bluuebunny/crossref_metadata_2025_split huggingface dataset:
Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV
Authors: Yannoni Yvonne M. , White Kalpana 
Published: April 2021

ABSTRACT
The neuron specific DrosophilaELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333-374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.
Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells
Authors: Sai Yoshimichi , Nies Anne T. , Arias Irwin M. 
Published: April 2021

ABSTRACT
The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line.
Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin.
WIF-B9 cells were transiently and stably transfected with a mdr1-GFPconstruct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basalateral plasma membrane. At 15°C, mdr1-GFP accumulated in Golgi; after a shift to 37°C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles.
In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocyes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFPprovide an important model in which to study trafficking and regulation of canalicular transporters.
Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias/Sai_F9.htm
TGF-β type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells
Authors: Piek Ester , Moustakas Aristidis , Kurisaki Akira , Heldin Carl-Henrik , Dijke Peter ten 
Published: April 2021

ABSTRACT
The capacities of different transforming growth factor-β (TGF-β) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-β1, but not activin A or osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7), was able to induce morphological transformation of NMuMG cells as shown by reorganisation of the actin cytoskeleton and relocalisation/downregulation of E-cadherin and β-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-β1 bound to TGF-β type I receptor (TβR-I)/ALK-5 and TβR-II, but not to activin type I receptor (ActR-I)/ALK-2. Activin A bound to ActR-IB/ALK-4 and ActR-II, and BMP-7 bound to ActR-I/ALK-2, BMP type I receptor (BMPR-I)/ALK-3, ActR-II and BMPR-II. TGF-β1 and BMP-7 activated the Smad-binding element (SBE)4promoter with equal potency, whereas activin A had no effect. Transfection of constitutively active (CA)-ALK-4 activated the 3TP promoter to the same extent as TGF-β1 and CA-ALK-5 indicating that activin signalling downstream of type I receptors was functional in NMuMG cells. In agreement with this, activin A induced low levels of plasminogen activator inhibitor I expression compared to the high induction by TGF-β1. In contrast to activin A and BMP-7, TGF-β1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-β1 induced the nuclear accumulation of Smad2 and/or Smad3. In addition, NMuMG cells transiently infected with adenoviral vectors expressing high level CA-ALK-5 exhibited full transdifferentiation. On the other hand, infections with low level CA-ALK-5, which alone did not result in transdifferentiation, together with Smad2 and Smad4, or with Smad3 and Smad4 led to transdifferentiation. In conclusion, TGF-β1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-β type I receptor (ALK-5) and its effector Smad proteins mediate the epithelial to mesenchymal transition. Activin A does not induce mesenchymal transformation, presumably because the number of activin receptors is limited, while BMP-7-initiated signalling cannot mediate transdifferentiation.
Colocalization of intranuclear lamin foci with RNA splicing factors
Authors: Jagatheesan G. , Thanumalayan S. , Muralikrishna Bh. , Rangaraj Nandini , Karande Anjali A. , Parnaik Veena K. 
Published: April 2021

ABSTRACT
The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.
Characterisation of the tumour necrosis factor (TNF)-α response elements in the human ICAM-2 promoter
Authors: McLaughlin Fiona , Ludbrook Valerie J. , Kola Ismail , Campbell Callum J. , Randi Anna M. 
Published: April 2021

ABSTRACT
ICAM-2 is a cell surface adhesion molecule constitutively expressed on the endothelium, involved in leukocyte recruitment into tissues. We recently showed that pro-inflammatory cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β down-regulate ICAM-2 expression at the transcriptional level. Here we investigate the elements in the ICAM-2 promoter required for the TNF-α-mediated down-regulation. Site directed mutagenesis of the ICAM-2 promoter implicated three consensus sites for Ets transcription factors in basal activity; two of these sites were also involved in the TNF-α-induced down-regulation. Electrophoretic mobility shift assays (EMSA) performed in human umbilical vein endothelial cells (HUVEC) showed that all three Ets binding sites (EBS) bind nuclear proteins.
TNF-α treatment (10 ng/ml for 24 hours) decreased binding to the double −135/−127EBS, but not to the −44EBS. The Ets family member Erg was found to be constitutively expressed in HUVEC, and TNF-α down-regulated Erg protein levels. Furthermore, an Erg cDNA transactivated the ICAM-2 promoter when transiently transfected into both HeLa cells and HUVEC. Protein expression of ICAM-2 and Erg was found to be similarly regulated by TNF-α in an ex vivo artery model. These data suggest that constitutive endothelial genes ICAM-2 and Erg are on the same pathway of cytokine-dependent regulation of gene expression.
Isolation and characterization of monoclonal antibodies directed against novel components of macrophage phagosomes
Authors: Morrissette Naomi S. , Gold Elizabeth S. , Guo Jian , Hamerman Jessica A. , Ozinsky Adrian , Bedian Vahe , Aderem Alan A. 
Published: April 2021

ABSTRACT
In order to identify novel proteins associated with various stages of macrophage phagocytosis, we have generated monoclonal antibodies that recognize phagosomes. Purified Fc receptor-mediated phagosomes, isolated by feeding IgG-conjugated magnetic beads to LPS-primed murine peritoneal macrophages, were used as the immunogen. An immunofluorescence screen was used to isolate and single-cell clone ∼150 monoclonal antibodies that recognize mouse macrophage phagosomes as well as labeling other cellular components in patterns which are frequently distinct from those observed with previously characterized phagosome-associated proteins. Predominant morphological categories (in addition to phagosome labeling) include staining of one or more of the following: cytoskeletal patterns, vesicular patterns and plasma membrane localization. In this paper, we describe the antibody screen, preliminary characterization of the antibodies and our identification of the antigens for three representative monoclonal antibodies.
These antibodies identify a plasma membrane associated receptor (Mac-1, a subunit of the complement receptor), an actin binding protein (coronin-2) and a vesicular protein (amphiphysin II). Some of the antibodies recognize many cell types, whereas other antibodies are apparently macrophage specific as assessed by flow cytometry and histology. Remarkably, several of the antibodies cross-react with the phagocytic slime mold, Dictyostelium discoideum, recognizing phagosomes and other cellular elements as assessed by immunofluorescence and immunoblots. These results indicate that macrophage phagocytosis has both conserved ancestral features and unique specialized aspects associated with the role of these phagocytes in immunity.
Abnormal synapse formation in agrin-depleted hippocampal neurons
Authors: Ferreira Adriana 
Published: April 2021

ABSTRACT
Agrin, a 200 kDa extracellular matrix protein, participates in the maturation of the postsynaptic target at the neuromuscular junction. Although agrin has also been detected in central neurons, little is known about its role in the formation of their synapses. In the present study, the pattern of expression, localization and function of agrin in developing hippocampal neurons were analyzed. The results indicate that an increase in agrin protein levels precedes synaptogenesis in cultured hippocampal neurons. This increase in agrin expression is accompanied by its extracellular deposition along the distal third of the axon. To investigate whether agrin plays a role during synapse formation, its expression in cultured hippocampal neurons was suppressed by means of antisense oligonucleotide treatment. The suppression of agrin expression results in the impairment of dendritic development and the formation of fewer synapses than in non-treated or sense-treated neurons. Moreover, this decreased synaptic density is accompanied by a selective inhibition of the clustering of GABA receptors. These results lead to the conclusion that agrin may be an important regulator of the maturation of dendrites and synaptogenesis in central neurons.
A potential role for the plasmin(ogen) system in the posttranslational cleavage of the neural cell adhesion molecule L1
Authors: Nayeem Naushaba , Silletti Steve , Yang Xiu-Ming , Lemmon Vance P. , Reisfeld Ralph A. , Stallcup William B. , Montgomery Anthony M. P. 
Published: April 2021

ABSTRACT
L1 is a neural recognition molecule that promotes neural developmental and regenerative processes. Posttranslational cleavage of L1 is believed to be important for regulating its function in vivo, but little is known of the proteolytic systems responsible. In this study we present evidence that plasmin can regulate both L1 expression and function. The addition of plasmin to cell lines results in a dose-dependent loss of surface L1 expression, with the simultaneous appearance of soluble L1 species. The addition of plasminogen to primary neurons and melanoma cells also resulted in the generation of plasmin and the concomitant release of L1. One product of plasmin-mediated cleavage is an amino-terminal fragment of approximately 140 kDa that has been previously described as a natural posttranslational cleavage product in vivo. This fragment was confirmed to result from cleavage at two sites in the middle of the third fibronectin-like domain of L1. Cleavage at a further site, proximal to the transmembrane domain of L1, was also observed at higher plasmin concentrations. Plasmin was further confirmed to abrogate homophilic L1 interactions required for cellular aggregation. Based on these findings we propose that plasmin is likely to be an important regulator of L1-mediated processes including those documented in the nervous system.
Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells
Authors: Gasman Stépha , Chasserot-Golaz Sylvette , Popoff Michel R. , Aunis Dominique , Bader Marie-France 
Published: April 2021

ABSTRACT
The Rho GTPase family, including Rho, Rac and Cdc42 proteins, is implicated in various cell functions requiring the reorganization of actin-based structures. In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis. We previously described that, in chromaffin cells, the trimeric granule-bound Go protein controls peripheral actin and prevents exocytosis in resting cells through the regulation of RhoA. To provide further insight into the function of Rho proteins in exocytosis, we focus here on their intracellular distribution in chromaffin cells. By confocal immunofluorescence analysis, we found that Rac1 and Cdc42 are exclusively localized in the subplasmalemmal region in both resting and nicotine-stimulated cells. In contrast, RhoA is associated with the membrane of secretory granules. We then investigated the effects of clostridial toxins, which differentially impair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion. Clostridium difficiletoxin B, which inactivates Rho, Rac and Cdc42, markedly altered the distribution of peripheral actin filaments. Neither Clostridium botulinumC3 toxin, which selectively ADP-ribosylates Rho, nor Clostridium sordelliilethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in the organization of subplasmalemmal actin. Indeed, toxin B strongly reduced secretagogue-evoked catecholamine release. This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochalasin E or Clostridium botulinumC2 toxin, suggesting that the inhibition of secretion by toxin B is entirely linked to the disorganization of actin. C. sordelliilethal toxin also inhibited catecholamine secretion, but this effect was not related to the actin cytoskeleton as seen in cells pretreated with cytochalasin E or C2 toxin. In contrast, C3 exoenzyme did not affect secretion. We propose that Cdc42 plays an active role in exocytosis by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.
Biochemical analysis of distinct Rab5- and Rab11-positive endosomes along the transferrin pathway
Authors: Trischler Martin , Stoorvogel Willem , Ullrich Oliver 
Published: April 2021

ABSTRACT
Rab GTPases are associated with distinct cellular compartments and function as specific regulators of intracellular transport. In the endocytic pathway, it is well documented that Rab5 regulates transport from plasma membrane to early (sorting) endosomes. In contrast, little is known about the precise localization and function of Rab4 and Rab11, which are believed to control endocytic recycling. In the present study we have analysed the protein composition of Rab5- and Rab11-carrying endosomes to gain further insight into the compartmental organization of the endocytic and recycling pathway. Endosome populations of this transport route were purified by immunoadsorption from endosome-enriched subcellular fractions using antibodies directed against the cytoplasmic tail of the transferrin receptor, Rab5 or Rab11. Endocytosed transferrin moved sequentially through compartments that could be immunoadsorbed with anti-Rab5 and anti-Rab11, consistent with the theory that Rab5 and Rab11 localise to sorting and recycling endosomes, respectively. These compartments exhibited morphological differences, as determined by electron microscopy. Although their overall protein compositions were very similar, some proteins were found to be selectively enriched. While Rab4 was present on all endosome populations, Rab5 and Rab11 were strikingly segregated. Furthermore, the Rab11-positive endosomes were rich in annexin II, actin and the t-SNARE syntaxin 13, compared to Rab5-containing endosomes. In an in vitro assay, the Rab5 effector protein EEA1 was preferentially recruited by Rab5-positive endosomes. Taken together, our data suggest an organization of the transferrin pathway into distinct Rab5- and Rab11-positive compartments.
Induction of DNA replication by peroxisome proliferators is independent of both tumour necrosis factor α priming and EGF-receptor tyrosine kinase activity
Authors: Chevalier Stephan , Macdonald Neil , Roberts Ruth A. 
Published: April 2021

ABSTRACT
Peroxisome proliferators (PPs) cause hepatocyte proliferation and tumorigenesis in rodent liver. PPs induce hepatocyte DNA synthesis although the mechanism is unclear. Tumour necrosis factor α (TNFα) and epidermal growth factor (EGF) have been implicated in mediating this growth response since these factors induce a threefold and 17.2-fold increase, respectively, in DNA synthesis in rat primary hepatocyte cultures. Previously, others have suggested that TNFα acts as a primer to sensitise hepatocytes to the proliferative effects of growth factors. Indeed, here we show that costimulation with TNFα and a suboptimal (4-20% of optimal) concentration of EGF permits an 11.7-fold increase in DNA synthesis in rat primary hepatocyte cultures. The PP nafenopin induced a 2.3-fold increase in DNA synthesis but there was no further increase upon co-administration of either TNFα or a suboptimal concentration of EGF. Furthermore, there was no gross dysregulation of the CDK and cyclin protein expression profile upon stimulation with nafenopin. Using a specific epidermal growth factor receptor tyrosine kinase inhibitor (4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-[1-pyrolidino])-propoxyquinazoline, EGFR-TKI), we show that signalling through EGF-R is not required for nafenopin-induced DNA synthesis. The EGFR-TKI also prevented progression into S phase upon stimulation with TNFα, but DNA synthesis was not reduced to control levels, indicating that TNFα has a mitogenic activity in the absence of EGF signalling. Therefore, although TNFα can act as a priming factor for growth factors such as EGF, nafenopin does not appear to act via this mechanism.
Participation of small GTPases in dorsal closure of the Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial morphogenesis
Authors: Harden Nicholas , Ricos Michael , Ong Yong Mei , Chia William , Lim Louis 
Published: April 2021

ABSTRACT
The Rho subfamily of Ras-related small GTPases participates in a variety of cellular events including organization of the actin cytoskeleton and signalling by c-Jun N-terminal kinase and p38 kinase cascades. These functions of the Rho subfamily are likely to be required in many developmental events. We have been studying the participation of the Rho subfamily in dorsal closure of the Drosophila embryo, a process involving morphogenesis of the epidermis. We have previously shown that Drac1, a Rho subfamily protein, is required for the presence of an actomyosin contractile apparatus believed to be driving the cell shape changes essential to dorsal closure. Expression of a dominant negative Drac1 transgene causes a loss of this contractile apparatus from the leading edge of the advancing epidermis and dorsal closure fails. We now show that two other Rho subfamily proteins, Dcdc42 and RhoA, as well as Ras1 are also required for dorsal closure. Dcdc42 appears to have conflicting roles during dorsal closure: establishment and/or maintenance of the leading edge cytoskeleton versus its down regulation. Down regulation of the leading edge cytoskeleton may be controlled by the serine/threonine kinase DPAK, a potential Drac1/Dcdc42 effector. RhoA is required for the integrity of the leading edge cytoskeleton specifically in cells flanking the segment borders. We have begun to characterize the interactions of the various small GTPases in regulating dorsal closure and find no evidence for the hierarchy of Rho subfamily activity described in some mammalian cell types. Rather, our results suggest that while all Rho subfamily p21s tested are required for dorsal closure, they act largely in parallel.