bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dystrophic phenotype induced in vitro by antibody blockade of muscle α-dystroglycan-laminin interaction
Authors: Brown Susan C. , Fassati Ariberto , Popplewell Linda , Page Anton M. , Henry Michael D. , Campbell Kevin P. , Dickson George 
Published: April 2021

ABSTRACT
Alpha-dystroglycan is a glycoprotein expressed on the surface of skeletal muscle fibres and other cell types. In muscle, α-dystroglycan provides a link between the myofibre cytoskeleton through its indirect binding to dystrophin, and the basal lamina through its binding to laminin-2, a protein of the extracellular matrix. The disruption of this linkage between the myofibre cytoskeleton and the extracellular matrix is a common feature of Duchenne and other muscular dystrophies, though the pathogenic mechanisms leading to muscle wasting remain unknown. By treating primary mouse muscle cultures with a monoclonal antibody which blocks α-dystroglycan binding to laminin, we show here the induction of a dystrophic phenotype in vitro. The phenotype is inducible in differentiated cultures only, is characterised by reduced myotube size, myofibril disorganisation, loss of contractile activity, reduced spontaneous clustering of acetylcholine receptors and is reversed by addition of excess exogenous laminin-2. Thus, α-dystroglycan may be part of a signalling pathway for the maturation and maintenance of skeletal myofibres. Detailed knowledge of this signalling pathway may provide insights into the molecular pathology of the various inherited muscular dystrophies, and identify valuable pharmacological targets and new therapeutic strategies.
Amino acid motifs required for isolated β cytoplasmic domains to regulate ‘in trans’ β1 integrin conformation and function in cell attachment
Authors: Mastrangelo Anthony M. , Homan Suzanne M. , Humphries Martin J. , LaFlamme Susan E. 
Published: April 2021

ABSTRACT
The role of β cytoplasmic domains in regulating β1 integrin conformation and function in cell attachment is not fully understood. In this study, we tested the ability of transiently expressed β cytoplasmic domains connected to an extracellular reporter domain to regulate ‘in trans’ the conformation of endogenous β1 integrins, and compared these effects on cell attachment. We found that chimeric receptors containing either the β1, β3 or β5 cytoplasmic domains inhibited the expression of the conformationally dependent 9EG7 and 12G10 epitopes on endogenous β1 integrins. In contrast, chimeric receptors containing the β4 or α5 cytoplasmic domain, or a control receptor lacking a cytoplasmic domain, had no effect. This inhibition occurred in a dose-dependent manner that required high levels of expression of the chimeric receptor. These results suggest that β1 integrin conformation can be regulated by conserved cytosolic interactions involving β cytoplasmic domains. This is further supported by our findings that mutations within amino acid motifs conserved among these β cytoplasmic domains, specifically the NXXY, NPXY and TST-like motifs, reduced the ability of these chimeric receptors to regulate β 1 integrin conformation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST-like motifs. The β1 chimera also inhibited the binding of soluble fibronectin to endogenous β1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9EG7 and 12G10 epitopes correlates with integrin function. However, Mn2+, which is an extracellular activator of integrin function, increased 9EG7 expression to basal levels in the presence of the β1 chimera, but did not rescue cell attachment to the same extent. Thus, although the β1 integrin conformation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the β chimeras may be inhibiting both ligand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attachment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function. This finding, together with the requirement for high levels of the chimeric receptors and the fact that mutations in the same conserved motifs in heterodimeric β1 integrins have been reported to regulate β1 integrin conformation and function in cell attachment, suggest that β cytoplasmic domains regulate these processes by interacting with cytosolic factors and that the regulatory effect of the chimeras may be due to their ability to titrate proteins from endogenous integrins.
Control of morphology, cytoskeleton and migration by syndecan-4
Authors: Longley Robert L. , Woods Anne , Fleetwood Allison , Cowling Graham J. , Gallagher John T. , Couchman John R. 
Published: April 2021

ABSTRACT
Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.
Overexpression of normal and mutant Arp1α (centractin) differentially affects microtubule organization during mitosis and interphase
Authors: Clark Imran B. , Meyer David I. 
Published: April 2021

ABSTRACT
Dynactin is a large multisubunit complex that regulates cytoplasmic dynein-mediated functions. To gain insight into the role of dynactin’s most abundant component, Arp1α was transiently overexpressed in mammalian cells. Arp1α overexpression resulted in a cell cycle delay at prometaphase. Intracellular dynactin, dynein and nuclear/mitotic apparatus (NuMA) protein were recruited to multiple foci associated with ectopic cytoplasmic aggregates of Arp1α in transfected cells. These ectopic aggregates nucleated supernumerary microtubule asters at prometaphase. Point mutations were generated in Arp1α that identified specific amino acids required for the prometaphase delay and for the formation of supernumerary microtubule asters. The mutant Arp1α proteins formed aggregates in cells that colocalized with dynactin and dynein peptides, but in contrast to wild-type Arp1α, NuMA localization remained unaffected. Although expression of mutant Arp1α proteins had no effect on mitotic cells, in interphase cells expression of the mutants resulted in disruption of the microtubule network. Immunoprecipitation studies demonstrated that overexpressed Arp1α interacts with dynactin and NuMA proteins in cell extracts, and that these interactions are destabilized in the Arp1α mutants. We conclude that the amino acids altered in the Arp1α mutant proteins participate in stabilizing interactions between overexpressed Arp1α and components of the endogenous dynactin complex as well as the NuMA protein.
Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation
Authors: Ebisawa Takanori , Tada Keiichiro , Kitajima Isao , Tojo Katsuyoshi , Sampath T. Kuber , Kawabata Masahiro , Miyazono Kohei , Imamura Takeshi 
Published: April 2021

ABSTRACT
Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-β superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.
A fraction of mouse sperm chromatin is organized in nucleosomal hypersensitive domains enriched in retroposon DNA
Authors: Pittoggi C. , Renzi L. , Zaccagnini G. , Cimini D. , Degrassi F. , Giordano R. , Magnano A. R. , Lorenzini R. , Lavia P. , Spadafora C. 
Published: April 2021

ABSTRACT
We have characterized a nuclease hypersensitive chromatin fraction from murine spermatozoa. Endogenous nuclease activity can be induced in mouse epididymal spermatozoa by appropriate stimuli and cause the localized degradation of chromosomal DNA. Based on these observations, we have isolated nuclease hypersensitive chromatin regions released from spermatozoa in the supernatant of pelleted sperm cells, and have cloned and characterized the DNA. Gel electrophoresis of end-labelled released DNA fragments showed a typical nucleosomal distribution. Peripherally distributed nucleohistones were visualized by immunofluorescence in sperm nuclei, and histones were identified by western blot in sperm chromatin. Moreover, the released DNA is enriched in retroposon DNA from a variety of families. FISH and immunofluorescence analysis showed that retroposon DNA and nucleohistone chromatin co-localize and are both peripherically distributed in nuclei of spermatozoa. In contrast, a major satellite DNA probe, used for control, co-localizes with highly condensed chromatin in the central region of sperm nuclei. The nuclear Ran and RCC1 proteins were also visualized in the dorsal margin of sperm nuclei, and were abundantly released with the hypersensitive chromatin fraction. Together, these results indicate that nucleohistone chromatin fraction(s) with typical features of ‘active’ chromatin are present in murine spermatozoa, are hypersensitive to nuclease cleavage, enriched in retroposon DNA and organized in nucleosomal domains. These observations suggest that nucleohistone domains identify a fraction of the sperm genome which may be functional during early embryogenesis.
VAMP (synaptobrevin) is present in the plasma membrane of nerve terminals
Authors: Taubenblatt P. , Dedieu J. C. , Gulik-Krzywicki T. , Morel N. 
Published: April 2021

ABSTRACT
Synaptic vesicle docking and exocytosis require the specific interaction of synaptic vesicle proteins (such as VAMP/synaptobrevin) with presynaptic plasma membrane proteins (such as syntaxin and SNAP 25). These proteins form a stable, SDS-resistant, multimolecular complex, the SNARE complex. The subcellular distribution of VAMP and syntaxin within Torpedo electric organ nerve endings was studied by immunogoldlabeling of SDS-digested freeze-fracture replicas (Fujimoto, 1995). This technique allowed us to visualize large surface areas of the presynaptic plasma membrane and numerous synaptic vesicles from rapidly frozen nerve endings and synaptosomes. VAMP was found associated with synaptic vesicles, as also shown by conventional electron microscopy immunolabeling, and to the presynaptic plasma membrane (P leaflet). Syntaxin was also detected in the nerve ending plasma membrane, without gold labeling of synaptic vesicles. Comparison of gold particle densities suggests that the presynaptic plasma membrane contains 3 VAMP molecules per molecule of syntaxin.
After biotinylation of intact synaptosomes, the synaptosomal plasma membrane was isolated on Streptavidin coated magnetic beads. Its antigenic content was compared to that of purified synaptic vesicles. VAMP was present in both membranes whereas syntaxin and SNAP 25 were highly enriched in the synaptosomal plasma membrane. This membrane has a low content of classical synaptic vesicle proteins (synaptophysin, SV2 and the vesicular acetylcholine transporter). The VAMP to syntaxin stoechiometry in the isolated synaptosomal membrane was estimated by comparison with purified antigens and close to 2, in accordance with morphological data. SDS-resistant SNARE complexes were detected in the isolated presynaptic membrane but absent in purified synaptic vesicles. Taken together, these results show that the presence of VAMP in the plasma membrane of nerve endings cannot result from exocytosis of synaptic vesicles, a process which could, as far as SNAREs are concerned, very much ressemble homotypic fusion.
High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion
Authors: Joberty Gérard , Stabila Paul F. , Coppola Thierry , Macara Ian G. , Regazzi Romano 
Published: April 2021

ABSTRACT
Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca2+-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic α-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca2+-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion.
High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca2+-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.
Ca2+/calmodulin and p85 cooperatively regulate an initiation of cytokinesis in Tetrahymena
Authors: Gonda Kohsuke , Katoh Mariko , Hanyu Kazuko , Watanabe Yoshio , Numata Osamu 
Published: April 2021

ABSTRACT
Tetrahymena p85 differs in mobility in two-dimensional SDS-polyacrylamide gel electrophoresis between wild-type and temperature-sensitive cell-division-arrest mutant cdaA1 cell extracts, and is localized to the presumptive division plane before the formation of the division furrow. The p85 contained three identical sequences which show homology to the calmodulin binding site of Ca2+/calmodulin dependent protein kinase Type II in Saccharomyces cerevisiae. We found the p85 directly interacts with Tetrahymena calmodulin in a Ca2+- dependent manner, using a co-sedimentation assay. We next examined the localization of p85 and calmodulin during cytokinesis using indirect immunofluorescence. The results showed that both proteins colocalize in the division furrow. This is the first observation that calmodulin is localized in the division furrow. Moreover, the direct interaction between p85 and Ca2+/calmodulin was inhibited by Ca2+/calmodulin inhibitor N-(6-aminohexyl)- 5-chloro-1-naphthalenesulfonamide HCl. When the cells were treated with the drug just before the beginning of cytokinesis, the drug also inhibited the localization of p85 and calmodulin to the division plane, and the formation of the contractile ring and division furrow. Therefore, we propose that the Ca2+/calmodulin signal and its target protein p85 cooperatively regulate an initiation of cytokinesis and may be also concerned with the progression of cytokinesis in Tetrahymena.
Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes
Authors: Smith Susan , de Lange Titia 
Published: April 2021

ABSTRACT
Tankyrase is a human poly(ADP-ribose) polymerase that was initially identified through its interaction with the telomeric protein TRF1, a negative regulator of telomere length. In vitro poly(ADP-ribosyl)ation by tankyrase inhibits TRF1 binding to telomeric DNA suggesting a role for tankyrase in telomere function. We previously demonstrated that tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase. Here we show that tankyrase localizes to additional subcellular sites in a cell cycle dependent manner. In interphase, tankyrase colocalized with TRF1 to telomeres, but in addition was found to reside at nuclear pore complexes, as evidenced by indirect immunofluorescence, subcellular fractionation and immunoelectron microscopy. At mitosis, concomitant with nuclear envelope breakdown and nuclear pore complex disassembly, tankyrase was found to relocate around the pericentriolar matrix of mitotic centrosomes. This complex staining pattern along with the observation that tankyrase did not contain a nuclear localization signal suggested that its telomeric localization might be regulated, perhaps by TRF1. Indeed, localization of exogenously-expressed tankyrase to telomeres was dependent upon co-transfection with TRF1. These data indicate that the subcellular localization of tankyrase can be regulated by both the cell cycle and TRF1.
A targeted gene silencing technique shows that Drosophila myosin VI is required for egg chamber and imaginal disc morphogenesis
Authors: Deng Wu-Min , Leaper Kevin , Bownes Mary 
Published: April 2021

ABSTRACT
We report that Drosophila unconventional myosin VI, encoded by Myosin heavy chain at 95F (Mhc95F), is required for both imaginal disc and egg chamber morphogenesis. During oogenesis, Mhc95F is expressed in migrating follicle cells, including the border cells, which migrate between the nurse cells to lie at the anterior of the oocyte; the columnar cells that migrate over the oocyte; the centripetal cells that migrate between the oocyte and nurse cells; and the dorsal-anterior follicle cells, which migrate to secrete the chorionic appendages. Its function during development has been studied using a targeted gene silencing technique, combining the Gal4-UAS targeted expression system and the antisense RNA technique. Antibody staining shows that the expression of myosin 95F is greatly decreased in follicle cells when antisense Mhc95F RNA is expressed. Interfering with expression of Drosophila myosin VI at various developmental stages frequently results in lethality. During metamorphosis it results in adult flies with malformed legs and wings, indicating that myosin VI is essential for imaginal disc morphogenesis. During oogenesis, abnormal follicle cell shapes and aberrant follicle cell migrations are observed when antisense Mhc95F is expressed in follicle cells during stages 9 to 10, suggesting that the Drosophila myosin VI is required for follicle cell epithelial morphogenesis.
The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites
Authors: Severt W. L. , Biber T. U. L. , Wu X.-Q. , Hecht N. B. , DeLorenzo R. J. , Jakoi E. R. 
Published: April 2021

ABSTRACT
Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing α-calmodulin dependent kinase II (α-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of α-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.
VAP-33 localizes to both an intracellular vesicle population and with occludin at the tight junction
Authors: Lapierre Lynne A. , Tuma Pamela L. , Navarre Jennifer , Goldenring James R. , Anderson James M. 
Published: April 2021

ABSTRACT
Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin’s role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin’s localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.