bluuebunny/crossref_metadata_2025 huggingface dataset:
Uroplakin I: a 27-kD protein associated with the asymmetric unit membrane of mammalian urothelium.
Authors: Yu J , Manabe M , Wu X R , Xu C , Surya B , Sun T T
Published: May 2004

The luminal surface of mammalian urothelium is covered with numerous plaques (also known as the asymmetric unit membrane or AUM) composed of semi-crystalline, hexagonal arrays of 12-nm protein particles. Despite the presumed importance of these plaques in stabilizing the urothelial surface during bladder distention, relatively little is known about their protein composition. Using a mouse mAb, AE31, we have identified a 27-kD protein that is urothelium-specific and is differentially expressed in superficial umbrella cells. This protein (pI approximately 5.8) partitions into the detergent phase during Triton X-114 phase separation. Pulse-chase experiments using cultured bovine urothelial cells showed that this protein is synthesized as a 32-kD precursor that is processed through a 30-kD intermediate, to the mature 27-kD form. In cytoplasmic vesicles containing immature AUM, the AE31 epitope is detected in patches on the cytoplasmic side, but in mature, apical AUM it is detected exclusively on the luminal side. This suggests an unusual translocation of the AE31 epitope during AUM maturation; more data are required, however, to substantiate this interpretation. Immunoaffinity purification of the 27-kD protein results in the copurification in approximately molar ratio of a 15-kD protein, as well as a small and variable amount of a 47-kD protein. Immunoblotting data indicate that these three proteins are immunologically distinguishable. This copurified 15-kD protein is relative basic (pI approximately 8.0). Like the 27-kD protein, it is urothelium-specific and is present mainly in the umbrella cells. Together, our data indicate that a 27-kD protein is urothelial plaque-associated (uroplakin I). Based on complex formation data, we provisionally name the 15-kD protein uroplakin II; additional data will be required to determine whether this and the 47-kD protein are integral parts of AUM. The identification of these AUM-associated and -related proteins, plus the availability of a culture system capable of synthesizing and processing some of these molecules, offer new opportunities for studying the detailed structure, assembly, and function of asymmetrical unit membrane.
Direct demonstration of the lectin activity of gp90MEL, a lymphocyte homing receptor.
Authors: Imai Y , True D D , Singer M S , Rosen S D
Published: May 2004

Considerable evidence implicates gp90MEL as a lymphocyte homing receptor mediating lymphocyte attachment to high endothelial venules of lymph nodes in mouse. The protein appears to function as a calcium-dependent, lectin-like receptor as inferred primarily by the ability of specific carbohydrates to block its function and by the presence of a calcium-type lectin domain in its primary sequence. An ELISA assay is described which provides the first demonstration that the isolated protein has lectin activity and allows a further definition of its carbohydrate specificity. In addition to the monosaccharides mannose-6-phosphate and fructose-1-phosphate, ligand activity is shown for the sulfated glycolipid, sulfatide, and for two sulfated fucose-containing polysaccharides (fucoidin and egg jelly coat) from nonmammalian sources.
Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin.
Authors: Kramer R H , Cheng Y F , Clyman R
Published: May 2004

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.
The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.
Authors: Schnabel E , Anderson J M , Farquhar M G
Published: May 2004

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.
Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules.
Authors: Sorokin L , Sonnenberg A , Aumailley M , Timpl R , Ekblom P
Published: May 2004

It has been previously shown that A-chain and domain(E8)-specific antibodies to laminin that inhibit cell adhesion also interfere with the establishment of epithelial cell polarity during kidney tubule development (Klein, G., M. Langegger, R. Timpl, and P. Ekblom. 1988. Cell. 55:331-341). A monoclonal antibody specific for the integrin alpha 6 subunit, which selectively blocks cell binding to E8, was used to study the receptors involved. Immunofluorescence staining of embryonic kidneys and of organ cultures of metanephric mesenchyme demonstrated coappearance of the integrin alpha 6 subunit and the laminin A-chain in regions where nonpolarized mesenchymal cells convert into polarized epithelial cells. Both epitopes showed marked colocalization in basal areas of tubules, while an exclusive immunostaining for alpha 6 was observed in lateral and apical cell surfaces of the tubular epithelial cells. Organ culture studies demonstrated a consistent inhibition of kidney epithelium development by antibodies against the alpha 6 subunit. The data suggest that the recognition of E8 cell-binding site of laminin by a specific integrin is crucial for the formation of kidney tubule epithelium from undifferentiated mesenchymal stem cells. In some other cell types (endothelium, some ureter cells) an exclusive expression of alpha 6 with no apparent colocalization of laminin A-chain in the corresponding basement membrane was seen. Thus, in these cells, integrins possessing the alpha 6 subunit may bind to laminin isoforms that differ from those synthesized by developing tubules.
Evidence for the presence of a proton pump of the vacuolar H(+)-ATPase type in the ruffled borders of osteoclasts.
Authors: Väänänen H K , Karhukorpi E K , Sundquist K , Wallmark B , Roininen I , Hentunen T , Tuukkanen J , Lakkakorpi P
Published: May 2004

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H(+)-ATPase.
c-fos expression precedes osteogenic differentiation of cartilage cells in vitro.
Authors: Closs E I , Murray A B , Schmidt J , Schön A , Erfle V , Strauss P G
Published: May 2004

We have investigated the temporal pattern of expression of c-fos in cartilage cells in mouse mandibular condyles. During in vitro cultivation, the progenitor cells in this organ differentiate to osteoblasts, and hypertrophic chondrocytes start to show features indicative of osteogenic differentiation. Prior to these processes we observed two distinct patterns of c-fos expression. High, transient c-fos expression was found in the entire tissue within 30 min of culture. This type of c-fos expression appeared to result from mechanical forces applied during dissection. The second type of c-fos expression appeared in individual cells in the zone of hypertrophic chondrocytes. A varying number of formerly quiescent chondrocytes expressed high levels of c-fos mRNA after between 30 min and 10 d in culture, with a peak in the number of cells between days 1 and 3. c-fos expression in these cartilage cells was followed by DNA replication and expression of genes typifying osteoblastic differentiation. After 7 d in culture, groups of cells with the typical ultrastructural features of osteoblasts, and surrounded by an osteoid-like matrix, were observed in single chondrocyte-type lacunae, suggesting division of chondrocytes and differentiation to osteoblasts. The data suggest that c-fos may play a crucial role in the perturbation of determined pathways of skeletoblast differentiation and in the regulation of endochondral bone formation.
The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin.
Authors: Athey B D , Smith M F , Rankert D A , Williams S P , Langmore J P
Published: May 2004

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.
Immunoglobulin heavy chain and binding protein complexes are dissociated in vivo by light chain addition.
Authors: Hendershot L M
Published: May 2004

Immunoglobulin heavy chain binding protein (BiP, GRP78) associates stably with the free, nonsecreted Ig heavy chains synthesized by Abelson virus transformed pre-B cell lines. In cells synthesizing both Ig heavy and light chains, the Ig subunits assemble rapidly and are secreted. Only incompletely assembled Ig molecules can be found bound to BiP in these cells. In addition to Ig heavy chains, a number of mutant and incompletely glycosylated transport-defective proteins are stably complexed with BiP. When normal proteins are examined for combination with BiP, only a small fraction of the intracellular pool of nascent, unfolded, or unassembled proteins can be found associated. It has been difficult to determine whether these BiP-associated molecules represent assembly intermediates which will be displaced from BiP and transported from the cell, or whether these are aberrant proteins that are ultimately degraded. In order for BiP to monitor and aid in normal protein transport, its association with these proteins must be reversible and the released proteins should be transport competent. In the studies described here, transient heterokaryons were formed between a myeloma line producing BiP-associated heavy chains and a myeloma line synthesizing the complementary light chain. Introduction of light chain synthesis resulted in assembly of prelabeled heavy chains with light chains, displacement of BiP from heavy chains, and secretion of Ig into the culture supernatant. These data demonstrate that BiP association can be reversible, with concordant release of transportable proteins. Thus, BiP can be considered a component of the exocytic secretory pathway, regulating the transport of both normal and abnormal proteins.
Invariant chain trimers are sequestered in the rough endoplasmic reticulum in the absence of association with HLA class II antigens.
Authors: Marks M S , Blum J S , Cresswell P
Published: May 2004

HLA class II antigens are heterodimeric cell surface glycoproteins that interact with antigenic peptides to form complexes recognizable by CD4-positive T cells. During their biosynthesis, class II antigens are retained in a post-Golgi compartment in association with the invariant chain, which dissociates before class II cell surface expression. To address whether the invariant chain mediates this post-Golgi retention, its transport and assembly were examined in cells that do not express HLA class II antigens. Pulse-chase analysis and endoglycosidase digestions showed that very little invariant chain proceeded as far as the trans-Golgi in class II-negative cell lines. Immunofluorescence studies suggested that in these cells the invariant chain is sequestered in the RER. Gel filtration and cross-linking data showed that RER-localized invariant chain is present as trimers or aggregated trimers. Multimerization is mediated by lumenal interactions; a proteolytic fragment of the invariant chain corresponding to the lumenal domain remained trimeric as determined by cross-linking analysis. Similar transport and structural characteristics were observed for a pool of excess invariant chain in class II-positive cells, suggesting that an excess of invariant chain in the ER may be important for class II antigen function. These results have important implications for the transport of cellular proteins in general and for the role of the invariant chain in class II antigen biosynthesis.
Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle.
Authors: Raymond C K , O'Hara P J , Eichinger G , Rothman J H , Stevens T H
Published: May 2004

vps3 mutants of the yeast Saccharomyces cerevisiae are impaired in the sorting of newly synthesized soluble vacuolar proteins and in the acidification of the vacuole (Rothman, J. H., and T. H. Stevens. Cell. 47:1041-1051; Rothman, J. H., C. T. Yamashiro, C. K. Raymond, P. M. Kane, and T. H. Stevens. 1989. J. Cell Biol. 109:93-100). The VPS3 gene, which was cloned using a novel selection procedure, encodes a low abundance, hydrophilic protein of 117 kD that most likely resides in the cytoplasm. Yeast strains bearing a deletion of the VPS3 gene (vps3-delta 1) are viable, yet their growth rate is significantly reduced relative to wild-type cells. Temperature shift experiments with strains carrying a temperature conditional vps3 allele demonstrate that cells rapidly lose the capacity to sort the vacuolar protein carboxypeptidase Y upon loss of VPS3 function. Vacuolar morphology was examined in wild-type and vps3-delta 1 yeast strains by fluorescence microscopy. The vacuoles in wild-type yeast cells are morphologically complex, and they appear to be actively partitioned between mother cells and buds during an early phase of bud growth. Vacuolar morphology in vps3-delta 1 mutants is significantly altered from the wild-type pattern, and the vacuolar segregation process seen in wild-type strains is defective in these mutants. With the exception of a vacuolar acidification defect, the phenotypes of vps3-delta 1 strains are significantly different from those of mutants lacking the vacuolar proton-translocating ATPase. These data demonstrate that the acidification defect in vps3-delta 1 cells is not the primary cause of the pleiotropic defects in vacuolar function observed in these mutants.
Exocytosis in mast cells by basic secretagogues: evidence for direct activation of GTP-binding proteins.
Authors: Aridor M , Traub L M , Sagi-Eisenberg R
Published: May 2004

Histamine release induced by the introduction of a nonhydrolyzable analogue of GTP, GTP-gamma-S, into ATP-permeabilized mast cells, is associated with phosphoinositide breakdown, as evidenced by the production of phosphatidic acid (PA) in a neomycin-sensitive process. The dependency of both PA formation and histamine secretion on GTP-gamma-S concentrations is bell shaped. Whereas concentrations of up to 0.1 mM GTP-gamma-S stimulate both processes, at higher concentrations the cells' responsiveness is inhibited. At a concentration of 1 mM, GTP-gamma-S self-inhibits both PA formation and histamine secretion. Inhibition of secretion can, however, be overcome by the basic secretagogues compound 48/80 and mastoparan that in suboptimal doses synergize with 1 mM GTP-gamma-S to potentiate secretion. Secretion under these conditions is not accompanied by PA formation and is resistant both to depletion of Ca2+ from internal stores and to pertussis toxin (PtX) treatment. In addition, 48/80, like mastoparan, is capable of directly stimulating the GTPase activity of G-proteins in a cell-free system. Together, our results are consistent with a model in which the continuous activation of a phosphoinositide-hydrolyzing phospholipase C (PLC) by a stimulatory G-protein suffices to trigger histamine secretion. Basic secretagogues of mast cells, such as compound 48/80 and mastoparan, are capable of inducing secretion in a mechanism that bypasses PLC by directly activating a G-protein that is presumably located downstream from PLC (GE). Thereby, these secretagogues induce histamine secretion in a receptor-independent manner.
Changes in the state of actin during the exocytotic reaction of permeabilized rat mast cells.
Authors: Koffer A , Tatham P E , Gomperts B D
Published: May 2004

The major part of mast cell actin is Triton-soluble and behaves as a monomer in the DNase I inhibition assay. Thus, actin exists predominantly in monomeric or short filament form, through filamentous actin is clearly apparent in the cortical region after rhodamine-phalloidin (RP) staining. The minimum actin content is estimated to be approximately 2.5 micrograms/10(6) cells (cytosolic concentration approximately 110 microM. After permeabilization of mast cells by the bacterial cytolysin streptolysin-O, approximately 60% of the Triton-soluble actin leaks out within 10 min. However, the staining of the cortical region by RP remains undiminished, and the cells are still capable of exocytosis when stimulated by GTP-gamma-S together with Ca2+. In the presence of cytochalasin E the requirement for Ca2+ is decreased, indicating that disassembly of the cytoskeleton may be a prerequisite for exocytosis. This disassembly is likely to be controlled by Ca2(+)-dependent actin regulatory proteins; their presence is indicated by a Ca2(+)-dependent inhibition of polymerization of extraneous pyrene-G-actin by a Triton extract of mast cells. The effect of cytochalasin E on secretion is similar to that of phorbol myristate acetate, an activator of protein kinase C; both agents enhance the apparent affinity for Ca2+ and cause variable extents of Ca2(+)-independent secretion. Exposing the permeabilized cells to increasing concentrations of Ca2+ caused a progressive decrease in F-actin levels as measured by flow cytometry of RP-stained cells. In this respect, both cytochalasin E and phorbol ester mimicked the effects of calcium. GTP-gamma-S was not required for the Ca2(+)-dependent cortical disassembly. Thus, since conditions have not yet been identified where secretion can occur in its absence, cortical disassembly may be essential (though it is not sufficient) for exocytosis to occur.
Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages.
Authors: Tabas I , Lim S , Xu X X , Maxfield F R
Published: May 2004

Hypercholesterolemic rabbit beta-VLDL and human LDL are both internalized by mouse peritoneal macrophages by receptor-mediated endocytosis. However, only beta-VLDL (which binds to the cells with a much higher affinity than LDL) markedly stimulates acyl-CoA/cholesterol acyl transferase (ACAT) and induces foam cell formation in these cells. As an initial step to test whether the two lipoproteins might be targeted to different organelles (which might differ in their ability to deliver cholesterol to microsomal ACAT), we studied the endocytic pathways of beta-VLDL and LDL. Lipoproteins were labeled with the non-transferable fluorescent label, DiI. When the macrophages were incubated with DiI-LDL for 10 min at 37 degrees C, the fluorescence was concentrated near the center of the cell both in heavily labeled vesicles and in a diffuse pattern. The pattern with DiI-beta-VLDL was quite different: an array of bright vesicles throughout the cytoplasm was the predominant feature. Differences in distribution were seen as early as 2 min of incubation and persisted throughout a 10-min chase period. By using a procedure in which photobleaching of DiI fluorescence converts diaminobenzidine into an electron-dense marker, we were able to identify at the ultrastructural level vesicles containing electron-dense material in cells incubated with DiI-beta-VLDL. Human E2/E2 beta-VLDL (from a patient with familial dysbetalipoproteinemia), which has a binding affinity and ACAT-stimulatory potential similar to LDL, gave a pattern of fluorescence virtually identical to LDL. Pulse-chase studies with 125I-labeled and [3H]cholesteryl ester-labeled lipoproteins disclosed that both protein degradation and cholesteryl ester hydrolysis were markedly retarded in beta-VLDL compared with LDL. Thus, in mouse peritoneal macrophages, endocytosed beta-VLDL appears in a distinct set of widely-distributed vesicles not seen with LDL (or with E2-beta-VLDL) and, compared with LDL, has a markedly diminished rate of protein degradation and cholesteryl ester hydrolysis. The differential routing of LDL and beta-VLDL may provide a mechanism for differences in ACAT-stimulatory potential between the two lipoproteins.
Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.
Authors: Kopitz J , Kisen G O , Gordon P B , Bohley P , Seglen P O
Published: May 2004

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.
Temperature-sensitive yeast mutants defective in mitochondrial inheritance.
Authors: McConnell S J , Stewart L C , Talin A , Yaffe M P
Published: May 2004

The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.
Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane.
Authors: van 't Hof W , van Meer G
Published: May 2004

Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below 16 degrees C, where vesicular traffic is essentially blocked, each fluorescent product accumulated in the Golgi area. At 37 degrees C, 50% had been transported to the cell surface within 0.5 h, as measured by selective extraction of the fluorescent lipids onto BSA in the medium ("back-exchange") at 10 degrees C. Transport to the two surfaces could be assayed separately, as a diffusion barrier existed for both NBD-lipids and BSA. C6-NBD-glucosylceramide was enriched twofold apically, whereas C6-NBD-sphingomyelin was equally distributed over both domains. Polarities did not decrease when 37 degrees C incubations were carried out in the presence of increasing BSA concentrations to trap the fluorescent lipids immediately after their arrival at the cell surface. Within 10 min from the start of synthesis, both products displayed their typical surface polarity. Lipid transcytosis displayed a half time of hours. In conclusion, newly synthesized sphingolipids in Caco-2 cells are sorted before reaching the cell surface. Transcytosis is not required for generating the in vivo lipid polarity.
Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells.
Authors: Wandinger-Ness A , Bennett M K , Antony C , Simons K
Published: May 2004

Immunoisolation techniques have led to the purification of apical and basolateral transport vesicles that mediate the delivery of proteins from the trans-Golgi network to the two plasma membrane domains of MDCK cells. We showed previously that these transport vesicles can be formed and released in the presence of ATP from mechanically perforated cells (Bennett, M. K., A. Wandinger-Ness, and K. Simons, 1988. EMBO (Euro. Mol. Biol. Organ.) J. 7:4075-4085). Using virally infected cells, we have monitored the purification of the trans-Golgi derived vesicles by following influenza hemagglutinin or vesicular stomatitis virus (VSV) G protein as apical and basolateral markers, respectively. Equilibrium density gradient centrifugation revealed that hemagglutinin containing vesicles had a slightly lower density than those containing VSV-G protein, indicating that the two fractions were distinct. Antibodies directed against the cytoplasmically exposed domains of the viral spike glycoproteins permitted the resolution of apical and basolateral vesicle fractions. The immunoisolated vesicles contained a subset of the proteins present in the starting fraction. Many of the proteins were sialylated as expected for proteins existing the trans-Golgi network. The two populations of vesicles contained a number of proteins in common, as well as components which were enriched up to 38-fold in one fraction relative to the other. Among the unique components, a number of transmembrane proteins could be identified using Triton X-114 phase partitioning. This work provides evidence that two distinct classes of vesicles are responsible for apical and basolateral protein delivery. Common protein components are suggested to be involved in vesicle budding and fusion steps, while unique components may be required for specific recognition events such as those involved in protein sorting and vesicle targeting.
Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.
Authors: Zanetti M , Litteri L , Gennaro R , Horstmann H , Romeo D
Published: May 2004

Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.
Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells.
Authors: Hanukoglu I , Suh B S , Himmelhoch S , Amsterdam A
Published: May 2004

After ovulation of an oocyte, granulosa cells of the ovarian follicle differentiate into luteal cells and become a major factor dedicated to the synthesis of the steroid hormone progesterone. We recently established granulosa cell lines by cotransfection of granulosa cells with SV-40 and Ha-ras oncogene. In these cells progesterone secretion can be induced by cAMP as in normal rat granulosa cells. The induction of progesterone secretion is observed only after approximately 24 h and closely follows the delayed but quantitatively dramatic induction of the mitochondrial cytochrome P450scc which catalyzes the first step in steroid hormone biosynthesis. The mitochondrial P450 system electron transport proteins, adrenodoxin and adrenodoxin reductase, are also induced but adrenodoxin shows a faster induction. Immunofluorescence studies show that the three enzymes are induced in all cells and incorporated into all mitochondria uniformly. Electron microscopic examination using immunogold technique further confirms this and reveals that adrenodoxin is predominantly located on the matrix side of the inner mitochondrial membrane. Thus, adrenodoxin, which is a small highly charged protein, shows a distribution similar to P450scc which is an integral membrane protein. The uniformity of the response of the cells provides further evidence for the homogeneity of the cell line and makes this new granulosa cell line a highly promising system for the study of the molecular mechanisms involved in changes in gene expression during the process of granulosa cell differentiation.
Predominance of clathrin light chain LCb correlates with the presence of a regulated secretory pathway.
Authors: Acton S L , Brodsky F M
Published: May 2004

Two forms of clathrin light chains, LCa and LCb, are expressed in all mammalian and avian tissues that have been examined, whereas only one type is found in yeast. Regions of structural dissimilarity between LCa and LCb indicate possible functional diversity. To determine how LCa and LCb might differentially influence clathrin function, light chain expression patterns and turnover were investigated. Relative expression levels of the two light chains were determined in cells and tissues with and without a regulated secretory pathway. LCa/LCb ratios ranged from 5:1 to 0.33:1. A higher proportion of LCb was observed in cells and tissues that maintain a regulated pathway of secretion, suggesting a specialized role for the LCb light chain in this process. The ratio of light chains in assembled clathrin was found to reflect the levels of total light chains expressed in the cell, indicating no preferential incorporation into triskelions or coated vesicles. The half-lives of LCa, LCb, and clathrin heavy chain were determined to be 24, 45, and 50 h, respectively. Thus, LCa is turned over independently of the other subunits. However, the half-lives of all three subunits are sufficiently long to allow triskelions to undergo many rounds of endocytosis, minimizing the possibility that turnover contributes to regulation of clathrin function. Rather, differential levels of LCa and LCb expression may influence tissue specific clathrin regulation, as suggested by the predominance of LCb in cells maintaining a regulated secretory pathway.
Yeast clathrin has a distinctive light chain that is important for cell growth.
Authors: Silveira L A , Wong D H , Masiarz F R , Schekman R
Published: May 2004

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.
The expression of myosin genes in developing skeletal muscle in the mouse embryo.
Authors: Lyons G E , Ontell M , Cox R , Sassoon D , Buckingham M
Published: May 2004

Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and accumulation. The data presented are the first detailed study of myosin gene expression at these early stages of skeletal muscle development.
A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.
Authors: Brink M , Gerisch G , Isenberg G , Noegel A A , Segall J E , Wallraff E , Schleicher M
Published: May 2004

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.
UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.
Authors: Spurck T P , Stonington O G , Snyder J A , Pickett-Heaps J D , Bajer A , Mole-Bajer J
Published: May 2004

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation. After this enlargement of the ARB, metaphase spindles recovered rapidly as the detached pole moved back towards the chromosomes, reestablishing spindle fibers as the ARB closed; this happened when the ARB cut a few fibers or across the entire half spindle. We never detected elongation of the cut kinetochore fibers. Rather, astral fibers growing from the pole appeared to bridge and then close the ARB, just before the movement of the pole toward the chromosomes. When a second irradiation was directed into the closing ARB, the polewards movement again stopped before it restarted. In all metaphase cells, once the pole had reestablished connection with the chromosomes, the unirradiated half spindle then also shortened to create a smaller symmetrical spindle capable of normal anaphase later. Anaphase cells did not recover this way; the severed pole remained detached but the chromosomes continued a modified form of movement, clumping into a telophase-like group. The results are discussed in terms of controls operating on spindle microtubule stability and mechanisms of mitotic force generation.
The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains.
Authors: Worman H J , Evans C D , Blobel G
Published: May 2004

The lamin B receptor is a previously identified integral membrane protein in the nuclear envelope of turkey erythrocytes that associates with the nuclear intermediate filament protein lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531-8534). In the present report, we use cell fractionation and antibodies against the lamin B receptor to localize it to an 8-M urea-extracted membrane fraction of chicken liver nuclei, supporting an inner nuclear membrane localization. We deduced the amino acid sequence of the chicken lamin B receptor from overlapping clones obtained by screening cDNA libraries with a probe generated by the polymerase chain reaction with primers based on the partial protein sequence of the isolated protein. The mature lamin B receptor has a calculated molecular mass of 73,375 D and eight segments of hydrophobic amino acids that could function as transmembrane domains as determined by hydropathy analysis. Preceding the first putative transmembrane segment is a highly charged 204-residue-long amino terminal region that contains two consensus sites for phosphorylation by protein kinase A. Since the lamin B receptor has been shown to be phosphorylated by protein kinase A in vitro and in vivo and this phosphorylation affects lamin B binding (Applebaum, J., G. Blobel, and S. D. Georgatos. 1990. J. Biol. Chem. 265:4181-4185), it is likely that this amino terminal region faces the nucleoplasm. The amino terminal region also contains three DNA-binding motifs that are found in gene regulatory proteins and histones, suggesting that the lamin B receptor may additionally play a role in gene regulation and/or chromatin organization.