bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic changes in the organization of cell adhesions and the associated cytoskeleton, induced by stimulation of tyrosine phosphorylation in bovine aortic endothelial cells
Authors: Ayalon Oran , Geiger Benjamin
Published: April 2021

In this study we have investigated the relationships between the stimulation of tyrosine-specific protein phosphorylation and the state of assembly of cell-cell and cell-matrix adherens-type junctions. Bovine aortic endothelial (BAE) cells were treated with either the phosphotyrosine phosphatase inhibitor pervanadate or with epidermal growth factor (EGF), and the effect of the treatment on the organization of cell contacts and the actin cytoskeleton was evaluated by digital immunomicroscopy. We show here that pervanadate induced a dramatic (about 40-fold) increase in the level of phosphotyrosine labeling of cell-cell junctions, which reached maximal values following 20 minutes of incubation. Concomitantly, the junctional levels of vinculin, actin and plakoglobin increased, followed by a slower recruitment of cadherins to these sites. Upon longer incubation cell-cell junctions deteriorated and stress fibers and focal adhesions were formed. EGF stimulation of serum-starved BAE cells induced a rapid ‘wave’ of junctional tyrosine phosphorylation, followed by cyclic changes in the local levels of phosphotyrosine labeling. Periodic changes were also found in the intensity of labeling of junctional actin, vinculin and cadherins.

These results suggest that tyrosine phosphorylation and the assembly of cell-cell adherens junctions are interdependent processes, and raise the possibility that the cross-talk between the two is responsible both for the regulation of junction formation and for adhesion-mediated signaling.
Localization of fibronectin matrix assembly sites on fibroblasts and endothelial cells
Authors: Christopher Renee A. , Kowalczyk Andrew P. , McKeown-Longo Paula J.
Published: April 2021

Polymerization of soluble fibronectin into extracellular matrix fibers occurs through the interaction between the amino terminus of fibronectin contained within a 70 kDa fragment and ‘matrix assembly sites’ on the cell surface. The present studies were performed to localize the ‘matrix assembly sites’ (defined by 70 kDa binding sites) on newly adherent cells and on cells containing preformed fibronectin matrix. Matrix nucleation sites on newly spread cells were visualized using Texas Red conjugated 70 kDa fragment and were found to colocalize with vinculin and substrate fibronectin fibrils. Cells plated onto vitronectin coated coverslips did not exhibit any 70 kDa binding sites although these cells were well-spread with fully developed focal adhesions. Time course studies indicated that 70 kDa binding sites could be detected on newly adherent cells within 30-40 minutes following cell plating onto fibronectin coated coverslips, prior to the reorganization of substrate fibronectin into fibrils. Similarly, exogenous fibronectin conjugated with Texas Red was also colocalized with vinculin when added to newly adherent cells. The disruption of actin filaments with cytochalasin D both prevented the expression of 70 kDa binding sites and also resulted in the loss of established 70 kDa binding sites on newly spread cells. After 3 days in culture, cells organized an extensive fibronectin matrix and 70 kDa was colocalized with two distinct types of matrix fibronectin fibers: fine linear cell-associated fibers which co-stained with the β1integrin and coarse extracellular fibers which did not stain for the β1integrin. There was also a third type of fibronectin fiber which was organized into a meshwork structure. There was no localization of either β1or 70 kDa to these structures. Treatment of 3-day cells with cytochalasin D resulted in the disruption of cell-matrix fibers and cell-associated 70 kDa binding sites. In contrast, the coarse extracellular matrix fibers as well as the meshwork fibers were unaffected by cytochalasin. In the presence of cytochalasin D, 70 kDa bound to sites which colocalized with the coarse extracellular matrix fibers. These data suggest that de novo assembly of fibronectin matrix occurs at sites of focal adhesion and as fibronectin polymerization proceeds, matrix nucleation sites colocalize along cell associated fibronectin fibers. At later times 70 kDa is localized to a subset of more mature fibronectin-containing fibers. These results suggest that there are at least three morphologically distinct 70 kDa binding sites on adherent cells: one which colocalizes with β1to focal adhesions, a second which colocalizes with β1and fibronectin in matrix contacts, and a third which localizes to extracellular matrix fibers.
Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts
Authors: Sato Takuya , Ovejero Maria del Carmen , Hou Peng , Heegaard Anne-Marie , Kumegawa Masayoshi , Tækker Foged Niels , Delaissé Jean-Marie
Published: April 2021

The osteoclasts are the cells responsible for bone resorption. Matrix metalloproteinases (MMPs) appear crucial for this process. To identify possible MMP expression in osteoclasts, we amplified osteoclast cDNA fragments having homology with MMP genes, and used them as a probe to screen a rabbit osteoclast cDNA library. We obtained a cDNA of 1,972 bp encoding a polypeptide of 582 amino acids that showed more than 92% identity to human, mouse, and rat membrane-type 1 MMP (MT1-MMP), a cell surface proteinase believed to trigger cancer cell invasion. By northern blotting, MT1-MMP was found to be highly expressed in purified osteoclasts when compared with alveolar macrophages and bone stromal cells, as well as with various tissues. In situ hybridization on bone sections showed that MT1-MMP is expressed also in osteoclasts in vivo. Antibodies recognizing MT1-MMP reacted with specific plasma membrane areas corresponding to lamellipodia and podosomes involved, respectively, in migratory and attachment activities of the osteoclasts. These observations highlight how cells might bring MT1-MMP into contact with focal points of the extracellular matrix, and are compatible with a role of MT1-MMP in migratory and attachment activities of the osteoclast.
Increased degranulation and phospholipase A2, C, and D activity in RBL cells stimulated through FcepsilonR1 is due to spreading and not simply adhesion
Authors: Apgar J.R.
Published: April 2021

Rat basophilic leukemia cells will adhere to and spread out on fibronectin coated surfaces in an integrin dependent manner. Adhesion and spreading on fibronectin leads to increased degranulation, inositol phosphate production, phospholipase D activation, and increased production of prostaglandin D2 and leukotriene C4 when the cells are activated through the high affinity IgE receptor. Rat basophilic leukemia cells will also adhere to surfaces coated with anti-rat class I antibodies, poly-L-lysine, and a lectin purified from Tetragonolobus purpureas. In all cases, antigen activated cells, which were adherent, displayed increased signaling, degranulation and eicosanoid production as compared to cells which were non-adherent. Cells which adhere to either anti-rat class I antibodies or poly-L-lysine also spread even though this is not mediated through integrins. In contrast, adhesion to the lectin from Tetragonolobus did not cause any appreciable spreading unless the cells were also triggered through the IgE receptor. Cells were also able to bind to fibronectin immobilized on polystyrene beads which mimics adhesion but does not allow spreading. However, these cells exhibited no increased signaling, degranulation, or eicosanoid production. Furthermore, rat basophilic leukemia cells can be modified by incubating them in the presence of biotinylated-phosphatidylserine which becomes incorporated into the membrane. These modified cells will adhere to streptavidin coated plates while unmodified cells will not. However, these modified cells do not spread, even after activation with antigen, and they show no increased degranulation or production of eicosanoids. These results indicate that adhesion itself is not sufficient for upregulation of the cells in response to antigen and that spreading of the cells may be the critical component.
Sphingomyelin synthase is absent from endosomes
Authors: van Helvoort A. , Stoorvogel W. , van Meer G. , Burger N.J.
Published: April 2021

Both the Golgi and the endosomes have recently been proposed as the main site of SM-synthase, the enzyme responsible for sphingomyelin (SM) biosynthesis. To settle this confusion, we studied the subcellular distribution of SM-synthase in human liver-derived HepG2 and baby hamster kidney BHK-21 cells. To discriminate between Golgi and endosomes we made use of 3,3-diaminobenzidine (DAB) cytochemistry. Cells were incubated with a conjugate of transferrin (Tf) and horseradish peroxidase (HRP), or with unconjugated HRP, to label the recycling pathway and the complete endocytic pathway (including lysosomes) with peroxidase activity, respectively. After cell homogenization, the peroxidase activity was used to induce a local deposition of DAB-polymer. The total SM-synthase activity was not affected by this procedure, and, in contrast to endosomes labeled with (125)I-Tf, organelles containing SM-synthase did not increase in buoyant density as determined by Percoll density gradient fractionation. Thus, little, if any, SM-synthase localizes to the endocytic pathway of HepG2 and BHK-21 cells. In experiments performed at low temperature to inhibit vesicular transport, we found less than 10% of newly synthesized short-chain SM at the cell surface. We conclude that most SM-synthase activity is present in the Golgi, and to a small extent at the cell surface.
Proteinase-activated receptor-2: expression by human neutrophils
Authors: Howells Gareth L. , Macey Marion G. , Chinni Carla , Hou Linda , Fox Mark T. , Harriott Patrick , Stone Stuart R.
Published: April 2021

Neutrophils were shown to express the proteinaseactivated receptor-2 (PAR-2), a seven transmembrane domain receptor, which is activated by cleavage by trypsin. Granulocytes from 14 donors stained positively for PAR-2 with affinity-purified rabbit antibodies raised against a peptide corresponding to the trypsin cleavage site of human PAR-2. Neutrophil activation in response to a receptor activating peptide (RAP) varied between donors. RAP (Ser-Leu-Ile-Gly-Lys-Val-NH2) alone induced an increase in the forward and side light scatter after 5-10 minutes and a small increase in the expression of the activation molecule CD11b. The increased expression of CD11b induced by RAP was markedly enhanced by priming the neutrophils with a low concentration (1 nM) of formyl-Leu-Met-Phe. Trypsin and RAP also induced an increase in intracellular calcium, but there were large variations in the magnitude of responses between donors also in this assay. The effects of RAP in the different assays were specific; acetylated RAP was completely without activity.
Novel functions of clathrin light chains: clathrin heavy chain trimerization is defective in light chain-deficient yeast
Authors: Huang Kristen M. , Gullberg Lisa , Nelson Karen K. , Stefan Christopher J. , Blumer Kendall , Lemmon Sandra K.
Published: April 2021

Clathrin is a major coat protein involved in sorting and retention of proteins at the late Golgi and in endocytosis from the cell surface. The clathrin triskelion contains three heavy chains, which provide the structural backbone of the clathrin lattice and three light chains, which are thought to regulate the formation or disassembly of clathrin coats. To better understand the function of the clathrin light chain, we characterized yeast strains carrying a disruption of the clathrin light chain gene (CLC1). Light chain-deficient cells showed phenotypes similar to those displayed by yeast that have a disruption in the clathrin heavy chain gene (CHC1). In clc1-Δ cells, the steady state level of the clathrin heavy chain was reduced to 20%-25% of wild-type levels and most of the heavy chain was not trimerized. If CHC1 was overexpressed in clc1-Δ cells, heavy chain trimers were detected and several clc1-Δ phenotypes were partially rescued. These results indicate that the light chain is important for heavy chain trimerization and the heavy chain still has some function in the absence of the light chain.

In yeast, deletion of CHC1 is lethal in strains carrying the scd1-i allele, while strains carrying the scd1-v allele can survive without the heavy chain. In previous studies we isolated several multicopy suppressors of inviability of chc1-Δ scd1-i cells. Surprisingly, one of these suppressors, SCD4, is identical to CLC1. Overexpression of CLC1 in viable chc1-Δ scd1-v strains rescued some but not all of the phenotypes displayed by these cells. In the absence of the heavy chain, the light chain was not found in a high molecular mass complex, but still associated with membranes. These results suggest that the light chain can function independently of the clathrin heavy chain in yeast.
Functional analysis of human RPS14 null alleles
Authors: Roufa Donald J. , Martín-Nieto José
Published: April 2021

Previously we described a large collection of cloned human DNAs that encode chemically defined missense mutations within the ribosomal protein S14 sequence. We determined that biologically inactive (i.e. null) alleles resulted primarily from point mutations targeted to two internal segments of the S14-coding sequence and designated these functionally critical regions as domains B and D. Further, we inferred that structural determinants within domains B and D are required for proper incorporation of the S14 protein into nascent 40 S ribosomal particles and/or for the normal function of mature cytoplasmic ribosomes. In this study we have used immunofluorescence to monitor the intracellular trafficking of epitopically labeled human S14 protein isoforms transiently expressed by cultured Chinese hamster cells. Data obtained distinguish null alleles of RPS14 which encode proteins that are not incorporated into pre-ribosomal subunit particles from null alleles whose products are compatible with normal ribosome assembly processes but result in functionally inactive cytoplasmic 40 S ribosomal subunits. Mutations assigned to the first allele class involve amino acid replacements located within S14 domains B and D; whereas mutations assigned to the second class are distributed throughout the S14 proteincoding sequence.
Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue
Authors: Klauke Norbert , Plattner Helmut
Published: April 2021

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (<1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28°C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within ≥20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exo-cytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i >1 to 10 μM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.
Sec12p requires Rer1p for sorting to coatomer (COPI)-coated vesicles and retrieval to the ER
Authors: Boehm Johannes , Letourneur François , Ballensiefen Wolfgang , Ossipov Dmitri , Démollière Corinne , Schmitt Hans Dieter
Published: April 2021

In Saccharomyces cerevisiae cells lacking the Rer1 protein (Rer1p), the type II transmembrane protein Sec12p fails to be retained in the ER. The transmembrane domain of Sec12p is sufficient to confer Rer1p-dependent ER retention to other membrane proteins. In rer1 mutants a large part of the Sec12-derived proteins can escape to the late Golgi. In contrast, rer3 mutants accumulate Sec12-derived hybrid proteins carrying early Golgi modifications. We found that rer3 mutants harbour unique alleles of the α-COP-encoding RET1 gene. ret1 mutants, along with other coatomer mutants, fail to retrieve KKXX-tagged type I transmembrane proteins from the Golgi back to the ER. Surprisingly rer3-11(=ret1-12) mutants do not affect this kind of ER recycling. Pulse-chase experiments using these mutants show that α-COP and Rer1p function together in a very early Golgi compartment to initiate the recycling of Sec12p-derived hybrid proteins. Rer1p protein may be directly involved in the retrieval process since it also recycles between the early Golgi and ER in a coatomer (COPI)-dependent manner. Rer1p may act as an adapter coupling the recycling of non-KKXX transmembrane proteins like Sec12p to the coatomer (COPI)-mediated backward traffic.
The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi
Authors: Nothwehr Steven F. , Hindes Anna E.
Published: April 2021

Genetic analysis of late Golgi membrane protein localization in Saccharomyces cerevisiae has uncovered a large number of genes (called GRD) that are required for retention of A-ALP, a model late Golgi membrane protein. Here we describe one of the GRD genes, VPS5/GRD2, that encodes a hydrophilic protein similar to human sorting nexin-1, a protein involved in trafficking of the epidermal growth factor receptor. In yeast cells containing a vps5 null mutation the late Golgi membrane proteins A-ALP and Kex2p were rapidly mislocalized to the vacuolar membrane. A-ALP was delivered to the vacuole in vps5 mutants in a manner independent of a block in the early endocytic pathway. vps5 null mutants also exhibited defects in both vacuolar morphology and in sorting of a soluble vacuolar protein, carboxypeptidase Y. The latter defect is apparently due to an inability to localize the carboxypeptidase Y sorting receptor, Vps10p, to the Golgi since it is rapidly degraded in the vacuole in vps5 mutants. Fractionation studies indicate that Vps5p is distributed between a free cytosolic pool and a particulate fraction containing Golgi, transport vesicles, and possibly endosomes, but lacking vacuolar membranes. Immunofluorescence microscopy experiments show that the membrane-associated pool of Vps5p localizes to an endosome-like organelle that accumulates in the class E vps27 mutant. These results support a model in which Vps5p is required for retrieval of membrane proteins from a prevacuolar/late endosomal compartment back to the late Golgi apparatus.
A nematode gene required for sperm vesicle fusion
Authors: Achanzar William E. , Ward Samuel
Published: April 2021

During maturation of spermatids to motile spermatozoa in Caenorhabditis elegans, large vesicles called membranous organelles (MOs) fuse with the spermatid plasma membrane. Mutations in the gene fer-1 cause abnormal spermatozoa in which the MOs do not fuse, although they abut the plasma membrane normally. Here we describe the fer-1 gene, which we found to be approximately 8.6 kb in length and to encode a 6.2 kb transcript whose expression is limited to the primary spermatocytes, the cells in which the MOs form. fer-1 is predicted to encode a 235 kDa protein which is highly charged except for a putative transmembrane domain near the C terminus. We identified the mutations associated with five fer-1 alleles, all of which are missense mutations causing single amino acid changes. FER-1 is not similar to any characterized proteins in sequence databases, nor does it contain known functional motifs other than the predicted transmembrane domain. The C-terminal transmembrane domain makes FER-1 resemble some viral fusion proteins, suggesting it may play a direct role in MO-plasma membrane fusion. FER-1 does show significant sequence similarity to several predicted human proteins of unknown function. Two of the identified fer-1 mutations are located in regions of similarity between FER-1 and two of these predicted proteins. This strengthens the biological significance of these similarities and suggests these regions of similarity represent functionally important domains of FER-1 and the human proteins.
Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion
Authors: Bonavaud Sylvie , Charrière-Bertrand Cécile , Rey Christiane , Leibovitch Marie-Pierre , Pedersen Nina , Frisdal Eric , Planus Emmanuelle , Blasi Francesco , Gherardi Romain , Barlovatz-Meimon Georgia
Published: April 2021

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA7PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA7PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.
Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading
Authors: Planus Emmanuelle , Barlovatz-Meimon Georgia , Rogers Rick A. , Bonavaud Sylvie , Ingber Donald E. , Wang Ning
Published: April 2021

Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen activator inhibitor type-1 (PAI-1) is often associated with the extracellular matrix. The spatial localization of these three molecules could account for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical force transmission across the cell surface to the cytoskeleton. Here we investigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matrix ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-1 at increasing concentrations. The number of adherent cells and their projected area were quantitated after 3 hours of plating. PAI-1 promoted cell adhesion and spreading in a dose dependent manner. Addition of antibodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dose dependent way. The PAI-1 mediated cell adhesion required the presence of urokinase at the cell surface. Removal of the glycosylphos-phatidylinositol (GPI)-linked proteins abolished cell adhesion on PAI-1 dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against αvβ3 integrin completely inhibited cell adhesion on PAI-1, suggesting that αvβ3 might be the transmembrane molecule that physically connects the complex of PAI-1, urokinase, and urokinase receptor to the cytoskeleton. Visualization of spread cells stained for filamentous actin with confocal microscopy showed a dose-dependent increase of filopodia on PAI-1 coated dishes and cytoskeletal reorganization, suggesting a migratory profile. These data indicate that PAI-1 plays a direct role in dynamic cell adhesion particularly at the leading edge, where increased levels of urokinase plasminogen activator (uPA) and its receptor (uPAR) are localized in migrating cells. Immobilized PAI-1 could therefore serve to bridge the cell surface with the extracellular matrix via the formation of a multimolecular complex that includes αvβ3 integrins in myogenic cells.
Occludin confers adhesiveness when expressed in fibroblasts
Authors: Itallie Christina M. Van , Anderson James Melvin
Published: April 2021

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cellcell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.
Myeloperoxidase mediates cell adhesion via the αMβ2 integrin (Mac-1, CD11b/CD18)
Authors: Johansson Mats W. , Patarroyo Manuel , Öberg Fredrik , Siegbahn Agneta , Nilsson Kenneth
Published: April 2021

Myeloperoxidase is a leukocyte component able to generate potent microbicidal substances. A homologous invertebrate blood cell protein, peroxinectin, is not only a peroxidase but also a cell adhesion ligand. We demonstrate in this study that human myeloperoxidase also mediates cell adhesion. Both the human myeloid cell line HL-60, when differentiated by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid, and human blood leukocytes, adhered to myeloperoxidase; however, undifferentiated HL-60 cells showed only minimal adhesion. No cells adhered to horseradish peroxidase, and cell adhesion to myeloperoxidase was not decreased by catalase, thus showing that peroxidase activity, per se, was neither sufficient nor necessary for the adhesion activity. Mannan, which has been reported to inhibit the binding of peroxidases to cells, did not affect adhesion to myeloperoxidase. However, adhesion to myeloperoxidase was inhibited by monoclonal antibodies to αM (CD11b) or to β2 (CD18) integrin subunits, but not by antibodies to αb (CD11a), αx (CD11c), or to other integrins. Native myeloperoxidase mediated dosedependent cell adhesion down to relatively low concentrations, and denaturation abolished the adhesion activity. It is evident that myeloperoxidase supports cell adhesion, a function which may be of considerable importance for leukocyte migration and infiltration in inflammatory reactions, that αMβ2 integrin (Mac-1 or CD11b/CD18) mediates this adhesion, and that the αMβ2 integrin-mediated adhesion to myeloperoxidase is distinct from the previously reported ability of this integrin to bind to certain denatured proteins at high concentrations.
Mutational analysis of regulated exocytosis in Tetrahymena
Authors: Melia Sharon M. , Cole Eric S. , Turkewitz Aaron P.
Published: April 2021

Genetic analysis of regulated exocytosis can be accomplished in ciliates, since mutants defective in stimulus-dependent secretion of dense-core vesicles can be identified. In Tetrahymena thermophila, secretion in wild-type cells can result in their encapsulation by the proteins released from vesicle cores. Cells with defects in secretion were isolated from mutagenized homozygous cells that were generated using a highly efficient method. Screening was based both on a visual assay for encapsulation, and on a novel panning step using differential centrifugation to take advantage of the selective mobility of mutants that fail to encapsulate upon stimulation. 18 mutants with defects in several ordered steps have been identified. Defects in a set of these could be localized to three stages: granule formation, transport to cell surface docking sites, and exocytosis itself. Mutants with defects in this last stage can be ordered into successive steps based on several criteria, including their responsiveness to multiple secretagogues and Ca2+ ionophores. The results of both somatic and genetic complementation on selected pairs also help to characterize the defective factors.
Degradation of phagosomal components in late endocytic organelles
Authors: Tjelle Torunn Elisabeth , Saigal Babita , Frøystad Marianne , Berg Trond
Published: April 2021

Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the ‘kiss- and-run’ hypothesis.

We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction).

We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.
Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis
Authors: Jenne Nicole , Rauchenberger Robert , Hacker Ulrike , Kast Thomas , Maniak Markus
Published: April 2021

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B− mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.
The role of NuMA in the interphase nucleus
Authors: Merdes Andreas , Cleveland Don W.
Published: April 2021

NuMA is an essential protein for the formation of spindle poles in mitosis. During interphase, NuMA is transported into the nucleus where it resides until prometaphase of the next mitotic cycle. We tested for a potential function of NuMA in interphase nuclei that were assembled from human sperm DNA using frog egg extract immunodepleted of NuMA. Despite the absence of NuMA, nuclei formed without visible changes of the chromatin structure, surrounded by an intact nuclear membrane containing pores and nuclear lamins. These nuclei were fully competent to import nuclear substrates and to replicate their DNA. By screening tissue sections of various organs, absence of NuMA from the nucleus was observed in a number of cell types, including sperm, granulocytes in the blood, and differentiated smooth and skeletal muscle fibers. Experiments on cultured myoblasts indicated that NuMA is degraded during muscle cell differentiation. The absence of NuMA in interphase nuclei of the tissues tested correlated with a non-spherical, elongated or beaded nuclear morphology, suggesting that during interphase NuMA may act as a non-essential nucleoskeletal element.
Identification and functional characterization of a novel human protein highly related to the yeast dynamin-like GTPase Vps1p
Authors: Imoto Mami , Tachibana Issei , Urrutia Raul
Published: April 2021

Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynaminrelated GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis -Golgi GTPase Rab1A, but not to the staining for the trans -Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1K38E) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.
Inhibition of GSK-3β leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium
Authors: Lucas Fiona R. , Goold Robert G. , Gordon-Weeks Phillip R. , Salinas Patricia C.
Published: April 2021

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3β, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3β, mimics WNT-7a in granule cells. Here we examined further the effect of GSK-3β inhibition on cytoskeletal re-organisation. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3β. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. Our data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3β which in turn lead to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3β and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3β and WNT.
Fibronectin is a survival factor for differentiated osteoblasts
Authors: Globus R. K. , Doty S. B. , Lull J. C. , Holmuhamedov E. , Humphries M. J. , Damsky C. H.
Published: April 2021

The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-β1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-β1, may affect bone formation, in part by regulating the survival of osteoblasts.
Modulation of gap junction expression during transient hyperplasia of rat epidermis
Authors: Risek Boris , Pozzi Ambra , Gilula Norton B.
Published: April 2021

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of α1 (Cx43) and β2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, α1 is expressed in the lower, less differentiated portion, while β2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in α1 and β2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with β2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in α1 and β2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.
Expression of specific tau exons in normal and tumoral pancreatic acinar cells
Authors: Vanier Marie-Thérèse , Neuville Pascal , Michalik Liliane , Launay Jean-François
Published: April 2021

Tau is a neuron-specific microtubule-associated protein (MAP) that is required for the development and maintaining neuronal cell polarity. Tau is encoded by a single gene, while its transcript undergoes a complex and regulated alternative splicing. We have recently reported that tau-like MAPs of 48-55 kDa, corresponding to 6 kb mRNA on northern blots, are expressed in pancreatic acinar cells. In the present study, the expression of tau exons in normal and tumoral pancreatic acinar cells was investigated by RT-PCR and cDNA sequencing. Tau isoforms with four tubulin-binding motifs containing either none, one or two N-terminal inserts (exons 2, 3) are indiscriminately expressed in normal and tumoral cells.

However, tau transcripts containing the sequence encoded by exon 6 are specifically expressed in pancreatic tumoral cells from exocrine origin. By immunofluorescence and electron microscopy, we have identified in cellular extensions of tumoral cells, tau-decorated microtubules arranged in bundles like those found in neuronal processes. Tau antisense oligonucleotides inhibit the development of these cellular processes and the expression of the 55 kDa tau isoform.
Commentary: the fission yeast microtubule cytoskeleton
Authors: Hagan Iain M.
Published: April 2021

The Schizosaccharomyces pombe genome sequencing project (http://www.sanger.ac.uk/Projects/S_pombe/) is nearly complete, and this is likely to generate interest in fission yeast as a model system beyond its traditional strongholds in the study of the cell cycle and sexual differentiation. In many fields S. pombe will offer a useful complement to the more widely studied Saccharomyces cerevisiae, but in some areas the impact of S. pombe may well rival or exceed that of this budding yeast in terms of relevance to higher systems. Because of the considerable differences from the S. cerevisiae microtubule cytoskeleton, studying microtubules in S. pombe is likely to enhance the contribution of model systems to our understanding of the principles and practices of microtubule organisation in eukaryotes in general.
Patterns of free calcium in zebrafish embryos
Authors: Créton Robbert , Speksnijder Johanna E. , Jaffe Lionel F.
Published: April 2021

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 µm/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation.