bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a novel C-terminal variant of βII spectrin: two isoforms of βII spectrin have distinct intracellular locations and activities
Authors: Hayes Nandini V. L. , Scott Catherine , Heerkens Egidius , Ohanian Vasken , Maggs Alison M. , Pinder Jennifer C. , Kordeli Ekaterini , Baines Anthony J. 
Published: April 2021

ABSTRACT
It is established that variations in the structure and activities of βI spectrin are mediated by differential mRNA splicing. The two βI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of βII spectrins? Thus far, only a long form of βII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of βII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7.0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of βII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of βII spectrin isoforms correlates with their differential intracellular targeting.
Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells
Authors: Stringa Emanuela , Knäuper Vera , Murphy Gillian , Gavrilovic Jelena 
Published: April 2021

ABSTRACT
Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35°C to mimic physiological conditions. PDGF-BB pre- treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by α2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on αvβ3 integrin. αvβ3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-β tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix.
Expression from the human occludin promoter is affected by tumor necrosis factor α and interferon γ
Authors: Mankertz J. , Tavalali S. , Schmitz H. , Mankertz A. , Riecken E. O. , Fromm M. , Schulzke J. D. 
Published: April 2021

ABSTRACT
The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation.
Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor α and another cytokine involved in inflammation, interferon γ, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor α and interferon γ diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier.
In conclusion, proinflammatory cytokines such as tumor necrosis factor α and interferon γ can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.
Bi-directional trafficking between the trans-Golgi network and the endosomal/lysosomal system
Authors: Rohn Wolfgang M. , Rouillé Yves , Waguri Satoshi , Hoflack Bernard 
Published: April 2021

ABSTRACT
Protein transport in the secretory and endocytic pathways of eukaryotic cells is mediated by vesicular transport intermediates. Their formation is a tightly controlled multistep process in which coat components are recruited onto specific membranes, and cargo, as well as targeting molecules, become segregated into nascent vesicles. At the trans-Golgi network, two transport systems deliver cargo molecules to the endosomal system. They can be distinguished with regard to coat components that select cargo molecules. AP-1 assembly proteins mediate transport of MPRs and furin, whereas AP-3 adaptors mediate transport of lysosomal membrane glycoproteins to the endosomal/lysosomal system. The molecular basis for protein-specific sorting lies within sorting signals that are present in the cytoplasmic tails of cargo proteins and allow specific interactions with individual coat components. In order to maintain cellular homeostasis, some proteins are retrieved from endosomal compartments and transported back to the trans-Golgi network. Distinct points for protein retrieval exist within the endosomal system, retrieval occurring from either early or late endosomes. Whereas significant progress has been made in recent years in identifying anterograde and retrograde transport pathways, the molecular mechanisms underlying protein sorting and retrieval are only poorly defined. Recently, however, novel vesicle coats (e.g. AP-4) and proteins that might be involved in sorting (e.g. PACS-1 and TIP47) have been described, and the interactions between assembly proteins and sorting signals are becoming increasingly well defined.
Subcellular localization of Bic-D::GFP is linked to an asymmetric oocyte nucleus
Authors: Paré Chantal , Suter Beat 
Published: April 2021

ABSTRACT
Bicaudal-D (Bic-D) is essential for the establishment of oocyte fate and subsequently for polarity formation within the developing Drosophila oocyte. To find out where in the germ cells Bic-D performs its various functions we made transgenic flies expressing a chimeric Bic-D::GFP fusion protein. Once Bic-D::GFP preferentially accumulates in the oocyte, it shows an initial anterior localization in germarial region 2. In the subsequent egg chamber stages 1-6 Bic-D::GFP preferentially accumulates between the oocyte nucleus and the posterior cortex in a focus that is consistently aligned with a crater-like indentation in the oocyte nucleus. After stage 6 Bic-D::GFP fluorescent signal is predominantly found between the oocyte nucleus and the dorso-anterior cortex. During the different phases several genes have been found to be required for the establishment of the new Bic-D::GFP distribution patterns. Dynein heavy chain (Dhc), spindle (spn) genes and maelstrom (mael) are required for the re-localization of the Bic-D::GFP focus from its anterior to its posterior oocyte position. Genes predicted to encode proteins that interact with RNA (egalitarian and orb) are required for the normal subcellular distribution of Bic-D::GFP in the germarium, and another potential RNA binding protein, spn-E, is required for proper transport of Bic-D::GFP from the nurse cells to the oocyte in later oogenesis stages. The results indicate that Bic-D requires the activity of mRNA binding proteins and a negative-end directed microtubule motor to localize to the appropriate cellular domains. Asymmetric subcellular accumulation of Bic-D and the polarization of the oocyte nucleus may reflect the function of this localization machinery in vectorial mRNA localization and in tethering of the oocyte nucleus. The subcellular polarity defined by the Bic-D focus and the nuclear polarity marks some of the first steps in antero-posterior and subsequently in dorso-ventral polarity formation.
Movies available on-line: http://www.biologists.com/JCS/movies/jcs1338.html
Integrin α2β1-dependent EGF receptor activation at cell-cell contact sites
Authors: Yu Xiaochun , Miyamoto Shingo , Mekada Eisuke 
Published: April 2021

ABSTRACT
Certain integrins including α2β1 and α3β1 localize to intercellular binding sites, and thus may participate in cell-cell interactions. We demonstrated here the physical and functional associations of integrin α2β1 with epidermal growth factor receptor (EGFR) at intercellular adhesion sites. Immunoprecipitation with anti-integrin α2 antibodies or anti-integrin β1 antibody resulted in preferential coprecipitation of EGFR from A431 cell lysates, while anti-EGFR antibody coprecipitated integrin α2β1. Chemical crosslinking confirmed the association of integrin α2β1 and EGFR. Colocalization of integrin α2β1 and EGFR at cell-cell contact sites was observed by double immunofluorescence staining of A431 cells. EGF-induced EGFR stimulation did not affect the association of integrin α2β1 and EGFR. However, immunostaining with the antibody specific to activated-EGFR revealed that EGFR localized at cell-cell contact sites are phosphorylated even in serum-depleted conditions, while EGFR localized to other sites is totally dephosphorylated in the same conditions. The EGFR phosphorylation in cell-cell contact sites observed in a serum-depleted culture was abrogated with a function-blocking antibody of integrin α2, but not with a non-function-blocking α2 antibody or function-blocking α3 antibody. Moreover, the EGFR phosphorylation in serum-depleted conditions was not observed in suspended cells, or largely abrogated in sparse cells, indicating that cell-cell adhesion is required for EGFR phosphorylation. These results indicate that integrin α2β1 not only physically associates with EGFR but also functions in serum-independent EGFR activation at cell-cell contact sites. The present results shed a new light on the role of intercellular integrins in cell-cell interactions.
Arrested maturing multivesicular endosomes observed in a Chinese hamster ovary cell mutant, LEX2, isolated by repeated flow-cytometric cell sorting
Authors: Ohashi Masato , Miwako Ishido , Yamamoto Akitsugu , Nagayama Kuniaki 
Published: April 2021

ABSTRACT
Chinese hamster ovary (CHO) cell mutants defective in the disintegration of endocytosed low-density lipoprotein (LDL) were isolated from mutagenized cells by repeated flow-cytometric cell sorting. After seven rounds of cell sorting, we obtained mutant pools, from which nine mutant clones were established. These mutant strains were all recessive, and were categorized into three complementation groups A, B, and C. The previously established CHO mutant, LEX1 (Lysosome-Endosome X1), fell into the complementation group A. One of the newly isolated mutants, LEX2, fell into the complementation group B, and showed slower degradation of RET-LDL than LEX1 cells. LEX2 showed prominence of well-elaborated multivesicular bodies (MVBs), positive for lysosomal glycoprotein-B/cathepsin D and cation-independent mannose 6-phosphate receptor (CI-MPR), yet negative for transferrin receptor or rab7. Endocytosed intact LDL accumulated in these CI-MPR-positive structures starting at 10-15 minutes of internalization and the accumulation reached completion at 20 minutes. Intermixing of separately internalized fluorescent LDLs between the LEX2 MVBs was slow and saturable at a lower level than observed between late endosomes/lysosomes in wild-type or in LEX1 cells. The receptor recycling pathway to the plasma membrane and the acidification of intracellular compartments were normal in LEX2 cells. These results are consistent with the idea that LEX2 cells are defective in the segregation and sequestration of contents at compartments equivalent to the transport intermediates, previously referred to as endosomal carrier vesicles or maturing MVBs. This MVB stage is likely to be an earlier stage than rab7-positive, lysosome-interacting late endosomes observed in LEX1 cells. Thus, LEX1 and LEX2 mutations could be considered as landmarks for these distinct late endocytic stages, and use of these cells in biochemical and molecular genetic analyses would help to understand the as yet unidentified details of late endocytic pathways including the MVB dynamics.
Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBFα/AML/PEBP2α) dependent activation of tissue-specific gene transcription
Authors: Javed Amjad , Guo Bo , Hiebert Scott , Choi Je-Yong , Green Jack , Zhao Shan-Chuan , Osborne Mark A. , Stifani Stefano , Stein Janet L. , Lian Jane B. , van Wijnen André J. , Stein Gary S. 
Published: April 2021

ABSTRACT
The Runt related transcription factors RUNX (AML/CBFα/PEBP2α) are key regulators of hematopoiesis and osteogenesis. Using co-transfection experiments with four natural promoters, including those of the osteocalcin (OC), multi drug resistance (MDR), Rous Sarcoma Virus long terminal repeat (LTR), and bone sialoprotein (BSP) genes, we show that each of these promoters responds differently to the forced expression of RUNX proteins. However, the three RUNX subtypes (i.e. AML1, AML2, and AML3) regulate each promoter in a similar manner. Although the OC promoter is activated in a C terminus dependent manner, the MDR, LTR and BSP promoters are repressed by three distinct mechanisms, either independent of or involving the AML C terminus, or requiring only the conserved C-terminal pentapeptide VWRPY. Using yeast two hybrid assays we find that the C terminus of AML1 interacts with a Groucho/TLE/R-esp repressor protein. Co-expression assays reveal that TLE proteins repress AML dependent activation of OC gene transcription. Western and northern blot analyses suggest that TLE expression is regulated reciprocally with the levels of OC gene expression during osteoblast differentiation. Digital immunofluorescence microscopy results show that TLE1 and TLE2 are both associated with the nuclear matrix, and that a significant subset of each colocalizes with AML transcription factors. This co-localization of TLE and AML proteins is lost upon removing the C terminus of AML family members. Our findings indicate that suppression of AML-dependent gene activation by TLE proteins involves functional interactions with the C terminus of AML at the nuclear matrix in situ. Our data are consistent with the concept that the C termini of AML proteins support activation or repression of cell-type specific genes depending on the regulatory organization of the target promoter and subnuclear localization.
Hrs interacts with SNAP-25 and regulates Ca2+-dependent exocytosis
Authors: Kwong Jeffrey , Roudabush Francine L. , Moore P. Hutton , Montague Michael , Oldham William , Li Yankun , Chin Lih-Shen , Li Lian 
Published: April 2021

ABSTRACT
Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca2+-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.
Functional analysis of CLIP-115 and its binding to microtubules
Authors: Hoogenraad Casper C. , Akhmanova Anna , Grosveld Frank , Zeeuw Chris I. De , Galjart Niels 
Published: April 2021

ABSTRACT
Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures. We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations. Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions. This head domain is connected to the C-terminal tail through a long coiled-coil structure. The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150Glued. Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs. In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends. However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently. This might be partly due to the divergent C-termini of the two proteins. We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges. Cysteine391 of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules. Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules.
Evidence for a myogenic stem cell that is exhausted in dystrophic muscle
Authors: Heslop L. , Morgan J. E. , Partridge T. A. 
Published: April 2021

ABSTRACT
Injection of the myotoxin notexin, was found to induce regeneration in muscles that had been subjected to 18 Gy of radiation. This finding was unexpected as irradiation doses of this magnitude are known to block regeneration in dystrophic (mdx) mouse muscle. To investigate this phenomenon further we subjected mdx and normal (C57Bl/10) muscle to irradiation and notexin treatment and analysed them in two ways. First by counting the number of newly regenerated myofibres expressing developmental myosin in cryosections of damaged muscles. Second, by isolating single myofibres from treated muscles and counting the number of muscle precursor cells issuing from these over 2 day and 5 day periods. After irradiation neither normal nor dystrophic muscles regenerate to any significant extent. Moreover, single myofibres cultured from such muscles produce very few muscle precursor cells and these undergo little or no proliferation. However, when irradiated normal and mdx muscles were subsequently treated with notexin, regeneration was observed. In addition, some of the single myofibres produced rapidly proliferative muscle precursor cells when cultured. This occurred more frequently, and the myogenic cells proliferated more extensively, with fibres cultured from normal compared with dystrophic muscles. Even after 25 Gy, notexin induced some regeneration but no proliferative myogenic cells remained associated with the muscle fibres. Thus, skeletal muscles contain a number of functionally distinct populations of myogenic cells. Most are radiation sensitive. However, some survive 18 Gy as proliferative myogenic cells that can be evoked by extreme conditions of muscle damage; this population is markedly diminished in muscles of the mdx mouse. A small third population survives 25 Gy and forms muscle but not proliferative myogenic cells.
Meltrin γ (ADAM-9) mediates cellular adhesion through α6β1 integrin, leading to a marked induction of fibroblast cell motility
Authors: Nath Deepa , Slocombe Patrick M. , Webster Ailsa , Stephens Paul E. , Docherty Andrew J. P. , Murphy Gillian 
Published: April 2021

ABSTRACT
The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin γ (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin γ can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin γ-Fc (Melγ-Fc) fusion protein binds to the integrin α6β1 on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since α6β1 is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Melγ-Fc displayed a rounded morphology and a marked increase (eight-to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin γ but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin γ may play a role in regulating the motility of cells by binding to α6β1 integrin and this may be important during a variety of biological and pathological processes.