bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmalemmal proteins of cultured vascular endothelial cells exhibit apical-basal polarity: analysis by surface-selective iodination.
Authors: Muller W A , Gimbrone M A
Published: May 2004

Vascular endothelium in vivo appears to function as a polarized epithelium. To determine whether cellular polarity exists at the level of the plasma membrane, we have examined cultured endothelial monolayers for evidence of differential distribution of externally disposed plasmalemmal proteins at apical and basal cell surfaces. Lactoperoxidase beads were used to selectively label the apical surfaces of confluent endothelial monolayers, the total surfaces of nonenzymatically resuspended cells, and the basal surfaces of monolayers inverted on poly-L-lysine-coated coverslips, while maintaining greater than 98% viability in all samples. Comparison of the SDS PAGE radioiodination patterns obtained for each surface revealed a number of specific bands markedly enriched on either apical or basal surface. This polarized distribution involved membrane-associated as well as integral membrane proteins and was observed in several strains of bovine aortic endothelial cells, as well as in both primary and passaged human umbilical vein endothelial cells. In contrast, two morphologically nonpolarized cell types, bovine aortic smooth muscle and mouse peritoneal macrophages, did not display differential localization of integral membrane proteins. Polarized distribution of integral membrane proteins was established before the formation of a confluent monolayer. When inverted (basal-side-up) monolayers were returned to culture, the apical-side-up pattern was reexpressed within a few days. These results demonstrate that cell surface-selective expression of plasmalemmal proteins is an intrinsic property of viable endothelial cells in vitro. This apical/basal asymmetry of membrane structure may provide a basis for polarized endothelial functions in vivo.
Integrin (the CSAT antigen): functionality requires oligomeric integrity.
Authors: Buck C A , Shea E , Duggan K , Horwitz A F
Published: May 2004

Integrin, the cell-substrate attachment (CSAT) antigen, is a complex of integral membrane glycoproteins whose apparent function is to mediate cell-substratum adhesion by serving as a transmembrane link between the extracellular matrix and elements of the cytoskeleton. Previous attempts to separate the members of this complex under nondenaturing conditions have been successful. We have now produced a monoclonal antibody "G" that is specific for the lower molecular mass cysteine-rich band 3 of the complex. Using an antibody affinity column containing this monoclonal antibody, it is possible to dissociate integrin into two fractions, one containing band 3, the other containing bands 1 plus 2. Neither fraction will by itself bind fibronectin, laminin, or talin. However, when the fractions are combined, the reconstituted integrin elutes from a gel filtration column in the same position as the native complex, and binding activity to these molecules returns. Further, it is shown by gel filtration that the recognition site for the adhesion-disrupting monoclonal antibodies CSAT and JG22 is on band 3, supporting the contention that integrin is an oligomer. The data presented here is consistent with integrin being either a mixture of heterodimers, each with a common subunit and reacting with a particular extracellular matrix molecule, or a single heterotrimer capable of binding to several different extracellular matrix molecules.
Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve.
Authors: Martini R , Schachner M
Published: May 2004

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non-myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt-Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N-CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.
Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.
Authors: Engvall E , Davis G E , Dickerson K , Ruoslahti E , Varon S , Manthorpe M
Published: May 2004

Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.
Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils.
Authors: Sakai L Y , Keene D R , Engvall E
Published: May 2004

A new connective tissue protein, which we call fibrillin, has been isolated from the medium of human fibroblast cell cultures. Electrophoresis of the disulfide bond-reduced protein gave a single band with an estimated molecular mass of 350,000 D. This 350-kD protein appeared to possess intrachain disulfide bonds. It could be stained with periodic acid-Schiff reagent, and after metabolic labeling, it contained [3H]glucosamine. It could not be labeled with [35S]sulfate. It was resistant to digestion by bacterial collagenase. Using mAbs specific for fibrillin, we demonstrated its widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule. Electron microscopic immunolocalization with colloidal gold conjugates specified its location to a class of extracellular structural elements described as microfibrils. These microfibrils possessed a characteristic appearance and averaged 10 nm in diameter. Microfibrils around the amorphous cores of the elastic fiber system as well as bundles of microfibrils without elastin cores were labeled equally well with antibody. Immunolocalization suggested that fibrillin is arrayed periodically along the individual microfibril and that individual microfibrils may be aligned within bundles. The periodicity of the epitope appeared to match the interstitial collagen band periodicity. In contrast, type VI collagen, which has been proposed as a possible microfibrillar component, was immunolocalized with a specific mAb to small diameter microfilaments that interweave among the large, banded collagen fibers; it was not associated with the system of microfibrils identified by the presence of fibrillin.
Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.
Authors: Navone F , Jahn R , Di Gioia G , Stukenbrok H , Greengard P , De Camilli P
Published: May 2004

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.
Immunogold electron microscopy of phytochrome in Avena: identification of intracellular sites responsible for phytochrome sequestering and enhanced pelletability.
Authors: McCurdy D W , Pratt L H
Published: May 2004

Using monoclonal antibodies to the plant photoreceptor, phytochrome, we have investigated by immunogold electron microscopy the rapid, red light-induced, intracellular redistribution (termed "sequestering") of phytochrome in dark-grown Avena coleoptiles. Pre-embedding immunolabeling of 5-micron-thick cryosections reveals that sequestered phytochrome is associated with numerous, discrete structures of similar morphology. Specific labeling of these structures was also achieved by post-embedding ("on-grid") immunostaining of LR-White-embedded tissue, regardless of whether the tissue had been fixed chemically or by freeze substitution. The phytochrome-associated structures are globular to oval in shape, 200-400 nm in size, and are composed of amorphous, granular material. No morphologically identifiable membranes are present either surrounding or within these structures, which are often present as apparent aggregates that approach several micrometers in size. An immunogold labeling procedure has also been developed to identify the particulate, subcellular component with which phytochrome is associated in vitro as a consequence of irradiation of Avena coleoptiles before their homogenization. Structures with appearance similar to those identified in situ are the only components of the pelletable material that are specifically labeled with gold. We conclude that the association of phytochrome with these structures in Avena represents the underlying molecular event that ultimately is expressed both as red light-induced sequestering in vivo and enhanced pelletability of phytochrome detected in vitro.
Topoisomerase II: A specific marker for cell proliferation.
Authors: Heck M M , Earnshaw W C
Published: May 2004

We have used an antibody probe to measure the levels of topoisomerase II in several transformed and developmentally regulated normal cell types. Transformed cells contain roughly 1 X 10(6) copies of the enzyme. During erythropoiesis in chicken embryos the enzyme level drops from 7.8 X 10(4) copies per erythroblast to less than 300 copies per erythrocyte concomitant with the cessation of mitosis in the blood. Cultured myoblasts also lose topoisomerase II upon fusion into nonproliferating myotubes. When peripheral blood lymphocytes (which lack detectable topoisomerase II) commence proliferation, they express topoisomerase II de novo. Appearance of the enzyme exactly parallels the onset of DNA replication. These results suggest that topoisomerase II is not required for transcription in higher eukaryotes, but that it may function during DNA replication. Furthermore, topoisomerase II is a sensitive and specific marker for proliferating cells.
Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes.
Authors: Rentrop M , Knapp B , Winter H , Schweizer J
Published: May 2004

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.
Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices.
Authors: Larkin J M , Donzell W C , Anderson R G
Published: May 2004

Previous studies have shown that when human fibroblasts are depleted of intracellular K+, coated pits disappear from the cell surface and the receptor-mediated endocytosis of low density lipoprotein (LDL) is inhibited. We have now used the K+ depletion protocol to study several aspects of coated pit function. First, since coated pits rapidly form when K+-depleted fibroblasts are incubated in the presence of 10 mM KCl, we studied the sequence of assembly of coated pits as visualized in carbon-platinum replicas of inner membrane surfaces from cells that had been incubated in the presence of K+ for various times. New coated pits initially appeared as planar clathrin lattices that increased in size by the formation of polygons at the margin of the lattice. Once the lattice reached a critical size it invaginated to form coated vesicles. Second, we determined that LDL-ferritin can induce clustering of LDL receptors over noncoated membrane on the surface of K+-depleted fibroblasts; however, when these cells are subsequently incubated in the presence of K+, these clusters become associated with newly formed coated pits and are internalized. Finally, we determined that K+ depletion inhibits the assembly of coated pits, but that existing coated pits in K+-depleted cells are able to internalize LDL. These results suggest that the clathrin lattice of coated pits is actively involved in membrane shape change during endocytosis and that the structural proteins of the lattice are cyclically assembled and disassembled in the process.
An actin-nucleating activity in polymorphonuclear leukocytes is modulated by chemotactic peptides.
Authors: Carson M , Weber A , Zigmond S H
Published: May 2004

We examined the actin-nucleating activity in polymorphonuclear leukocyte lysates prepared at various times after chemotactic peptide addition. The actin nucleation increases two- to threefold within 15 s after peptide addition, decays to basal levels within 90 s, and is largely independent of cytoplasmic calcium fluxes. The peptide-induced nucleation sites behave as free barbed ends and therefore may increase the level of polymerized actin in vivo. The new nucleation sites may also determine the cellular sites of actin polymerization. This localization of actin polymerization could be important for the directional extension of lamellipodia during chemotaxis.
Homing of hemopoietic precursor cells to the embryonic thymus: characterization of an invasive mechanism induced by chemotactic peptides.
Authors: Savagner P , Imhof B A , Yamada K M , Thiery J P
Published: May 2004

During embryonic development, T cell precursors migrate to the thymus, where immunocompetency is acquired. Our previous studies have shown that avian hemopoietic precursor cells are recruited to the thymus by chemotactic peptides secreted by thymic epithelial cells (Champion, S., B. A. Imhof, P. Savagner, and J. P. Thiery, 1986, Cell, 44:781-790). In this study, we have characterized the homing of these precursor cells to the thymus in vivo by electron and light microscopy. Hemopoietic precursors could be seen to extravasate from blood or lymphatic vessels, migrate in the mesenchyme, traverse the perithymic basement membrane, and finally intercalate into the thymic epithelium. Labeled hemopoietic precursors injected into the blood circulation also followed the same pathway. Migrating hemopoietic precursor cells were found to express the fibronectin receptor complex. In the presence of thymic chemotactic peptides, hemopoietic precursors traverse a human amniotic basement membrane. This invasive process was inhibited by antibodies to laminin or to fibronectin, two major glycoproteins of the amniotic membrane, by monovalent Fab' fragments of antibodies to the fibronectin receptor, and, finally by synthetic peptides that contain the cell-binding sequence Arg-Gly-Asp-Ser of fibronectin. These results indicate that hemopoietic precursors respond to thymic chemotactic peptides by invasive behavior. Direct interactions between basement membrane components and fibronectin receptors appear to be required for this developmentally regulated invasion process.
Tau protein function in living cells.
Authors: Drubin D G , Kirschner M W
Published: May 2004

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.
Micromanipulated bivalents can trigger mini-spindle formation in Drosophila melanogaster spermatocyte cytoplasm.
Authors: Church K , Nicklas R B , Lin H P
Published: May 2004

Single (individual) bivalents in cultured Drosophila melanogaster primary spermatocytes were detached from the spindle with a micromanipulation needle and placed in the cytoplasm. Such bivalents are prevented from rejoining the spindle by a natural membrane barrier that surrounds the spindle, but they quickly orient as if on a spindle of their own and the half-bivalents separate in anaphase. Serial section electron microscopy shows that a mini-spindle forms around the cytoplasmic bivalent, i.e., the microtubule density in the vicinity of the bivalent is much greater than in other cytoplasmic regions. This microtubule population cannot be accounted for solely by kinetochore nucleation and/or capture of microtubules. Furthermore, the mini-spindles frequently form at odd angles to the main spindle, so that at least one pole has no relationship to the poles of the main spindle. We conclude that a bivalent, or factors that become associated with the bivalent as a result of the manipulation, can either stabilize microtubules or promote their assembly. The bivalent activates latent microtubule organizing centers, or alternatively, polar organizing material has been passively transported from the main spindle to the cytoplasm by the micromanipulation procedure.
A monoclonal antibody against the nucleus reveals the presence of a common protein in the nuclear envelope, the perichromosomal region, and cytoplasmic vesicles.
Authors: Wataya-Kaneda M , Kaneda Y , Sakurai T , Sugawa H , Uchida T
Published: May 2004

A monoclonal antibody that recognizes antigenic determinants on the nucleus of cultured mammalian cells was isolated. Immunofluorescence studies using this antibody showed that the recognized antigen was present not only on the nucleus but also in cytoplasmic vesicles of interphase cells and in the perichromosomal region of mitotic cells. Premature chromosome condensation analysis showed that the reactive site for this monoclonal antibody could be detected in the perichromosomal region during the G2 and M phases, but not during the G1 and S phases. Finally, immunoblot analysis showed that this monoclonal antibody prepared against the nucleus recognized a protein of approximately 40 kD both in the cytoplasm and in the perichromosomal regions.
Localization of cell surface sites involved in fibronectin fibrillogenesis.
Authors: Peters D M , Mosher D F
Published: May 2004

Fibronectin binding sites on cultured human fibroblasts were localized by high voltage electron microscopy using either 5- or 18-nm colloidal gold beads (Au5 or Au18) bound to intact fibronectin, the 70-kD amino-terminal fragment of fibronectin that blocks incorporation of exogenous fibronectin into extracellular matrix, or 160-180-kD fragments of fibronectin with cell adhesion and heparin-binding activities. Binding sites for Au18-fibronectin on the cell surface were localized to specific regions along the edge of the fibroblast and on retraction fibers. Au18-fibronectin complexes at these sites were initially localized in clusters that co-aligned with intracellular microfilament bundles. With longer incubations, Au18-fibronectin complexes were arranged into long fibrillar networks on the cell surface and in the extracellular space. The appearance of Au18-fibronectin in these fibrillar networks and disappearance of clusters of Au18-fibronectin suggest that Au18-fibronectin complexes are arranged into matrix at specific regions of the cell surface. Au18-70-kD fragment complexes initially had a similar distribution to Au18-fibronectin complexes. With longer incubations, Au18-70-kD fragment complexes were found in long linear arrangements on the cell surface. Double labeling experiments using Au18-70-kD fragment and Au5-160-180-kD fragments showed that the 70-kD fragment and the 160-180-kD fragments bind to different regions of the cell.
Platelet thrombospondin mediates attachment and spreading of human melanoma cells.
Authors: Roberts D D , Sherwood J A , Ginsburg V
Published: May 2004

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.
Selection of chemotaxis mutants of Dictyostelium discoideum.
Authors: Segall J E , Fisher P R , Gerisch G
Published: May 2004

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant-degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described.
Isolation and characterization of the gene for myosin light chain two of Drosophila melanogaster.
Authors: Toffenetti J , Mischke D , Pardue M L
Published: May 2004

A recombinant lambda-phage DNA clone containing Drosophila melanogaster sequences encoding the gene for myosin light chain (MLC) two has been isolated from a library of randomly sheared DNA. The Drosophila MLC2 gene is located in region 99E1-3 on the right arm of chromosome 3, several bands removed from the site reported for the other myosin light chain gene at 98B. The MLC2 sequence at 99E1-3 appears to encode all of the isoforms of Drosophila MLC2. The polypeptide encoded at 99E was identified as MLC2 by the following criteria: the in vitro translation product is identical in size to MLC2 isolated from Drosophila muscle, and on two-dimensional gels the in vitro translation product can be separated into two or more peptides that co-migrate with isoforms of larval and thoracic MLC2. RNA encoding the polypeptide was detected in embryos only after the onset of muscle differentiation and was also abundant in adult thoracic muscle. The nucleotide sequence of cDNA generated from late embryonic RNA would be translated to yield a protein sequence with multiple regions of homology to vertebrate MLC2. (There are shorter regions of homology to vertebrate MLC1). Like a number of vertebrate muscle proteins, Drosophila MLC2 has an acetylated amino-terminus.
Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins.
Authors: Burgess D R , Broschat K O , Hayden J M
Published: May 2004

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.
Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo.
Authors: Kim S , Magendantz M , Katz W , Solomon F
Published: May 2004

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.
Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum.
Authors: Ibrahimi I
Published: May 2004

The translocation of prepromelittin (pPM) across mammalian endoplasmic reticulum was studied in both wheat germ and reticulocyte lysate. In the wheat germ system, signal recognition particle (SRP) caused a transient arrest in the synthesis of pPM. This was indicated by a slowdown in the rate of synthesis of pPM in the presence of SRP. The arrest was specific, dependent on the concentration of SRP, and more effective at early incubation time. In a tightly synchronized translation system, SRP had no apparent effect on the elongation of pPM, indicating that the effect of SRP on pPM chain synthesis might be at the final stages of chain elongation and release from the ribosome. This was reflected in a transient accumulation of pPM as peptidyl tRNA. Because pPM is composed of only 70 amino acids, arrest by SRP may be very close to chain termination. Arrest at this stage of chain synthesis seems to be unstable and the nascent chain gets terminated and released from the ribosome after a transient delay. The translocation of pPM was shown to be dependent on both SRP and docking protein. The difference in the translocation efficiency of pPM in reticulocyte and wheat germ lysates may reflect a difference in the targeting process in the two systems.
Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends.
Authors: Gorbsky G J , Sammak P J , Borisy G G
Published: May 2004

During the movement of chromosomes in anaphase, microtubules that extend between the kinetochores and the poles shorten. We sought to determine where subunits are lost from these microtubules during their shortening. Prophase or prometaphase cells on coverslips were injected with fluoresceinated tubulin and allowed to progress through mitosis. Immediately after the onset of anaphase, a bar-shaped beam of laser light was used to mark a domain on the kinetochore fibers by photobleaching a band, approximately 1.0 micron wide, across the spindle. In different cells, spindles were photobleached at varying distances from the chromosomes. Cells were allowed to continue in anaphase until the chromosomes had further separated. They were then lysed, fixed, and prepared for double-label immunofluorescence with an antibody to fluorescein that does not bind appreciably to bleached fluorescein, and with an antibody to tubulin. Photobleached domains of microtubules appeared as bands of reduced fluorescence in the anti-fluorescein image. However, the anti-tubulin labeling revealed that microtubules were present and continuous through the photobleached domains. In all cases, the chromosomes approached and invaded the bleached domain while the bleached domain itself remained stationary with respect to the near pole. These results demonstrate that the chromosomes move along stationary kinetochore microtubules and that depolymerization of these microtubules during anaphase takes place at the kinetochore. In contrast to the generally accepted older view that chromosomes are passive objects pulled by "traction fibers," we suggest that the kinetochore is an active participant in generating the motive force that propels the chromosome to the pole.
Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages.
Authors: Pumplin D W , Bloch R J
Published: May 2004

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.
Control of hsp70 RNA levels in human lymphocytes.
Authors: Kaczmarek L , Calabretta B , Kao H T , Heintz N , Nevins J , Baserga R
Published: May 2004

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.
Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
Authors: Finlay D R , Newmeyer D D , Price T M , Forbes D J
Published: May 2004

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.
Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.
Authors: Wiedmann M , Kurzchalia T V , Bielka H , Rapoport T A
Published: May 2004

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.
Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.
Authors: Capony F , Morisset M , Barrett A J , Capony J P , Broquet P , Vignon F , Chambon M , Louisot P , Rochefort H
Published: May 2004

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.