bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors
Authors: Trinkle-Mulcahy Laura , Ajuh Paul , Prescott Alan , Claverie-Martin Felix , Cohen Stanley , Lamond Angus I. , Cohen Philip 
Published: April 2021

ABSTRACT
Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.
Characterization of a focal adhesion protein, Hic-5, that shares extensive homology with paxillin
Authors: Thomas Sheila M. , Hagel Margit , Turner Christopher E. 
Published: April 2021

ABSTRACT
Paxillin is a focal adhesion scaffolding protein which was originally identified as a substrate of the oncogenic tyrosine kinase, v-src. Paxillin has been proposed to be involved in regulation of focal adhesion dynamics. Two alternatively spliced mouse paxillin cDNAs were cloned and in the process, a paxillin-related protein, Hic-5, was also identified. Cloning and characterization of Hic-5 indicates that this protein shares extensive homology with paxillin. Although Hic-5 was originally characterized as a TGF-β-inducible gene and proposed to be a transcription factor involved in senescence, the studies here demonstrate that Hic-5 is localized to focal adhesion in REF52 cells and can interact with the focal adhesion proteins, Fak, Frnk, and vinculin. In addition, like paxillin, Hic-5 can bind to a negative regulator of Src PTKs, csk but does not bind to the adaptor protein Crk. Like paxillin, localization of this protein to focal adhesions is mediated primarily by the LIM domains; however, sequences outside the LIM domains also play a minor role in focal adhesion targeting. These results suggest that Hic-5 like paxillin could be involved in regulation of focal adhesion dynamics and raise the possibility that Hic-5 and paxillin could have overlapping or opposing functions in the overall regulation of cell growth and differentiation.
Role of collagenase in mediating in vitro alveolar epithelial wound repair
Authors: Planus Emmanuelle , Galiacy Stéphane , Matthay Michael , Laurent Valérie , Gavrilovic Jelena , Murphy Gillian , Clérici Christine , Isabey Daniel , Lafuma Chantal , d’Ortho Marie-Pia 
Published: April 2021

ABSTRACT
Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases.
Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.
Induction of antisense Pax-3 expression leads to the rapid morphological differentiation of neuronal cells and an altered response to the mitogenic growth factor bFGF
Authors: Reeves F. C. , Burdge G. C. , Fredericks W. J. , Rauscher F. J. , Lillycrop K. A. 
Published: April 2021

ABSTRACT
Mutations within the Pax-3 gene lead to a range of developmental abnormalities in both humans and mice. In this report, we have investigated the role that Pax-3 plays in neuronal cell development by specifically downregulating Pax-3 expression within a neuronal cell line. This was achieved by stably transfecting the neuronal cell line ND7 with an expression vector in which antisense Pax-3 RNA was produced under the control of the inducible MMTV promoter. In the stable transfectants, we found that the addition of dexamethasone led to the induction of antisense Pax-3 RNA and a rapid downregulation in endogenous Pax-3 protein expression. The decrease in endogenous Pax-3 protein expression corresponded with a dramatic change in the morphology of the cell: the normally rounded ND7 cells exhibited increased cell to substrate adhesion, extended long neurite processes and expressed genes such as snap-25 that are characteristic of a mature neuron. The morphological differentiation induced by a reduction in Pax-3 expression was followed 24-48 hours later by a cessation in cell proliferation. Interestingly the morphological differentiation and cessation in cell proliferation inducted in the cell lines lacking Pax-3 could be reversed by the addition of the mitogenic growth factor EGF but not by bFGF, whose receptor was downregulated in these cells. These results suggest that the expression of Pax-3 is essential to maintain the undifferentiated phenotype of these immature neuronal cells, and in its absence the cells acquire many of the characteristics of a mature neuronal cell. The slow onset of cell cycle arrest in the cells lacking Pax-3 argues against this transcription factor playing a direct role in the regulation of neuronal cell proliferation.
Syndecan-4 and integrins: combinatorial signaling in cell adhesion
Authors: Couchman John R. , Woods Anne 
Published: April 2021

ABSTRACT
It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding site for protein kinase Cα and phosphatidylinositol 4,5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein is overexpressed; this suggests that oligomerization of syndecan-4 plays a major role in signaling from the extracellular matrix in adhesion.
Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers
Authors: Echtermeyer Frank , Baciu Peter C. , Saoncella Stefania , Ge Yimin , Goetinck Paul F. 
Published: April 2021

ABSTRACT
The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through β1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.
A dynamic connection between centromeres and ND10 proteins
Authors: Everett Roger D. , Earnshaw William C. , Pluta Ann F. , Sternsdorf Thomas , Ainsztein Alexandra M. , Carmena Mar , Ruchaud Sandrine , Hsu Wei-Li , Orr Anne 
Published: April 2021

ABSTRACT
ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G2 phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.
Distribution of thimet oligopeptidase (E.C. 3.4.24.15) in human and rat testes
Authors: Pineau Charles , McCool Suzan , Glucksman Marc J. , Jégou Bernard , Pierotti Adrian R. 
Published: April 2021

ABSTRACT
Thimet oligopeptidase (TOP:E.C. 3.4.24.15) is a thiol sensitive metalloendopeptidase which is widely distributed and active in most tissues including testis, brain and pituitary. In the median eminence it is postulated to play a role in the degradation of GnRH released from the hypothalamus and thus to modulate LH levels. In the rat and human, the testis is the richest source of TOP activity with levels 3-to 5-fold higher than that of the brain. In order to define the exact localisation of this enzyme within the rat and human testis, the distribution of TOP in the developing and adult gonad was examined in situ and in isolated cells by immunohistochemistry, western blotting and northern blotting analysis.
Ontogeny studies have demonstrated that TOP is detectable by western blotting from 9 days with levels of expression increasing with the age of the animal. Immunolocalisation of the protein in the interstitium was positive from 9 days onwards but was negative within the seminiferous tubules before 35 days of age, whereas TOP mRNA was not detected within the testis until 35 days of age with subsequent stable expression levels up to 90 days. In the adult rat testis, a strong TOP immunoreactivity was observed within seminiferous tubules, in elongating and elongated spermatids and residual bodies. In the interstitial compartment, immunoreactivity was also observed in Leydig cells and throughout the interstitial space. Western blot analyses confirmed the distribution of expression observed using immunochemistry, however Leydig cells display a lower signal than expected from the immunohistochemical data. Northern hybridization showed that the transcript is present in pachytene spermatocytes, early spermatids, and residual bodies, whereas its presence was not observed in Leydig cells probably due to very low levels of expression of the message. Analyses of various human tissue extracts showed that the testis displays the highest levels of TOP mRNA, with immunohistochemical experiments revealing that, as in the rat, the protein is principally expressed in elongated spermatids/residual bodies, and in Leydig cells.
It is concluded that in the human and rat testes, TOP is highly expressed, in particular in post-meiotic germ cells and Leydig cells. The possible involvement of TOP in proteolytic events associated with the process of spermiogenesis and Leydig cell function is currently under investigation.
Studies on the inhibition of endosome fusion by GTPγS-bound ARF
Authors: Jones Arwyn T. , Spiro David J. , Kirchhausen Tomas , Melançon Paul , Wessling-Resnick Marianne 
Published: April 2021

ABSTRACT
Using a cell free assay, we have previously shown that ARF is not required for endosome fusion but that inhibition of fusion by GTPγS is dependent on a cytosolic pool of ARFs. Since ARF is proposed to function in intracellular membrane traffic by promoting vesicle biogenesis, and components of clathrin- and COP-coated vesicles have been localized on endosomal structures, we investigated whether ARF-mediated inhibition of early endosome fusion involves the recruitment or irreversible association of these proteins onto endosomal membranes. We now report that depletion of components of clathrin coated vesicles (clathrin, AP-1 and AP-2) or COPI vesicles (β COP) does not affect the capacity of GTPγS-activated ARF to inhibit endosome fusion. Inhibition of fusion by activated ARF is also independent of endosomal acidification since assays performed in the presence of the vacuolar ATPase inhibitor bafilomycin A1 are equally sensitive to GTPγS-bound ARF. Finally, in contrast to reported effects on lysosomes, we demonstrate that ARF-GTPγS does not induce endosomal lysis. These combined data argue that sequestration of known coat proteins to membranes by activated ARF is not involved in the inhibition of early endosome fusion and that its capacity to inhibit fusion involves other specific interactions with the endosome surface. These results contrast with the mechanistic action of ARF on intra-Golgi transport and nuclear envelope assembly.
Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding
Authors: Schlöndorff Johannes , Blobel Carl P. 
Published: April 2021

ABSTRACT
Metalloprotease-disintegrins (ADAMs) have captured our attention as key players in fertilization and in the processing of the ectodomains of proteins such as tumor necrosis factor α (TNFα), and because of their roles in Notch-mediated signaling, neurogenesis and muscle fusion. ADAMs are integral membrane glycoproteins that contain a disintegrin domain, which is related to snake-venom integrin ligands, and a metalloprotease domain (which can contain or lack a catalytic site). Here, we review and critically discuss current topics in the ADAMs field, including the central role of fertilin in fertilization, the role of the TNFα convertase in protein ectodomain processing, the role of Kuzbanian in Notch signaling, and links between ADAMs and processing of the amyloid-precursor protein.
Substitution of flight muscle-specific actin by human β-cytoplasmic actin in the indirect flight muscle of Drosophila
Authors: Brault Véronique , Reedy Mary C. , Sauder Ursula , Kammerer Richard A. , Aebi Ueli , Schoenenberger Cora-Ann 
Published: April 2021

ABSTRACT
The human β-cytoplasmic actin differs by only 15 amino acids from Act88F actin which is the only actin expressed in the indirect flight muscle (IFM) of Drosophila melanogaster. To test the structural and functional significance of this difference, we ectopically expressed β- cytoplasmic actin in the IFM of Drosophila that lack endogenous Act88F. When expression of the heterologous actin was regulated by ∼1.5 kb of the 5′ promoter region of the Act88F gene, little β-cytoplasmic actin accumulated in the IFM of the flightless transformants. Including Act88F- specific 5′ and 3′ untranslated regions (UTRs) yielded transformants that expressed wild-type amounts of β-cytoplasmic actin. Despite the assembly of β-cytoplasmic actin containing thin filaments to which endogenous myosin crossbridges attached, sarcomere organization was deficient, leaving the transformants flightless. Rather than affecting primarily actin-myosin interactions, our findings suggest that the β-cytoplasmic actin isoform is not competent to interact with other actin-binding proteins in the IFM that are involved in the organization of functional myofibrils.
Trimeric G proteins modulate the dynamic interaction of PKAII with the Golgi complex
Authors: Martín María Esther , Hidalgo Josefina , Vega Francisco M. , Velasco Angel 
Published: April 2021

ABSTRACT
The Golgi complex represents a major subcellular location of protein kinase A (PKA) concentration in mammalian cells where it has been previously shown to be involved in vesicle-mediated protein transport processes. We have studied the factors that influence the interaction of PKA typeII subunits with the Golgi complex. In addition to the cytosol, both the catalytic (Cα) and regulatory (RIIα) subunits of PKAII were detected at both sides of the Golgi stack, particularly in elements of the cis- and trans-Golgi networks. PKAII subunits, in contrast, were practically absent from the middle Golgi cisternae. Cell treatment with either brefeldin A, AlF4- or at low temperature induced PKAII dissociation from the Golgi complex and redistribution to the cytosol. This suggested the existence of a cycle of association/dissociation of PKAII holoenzyme to the Golgi. The interaction of purified RIIα with Golgi membranes was studied in vitro and found not to be affected by brefeldin A while it was sensitive to modulators of heterotrimeric G proteins such as AlF4-, GTPγS, βγ subunits and mastoparan. RIIα binding was stimulated by recombinant, myristoylated Gαi3 subunit and inhibited by cAMP. Pretreatment of Golgi membranes with bacterial toxins known to catalyze ADP-ribosylation of selected Gα subunits also modified RIIα binding. Taken together the data support a regulatory role for Golgi-associated Gα proteins in PKAII recruitment from the cytosol.
Motogenic activity of IGD-containing synthetic peptides
Authors: Schor S. L. , Ellis I. , Banyard J. , Schor A. M. 
Published: April 2021

ABSTRACT
Although the IGD amino acid motif (iso-gly-asp) is a highly conserved feature of the fibronectin type I module, no biological activity has as yet been ascribed to it. We have previously reported that the gelatin-binding domain of fibronectin stimulates the migration of human skin fibroblasts into native, but not denatured, type I collagen substrata. Two IGD-containing type I modules are present within the gelatin-binding domain. The object of this study was to ascertain whether soluble synthetic peptides containing the IGD motif stimulate fibroblast migration. We found that IGD peptides stimulated fibroblast migration in the following order of activity: IGDS (as present in the ninth type I module) &gt; IGDQ (as present in the seventh type I module) &gt; IGD. The scrambled SDGI peptide and the well-characterised RGDS peptide were devoid of motogenic activity. The migratory response of fibroblasts to IGD-containing peptides consisted of two distinct phases: an initial period of peptide-mediated cell activation and a subsequent period of enhanced migration manifest in the absence of further IGD peptide. Cell activation was substratum-independent (occurring equally well on both native and denatured type I collagen substrata), whilst the manifestation of enhanced migration was persistent and substratum-dependent (being evident only by cells adherent to a native collagen substratum). Our data further indicated that cell activation (1) is elicited by a signal transduction cascade occurring within minutes of cell exposure to IGD-containing peptides, (2) is dependent upon integrin αvβ3 functionality, (3) involves the tyrosine phosphorylation of focal adhesion kinase (ppFAK125) and (4) is inhibited by signalling mediated through integrin α5β1. The expression of migration stimulating activity by soluble IGD-containing peptides clearly distinguishes them from their RGD counterparts. This is the first identified biological activity of the highly conserved IGD motif and provides a rational platform for the development of a novel family of therapeutic compounds designed to stimulate cell migration in relevant clinical situations, such as impaired wound healing.
Partial rescue of epithelial phenotype in integrin β4 null mice by a keratin-5 promoter driven human integrin β4 transgene
Authors: Neut Ronald van der , Cachaço Ana Sofia , Thorsteinsdóttir Sólveig , Janssen Hans , Prins Duncan , Bulthuis Jurjen , Valk Martin van der , Calafat Jero , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
Integrin β4 null mice exhibit extensive epidermal detachment, reminiscent of the human skin blistering disease junctional epidermolysis bullosa associated with pyloric atresia. Hemidesmosomes, the stable adhesion structures of squamous epithelia, are not formed in the absence of α6β4. Null mutant mice die shortly after birth, but apart from their striking epithelial phenotype, no obvious developmental defects have been observed. To elucidate the cause of death in these mice, we generated transgenic mice with a heterologous construct consisting of the squamous epithelial-specific keratin-5 promoter and a human integrin β4 subunit cDNA. The transgene was not expressed in the presence of endogenous β4, probably as a result of competition for a limited pool of α6 subunits. In a β4 null background, however, the transgene was expressed, and its expression pattern followed that of squamous epithelial-specific keratins. These rescued pups appeared healthy and ultrastructural analysis revealed that the interspecies heterodimer α6mouse/β4human was sufficient to trigger the assembly of hemidesmosomes. After a variable period of up to 48 hours after birth these animals began to exhibit haemorrhages at the plantar and palmar areas. We observed the formation of small blisters and found that the transgene was not detectably expressed in this region, which is devoid of hair follicles. The rescued neonates became increasingly cyanotic and died soon after the onset of this phenomenon. We performed a developmental study of the expression of β4 in the complete respiratory tract, but we found no correlation between the spatiotemporal distribution of β4 and the onset of the respiratory insufficiency. It became clear, however, that there was a gradual detachment of squamous epithelia in the oral and nasal cavities which led to obstruction of the respiratory tract, suggesting that in β4 null and rescued mice, neonatal death was a direct consequence of decreased adhesion properties of hairless squamous epithelia, rather than a developmental defect of the lungs.