bluuebunny/crossref_metadata_2025 huggingface dataset:
ERGIC-53 and traffic in the secretory pathway
Authors: Hauri Hans-Peter , Kappeler Felix , Andersson Helena , Appenzeller Christian
Published: April 2021

The ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53 is a mannose-specific membrane lectin operating as a cargo receptor for the transport of glycoproteins from the ER to the ERGIC. Lack of functional ERGIC-53 leads to a selective defect in secretion of glycoproteins in cultured cells and to hemophilia in humans. Beyond its interest as a transport receptor, ERGIC-53 is an attractive probe for studying numerous aspects of protein trafficking in the secretory pathway, including traffic routes, mechanisms of anterograde and retrograde traffic, retention of proteins in the ER, and the function of the ERGIC. Understanding these fundamental processes of cell biology will be crucial for the elucidation and treatment of many inherited and acquired diseases, such as cystic fibrosis, Alzheimer’s disease and viral infections.
Migration of human vascular smooth muscle cells involves serum-dependent repeated cytosolic calcium transients
Authors: Scherberich Arnaud , Campos-Toimil Manuel , Rondé Philippe , Takeda Kenneth , Beretz Alain
Published: April 2021

Migration of vascular smooth muscle cells (VSMC) is a key event in the formation of neointima during atherosclerosis. Fura-2 loaded VSMCs were used to investigate calcium homeostasis during cell migration. Multiple spontaneous transient increases in cytosolic free calcium [Ca2+]iwere observed in single human VSMCs migrating on type I collagen. Such [Ca2+]itransients were dependent on the presence of serum or PDGF-BB. Removal of serum, or loading cells with BAPTA, abolished the transients and decreased cell migration speed. The transients were not affected by disruption of cell polarization by dihydrocytochalasin B. Adhesion was used to investigate the specific role of cell-substrate interactions in the generation of transients. Transients are seen in VSMCs adhering either on collagen or on poly-L-lysine, suggesting that generation of transients is not strictly dependent on integrins. Buffering [Ca2+]iwith BAPTA led to accumulation of β1 integrins at the cellular tail, and to increased release of integrin on the extracellular matrix. These results demonstrate a role for [Ca2+]itransients in the rapid, serum-dependent migration of VSMCs. These [Ca2+]itransients are present in migrating VSMCs only when two simultaneous events occur: (1) substrate independent spreading and (2) stimulation of cells by serum components such as PDGF-BB.
The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins
Authors: Sun Wei-hsin , Hola Marek , Pedley Kevin , Tada Shusuke , Blow J. Julian , Todorov Ivan T. , Kearsey Stephen E. , Brooks Robert F.
Published: April 2021

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G1, the extent to which the license is retained when cells enter the quiescent state (G0) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopusegg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopusegg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.
Induced detachment of acentric chromatin from mitotic chromosomes leads to their cytoplasmic localization at G1and the micronucleation by lamin reorganization at S phase
Authors: Tanaka Tsubasa , Shimizu Noriaki
Published: April 2021

Acentric and atelomeric double minute chromatin found in human cancer cells are eliminated from cells by selective incorporation into the micronuclei. We showed previously that most of the micronuclei were formed at S phase and mediated by the nuclear bud-shaped structures that selectively entrap double minutes. In this paper, we have examined the behavior of double minutes in relation to the nuclear lamin protein in cell cycle-synchronized human COLO 320DM tumor cells. At the G1phase, we observed that a portion of double minutes was localized at the cytoplasm and showed no association with lamin. The frequency of this localization was increased by hydroxyurea, an inducer of micronuclei, if treated at the preceding S phase. The acentric double minutes were normally segregated to daughter cells by attaching to the mitotic chromosomes, and the hydroxyurea-treatment induced their detachment, possibly through the introduction of the double strand break. When the cells entered S phase, our data suggested that the lamin protein accumulated around the cytoplasmic double minutes at the proximity of the nucleus leading to the formation of the nuclear bud-shaped structure and the initiation of DNA replication. This association of cytoplasmic double minutes with lamin coincided with the large-scale rearrangement of the intranuclear lamin protein. The implication of these findings as well as their application to a broad spectrum of other acentric, atelomeric and autonomously replicating molecules are discussed.
The actin cytoskeleton of Dictyostelium: a story told by mutants
Authors: Noegel Angelika A. , Schleicher Michael
Published: April 2021

Actin-binding proteins are effectors of cell signalling and coordinators of cellular behaviour. Research on the Dictyostelium actin cytoskeleton has focused both on the elucidation of the function of bona fide actin-binding proteins as well as on proteins involved in signalling to the cytoskeleton. A major part of this work is concerned with the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding of the role of the actin cytoskeleton in growth and development. Furthermore, the studies have identified several cellular and developmental stages that are particularly sensitive to an unbalanced cytoskeleton. In addition, use of GFP fusion proteins is revealing the spatial and temporal dynamics of interactions between actin-associated proteins and the cytoskeleton.
Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice
Authors: Delplanque Aurélie , Coraux Christelle , Tirouvanziam Rabindra , Khazaal Ibrahim , Puchelle Edith , Ambros Peter , Gaillard Dominique , Péault Bruno
Published: April 2021

We have developed an in vivo assay for progenitor cells of the human tracheobronchial epithelium relying on the transplantation of human prenatal respiratory tissues into severe combined immunodeficiency mice. Engrafted embryonic or fetal open tracheobronchial rudiments are rapidly closed at each end by a neoformed membrane that we named the operculum. After 2-4 weeks, differentiated human respiratory epithelium covers both the native airway matrix and the new operculum. Human epithelial cells dissociated from either emerging embryonic lung primordia or mature xenografts were seeded in host human airway grafts, of which native epithelium had been eliminated by several cycles of freezing and thawing. All grafts seeded with donor epithelial cells and implanted back into SCID mice recovered a surface mucociliary epithelium expressing expected markers and secreting mucus. Spontaneous epithelium regrowth was never observed in control unseeded, denuded grafts. In some experiments, donor epithelial cells and host denuded airway were sex-mismatched and the donor origin of newly formed epithelial structures was confirmed by sex chromosome detection. After two rounds of seeding and reimplantation, a normal epithelium was observed to line the 3rd generation operculum. These observations substantiate a functional assay for human candidate airway epithelium stem cells.
Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes
Authors: Haraguchi Tokuko , Koujin Takako , Hayakawa Tomohiro , Kaneda Toru , Tsutsumi Chihiro , Imamoto Naoko , Akazawa Chihiro , Sukegawa Jun , Yoneda Yoshihiro , Hiraoka Yasushi
Published: April 2021

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.
The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells
Authors: Guillemain Ghislaine , Loizeau Martine , Pinçon-Raymond Martine , Girard Jean , Leturque Armelle
Published: April 2021

The hypothesis that the glucose transporter GLUT2 can function as a protein mediating transcriptional glucose signaling was addressed. To divert the putative interacting proteins from a glucose signaling pathway, two intracytoplasmic domains of GLUT2, the C terminus and the large loop located between transmembrane domains 6 and 7, were transfected into mhAT3F hepatoma cells. Glucose-induced accumulation of two hepatic gene mRNAs (GLUT2 and L-pyruvate kinase) was specifically inhibited in cells transfected with the GLUT2 loop and not with the GLUT2 C terminus. The dual effects of glucose were dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced transcription. This inhibition by the GLUT2 loop could be due to competitive binding of a protein that normally interacts with endogenous GLUT2. In addition, the GLUT2 loop, tagged with green fluorescent protein (GFP), was located within the nucleus, whereas the GFP and GFP-GLUT2 C-terminal proteins remained in the cytoplasm. In living cells, a fraction (50%) of the expressed GFP-GLUT2 loop translocated rapidly from the cytoplasm to the nucleus in response to high glucose concentration and conversely in the absence of glucose. We conclude that, via protein interactions with its large loop, GLUT2 may transduce a glucose signal from the plasma membrane to the nucleus.
Assembly of the exogenous extracellular matrix during basement membrane formation by alveolar epithelial cells in vitro
Authors: Furuyama Akiko , Mochitate Katsumi
Published: April 2021

We found that immortalized alveolar type II epithelial cells (SV40-T2 cells) that were cultured on dense fibrillar collagen supplemented with Matrigel gel formed a thin and continuous lamina densa beneath them. Immunohistochemical analysis of laminin-1, type IV collagen, entactin (nidogen) and perlecan in the culture indicated that all these components were integrated into a sheet structure of basement membrane beneath the cells. Analysis of the temporal and spatial distribution of the basement membrane macromolecules revealed that the initial deposits of laminin-1 and entactin were significantly greater in area in the presence of Matrigel. These globular deposits and the coarse mesh of basement membrane macromolecules developed into a flat membranous basement membrane. In the absence of Matrigel, the SV40-T2 cells failed to form a continuous lamina densa, and the deposits stayed in the coarse mesh. The major biotinylated Matrigel components that were integrated into the basement membrane were laminin-1 and entactin. Furthermore, SV40-T2 cells supplemented with exogenous laminin-1 alone as well as laminin-1 contaminated with entactin formed a continuous lamina densa. These results indicate that the laminin-1 and entactin supplied from the Matrigel were incorporated into a basement membrane beneath the SV40-T2 cells, and contributed to the formation of basement membrane. Therefore, we concluded that the alveolar epithelial cells synthesize laminin-1, entactin, type IV collagen, and perlecan, but that they also needed to assemble exogenous laminin-1 into the basement membrane to complete its formation in vitro.
Tying the knot: linking cytokinesis to the nuclear cycle
Authors: Balasubramanian Mohan K. , McCollum Dannel , Surana Uttam
Published: April 2021

For the survival of both the parent and the progeny, it is imperative that the process of their physical division (cytokinesis) be precisely coordinated with progression through the mitotic cell cycle. Recent studies in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are beginning to unravel the nature of the links between cytokinesis and the nuclear division cycle. The cyclin-dependent kinases and a novel surveillance mechanism that monitors cytokinesis and/or morphogenesis appear to play important regulatory roles in forging these links. It is becoming increasingly clear that the inactivation of the mitosis-promoting cyclin-dependent kinase, which marks the completion of the nuclear division cycle, is essential for actomyosin ring constriction and division septum assembly in both yeasts. Additionally, the spindle pole bodies are emerging as important transient locale for proteins that might play a key role in coupling the completion of mitosis to the onset of cytokinesis.
Effects of cytochalasin D and latrunculin B on mechanical properties of cells
Authors: Wakatsuki Tetsuro , Schwab Bill , Thompson Nathan C. , Elson Elliot L.
Published: April 2021

Actin microfilaments transmit traction and contraction forces generated within a cell to the extracellular matrix during embryonic development, wound healing and cell motility, and to maintain tissue structure and tone. Therefore, the state of the actin cytoskeleton strongly influences the mechanical properties of cells and tissues. Cytochalasin D and Latrunculin are commonly used reagents that, by different mechanisms, alter the state of actin polymerization or the organization of actin filaments. We have investigated the effect of a wide range of Cytochalasin D and Latrunculin B concentrations (from 40 pM to 10 μM) on the mechanical properties of the cells within fibroblast populated collagen matrices. Contractile force and dynamic stiffness were measured by uniaxial stress-strain testing. The range of effective concentrations of Cytochalasin D (200 pM-2 μM) was broader than that of Latrunculin B (20 nM-200 nM). Activating the cells by serum did not change the effective range of Cytochalasin D concentrations but shifted that of Latrunculin B upward by tenfold. Simple mathematical binding models based on the presumed mechanisms of action of Cytochalasin D and Latrunculin B simulated the concentration-dependent mechanical changes reasonably well. This study shows a strong dependence of the mechanical properties of cells and tissues on the organization and degree of polymerization of actin filaments.
Control of mitochondrial morphology by a human mitofusin
Authors: Santel Ansgar , Fuller Margaret T.
Published: April 2021

Although changes in mitochondrial size and arrangement accompany both cellular differentiation and human disease, the mechanisms that mediate mitochondrial fusion, fission and morphogenesis in mammalian cells are not understood. We have identified two human genes encoding potential mediators of mitochondrial fusion. The mitofusins (Mfn1 and Mfn2) are homologs of the Drosophila protein fuzzy onion (Fzo) that associate with mitochondria and alter mitochondrial morphology when expressed by transient transfection in tissue culture cells. An internal region including a predicted bipartite transmembrane domain (TM) is sufficient to target Mfn2 to mitochondria and requires hydrophobic residues within the TM. Co-expression of Mfn2 with a dominant interfering mutant dynamin-related protein (Drp1K38A) proposed to block mitochondrial fission resulted in long mitochondrial filaments and networks. Formation of mitochondrial filaments and networks required a wild-type Mfn2 GTPase domain, suggesting that the Mfn2 GTPase regulates or mediates mitochondrial fusion and that mitofusins and dynamin related GTPases play opposing roles in mitochondrial fusion and fission in mammals, as in yeast.
Evidence for a role of the α-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes
Authors: Vée Sophie , Lafanechère Laurence , Fisher Daniel , Wehland Jürgen , Job Didier , Picard André
Published: April 2021

Microinjected mAb YL1/2, an α-tubulin antibody specific for the tyrosinated form of the protein, blocks the cell cycle in developing oocytes. Here, we have investigated the mechanism involved in the mAb effect. Both developing starfish and Xenopus oocytes were injected with two different α-tubulin C terminus antibodies. The injected antibodies blocked cell entry into mitosis through specific inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized microtubules and was mimicked by injected synthetic peptides corresponding to the tyrosinated α-tubulin C terminus, whereas peptides lacking the terminal tyrosine were ineffective. These results indicate that tyrosinated α-tubulin, or another protein sharing the same C-terminal epitope, is involved in specific regulation of cyclin B synthesis in developing oocytes.
Effects of stathmin inhibition on the mitotic spindle
Authors: Iancu Camelia , Mistry Sucharita J. , Arkin Steven , Wallenstein Sylvan , Atweh George F.
Published: April 2021

Stathmin is a major cytosolic phosphoprotein that plays an important role in the regulation of microtubule dynamics during cell cycle progression. It has recently been proposed that the major function of stathmin is to promote depolymerization of the microtubules that make up the mitotic spindle. In this report, we tested the prediction that a deficiency in stathmin expression would result in constitutive stabilization of microtubules and lead to abnormalities in the organization of the mitotic spindle. Our studies demonstrate that antisense inhibition of stathmin expression in K562 erythroleukemic cells results in increased ratio of polymerized to depolymerized tubulin. These changes are associated with phenotypic abnormalities of the mitotic spindle and difficulty in completing mitosis. These studies also showed that inhibition of stathmin expression results in increased susceptibility of K562 leukemic cells to the pharmacological agents, like taxol, which are known to stabilize the mitotic spindle. In contrast, stathmin inhibition results in decreased sensitivity to vinblastine, an agent that destabilizes the mitotic spindle. Thus, our experimental findings are supportive of the model that stathmin is a microtubule-destabilizing factor that plays an important role in the regulation of the mitotic spindle. We also suggest a potential therapeutic approach for cancer based on the combination of stathmin inhibition with pharmacologic agents that stabilize the mitotic spindle.
Matrix metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices
Authors: Davis George E. , Allen Kristine A. Pintar , Salazar René , Maxwell Steven A.
Published: April 2021

Here, we describe a new function for plasmin and matrix metalloproteinases (MMPs), which is to regulate the regression of capillary tubes in three-dimensional extracellular matrix environments. Using a well-described capillary morphogenesis system in three-dimensional collagen matrices, a new model of capillary regression has been established by adding plasminogen to the culture medium. Plasminogen is converted to plasmin by endothelial cell plasminogen activators which then induces matrix metalloproteinase-dependent collagen gel contraction and capillary regression. Plasminogen addition results in activation of MMP-1 and MMP-9, which then results in collagen proteolysis followed by capillary regression. The endothelial cells undergo apoptosis following gel contraction as detected by flow cytometric analysis as well as by detectable caspase-3 cleavage and caspase-dependent cleavage of the actin cytoskeletal regulatory protein, gelsolin. In addition, directly correlating with the contraction response, tyrosine phosphorylation of p130cas, an adapter protein in the focal adhesion complex, is observed followed by disappearance of the protein. Proteinase inhibitors that block MMPs (TIMP-1 or TIMP-2), plasminogen activators (PAI-1) or plasmin (aprotinin) completely block the gel contraction and regression process. In addition, chemical inhibitors of MMPs that block capillary regression also block MMP-1 and MMP-9 activation suggesting that a key element in this regression response is the molecular control of MMP activation by endothelial cells. Blocking antibodies directed to MMP-1 or MMP-9 interfere with capillary regression while blocking antibodies directed to PAI-1 accelerate capillary regression suggesting that endogenous synthesis of PAI-1 negatively regulates this process. These data present a novel system to study a new mechanism that may regulate regression of capillary tubes, namely, plasmin and MMP-mediated degradation of extracellular matrix.
Integrin regulation of cell-cell adhesion during epithelial tubule formation
Authors: Ojakian George K. , Ratcliffe Don R. , Schwimmer Randi
Published: April 2021

The extracellular matrix plays an important role in regulation of epithelial development and organization. To determine more precisely the function of extracellular matrix in this process, the initial steps in collagen-mediated formation of epithelial tubules were studied using a model cell culture system. Previous studies have demonstrated that incubation of Madin-Darby canine kidney (MDCK) epithelial cells with a collagen gel overlay induces β1 integrin-regulated epithelial remodeling accompanied by extensive cell rearrangements and formation of epithelial tubules. During epithelial remodeling there was extensive disruption of the epithelial junctional complex. Progressive opening of tight junctions was observed over 8 hours using transepithelial resistance measurements and immunofluorescence microscopy demonstrated that tight and adherens junction proteins were dispersed throughout the apical and basolateral membranes. Junction complex disruption allowed the formation of apical cell extensions and subsequent migration of selected cell sheets from the epithelial monolayer. Confocal microscopy demonstrated the presence of adherens junction (E-cadherin, α-catenin, β-catenin, plakoglobin) and desmosomal (desmoplakin-1/2, plakoglobin) proteins on, and within, cell extensions demonstrating that cell junctions had undergone considerable disassembly. However, groups of cell extensions appeared to be associated by E-cadherin/ catenin-mediated interactions. Association of E-cadherin/ catenin complexes with the epithelial cytoskeleton was analyzed by differential detergent extraction. SDS-PAGE and immunoblot analysis demonstrated that adherens junction proteins were primarily cytoskeleton-associated in control cells. During integrin-regulated remodeling, there was a progressive reduction in the interaction of adherens junction proteins with the cytoskeleton suggesting that they play an important role in the maintenance of epithelial integrity. Since loss of transepithelial electrical resistance and disruption of junctional complexes were inhibited by an antifunctional integrin antibody, we propose that activation of integrin signaling pathways regulate junctional complex stability, cell-cell interactions and cell migration. These observations provide evidence that integrin-regulated MDCK epithelial tubule formation can serve as a model system for studying rearrangements of epithelial sheets which occur during development.
Phosphoinositide 3-kinase signalling pathways
Authors: Cantrell Doreen Ann
Published: April 2021

Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3′-OH position of the inositol ring of inositol phospholipids, producing three lipid products: PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These lipids bind to the pleckstrin homology (PH) domains of proteins and control the activity and subcellular localisation of a diverse array of signal transduction molecules. Three major classes of signalling molecule are regulated by binding of D-3 phosphoinositides to PH domains: guanine-nucleotide-exchange proteins for Rho family GTPases, the TEC family tyrosine kinases such as BTK and ITK in B and T lymphocytes, respectively, and the AGC superfamily of serine/threonine protein kinases. These molecules are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes, including cell cycle progression, cell growth, cell motility, cell adhesion and cell survival.
Initiating DNA synthesis: from recruiting to activating the MCM complex
Authors: Lei Ming , Tye Bik K.
Published: April 2021

The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.
Differential effect of GalNAcα-O-bn on intracellular trafficking in enterocytic HT-29 and Caco-2 cells: correlation with the glycosyltransferase expression pattern
Authors: Gouyer Valérie , Leteurtre Emmanuelle , Delmotte Philippe , Steelant Wim F. A. , Krzewinski-Recchi Marie-Ange , Zanetta Jean-Pierre , Lesuffleur Thécla , Trugnan Germain , Delannoy Philippe , Huet Guillemette
Published: April 2021

Our previous work has shown that long-term treatment of mucus-secreting HT-29 cells with 1-benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside (GalNAcα-O-bn), a competitive inhibitor of O-glycosylation, induced several phenotypic changes, in particular a blockade in the secretion of mucins, which are extensively O-glycosylated glycoproteins. Here, we have analyzed the effects of GalNAcα-O-bn upon the intracellular trafficking of basolateral and apical membrane glycoproteins at the cellular and biochemical levels in two types of cells, HT-29 G− and Caco-2, differentiated into an enterocyte-like phenotype. In HT-29 G− cells, but not in Caco-2 cells, DPP-IV and CD44 failed to be targeted to the apical or basolateral membrane, respectively, and accumulated inside intracytoplasmic vesicles together with GalNAcα-O-bn metabolites. We observed a strong inhibition of α2,3-sialylation of glycoproteins in HT-29 G− cells correlated to the high expression of α2,3-sialyltransferases ST3Gal I and ST3Gal IV. In these cells, DPP-IV and CD44 lost the sialic acid residue substituting the O-linked core 1 structure Galβ1-3GalNAc (T-antigen). In contrast, sialylation was not modified in Caco-2 cells, but a decrease of α1,2-fucosylation was observed, in correlation with the high expression of α1,2-fucosyltransferases Fuc-TI and Fuc-TII. In conclusion, in HT-29 G− cells, GalNAcα-O-bn induces a specific cellular phenotype, which is morphologically characterized by the formation of numerous intracellular vesicles, in which are accumulated defectively sialylated O-glycosylproteins originally targeted to basolateral or apical membranes, and GalNAcα-O-bn metabolites.
Avidin expression during chick chondrocyte and myoblast development in vitro and in vivo: regulation of cell proliferation
Authors: Zerega Barbara , Camardella Laura , Cermelli Silvia , Sala Roberta , Cancedda Ranieri , Cancedda Fiorella Descalzi
Published: April 2021

Avidin is a major [35S]methionine-labeled protein induced by bacterial lipopolysaccharide (LPS) and interleukin 6 (IL-6) in cultured chick embryo myoblasts and chondrocytes. It was identified by N-terminal sequencing of the protein purified from conditioned culture medium of LPS-stimulated myoblasts. In addition, avidin was secreted by unstimulated myoblasts and chondrocytes during in vitro differentiation; maximal expression being observed in differentiated myofibers and hypertrophic chondrocytes. In developing chick embryos, immunohistochemistry revealed avidin in skeletal muscles and growth plate hypertrophic cartilage. Avidin was secreted into culture as a biologically active tetramer. Exogenous avidin added to the medium of proliferating chondrocytes progressively inhibited cell proliferation, whereas addition of avidin to differentiating chondrocytes in suspension allowed full cell differentiation. No toxic effects for the cells were observed in both culture conditions. Western blots of samples from cytosolic extracts using alkaline-phosphatase-conjugated streptavidin showed three biotin-containing proteins. Acetyl-CoA carboxylase was identified by specific antibodies. Based on these data, we propose that avidin binds extracellular biotin and regulates cell proliferation by interfering with fatty acid biosynthesis during terminal cell differentiation and/or in response to inflammatory stimuli.
Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction
Authors: Aoki Hiromasa , Fujii Makiko , Imamura Takeshi , Yagi Ken , Takehara Kazuhiko , Kato Mitsuyasu , Miyazono Kohei
Published: April 2021

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β superfamily, which regulate the differentiation of osteoprogenitor cells. Here we show that among members of the BMP family, BMP-4 and growth/differentiation factor 5 (GDF-5) induce osteoblast differentiation through the activation of three receptor- regulated Smads (i.e. Smad1, Smad5 and Smad8). By contrast, BMP-6 and BMP-7 induce alkaline phosphatase activity through Smad1 and Smad5, but not through Smad8. Consistent with these findings, BMP-4 induced phosphorylation and nuclear translocation of Smad1, Smad5 and Smad8, but BMP-6 activated only Smad1 and Smad5. BMP-4 and GDF-5 are known to bind to activin receptor-like kinase 3 (ALK-3) and/or ALK-6 (also termed BMP type IA and type IB receptors, respectively), whereas BMP-6 and BMP-7 preferentially bind to ALK-2. Compared with the effects induced by only one of the type I receptors, the combination of constitutively active forms of ALK-2 and ALK-3 (or ALK-6) more strongly induced alkaline phosphatase activity in C2C12 cells. Moreover, addition of BMP-4 and BMP-6 to C2C12 cells resulted in higher alkaline phosphatase activity than that of only one of these BMPs. The combination of ALK-2 and ALK-3 also induced higher transcriptional activity than either receptor alone. Thus, ALK-2 and ALK-3 (or ALK-6) might synergistically induce osteoblast differentiation of C2C12 cells, possibly through efficient activation of downstream signaling pathways.
The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions
Authors: Herrmann Christine H. , Mancini Michael A.
Published: April 2021

TAK/P-TEFb is an elongation factor for RNA polymerase II-directed transcription that is thought to function by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II. TAK/P-TEFb is composed of Cdk9 and cyclin T and serves as the cellular cofactor for the human immunodeficiency virus transactivator Tat protein. In this study, we examined the subcellular distribution of Cdk9 and cyclin T1 using high resolution immunofluorescence microscopy and found that Cdk9 and cyclin T1 localized throughout the non-nucleolar nucleoplasm, with increased signal present at numerous foci. Both Cdk9 and cyclin T1 showed only limited colocalization with different phosphorylated forms of RNA polymerase II. However, significant colocalization with antibodies to several splicing factors that identify nuclear ‘speckles’ was observed for Cdk9 and especially for cyclin T1. The pattern of Cdk9 and cyclin T1 distribution was altered in cells treated with transcription inhibitors. Transient expression of cyclin T1 deletion mutants indicated that a region in the central portion of cyclin T1 is important for accumulation at speckles. Furthermore, cyclin T1 proteins that accumulated at speckles were capable of recruiting Cdk9 and the HIV Tat protein to this compartment in overexpression experiments. These results suggest that cyclin T1 functions to recruit its binding partners to nuclear speckles and raises the possibility that nuclear speckles are a site of TAK/P-TEFb function.
Pulsed DC electric fields couple to natural NAD(P)H oscillations in HT-1080 fibrosarcoma cells
Authors: Rosenspire Allen J. , Kindzelskii Andrei L. , Petty Howard R.
Published: April 2021

Previously, we have demonstrated that NAD(P)H levels in neutrophils and macrophages are oscillatory. We have also found that weak ultra low frequency AC or pulsed DC electric fields can resonate with, and increase the amplitude of, NAD(P)H oscillations in these cells. For these cells, increased NAD(P)H amplitudes directly signal changes in behavior in the absence of cytokines or chemotactic factors. Here, we have studied the effect of pulsed DC electric fields on HT-1080 fibrosarcoma cells. As in neutrophils and macrophages, NAD(P)H levels oscillate. We find that weak (∼10−5 V/m), but properly phased DC (pulsed) electric fields, resonate with NAD(P)H oscillations in polarized and migratory, but not spherical, HT-1080 cells. In this instance, electric field resonance signals an increase in HT-1080 pericellular proteolytic activity. Electric field resonance also triggers an immediate increase in the production of reactive oxygen metabolites. Under resonance conditions, we find evidence of DNA damage in HT-1080 cells in as little as 5 minutes. Thus the ability of external electric fields to effect cell function and physiology by acting on NAD(P)H oscillations is not restricted to cells of the hematopoietic lineage, but may be a universal property of many, if not all polarized and migratory eukaryotic cells.
Superactivation of integrin αvβ3 by low antagonist concentrations
Authors: Legler Daniel F. , Wiedle Guido , Ross F. Patrick , Imhof Beat A.
Published: April 2021

Integrins are implicated in cell adhesion, migration and homeostasis. An important feature is their ability to adopt different affinity states that can be regulated by a variety of intra- and extracellular factors. To study affinity modulation of the integrin ectodomain by extracellular factors, we produced a soluble recombinant form of mouse integrin αvβ3 in a mammalian expression system and isolated it to purity. We show that the two transmembrane truncated integrin subunits stably associate to form a functional receptor, soluble recombinant αvβ3. The affinity of this receptor for its ligands vitronectin, fibronectin and fibrinogen can be modulated by the divalent cations magnesium, calcium and manganese. Most importantly, we found that a cyclic RGD-peptide has a biphasic effect on rsαvβ3 and native purified αvβ3, with an antagonistic phase at high concentrations, and an agonistic phase at low concentrations. This integrin superactivation by low antagonist concentrations is shown in binding of srαvβ3 to immobilized ligands by ELISA, and in adhesion of cells that express the chimaeric integrin ligand KISS31 to immobilized rsαvβ3 and native purified αvβ3. Our results indicate that low concentrations of the ligand mimetic cyclo-RGD can result in superactivation of the extracellular domain of integrin αvβ3 to a comparable level as activation by manganese.
Phosphatidylinositol 3-kinase mediates integrin-dependent NF-κB and MAPK activation through separate signaling pathways
Authors: Reyes-Reyes Merit , Mora Nancy , Zentella Alejandro , Rosales Carlos
Published: April 2021

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, β1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via β1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor kB (NF-κB) activation was then analyzed. We found that integrins activated both NF-κ B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-kB activation. In contrast, a dominant negative mutant of Rac completely prevented NF-κ B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-κ B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Rho augmented NF-κ B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-κ B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.
Genome restructuring in rye affects the expression, organization and disposition of homologous rDNA loci
Authors: Caperta Ana D. , Neves Nuno , Morais-Cecílio Leonor , Malhó Rui , Viegas Wanda
Published: April 2021

The standard rye cultivar `Imperial' and a structural variant carrying an intact 1R chromosome and two telocentric 1R chromosomes (short and long arms)were used to investigate expression patterns of homologous rDNA loci, and the influence of chromosome structural change on their interphase organisation and relative disposition. Sequential silver staining and in situ hybridization with the rDNA probe pTa71, established a correspondence between the expression and organization patterns of rDNA domains in metaphase and interphase cells. In most cells of the cultivar Imperial, nucleolar organizer region (NOR)silver staining on metaphase chromosomes with equivalent numbers of rDNA genes revealed a size heteromorphism between homologous rDNA loci, resulting from their differential expression. NOR heteromorphism in the structural variant line was significantly reduced. The preferential activity of one NOR over its homologue was found to be random within cells and independent of parental origin. Nucleotypic modifications mediated by changes in the 1R chromosome structure include increased proximity between homologous rDNA loci in interphase, and an increase in the frequency of cells with intra-nucleolar ribosomal condensed chromatin. These results seem to indicate a `sequence recognition' process for the regulation of homologous loci.
A conserved C-terminal domain of EFA6-family ARF6-guanine nucleotide exchange factors induces lengthening of microvilli-like membrane protrusions
Authors: Derrien Valérie , Couillault Carole , Franco Michel , Martineau Stéphanie , Montcourrier Philippe , Houlgatte Rémi , Chavrier Philippe
Published: April 2021

We recently reported the identification of EFA6 (exchange factor for ARF6), a brain-specific Sec7-domain-containing guanine nucleotide exchange factor that works specifically on ARF6. Here, we have characterized the product of a broadly expressed gene encoding a novel 1056 amino-acid protein that we have named EFA6B. We show that EFA6B, which contains a Sec7 domain that is highly homologous to EFA6, works as an ARF6-specific guanine exchange factor in vitro. Like EFA6, which will be referred to as EFA6A from now on, EFA6B is involved in membrane recycling and colocalizes with ARF6 in actin-rich membrane ruffles and microvilli-like protrusions on the dorsal cell surface in transfected baby hamster kidney cells. Strikingly, homology between EFA6A and EFA6B is not limited to the Sec7 domain but extends to an adjacent pleckstrin homology (PH) domain and a ∼150 amino-acid C-terminal region containing a predicted coiled coil motif. Association of EFA6A with membrane ruffles and microvilli-like structures depends on the PH domain, which probably interacts with phosphatidylinositol 4,5-biphosphate. Moreover, we show that overexpression of the PH domain/C-terminal region of EFA6A or EFA6B in the absence of the Sec7 domain promotes lengthening of dorsal microvillar protrusions. This morphological change requires the integrity of the coiled-coil motif. Lastly, database analysis reveals that the EFA6-family comprises at least four members in humans and is conserved in multicellular organisms throughout evolution. Our results suggest that EFA6 family guanine exchange factors are modular proteins that work through the coordinated action of the catalytic Sec7 domain to promote ARF6 activation, through the PH domain to regulate association with specific subdomains of the plasma membrane and through the C-terminal region to control actin cytoskeletal reorganization.
A novel linker histone-like protein is associated with cytoplasmic filaments inCaenorhabditis elegans
Authors: Jedrusik Monika A. , Vogt Stefan , Claus Peter , Schulze Ekkehard
Published: April 2021

The histone H1 complement of Caenorhabditis elegans contains a single unusual protein, H1.X. Although H1.X possesses the globular domain and the canonical three-domain structure of linker histones, the amino acid composition of H1.X is distinctly different from conventional linker histones in both terminal domains. We have characterized H1.X in C. elegans by antibody labeling, green fluorescent protein fusion protein expression and RNA interference. Unlike normal linker histones, H1.X is a cytoplasmic as well as a nuclear protein and is not associated with chromosomes. H1.X is most prominently expressed in the marginal cells of the pharynx and is associated with a peculiar cytoplasmic cytoskeletal structure therein, the tonofilaments. Additionally H1.X::GFP is expressed in the cytoplasm of body and vulva muscle cells, neurons, excretory cells and in the nucleoli of embryonic blastomeres and adult gut cells. RNA interference with H1.X results in uncoordinated and egg laying defective animals, as well as in a longitudinally enlarged pharynx. These phenotypes indicate a cytoplasmic role of H1.X in muscle growth and muscle function.
Role of sulfated O-linked glycoproteins in zymogen granule formation
Authors: De Lisle Robert C.
Published: April 2021

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.
Membrane ruffling and macropinocytosis in A431 cells require cholesterol
Authors: Grimmer Stine , van Deurs Bo , Sandvig Kirsten
Published: April 2021

Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-β-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However,confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus,our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.