bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.
Authors: Werb Z , Tremble P M , Behrendtsen O , Crowley E , Damsky C H
Published: May 2004

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.
Adhesive and degradative properties of human placental cytotrophoblast cells in vitro.
Authors: Fisher S J , Cui T Y , Zhang L , Hartman L , Grahl K , Zhang G Y , Tarpey J , Damsky C H
Published: May 2004

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen
The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast.
Authors: Salminen A , Novick P J
Published: May 2004

SEC15 function is required at a late stage of the yeast secretory pathway. Duplication of the gene encoding the ras-like, GTP-binding protein, Sec4, can suppress the partial loss of function resulting from the sec15-l mutation, but cannot suppress disruption of sec15. Analysis of the SEC15 gene predicts a hydrophilic protein product of 105 kD. Anti-Sec15 antibody recognizes a protein of 116-kD apparent molecular mass which is associated with a microsomal fraction of yeast in a strongly pH dependent fashion. Overproduction of Sec15 protein interferes with the secretory pathway, resulting in the formation of a cluster of secretory vesicles, and a patch of Sec15 protein revealed by immunofluorescence. The sec4-8 and sec2-4l mutations, but not mutations in other SEC genes, prevent formation of the Sec15 protein patch. We propose that Sec15 protein responds to the function of the Sec4 protein to control vesicular traffic.
(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.
Authors: Marxer A , Stieger B , Quaroni A , Kashgarian M , Hauri H P
Published: May 2004

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.
Expression of multiple tau isoforms and microtubule bundle formation in fibroblasts transfected with a single tau cDNA.
Authors: Kanai Y , Takemura R , Oshima T , Mori H , Ihara Y , Yanagisawa M , Masaki T , Hirokawa N
Published: May 2004

Tau proteins are a class of low molecular mass microtubule-associated proteins that are specifically expressed in the nervous system. A cDNA clone of adult rat tau was isolated and sequenced. To analyze functions of tau proteins in vivo, we carried out transfection experiments. A fibroblast cell line, which was transfected with the cDNA, expressed three bands of tau, while six bands were expressed in rat brain. After dephosphorylation, one of the three bands disappeared, demonstrating directly that phosphorylation was involved in the multiplicity of tau. Morphologically, we observed a thick bundle formation of microtubules in the transiently and stably tau-gene-transfected cells. In addition, we found that the production of tubulin was prominently enhanced in the stably transfected cells. Thus, we suppose that tau proteins promote polymerization of tubulin, form bundles of microtubules in vivo, and play important roles in growing and maintaining nerve cell processes.
Centrosome movement in the early divisions of Caenorhabditis elegans: a cortical site determining centrosome position.
Authors: Hyman A A
Published: May 2004

In Caenorhabditis elegans embryos, early blastomeres of the P cell lineage divide successively on the same axis. This axis is a consequence of the specific rotational movement of the pair of centrosomes and nucleus (Hyman, A. A., and J. G. White. 1987. J. Cell Biol. 105:2123-2135). A laser has been used to perturb the centrosome movements that determine the pattern of early embryonic divisions. The results support a previously proposed model in which a centrosome rotates towards its correct position by shortening of connections, possibly microtubules, between a centrosome and a defined site on the cortex of the embryo.
Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis.
Authors: Pariset C , Feinberg J , Dacheux J L , Oyen O , Jahnsen T , Weinman S
Published: May 2004

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.
Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments.
Authors: Lankford K L , Letourneau P C
Published: May 2004

We investigated the effects of calcium removal and calcium ionophores on the behavior and ultrastructure of cultured chick dorsal root ganglia (DRG) neurons to identify possible mechanisms by which calcium might regulate neurite outgrowth. Both calcium removal and the addition of calcium ionophores A23187 or ionomycin blocked outgrowth in previously elongating neurites, although in the case of calcium ionophores, changes in growth cone shape and retraction of neurites were also observed. Treatment with calcium ionophores significantly increased growth cone calcium. The ability of the microtubule stabilizing agent taxol to block A23187-induced neurite retraction and the ability of the actin stabilizing agent phalloidin to reverse both A23187-induced growth cone collapse and neurite retraction suggested that calcium acted on the cytoskeleton. Whole mount electron micrographs revealed an apparent disruption of actin filaments in the periphery (but not filopodia) of growth cones that were exposed to calcium ionophores in medium with normal calcium concentrations. This effect was not seen in cells treated with calcium ionophores in calcium-free medium or cells treated with the monovalent cation ionophore monensin, indicating that these effects were calcium specific. Ultrastructure of Triton X-100 extracted whole mounts further indicated that both microtubules and microfilaments may be more stable or extraction resistant after treatments which lower intracellular calcium. Taken together, the data suggest that calcium may control neurite elongation at least in part by regulating actin filament stability, and support a model for neurite outgrowth involving a balance between assembly and disassembly of the cytoskeleton.
High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly.
Authors: Chandler D E , Whitaker M , Zimmerberg J
Published: May 2004

Recently, we have shown that high molecular weight polymers inhibit cortical granule exocytosis at total osmolalities only slightly higher than that of sea water (Whitaker, M., and J. Zimmerberg. 1987. J. Physiol. 389:527-539). In this study, we visualize the step at which this inhibition occurs. Lytechinus pictus and Strongylocentrotus purpuratus eggs were exposed to 0.8 M stachyose or 40% (wt/vol) dextran (average molecular mass of 10 kD) in artificial sea water, activated with 60 microM of the calcium ionophore A23187, and then either fixed with glutaraldehyde and embedded or quick-frozen and freeze-fractured. Stachyose (2.6 osmol/kg) appears to inhibit cortical granule exocytosis by eliciting formation of a granule-free zone (GFZ) in the egg cortex which pushes granules away from the plasma membrane thus preventing their fusion. In contrast, 40% dextran (1.58 osmol/kg) does not result in a GFZ and cortical granules undergo fusion. In some specimens, the pores joining granule and plasma membranes are relatively small; in other cases, the exocytotic pocket has been stabilized in an omega configuration and the granule matrix remains intact. These observations suggest that high molecular weight polymers block exocytosis because of their inability to enter the granule matrix: they retard the water entry that is needed for matrix dispersal.
A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.
Authors: Farach-Carson M C , Carson D D , Collier J L , Lennarz W J , Park H R , Wright G C
Published: May 2004

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.
An evolutionarily conserved enzyme degrades transforming growth factor-alpha as well as insulin.
Authors: Garcia J V , Gehm B D , Rosner M R
Published: May 2004

A single enzyme found in both Drosophila and mammalian cells is able to selectively bind and degrade transforming growth factor (TGF)-alpha and insulin, but not EGF, at physiological concentrations. These growth factors are also able to inhibit binding and degradation of one another by the enzyme. Although there are significant immunological differences between the mammalian and Drosophila enzymes, the substrate specificity has been highly conserved. These results demonstrate the existence of a selective TGF-alpha-degrading enzyme in both Drosophila and mammalian cells. The evolutionary conservation of the ability to degrade both insulin and TGF-alpha suggests that this property is important for the physiological role of the enzyme and its potential for regulating growth factor levels.
Thrombospondin modulates focal adhesions in endothelial cells.
Authors: Murphy-Ullrich J E , Höök M
Published: May 2004

We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin-binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.
In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney.
Authors: Laurie G W , Horikoshi S , Killen P D , Segui-Real B , Yamada Y
Published: May 2004

The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding.
Protein import through the nuclear pore complex is a multistep process.
Authors: Akey C W , Goldfarb D S
Published: May 2004

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.
Posttranslational events leading to the assembly of photosystem II protein complex: a study using photosynthesis mutants from Chlamydomonas reinhardtii.
Authors: de Vitry C , Olive J , Drapier D , Recouvreur M , Wollman F A
Published: May 2004

We studied the assembly of photosystem II (PSII) in several mutants from Chlamydomonas reinhardtii which were unable to synthesize either one PSII core subunit (P6 [43 kD], D1, or D2) or one oxygen-evolving enhancer (OEE1 or OEE2) subunit. Synthesis of the PSII subunits was analyzed on electrophoretograms of cells pulse labeled with [14C]acetate. Their accumulation in thylakoid membranes was studied on immunoblots, their chlorophyll-binding ability on nondenaturating gels, their assembly by detergent fractionation, their stability by pulse-chase experiments and determination of in vitro protease sensitivity, and their localization by immunocytochemistry. In Chlamydomonas, the PSII core subunits P5 (47 kD), D1, and D2 are synthesized in a concerted manner while P6 synthesis is independent. P5 and P6 accumulate independently of each other in the stacked membranes. They bind chlorophyll soon after, or concomitantly with, their synthesis and independently of the presence of the other PSII subunits. Resistance to degradation increases step by step: beginning with assembly of P5, D1, and D2, then with binding of P6, and, finally, with binding of the OEE subunits on two independent high affinity sites (one for OEE1 and another for OEE2 to which OEE3 binds). In the absence of PSII cores, the OEE subunits accumulate independently in the thylakoid lumen and bind loosely to the membranes; OEE1 was found on stacked membranes, but OEE2 was found on either stacked or unstacked membranes depending on whether or not P6 was synthesized.
Translocation arrest by reversible folding of a precursor protein imported into mitochondria. A means to quantitate translocation contact sites.
Authors: Rassow J , Guiard B , Wienhues U , Herzog V , Hartl F U , Neupert W
Published: May 2004

Passage of precursor proteins through translocation contact sites of mitochondria was investigated by studying the import of a fusion protein consisting of the NH2-terminal 167 amino acids of yeast cytochrome b2 precursor and the complete mouse dihydrofolate reductase. Isolated mitochondria of Neurospora crassa readily imported the fusion protein. In the presence of methotrexate import was halted and a stable intermediate spanning both mitochondrial membranes at translocation contact sites accumulated. The complete dihydrofolate reductase moiety in this intermediate was external to the outer membrane, and the 136 amino acid residues of the cytochrome b2 moiety remaining after cleavage by the matrix processing peptidase spanned both outer and inner membranes. Removal of methotrexate led to import of the intermediate retained at the contact site into the matrix. Thus unfolding at the surface of the outer mitochondrial membrane is a prerequisite for passage through translocation contact sites. The membrane-spanning intermediate was used to estimate the number of translocation sites. Saturation was reached at 70 pmol intermediate per milligram of mitochondrial protein. This amount of translocation intermediates was calculated to occupy approximately 1% of the total surface of the outer membrane. The morphometrically determined area of close contact between outer and inner membranes corresponded to approximately 7% of the total outer membrane surface. Accumulation of the intermediate inhibited the import of other precursor proteins suggesting that different precursor proteins are using common translocation contact sites. We conclude that the machinery for protein translocation into mitochondria is present at contact sites in limited number.
Mutagenesis and chimeric genes define determinants in the beta subunits of human chorionic gonadotropin and lutropin for secretion and assembly.
Authors: Matzuk M M , Spangler M M , Camel M , Suganuma N , Boime I
Published: May 2004

Chorionic gonadotropin (CG) and lutropin (LH) are members of a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. The glycoprotein hormone beta subunits share a high degree of amino acid homology that is most evident for the LH beta and CG beta subunits having greater than 80% sequence similarity. However, transfection studies have shown that human CG beta and alpha can be secreted as monomers and can combine efficiently to form dimer, whereas secretion and assembly of human LH beta is less efficient. To determine which specific regions of the LH beta and CG beta subunits are responsible for these differences, mutant and chimeric LH beta-CG beta genes were constructed and transfected into CHO cells. Expression of these subunits showed that both the hydrophobic carboxy-terminal seven amino acids and amino acids Trp8, Ile15, Met42, and Asp77 together inhibit the secretion of LH beta. The carboxy-terminal amino acids, along with Trp8, Ile15, Met42, and Thr58 are implicated in the delayed assembly of LH beta. These unique features of LH beta may also play an important role in pituitary intracellular events and may be responsible for the differential glycosylation and sorting of LH and FSH in gonadotrophs.
Plasma membrane association of Acanthamoeba myosin I.
Authors: Miyata H , Bowers B , Korn E D
Published: May 2004

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.
The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.
Authors: Sinard J H , Pollard T D
Published: May 2004

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.
Mapping myosin light chains by immunoelectron microscopy. Use of anti-fluorescyl antibodies as structural probes.
Authors: Katoh T , Lowey S
Published: May 2004

The two classes of light chains in vertebrate fast muscle myosin have been selectively labeled with the thiol specific reagent 5-(iodoacetamido) fluorescein to determine their location in the myosin head. The alkali light chains (A1 and A2) were labeled at a single cysteine residue near the COOH terminus, whereas the regulatory light chain (LC2) was reacted at either cysteine 125 or 154. The two cysteines of LC2 appear to be near each other in the tertiary structure as evidenced by the ease of formation of an intramolecular disulfide bond. Besides having favorable spectral properties, fluorescein is a potent haptenic immunogen for raising high affinity antibodies. When anti-fluorescyl antibodies were added to the fluorescein-labeled light chains, the fluorescence was quenched by greater than 90%, thereby providing a simple method for determining an association constant. The interaction with antibody was the same for light chains exchanged into myosin as for free light chains. Complexes of antibody bound to light chain could be visualized in the electron microscope by rotary shadowing with platinum. By this approach we have shown that the COOH-terminal regions of the two classes of light chains are widely separated in myosin: the cysteine residues of LC2 lie close to the head/rod junction, whereas the single cysteine of A1 or A2 is located approximately 90 A distal to the junction. These sites correspond to the positions of the NH2 termini of the light chains mapped in earlier studies (Winkelmann, D. A., and S. Lowey. 1986. J. Mol. Biol. 188:595-612; Tokunaga, M., M. Suzuki, K. Saeki, and T. Wakabayashi. 1987b. J. Mol. Biol. 194:245-255). We conclude that the two classes of light chains do not lie in a simple colinear arrangement, but instead have a more complex organization in distinct regions of the myosin head.
Polymorphonuclear leukocyte adherence induces actin polymerization by a transduction pathway which differs from that used by chemoattractants.
Authors: Southwick F S , Dabiri G A , Paschetto M , Zigmond S H
Published: May 2004

Nitrobenzoxadiazole-phallacidin in combination with quantitative fluorescent microscopy have been used to measure F-actin concentrations in human polymorphonuclear leukocytes (PMN) as they adhere to a plastic surface. Like stimulation with chemoattractants, adherence is associated with a twofold rise in F-actin content. However unlike the rapid rise in F-actin induced by chemoattractants which peaks within 30 s, actin assembly induced by adherence is slower, maximum F-actin values not being observed until 10 min. Furthermore the rise in F-actin induced by adherence is persistent, remaining constant over 60 min while F-actin returns to near basal levels after 20 min exposure to chemoattractant. The combination of adherence (5 min) followed by chemoattractant (FMLP 5 x 10(-8) M for 40 s) resulted in an additive rise in F-actin content to greater than threefold over unstimulated values. Unlike chemoattractant induced actin assembly, adherence-associated PMN actin polymerization was not inhibited by pertussis toxin, but was markedly reduced by lowering extracellular Ca2+. Fluorescent micrographs of adherent PMN stained with nitrobenzoxadiazole-phallacidin revealed F-actin in the lamellipodia and in small foci on the adherent surface. These findings suggest that the transduction mechanisms by which adherence induces PMN actin polymerization differ from those used by chemoattractant receptors.
Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation.
Authors: Hartwig J H , Chambers K A , Hopcia K L , Kwiatkowski D J
Published: May 2004

Profilin is a conserved, widely distributed actin monomer binding protein found in eukaryotic cells. Mammalian profilin reversibly sequesters actin monomers in a high affinity profilactin complex. In vitro, the complex is dissociated in response to treatment with the polyphosphoinositides, phosphatidylinositol monophosphate, and phosphatidylinositol 4,5-bisphosphate. Here, we demonstrate the ultrastructural immunolocalization of profilin in human leukocytes and platelets. In both cell types, a significant fraction of profilin is found associated with regions of cell membrane devoid of actin filaments and other discernible structures. After platelet activation, the membrane association of profilin reversibly increases. This study represents the first direct evidence for an interaction between profilin and phospholipids in vivo.
Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes.
Authors: Tilney L G , Portnoy D A
Published: May 2004

Listeria monocytogenes was used as a model intracellular parasite to study stages in the entry, growth, movement, and spread of bacteria in a macrophage cell line. The first step in infection is phagocytosis of the Listeria, followed by the dissolution of the membrane surrounding the phagosome presumably mediated by hemolysin secreted by Listeria as nonhemolytic mutants remain in intact vacuoles. Within 2 h after infection, each now cytoplasmic Listeria becomes encapsulated by actin filaments, identified as such by decoration of the actin filaments with subfragment 1 of myosin. These filaments are very short. The Listeria grow and divide and the actin filaments rearrange to form a long tail (often 5 microns in length) extending from only one end of the bacterium, a "comet's tail," in which the actin filaments appear randomly oriented. The Listeria "comet" moves to the cell surface with its tail oriented towards the cell center and becomes incorporated into a cell extension with the Listeria at the tip of the process and its tail trailing into the cytoplasm behind it. This extension contacts a neighboring macrophage that phagocytoses the extension of the first macrophage. Thus, within the cytoplasm of the second macrophage is a Listeria with its actin tail surrounded by a membrane that in turn is surrounded by the phagosome membrane of the new host. Both these membranes are then solubilized by the Listeria and the cycle is repeated. Thus, once inside a host cell, the infecting Listeria and their progeny can spread from cell to cell by remaining intracellular and thus bypass the humoral immune system of the organism. To establish if actin filaments are essential for the spread of Listeria from cell to cell, we treated infected macrophages with cytochalasin D. The Listeria not only failed to spread, but most were found deep within the cytoplasm, rather than near the periphery of the cell. Thin sections revealed that the net of actin filaments is not formed nor is a "comet" tail produced.
Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin.
Authors: Byers T J , Husain-Chishti A , Dubreuil R R , Branton D , Goldstein L S
Published: May 2004

We used chicken alpha spectrin as a ligand probe to isolate Drosophila beta spectrin cDNA sequences from a lambda gt11 expression library. Analysis of 800 residues of deduced amino acid sequence at the amino-terminal end revealed a strikingly conserved domain of integral of 230 residues that shows a high degree of sequence similarity to the amino-terminal domains of alpha actinin and dystrophin. This conserved domain constitutes a new diagnostic criterion for spectrin-related proteins and allows the known properties of one of these proteins to predict functional properties of the others. The conservation of the amino-terminal domain, and other regions in spectrin, alpha actinin, and dystrophin, demonstrates that a common set of domains were linked in different combinations through evolution to generate the distinctive members of the spectrin superfamily.
Overexpression of the vimentin gene in transgenic mice inhibits normal lens cell differentiation.
Authors: Capetanaki Y , Smith S , Heath J P
Published: May 2004

To investigate the role of the intermediate filament protein vimentin in the normal differentiation and morphogenesis of the eye lens fiber cells, we generated transgenic mice bearing multiple copies of the chicken vimentin gene. In most cases, the vimentin transgene was overexpressed in the lenses of these animals, reaching up to 10 times the endogenous levels. This high expression of vimentin interfered very strongly with the normal differentiation of the lens fibers. The normal fiber cell denucleation and elongation processes were impaired and the animals developed pronounced cataracts, followed by extensive lens degeneration. The age of appearance and extent of these abnormalities in the different transgenic lines were directly related to the vimentin level. Electron microscopic analysis revealed that the accumulated transgenic protein forms normal intermediate filaments.
Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis.
Authors: Baribault H , Blouin R , Bourgon L , Marceau N
Published: May 2004

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with gamma 32P-ATP after treatment with EGF for 30 min to 3 h at 37 degrees C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.
Authors: Snell W J , Eskue W A , Buchanan M J
Published: May 2004

During fertilization in the biflagellated alga, Chlamydomonas reinhardtii, gametes of opposite mating types adhere to each other via agglutinin molecules located on their flagellar surfaces, generating a sexual signal that induces several cellular responses including cell wall release. This cell contact-generated signal is mediated by cAMP and release of the wall, which is devoid of cellulose and contains several hydroxyproline-rich glycoproteins, is due to the activation of a metalloprotease, lysin. Although we originally assumed that lysin would be stored intracellularly in a compartment structurally separate from its substrate, recently we showed that lysin is stored in the periplasm as an inactive, higher relative molecular mass precursor, prolysin (Buchanan, M.J., S. H. Imam, W. A. Eskue, and W. J. Snell. 1989. J. Cell Biol. 108:199-207). Here we show that conversion of prolysin to lysin is due to a cellular, nonperiplasmic enzyme that has the properties of a serine protease. Release of this serine protease into the periplasm is induced by incubation of gametes in dibutyryl cAMP. This may be one of the few examples of regulated secretion of a protease in a eucaryotic microorganism and a novel example of regulated secretion in a plant system.
The major component of the paraflagellar rod of Trypanosoma brucei is a helical protein that is encoded by two identical, tandemly linked genes.
Authors: Schlaeppi K , Deflorin J , Seebeck T
Published: May 2004

The flagellum of the parasitic hemoflagellate Trypanosoma brucei contains two major structures: (a) the microtubule axoneme, and (b) a highly ordered, filamentous array, the paraflagellar rod (PFR). This is a complex, three-dimensional structure, of yet unknown function, that extends along most of the axoneme and is closely linked to it. Its major structural component is a single protein of 600 amino acids. This PFR protein can assume two different conformations, resulting in two distinct bands of apparent molecular masses of 73 and 69 kD in SDS-gel electrophoresis. Secondary structure predictions indicate a very high helix content. Despite its biochemical similarity to the intermediate filament proteins (solubility properties, amino acid composition, and high degree of helicity), the PFR protein does not belong in this class of cytoskeletal proteins. The PFR protein is coded for by two tandemly linked genes of identical nucleotide sequence. Both genes are transcribed into stable mRNAs of very similar length that carry the mini-exon sequence at their 5' termini.
Presence of a protein immunologically related to lamin B in the postsynaptic membrane of Torpedo marmorata electrocyte.
Authors: Cartaud A , Courvalin J C , Ludosky M A , Cartaud J
Published: May 2004

The Torpedo electrocyte is a flattened syncytium derived from skeletal muscle, characterized by two functionally distinct plasma membrane domains. The electrocyte is filled up with a transversal network of intermediate filaments (IF) of desmin which contact in an end-on fashion both sides of the cell. In this work, we show that polyclonal antibodies specific for lamin B recognizes a component of the plasma membrane of Torpedo electrocyte. This protein which thus shares epitopes with lamin B has a relative molecular mass of 54 kD, an acidic IP of 5.4. It is localized exclusively on the cytoplasmic side of the innervated membrane of the electrocyte at sites of IF-membrane contacts. Since our previous work showed that the noninnervated membrane contains ankyrin (Kordeli, E., J. Cartaud, H. O. Nghiêm, L. A. Pradel, C. Dubreuil, D. Paulin, and J.-P. Changeux. 1986. J. Cell Biol. 102:748-761), the present results suggest that desmin filaments may be anchored via the 54-kD protein to the innervated membrane and via ankyrin to the noninnervated membrane. These findings would represent an extension of the model proposed by Georgatos and Blobel (Georgatos, S. D., and G. Blobel. 1987a. J. Cell Biol. 105:105-115) in which type III intermediate size filaments are vectorially inserted to plasma and nuclear membranes by ankyrin and lamin B, respectively.