doses result in a steep, far-reaching gradient and patterns with additional digits 3 and 4. The experimentally measured TTNPB distribution along the anteroposterior axis, can be modeled by a local source and a dispersed sink. This model correctly predicts the site of specification of digit 2, and provides an empirical estimate of the diffusion coefficient (D) of retinoids in embryonic limb tissue. The numerical value of approximately 10(-7) cm2s-1 for D suggests that retinoids are not freely diffusible in the limb rudiment, but interact with the previously identified cellular retinoic acid binding protein. In addition, D affords an estimate of the time required to establish a diffusion gradient as 3 to 4 h. This time span is in a range compatible with the time scale of pattern specification in developing vertebrate limbs. Our studies support the view that diffusion of morphogenetic substances is a plausible mechanism of pattern formation in secondary embryonic fields.
Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.
Authors: Dermietzel R , Yancey S B , Traub O , Willecke K , Revel J P
Published: May 2004

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.
Clathrin assembly proteins: affinity purification and a model for coat assembly.
Authors: Keen J H
Published: May 2004

Assembly protein (AP) preparations from bovine brain coated vesicles have been fractionated by clathrin-Sepharose affinity chromatography. Two distinct fractions that possess coat assembly activity were obtained and are termed AP-1 and AP-2. The AP-1, not retained on the resin, has principal components with molecular weights of 108,000, 100,000, 47,000, and 19,000. The AP-2, bound to the resin and eluted by Tris-HCl at a concentration that parallels the latter's effect on coat disassembly, corresponds to the active complex described previously (Zaremba, S., and J. H. Keen, 1983, J. Cell Biol., 97:1339-1347). Its composition is similar to that of the AP-1 in that it contains 100,000-, 50,000-, and 16,000-mol-wt polypeptides in equimolar amounts; minor amounts of 112,000- and 115,000-mol-wt polypeptides are also present. Both are distinct from a recently described assembly protein of larger subunit molecular weight that we term AP-3. These results indicate the existence of a family of assembly proteins within cells. On incubation with clathrin both AP-1 and AP-2 induce the formation of coat structures, those containing AP-1 slightly smaller (mean diameter = 72 nm) than those formed in the presence of AP-2 (mean diameter = 79 nm); both structures have been detected previously in coated vesicle preparations from brain. Coats formed in the presence of AP-2 consistently contain approximately one molecule each of the 100,000-, 50,000-, and 16,000-mol-wt polypeptides per clathrin trimer. By low angle laser light scattering the molecular weight of native AP-2 was determined to be approximately 343,000, indicating that it is a dimer of each of the three subunits, and implying that it is functionally bivalent in clathrin binding. A model for AP-mediated coat assembly is proposed in which a bivalent AP-2 molecule bridges the distal legs or terminal domains of two clathrin trimers that are destined to occupy adjacent vertices in the assembled coat. Binding of a second AP-2 molecule locks these two trimers in register for assembly and further addition of AP-2 to free trimer legs promotes completion of the clathrin lattice. Effects of AP binding on the angle and flexibility of the legs at the hub of the trimer (the "pucker") are suggested to account for the characteristic size distributions of coats formed under varied conditions and, more speculatively, to contribute to the transformation of flat clathrin lattices to curved coated vesicles that are thought to occur during endocytosis.
Deep-etch visualization of 27S clathrin: a tetrahedral tetramer.
Authors: Heuser J E , Keen J H , Amende L M , Lippoldt R E , Prasad K
Published: May 2004

It has recently been reported that 8S clathrin trimers or "triskelions" form larger 27S oligomers upon dialysis into low ionic strength buffers (Prasad, K., R. E. Lippoldt, H. Edelhoch, and M. S. Lewis, 1986, Biochemistry, 25:5214-5219). Here, deep-etch electron microscopy of the 27S species reveals that they are closed tetrahedra composed of four clathrin triskelions. This was determined by two approaches. First, standard quick-freezing and freeze-etching of unfixed 27S species suspended in 2 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer, pH 5.9, yielded unambiguous images of tetrahedra that measured 33 nm on each edge. Second, the technique of freeze-drying molecules on mica (Heuser, J. E., 1983, J. Mol. Biol., 169:155-195) was modified to overcome the low affinity of mica in 2 mM MES, by pretreating the mica with polylysine. Thereafter, 27S species adsorbed avidly to it and collapsed into characteristic configurations containing four globular domains, each linked to the others by three approximately 33-nm struts. The globular domains look like vertices of deep-etched clathrin triskelions and the links, numbering 12 in all, look like four sets of triskelion legs. New light scattering and equilibrium centrifugation data confirm that 27S polymer is four times as massive as one clathrin triskelion. We conclude that in conditions that do not favor the formation of standard clathrin cages, low affinity interactions lead to closed, symmetrical assemblies of four triskelions, each of which assumes a unique puckered, straight-legged configuration to create the edges of a tetrahedron. Tetrahedra are similar in construction to the cubic octomers of clathrin recently found in ammonium sulfate solutions (Sorger, P. K., R. A. Crowther, J. T. Finch, and B. M. F. Pearse, 1986, J. Cell Biol., 103:1213-1219) but are still smaller, involving only half as many clathrin triskelions.
Polarized secretion of a platelet-derived growth factor-like chemotactic factor by endothelial cells in vitro.
Authors: Zerwes H G , Risau W
Published: May 2004

Cultured bovine aortic endothelial cells secrete a potent migration-stimulating factor for vascular smooth muscle cells (SMCs) and adventitial fibroblasts. Vascular pericytes are 20-fold less responsive, and endothelial cells themselves do not respond at all. Checkerboard analysis of SMC migration in a micro-chemotaxis chamber assay shows that the factor is chemotactic. Chemotactic activity for SMCs and adventitial fibroblasts is specifically inhibited by antibodies against platelet-derived growth factor. Endothelial cells cultured on nitrocellulose filters secrete the platelet-derived growth factor-like factor almost exclusively into the basal compartment. We suggest that this factor plays an important role in the recruitment of vascular wall cells during the morphogenesis of blood vessels and pathological conditions, such as atherosclerosis.
Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences.
Authors: Parry G , Cullen B , Kaetzel C S , Kramer R , Moss L
Published: May 2004

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)
The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis.
Authors: Cooke C A , Heck M M , Earnshaw W C
Published: May 2004

We describe a novel set of polypeptide antigens that shows a dramatic change in structural localization during mitosis. Through metaphase these antigens define a new chromosomal substructure that is located between the sister chromatids. Because the antigens are concentrated in the pericentromeric region, we have provisionally termed them the INCENPs (inner centromere proteins). The INCENPs (two polypeptides of 155 and 135 kD) were identified with a monoclonal antibody that was raised against the bulk proteins of the mitotic chromosome scaffold fraction. These two polypeptides are the most tightly bound chromosomal proteins known. When scaffolds are prepared, 100% of the detectable INCENPs remain scaffold associated. We were therefore unprepared for the fate of the INCENPs at anaphase. As the sister chromatids separate, the INCENPs dissociate fully from them, remaining behind at the metaphase plate as the chromatids migrate to the spindle poles. During anaphase the INCENPs are found on coarse fibers in the central spindle, and also in close apposition to the cell membrane in the region of the forming contractile ring. During telophase, the INCENPs gradually become focused onto the forming midbody, together with which they are ultimately discarded. Several possible in vivo roles for the INCENPs are suggested by these data: regulation of sister chromatid pairing, stabilization of the plane of cleavage, and separation of spindle poles at anaphase.
Redistribution of nuclear ribonucleoprotein antigens during herpes simplex virus infection.
Authors: Martin T E , Barghusen S C , Leser G P , Spear P G
Published: May 2004

Infection of human epidermoid carcinoma No. 2 cells with herpes simplex virus type 1 (HSV-1) leads to a reorganization of antigens associated with both the small and heterogeneous nuclear ribonucleoprotein complexes (snRNP and hnRNP). The hnRNP core protein antigens remain associated with the host chromatin, which appears to collapse into internal aggregates and along the nuclear envelope. More striking is the formation of prominent clusters of snRNP antigens (both general and U1 snRNP specific), which appear to condense throughout the nucleus then migrate to the periphery. These snRNP clusters have been identified at the fine structure level by immuno-electron microscopy. The HSV-1 presumed transcriptional activator ICP4, DNA-binding protein ICP8, and two capsid proteins ICP5 and p40 are not detectably associated with the snRNP clusters. Similar reorganization of snRNP occurs with HSV-2 and upon infection of African green monkey VERO cells with HSV-1. We speculate that the snRNP clusters arise from an increase in size and density of the interchromatin granule region of the host cell as a result of the partial inactivation of snRNP and host pre-mRNA splicing.
Drosophila spectrin. II. Conserved features of the alpha-subunit are revealed by analysis of cDNA clones and fusion proteins.
Authors: Byers T J , Dubreuil R , Branton D , Kiehart D P , Goldstein L S
Published: May 2004

Drosophila alpha-spectrin cDNA sequences were isolated from a lambda gt11 expression library. These cDNA clones encode fusion proteins that include portions of the Drosophila alpha-spectrin polypeptide as shown by a number of structural and functional criteria. The fusion proteins elicited antibodies that reacted strongly with Drosophila and vertebrate alpha-spectrins and a comparison of cyanogen bromide peptide maps demonstrated a clear structural correspondence between one fusion protein and purified Drosophila alpha-spectrin. Alpha-spectrin fusion protein also displayed calcium-dependent calmodulin-binding activity in blot overlay experiments and one fusion protein bound specifically to both Drosophila and bovine brain beta-spectrin subunits on protein blots. A region of the Drosophila cDNA cross-hybridized at lowered stringency with an avian alpha-spectrin cDNA. Together these data show that the composition, structure, and binding properties of the spectrin family of proteins have been remarkably well conserved between arthropods and vertebrates. Drosophila cDNA hybridized to an mRNA of greater than or equal to 9 kb on blots of total Drosophila poly A+ RNA; and hybridized in situ to a single site in polytene region 62B, 1-7. This result and Southern blot analysis of genomic DNA indicate that the sequences are likely to be single copy in the Drosophila genome.
The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits.
Authors: Zokas L , Glenney J R
Published: May 2004

Calpactins are a family of related Ca++-regulated cytoskeletal proteins. To analyze the expression and cytoskeletal association of calpactins we raised monoclonal antibodies with specificity for the heavy or light chains of calpactin I or to calpactin II. Comparison of the tissue distribution of calpactin I heavy and light chains by Western blots revealed that these subunits are coordinately expressed. Both soluble and cytoskeletal forms of the heavy chain of calpactin I were detected in human fibroblasts whereas only a soluble pool of calpactin II was found. These two forms of the calpactin I heavy chain differed both in their state of association with the light chain and in their rate of turnover. Both the soluble pool of the calpactin I heavy chain and calpactin II turned over three to four times faster than the cytoskeletal pool of heavy and light chains. Immunofluorescence microscopy revealed that the calpactin I light chain was present exclusively in the cytoskeleton whereas the calpactin I heavy chain distribution was more diffuse. No difference in the amount of light chain or the cytoskeletal attachment of phosphorylated calpactin I heavy chain was found in Rous sarcoma virus-transformed chick embryo fibroblasts compared with their normal counterpart. The antibody to the light chain of calpactin I was microinjected into cultured fibroblasts and kidney epithelial cells. In many cases antibody clustering was observed with the concomitant aggregation of the associated calpactin I heavy chain. The distribution of fodrin and calpactin II in injected cells remained unchanged. These results are consistent with the existence of two functionally distinct pools of calpactin I which differ in their association with the cytoskeleton.
Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis.
Authors: Schmit A C , Lambert A M
Published: May 2004

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.
Posttranslational modification and microtubule stability.
Authors: Schulze E , Asai D J , Bulinski J C , Kirschner M
Published: May 2004

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.
Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.
Authors: Gard D L , Kirschner M W
Published: May 2004

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.