bluuebunny/crossref_metadata_2025 huggingface dataset:
Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism.
Authors: Dejana E , Lampugnani M G , Giorgi M , Gaboli M , Federici A B , Ruggeri Z M , Marchisio P C
Published: May 2004

Von Willebrand factor (vWF) is a constitutive and specific component of endothelial cell (EC) matrix. In this paper we show that, in vitro, vWF can induce EC adhesion and promote organization of microfilaments and adhesion plaques. In contrast, human vascular smooth muscle cells and MG63 osteosarcoma cells did not adhere and spread on vWF. Using antibodies to the beta chains of fibronectin (beta 1) and vitronectin (beta 3) receptors it was found that ECs adherent to vWF show clustering of both receptors. The beta 1 receptor antibodies are arranged along stress fibers at sites of extracellular matrix contact while the beta 3 receptor antibodies were sharply confined at adhesion plaques. ECs release and organize endogenous fibronectin early during adhesion to vWF. Upon blocking protein synthesis and secretion, ECs can equally adhere and spread on vWF but, while the beta 3 receptors are regularly organized, the beta 1 receptors remain diffuse. This suggests that the organization of the beta 1 receptors depend on the release of fibronectin and/or other matrix proteins operated by the same cell. Antibodies to the beta 3 receptors fully block EC adhesion to vWF and detach ECs seeded on this substratum. In contrast, antibodies to the beta 1 receptors are poorly active. Overall these results fit with an accessory role of beta 1 receptors and indicate a leading role for the beta 3 receptors in EC interaction with vWF. To identify the EC binding domain on vWF we used monoclonal antibodies produced against a peptide representing the residues Glu1737-Ser1750 of the mature vWF and thought to be important in mediating its binding to the platelet receptor glycoprotein IIb-IIIa. We found that the antibody that recognizes the residues 1,744-1,746, containing the Arg-Gly-Asp sequence, completely inhibit EC adhesion to vWF whereas a second antibody recognizing the adjacent residues 1,740-1,742 (Arg-Gly-Asp-free) is inactive. Both antibodies do not interfere with EC adhesion to vitronectin. This defines the molecular domain on vWF that is specifically recognized by ECs and reaffirms the direct role of the Arg-Gly-Asp sequence as the integrin receptor recognition site also in the vWF molecule.
High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.
Authors: Gustafson E J , Lukasiewicz H , Wachtfogel Y T , Norton K J , Schmaier A H , Niewiarowski S , Colman R W
Published: May 2004

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.
Functional tissue factor is entirely cell surface expressed on lipopolysaccharide-stimulated human blood monocytes and a constitutively tissue factor-producing neoplastic cell line.
Authors: Drake T A , Ruf W , Morrissey J H , Edgington T S
Published: May 2004

Tissue factor (TF) is an integral membrane glycoprotein which, as the receptor and essential cofactor for coagulation factors VII and VIIa (FVII and FVIIa, respectively), is the primary cellular activator of the coagulation protease cascade. Previous studies on the procoagulant activity of a variety of cell types (either lysed or in the intact state) have variously been interpreted as showing that TF is either stored intracellularly or is present in a cryptic form in the surface membrane. Using mAbs to TF, we have directly investigated the subcellular localization and functional activity of TF in lipopolysaccharide-stimulated blood monocytes and J82 bladder carcinoma cells. Blocking of surface TF of viable cells with inhibitory anti-TF mAbs abolished greater than 90% of TF activity of the intact cells as well as of lysed cells. Furthermore, quantitative analysis of the binding of FVII and anti-TF mAb to J82 cells demonstrated that all surface-expressed TF molecules were capable of binding the ligand, FVII. By immunoelectron microscopy, TF was present only in the surface membrane of monocytes and J82 cells, although the latter also contained apparently inactive TF antigen in multivesicular bodies. On the intact cell surface the catalytic activity of the TF-FVIIa complex was investigated and found to be markedly less relative to cell lysates. Membrane alterations that affect the cofactor activity of TF may be a means of regulating the extent of initiation of the coagulation protease cascade in various cellular settings.
The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain.
Authors: Takada Y , Hemler M E
Published: May 2004

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.
Platelet-derived growth factor and transforming growth factor-beta enhance tissue repair activities by unique mechanisms.
Authors: Pierce G F , Mustoe T A , Lingelbach J , Masakowski V R , Griffin G L , Senior R M , Deuel T F
Published: May 2004

Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) markedly potentiate tissue repair in vivo. In the present experiments, both in vitro and in vivo responses to PDGF and TGF-beta were tested to identify mechanisms whereby these growth factors might each enhance the wound-healing response. Recombinant human PDGF B-chain homodimers (PDGF-BB) and TGF-beta 1 had identical dose-response curves in chemotactic assays with monocytes and fibroblasts as the natural proteins from platelets. Single applications of PDGF-BB (2 micrograms, 80 pmol) and TGF-beta 1 (20 micrograms, 600 pmol) were next applied to linear incisions in rats and each enhanced the strength required to disrupt the wounds at 5 d up to 212% of paired control wounds. Histological analysis of treated wounds demonstrated an in vivo chemotactic response of macrophages and fibroblasts to both PDGF-BB and to TGF-beta 1 but the response to TGF-beta 1 was significantly less than that observed with PDGF-BB. Marked increases of procollagen type I were observed by immunohistochemical staining in fibroblasts in treated wounds during the first week. The augmented breaking strength of TGF-beta 1 was not observed 2 and 3 wk after wounding. However, the positive influence of PDGF-BB on wound breaking strength persisted through the 7 wk of testing. Furthermore, PDGF-BB-treated wounds had persistently increased numbers of fibroblasts and granulation tissue through day 21, whereas the enhanced cellular influx in TGF-beta 1-treated wounds was not detectable beyond day 7. Wound macrophages and fibroblasts from PDGF-BB-treated wounds contained sharply increased levels of immunohistochemically detectable intracellular TGF-beta. Furthermore, PDGF-BB in vitro induced a marked, time-dependent stimulation of TGF-beta mRNA levels in cultured normal rat kidney fibroblasts. The results suggest that TGF-beta transiently attracts fibroblasts into the wound and may stimulate collagen synthesis directly. In contrast, PDGF is a more potent chemoattractant for wound macrophages and fibroblasts and may stimulate these cells to express endogenous growth factors, including TGF-beta, which, in turn, directly stimulate new collagen synthesis and sustained enhancement of wound healing over a more prolonged period of time.
Spatial segregation of the regulated and constitutive secretory pathways.
Authors: Rivas R J , Moore H P
Published: May 2004

Recent experiments using DNA transfection have shown that secretory proteins in AtT-20 cells are sorted into two biochemically distinct secretory pathways. These two pathways differ in the temporal regulation of exocytosis. Proteins secreted by the regulated pathway are stored in dense-core granules until release is stimulated by secretagogues. In contrast, proteins secreted by the constitutive pathway are exported continuously, without storage. It is not known whether there are mechanisms to segregate regulated and constitutive secretory vesicles spatially. In this study, we examined the site of insertion of constitutive vesicles and compared it with that of regulated secretory granules. Regulated granules accumulate at tips of processes in these cells. To determine whether constitutively externalized membrane proteins are inserted into plasma membrane at the cell body or at process tips, AtT-20 cells were infected with ts-O45, a temperature-sensitive mutant of vesicular stomatitis virus in which transport of the surface glycoprotein G is conditionally blocked in the ER. After switching to the permissive temperature, insertion of G protein was detected at the cell body, not at process tips. Targeting of constitutive and regulated secretory vesicles to distinct areas of the plasma membrane appears to be mediated by microtubules. We found that while disruption of microtubules by colchicine had no effect on constitutive secretion, it completely blocked the accumulation of regulated granules at special release sites. Colchicine also affected the proper packaging of regulated secretory proteins. We conclude that regulated and constitutive secretory vesicles are targeted to different areas of the plasma membrane, most probably by differential interactions with microtubules. These results imply that regulated secretory granules may have unique membrane receptors for selective attachment to microtubules.
Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.
Authors: Landry J , Chrétien P , Lambert H , Hickey E , Weber L A
Published: May 2004

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.
Pre-Golgi degradation of newly synthesized T-cell antigen receptor chains: intrinsic sensitivity and the role of subunit assembly.
Authors: Bonifacino J S , Suzuki C K , Lippincott-Schwartz J , Weissman A M , Klausner R D
Published: May 2004

The T cell antigen receptor (TCR) is a multisubunit complex composed of at least seven transmembrane chains. The predominant species in most T cells has the composition alpha beta gamma delta epsilon zeta 2. The roles of subunit assembly in transport out of the ER and in the recently described process of pre-Golgi degradation of newly synthesized TCR chains were analyzed in a T-cell line deficient in the synthesis of delta chains (delta 2) and in COS-1 fibroblasts transfected with genes encoding individual TCR chains. Studies with the delta-deficient T-cell line showed that, in the absence of delta, the other TCR chains were synthesized at normal rates, but, instead of being transported to the cell surface, they were retained in the ER. Analysis of the fate of TCR chains retained in the ER showed that they were degraded at vastly different rates by a nonlysosomal pathway. Whereas the alpha chains were degraded rapidly, gamma, zeta, and epsilon were relatively long-lived. To analyze whether this selective degradation was because of different intrinsic susceptibility of the individual chains to degradation or to the formation of resistant oligomers, TCR chains were expressed alone or in combinations in COS-1 fibroblasts. These studies showed that (a) individual TCR chains were degraded at different rates when expressed alone in COS-1 cells, and (b) sensitive chains could be stabilized by coexpression with a resistant chain. Taken together, these observations indicate that both intrinsic sensitivity and subunit assembly play a role in determining the rates at which newly synthesized TCR chains are degraded in the ER.
Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway.
Authors: Lucocq J M , Berger E G , Warren G
Published: May 2004

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.
Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells.
Authors: Breitfeld P P , Harris J M , Mostov K E
Published: May 2004

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.
Disrupted yeast mitochondria can import precursor proteins directly through their inner membrane.
Authors: Hwang S , Jascur T , Vestweber D , Pon L , Schatz G
Published: May 2004

Import of precursor proteins into the yeast mitochondrial matrix can occur directly across the inner membrane. First, disruption of the outer membrane restores protein import to mitochondria whose normal import sites have been blocked by an antibody against the outer membrane or by a chimeric, incompletely translocated precursor protein. Second, a potential- and ATP-dependent import of authentic or artificial precursor proteins is observed with purified inner membrane vesicles virtually free of outer membrane components. Third, import into purified inner membrane vesicles is insensitive to antibody against the outer membrane. Thus, while outer membrane components are clearly required in vivo, the inner membrane contains a complete protein translocation system that can operate by itself if the outer membrane barrier is removed.
Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly.
Authors: Fürst D O , Osborn M , Weber K
Published: May 2004

Antibodies to muscle-specific proteins were used in immunofluorescence to monitor the development of skeletal muscle during mouse embryogenesis. At gestation day (g.d.) 9 a single layer of vimentin filament containing cells in the myotome domain of cervical somites begins to stain positively for myogenic proteins. The muscle-specific proteins are expressed in a specific order between g.d. 9 and 9.5. Desmin is detected first, then titin, then the muscle specific actin and myosin heavy chains, and finally nebulin. At g.d. 9.5 fibrous desmin structures are already present, while for the other myogenic proteins no structure can be detected. Some prefusion myoblasts display at g.d. 11 and 12 tiny and immature myofibrils. These reveal a periodic pattern of myosin, nebulin, and those titin epitopes known to occur at and close to the Z line. In contrast titin epitopes, which are present in mature myofibrils along the A band and at the A-I junction, are still randomly distributed. We propose, that the Z line connected structures and the A bands (myosin filaments) assemble independently, and that the known interaction of the I-Z-I brushes with the A bands occurs at a later developmental stage. After fusion of myoblasts to myotubes at g.d. 13 and 14 all titin epitopes show the myofibrillar banding pattern. The predominantly longitudinal orientation of desmin filaments seen in myoblasts and in early myotubes is transformed at g.d. 17 and 18 to distinct Z line connected striations. Vimentin, still present together with desmin in the myoblasts, is lost from the myotubes. Our results indicate that the putative elastic titin filaments act as integrators during skeletal muscle development. Some developmental aspects of eye and limb muscles are also described.
Enhancement of vinculin synthesis by migrating stratified squamous epithelium.
Authors: Zieske J D , Bukusoglu G , Gipson I K
Published: May 2004

A 110-115-kD protein is present at levels 27-fold higher in migratory epithelium in the rat cornea than in stationary epithelium. This protein represents 2.7% of the total protein in migratory epithelium 6-h postabrasion wound and 0.1% of the total protein in stationary epithelium. Our findings demonstrate that this 110-115-kD protein is vinculin. In Western blots comparing proteins from migratory and control epithelium, antibody against vinculin cross-reacted with the 110-115-kD protein. Using immunoslot blots, vinculin was determined to be present at maximal levels 6 h postabrasion wound, at levels 22- and 8-fold higher than control at 18 and 48 h, respectively, returning to control levels 72 h postwounding. Vinculin was also localized by indirect immunohistochemistry in migrating corneal epithelium. 3-mm scrape wounds were allowed to heal in vivo for 20 h. In flat mounts of these whole wounded corneas, vinculin was localized as punctate spots in the leading edge of migrating epithelium. In cryostat sections, vinculin was localized as punctate spots along the basal cell membranes of the migrating sheet adjacent to the basement membrane and in patches between cells as well as diffusely throughout the cell. Only very diffuse localization with occasional punctate spots between adjacent superficial cells was present in stationary epithelium. The increased synthesis of vinculin during migration and the localization of vinculin at the leading edge of migratory epithelium suggest that vinculin may be involved in cell-cell and cell-substrate adhesion as the sheet of epithelium migrates to cover a wound.
Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.
Authors: Way M , Gooch J , Pope B , Weeds A G
Published: May 2004

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.
The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation.
Authors: Noegel A A , Rapp S , Lottspeich F , Schleicher M , Stewart M
Published: May 2004

The 120-kD gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are homodimers and have extended rod-like configurations that are respectively approximately 35 and 40 nm long. Here we report the complete cDNA sequence of the 120-kD gelation factor which codes for a protein of 857 amino acids. Its calculated molecular mass is 92.2 kD which is considerably smaller than suggested by its mobility in SDS-PAGE. Analysis of the sequence shows a region that is highly homologous to D. discoideum alpha-actinin, chicken fibroblast alpha-actinin, and human dystrophin. This conserved domain probably represents an actin binding site that is connected to the rod-forming part of the molecule via a highly charged stretch of amino acids. Whereas the sequence of alpha-actinin (Noegel, A., W. Witke, and M. Schleicher. 1987. FEBS [Fed. Eur. Biochem. Soc.] Lett. 221:391-396) suggests that the extended rod domain of the molecule is based on four spectrin-like repeats with high alpha-helix potential, the rod domain of the 120-kD gelation factor is constructed from six 100-residue repeats that have a high content of glycine and proline residues and which, in contrast to alpha-actinin, do not appear to have a high alpha-helical content. These repeats show a distinctive pattern of regions that have high beta-sheet potential alternating with short zones rich in residues with a high potential for turns. This observation suggests that each 100-residue motif has a cross-beta conformation with approximately nine sheets arranged perpendicular to the long axis of the molecule. In the high beta-potential zones every second residue is often hydrophobic. In a cross-beta structure, this pattern would result in one side of the domain having a surface rich in hydrophobic side chains which could account for the dimerization of the 120-kD gelation factor subunits.
Intracellular free calcium and mitosis in mammalian cells: anaphase onset is calcium modulated, but is not triggered by a brief transient.
Authors: Tombes R M , Borisy G G
Published: May 2004

Swiss 3T3 fibroblasts and LLC-PK epithelial cells in prometaphase or metaphase were either injected with fura-2 or loaded with the acetoxymethyl ester derivative of fura-2 (fura-2 AM) and monitored by microspectrofluorimetry. With both methods of loading, we observed two aspects of intracellular free calcium (Cai) metabolism. (a) Most fibroblasts and epithelial cells exhibited a gradual rise from 75 nM in metaphase to 185 nM during cleavage, returning to baseline by early G1. (b) Mitotic Swiss 3T3 cells exhibited rapid transient Cai changes, similar to those previously reported [Poenie, M., J. Alderton, R. Y. Tsien, R. A. Steinhardt. 1985. Nature (Lond.). 315:147-149; Poenie, M., J. Alderton, R. Steinhardt, and R. Tsien. 1986. Science (Wash. DC). 233:886-889; Ratan, R., and M. L. Shelanski. 1988. J. Cell Biol. 107:993]. These Cai transients occurred repetitively, often beginning in metaphase and continuing long after daughter cell formation. Eliminating serum or calcium from the medium abolished the transients, but delayed neither the gradual Cai elevation nor anaphase onset. Co-injection of EGTA or 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) with fura-2 in calcium-free medium, but not in calcium containing medium, blocked both anaphase and the sustained Cai elevation in almost all cases. Blocked cells were rescued by returning calcium to the medium, whereupon Cai slowly but steadily rose as the cell entered anaphase. Spindle microtubules persisted through the EGTA block. Depolymerization of spindle microtubules by nocodazole also reversibly blocked anaphase onset and the sustained Cai elevation, but did not block transients. This study has revealed the following: (a) anaphase in mammalian fibroblasts and epithelial cells is not triggered by brief calcium transients; (b) anaphase is a calcium-modulated event, usually accompanied by a sustained elevation of Cai above 50 nM; (c) the elevation of Cai is dependent upon an intact spindle; and (d) fibroblasts progress through mitosis by drawing upon either intracellular or extracellular sources of calcium.
Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence.
Authors: Mitchison T J
Published: May 2004

I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.
Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts.
Authors: Jaken S , Leach K , Klauck T
Published: May 2004

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.
The initial events in myelin synthesis: orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes.
Authors: Hudson L D , Friedrich V L , Behar T , Dubois-Dalcq M , Lazzarini R A
Published: May 2004

Proteolipid protein (PLP) is the most abundant transmembrane protein in myelin of the central nervous system. Conflicting models of PLP topology have been generated by computer predictions based on its primary sequence and experiments with purified myelin. We have examined the initial events in myelin synthesis, including the insertion and orientation of PLP in the plasma membrane, in rat oligodendrocytes which express PLP and the other myelin-specific proteins when cultured without neurons (Dubois-Dalcq, M., T. Behar, L. Hudson, and R. A. Lazzarini. 1986. J. Cell Biol. 102:384-392). These cells, identified by the presence of surface galactocerebroside, the major myelin glycolipid, were stained with six anti-peptide antibodies directed against hydrophilic or short hydrophobic sequences of PLP. Five of these anti-peptide antibodies specifically stained living oligodendrocytes. Staining was only seen approximately 10 d after PLP was first detected in the cytoplasm of fixed and permeabilized cells, suggesting that PLP is slowly transported from the RER to the cell surface. The presence of PLP domains on the extracellular surface was also confirmed by cleavage of such domains with proteases and by antibody-dependent complement-mediated lysis of living oligodendrocytes. Our results indicate that PLP has only two transmembrane domains and that the great majority of the protein, including its amino and carboxy termini, is located on the extracellular face of the oligodendrocyte plasma membrane. This disposition of the PLP molecule suggests that homophilic interactions between PLP molecules of apposed extracellular faces may mediate compaction of adjacent bilayers in the myelin sheath.
Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components.
Authors: Pumplin D W
Published: May 2004

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.
The mouse neuronal cell surface protein F3: a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin.
Authors: Gennarini G , Cibelli G , Rougon G , Mattei M G , Goridis C
Published: May 2004

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.
Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms.
Authors: Doherty P , Barton C H , Dickson G , Seaton P , Rowett L H , Moore S E , Gower H J , Walsh F S
Published: May 2004

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.
Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.
Authors: Akiyama S K , Yamada S S , Chen W T , Yamada K M
Published: May 2004

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.
Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat.
Authors: Ffrench-Constant C , Van de Water L , Dvorak H F , Hynes R O
Published: May 2004

The adhesive extracellular matrix glycoprotein fibronectin (FN) is thought to play an important role in the cell migration associated with wound healing. Immunolocalization studies show abundant FN in healing wounds; however, these studies cannot define the cellular site(s) of FN synthesis, nor do they distinguish the different and potentially functionally distinct forms of FN that can arise from alternative splicing of the primary gene transcript. To examine these questions of FN synthesis and splicing during wound healing, we have performed in situ hybridization with segment-specific probes on healing wounds in adult rat skin. We find that the FN gene is expressed at increased levels after wounding both in the cells at the base of the wound and in subjacent muscle and dermis lateral to the wound. Interestingly, however, the pattern of splicing of FN mRNA was different in these areas. In adjacent dermis and muscle, the splicing pattern remains identical with that seen in normal adult rat skin, with two of the three spliced segments (EIIIA and EIIIB) excluded from FN mRNA. In contrast, these two segments are included in the FN mRNA present in the cells at the base of the wound. As a result, the mRNA in this region is spliced in a pattern identical with that found during early embryogenesis. The finding that the pattern of FN splicing during wound healing resembles an embryonic pattern suggests that alternative splicing may be used during wound healing as a mechanism to generate forms of FN that may be functionally more appropriate for the cell migration and proliferation associated with tissue repair.