bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets
Authors: Roelofsen Han , Soroka Carol J. , Keppler Dietrich , Boyer James L. 
Published: April 2021

ABSTRACT
The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions. This transport function is markedly diminished in mutant rats that lack the transport protein. To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity. The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted. The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein. The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane. Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane. The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference. These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane. The relevance of this phenomenon for the intact liver is discussed.
Osteopontin is a ligand for the α4β1 integrin
Authors: Bayless Kayla J. , Meininger Gerald A. , Scholtz J. Martin , Davis George E. 
Published: April 2021

ABSTRACT
Recent work has shown that osteopontin expression is upregulated at sites of cardiovascular injury. It has been hypothesized that osteopontin provides an adhesive matrix for endothelial and smooth muscle cells during remodeling of the vascular wall following injury. Osteopontin has also been found to be synthesized by monocytes and macrophages within injury sites. Here, we present data showing that osteopontin can promote leukocyte adhesion through the α4β1 integrin. In the presence of physiologic concentrations of Mg2+ and Ca2+, osteopontin purified from bovine milk promoted cell-substrate adhesion of HL-60 and Ramos cells, two model leukocyte cell lines. As with other adhesive ligands, adhesion to osteopontin required leukocyte activation. Under these conditions, no adhesion to control substrates such as bovine serum albumin was observed. Leukocyte adhesion was inhibited by anti-integrin antibodies directed at either the α4 or β1 integrin subunits but not by control antibodies directed to other integrins. Further adhesion experiments revealed that leukocyte binding to osteopontin was completely inhibited by an α4β1-binding peptide containing the leucine-aspartate-valine (LDV) sequence, while a control, non-binding peptide containing leucine-glutamate-valine (LEV) had minimal effects. Affinity chromatography using either surface labeled HL-60 or Ramos cell extracts revealed that the α4β1 integrin specifically bound to osteopontin. Immunoprecipitation of eluted fractions from these columns positively identified the α4β1 integrin. In order to localize potential α4β1-binding sites within osteopontin, the protein was proteolytically cleaved with thrombin. A 30 kDa N-terminal osteopontin fragment purified using fast protein liquid chromatography promoted α4β1 dependent leukocyte adhesion in a manner similar to that of the intact protein. These data collectively demonstrate that the α4β1 integrin is a new adhesion receptor for osteopontin and that an α4β1 binding site exists in the NH2-terminal thrombin fragment of osteopontin.
Myofibrillar interaction of cytosolic creatine kinase (CK) isoenzymes: allocation of N-terminal binding epitope in MM-CK and BB-CK
Authors: Stolz Martin , Wallimann Theo 
Published: April 2021

ABSTRACT
The molecular origin of the isoenzyme-specific interaction of cytosolic creatine kinase isoenzymes, muscle-type creatine kinase and brain-type creatine kinase, with myofibrillar structures has been studied by confocal microscopy in an functional in situ binding assay with chemically skinned, unfixed skeletal muscle fibers using wild-type and chimeric creatine kinase isoproteins. The specific interaction of both wild-type isoforms with the sarcomeric structure resulted in a stable, isoform-characteristic labeling pattern with muscle-type creatine kinase bound exclusively and tightly to the sarcomeric M-band while brain-type creatine kinase was confined to the I-band region. Chimeric proteins of both muscle-type and brain-type creatine kinases were constructed to localize the corresponding binding domain(s). Exchanged domains included the N-terminal part (residues 1-234), the region containing an isoenzyme ‘diagnostic box’ (residues 235-285) and the C-terminal part (residues 286-380). The purified recombinant proteins were all fully intact and enzymatically active. All chimeric proteins containing the N-terminal region (amino acid 1-234) of muscle-type or brain-type creatine kinase were always specifically targeted to the sarcomeric M-band or I-band, respectively. We therefore propose that the relevant epitope(s), determining the isoenzyme-specific targeting in skeletal muscle, are entirely located within the N-terminal regions of both cytosolic creatine kinase isoforms.
Dynamic changes in the subnuclear organisation of pre-mRNA splicing proteins and RBM during human germ cell development
Authors: Elliott David J. , Oghene Kathy , Makarov Genia , Makarova Olga , Hargreave Timothy B. , Chandley Ann C. , Eperon Ian C. , Cooke Howard J. 
Published: April 2021

ABSTRACT
RBM is a germ-cell-specific RNA-binding protein encoded by the Y chromosome in all mammals, implying an important and evolutionarily conserved (but as yet unidentified) function during male germ cell development. In order to address this function, we have developed new antibody reagents to immunolocalise RBM in the different cell types in the human testis. We find that RBM has a different expression profile from its closest homologue hnRNPG. Despite its ubiquitous expression in all transcriptionally active germ cell types, RBM has a complex and dynamic cell biology in human germ cells. The ratio of RBM distributed between punctate nuclear structures and the remainder of the nucleoplasm is dynamically modulated over the course of germ cell development. Moreover, pre-mRNA splicing components are targeted to the same punctate nuclear regions as RBM during the early stages of germ cell development but late in meiosis this spatial association breaks down. After meiosis, pre-mRNA splicing components are differentially targeted to a specific region of the nucleus. While pre-mRNA splicing components undergo profound spatial reorganisations during spermatogenesis, neither heterogeneous ribonucleoproteins nor the transcription factor Sp1 show either developmental spatial reorganisations or any specific co-localisation with RBM. These results suggest dynamic and possibly multiple functions for RBM in germ cell development.
The distribution and translocation of the G protein ADP-Ribosylation Factor 1 in live cells is determined by its GTPase activity
Authors: Vasudevan Chandrasekaran , Han Weiping , Tan Yadi , Nie Yimin , Li Danqing , Shome Kuntala , Watkins Simon C. , Levitan Edwin S. , Romero Guillermo 
Published: April 2021

ABSTRACT
ADP-ribosylation factors (ARF) are small G proteins that play key roles in vesicular transport processes. We have studied the distribution of ARF1 in live cells using chimeras of ARF1 mutants (wild type (wt) ARF1; Q71L-ARF1 (reduced GTPase); T31N (low affinity for GTP); and ΔNwt (deletion of amino acids 2-18)) with green fluorescent protein (GFP). Confocal microscopy studies showed that the wt and Q71L proteins were localized in the Golgi and cytoplasm. The ΔNwt and the T31N mutants were exclusively cytoplasmic. The behavior of the wt and Q71L proteins was studied in detail. About 15% of wt-ARF1-GFP was bound to the Golgi. Bound wt-ARF1-GFP dissociated rapidly after addition of Brefeldin A (BFA). This process did not appear to be a consequence of BFA-induced disappearance of the Golgi. Photobleaching recovery showed that essentially all the ARF-GFP was mobile, although it diffused very slowly. In contrast, about 40-50% of the Q71L mutant was found in the Golgi, and its rate of dissociation in the presence of BFA was slow and biphasic. Q71L-ARF1-GFP diffused more slowly than the wt. We conclude that ARF1 proteins exist in a dynamic equilibrium between Golgi-bound and cytosolic pools, and that the translocation of ARF in live cells requires the hydrolysis of GTP by the Golgi-bound protein.
Nuclear envelope disassembly in mitotic extract requires functional nuclear pores and a nuclear lamina
Authors: Collas Philippe 
Published: April 2021

ABSTRACT
Using sea urchin embryonic and in-vitro-assembled nuclei incubated in sea urchin mitotic extract, I provide evidence for a requirement for functional nuclear pores and a nuclear lamina for nuclear envelope disassembly in vitro. In interphase gastrula nuclei, lamin B interacts with p56, an integral protein of inner nuclear membrane cross-reacting with antibodies to human lamin B receptor. Incubation of gastrula nuclei in mitotic cytosol containing an ATP-generating system rapidly induces hyperphosphorylation of p56 and lamin B. Subsequently, p56-lamin B interactions are weakened and the two proteins segregate into distinct nuclear envelope-derived vesicles upon disassembly of nuclear membranes and of the lamina. Nuclear disassembly is accompanied by chromatin condensation. Blocking nuclear pore function with wheat germ agglutinin or antibodies to nucleoporins prevents p56 and lamin B hyperphosphorylation, nuclear membrane breakdown and lamina solubilization. These events are not rescued by permeabilization of nuclear membranes to molecules of 150,000 Mr with lysolecithin. In-vitro-assembled nuclei containing nuclear membranes with functional pores but no lamina do not disassemble in mitotic cytosol in spite of p56 hyperphosphorylation. Nuclear import of soluble lamin B and reformation of a lamina in interphase extract restores nuclear disassembly in mitotic cytosol. The data indicate a role for functional nuclear pores in nuclear disassembly in vitro. They show that p56 hyperphosphorylation is not sufficient for nuclear membrane disassembly in mitotic cytosol and argue that the nuclear lamina plays a critical role in nuclear disassembly at mitosis.
Inhibition of adhesion and induction of epithelial cell invasion by HAV-containing E-cadherin-specific peptides
Authors: Noë Veerle , Willems Jean , Vandekerckhove Joël , Roy Frans Van , Bruyneel Erik , Mareel Marc 
Published: April 2021

ABSTRACT
The E-cadherin/catenin complex, an organizer of epithelial structure and function, is disturbed in invasive cancer. The HAV (histidine alanine valine) sequence in the first extracellular domain of E-cadherin is crucial for homophilic interactions between cadherins. We report that specific peptides containing an HAV sequence interfere with the functions of the E-cadherin/catenin complex. Cells either expressing specific cadherins or not were challenged with both cadherin and noncadherin peptides comprising a central HAV sequence. Specific E-cadherin peptides inhibited cell aggregation, disturbed the epithelial morphotype and were able to stimulate invasion of cells expressing E-cadherins. Conditioned medium, containing E-cadherin fragments, also stimulated invasion in contrast to conditioned medium from which the E-cadherin fragments were removed. Our studies show that E-cadherin functions are inhibited by homologous proteolytic HAV-containing fragments that are released in an autocrine manner and subsequently inhibit the E-cadherin/catenin complex. In this way such cadherin fragments may induce and support cancer invasion.
Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6
Authors: Schmitz Heinz , Fromm Michael , Bentzel Carl J. , Scholz Peter , Detjen Katharina , Mankertz Joachim , Bode Hagen , Epple Hans-Jörg , Riecken Ernst-Otto , Schulzke Jörg-Dieter 
Published: April 2021

ABSTRACT
Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFα) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNFα (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFα treatment. In freeze-fracture EM, TJ complexity was decreased by TNFα, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFα. A combination of TNFα with interferon-γ acted synergistically on the epithelial barrier. In conclusion, TNFα impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.
Effect of targeted expression of clusterin in photoreceptor cells on retinal development and differentiation
Authors: Jomary Catherine , Chatelain Gilles , Michel Denis , Weston Ann , Neal Michael J. , Jones Stephen E. 
Published: April 2021

ABSTRACT
Clusterin expression is increased in tissues undergoing apoptosis, including neurodegenerative retina, but the causal relationships remain to be clarified. To test the hypothesis that overexpression of clusterin could induce apoptosis in neurons, transgenic mice were generated in which rat clusterin transgene was expressed in photoreceptor cells under the transcriptional control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter. Photoreceptor cell death in the resulting transgenic mice was examined by histology and TUNEL techniques. The expression of the clusterin transgene was confirmed by in situ hybridization in the photoreceptor cells, and results in a complex pattern of clusterin protein distribution in the retina. A reduction in apoptotic staining in the transgenic retinas was observed from birth to postnatal day 15. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor cell death, but may relate to cytoprotective functions.
The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment
Authors: Greenfield Julia J. A. , High Stephen 
Published: April 2021

ABSTRACT
The heteromeric Sec61 complex is composed of α, β and γ subunits and forms the core of the mammalian ER translocon. Oligomers of the Sec61 complex form a transmembrane channel where proteins are translocated across and integrated into the ER membrane. We have studied the subcellular localisation of the Sec61 complex using both wild-type COS1 cells and cells transfected with GFP-tagged Sec61α. By double labelling immunofluorescence microscopy the GFP-tagged Sec61α was found in both the ER and the ER-Golgi intermediate compartment (ERGIC) but not in the trans-Golgi network. Immunofluorescence studies of endogenous Sec61β and Sec61γ showed that these proteins are also located in both the ER and the ERGIC. Using the alternative strategy of subcellular fractionation, we have shown that wild-type Sec61α, β and γ, and GFP-tagged Sec61α, are all present in both the ER and the ERGIC/Golgi fractions of the gradient. The presence of the Sec61 subunits in a post-ER compartment suggests that these proteins can escape the ER and be recycled back, despite the fact that none of them contain any known membrane protein retrieval signals such as cytosolic di-lysine or di-arginine motifs. We also found that another translocon component, the glycoprotein TRAM, was present in post-ER compartments as demonstrated by subcellular fractionation. Our data indicate that the core components of the mammalian ER translocon are not permanently resident in the ER, but rather that they are maintained in the ER by a specific retrieval mechanism.
A spliced variant of AE1 gene encodes a truncated form of Band 3 in heart: the predominant anion exchanger in ventricular myocytes
Authors: Richards Stephen M. , Jaconi Marisa E. , Vassort Guy , Pucéat Michel 
Published: April 2021

ABSTRACT
The anion exchangers (AE) are encoded by a multigenic family that comprises at least three genes, AE1, AE2 and AE3, and numerous splicoforms. Besides regulating intracellular pH (pHi) via the Cl−/HCO3− exchange, the AEs exert various cellular functions including generation of a senescent antigen, anchorage of the cytoskeleton to the membrane and regulation of metabolism. Most cells express several AE isoforms. Despite the key role of this family of proteins, little is known about the function of specific AE isoforms in any tissue, including the heart. We therefore chose isolated cardiac cells, in which a tight control of pHi is mandatory for the excitation-contraction coupling process, to thoroughly investigate the expression of the AE genes at both the mRNA and protein levels. RT-PCR revealed the presence of AE1, AE2 and AE3 mRNAs in both neonatal and adult rat cardiomyocytes. AE1 is expressed both as the erythroid form (Band 3 or eAE1) and a novel alternate transcript (nAE1), which was more specifically characterized using a PCR mapping strategy.
Two variants of AE2 (AE2a and AE2c) were found at the mRNA level. Cardiac as well as brain AE3 mRNAs were expressed in both neonatal and adult rat cardiomyocytes. Several AE protein isoforms were found, including a truncated form of AE1 and two AE3s, but there was no evidence of AE2 protein in adult rat cardiomyocytes. In cardiomyocytes transfected with an AE3 oligodeoxynucleotide antisense, AE3 immunoreactivity was dramatically decreased but the activity of the Cl−/HCO3− exchange was unchanged. In contrast, intracellular microinjection of blocking anti-AE1 antibodies inhibited the AE activity. Altogether, our findings suggest that a specific and novel AE1 splicoform (nAE1) mediates the cardiac Cl−/HCO3− exchange. The multiple gene and protein expression within the same cell type suggest numerous functions for this protein family.
A functional analysis of inscuteable and its roles during Drosophila:asymmetric cell divisions
Authors: Tio Murni , Zavortink Michael , Yang Xiaohang , Chia William 
Published: April 2021

ABSTRACT
Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localisation and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localisation and segregation of the determinant Numb during ganglion mother cell divisions ensure that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organise both neuroblast and ganglion mother cell asymmetric divisions is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localised to the apical cell cortex and is required both for the basal localisation of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. Here we define the functional domains of Inscuteable. We show that aa252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region, aa288-497, is necessary and sufficient for apical cortical localisation and for mitotic spindle (re)orientation along the apical/basal axis. A larger region aa288-540 is necessary and sufficient for asymmetric Numb localisation and segregation; however, correct localisation of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localisation (aa288-540). Our data suggest that apical localisation of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Finally, we show that although inscuteable RNA is normally apically localised, RNA localisation is not required for protein localisation or any aspects of inscuteable function.
Dynamic redistribution of calmodulin in HeLa cells during cell division as revealed by a GFP-calmodulin fusion protein technique
Authors: Li Chao-Jun , Heim Roger , Lu Pin , Pu Yongmei , Tsien Roger Y. , Chang Donald C. 
Published: April 2021

ABSTRACT
It has been suggested by many studies that Ca2+ signaling plays an important role in regulating key steps in cell division. In order to study the down stream components of calcium signaling, we have fused the gene of calmodulin (CaM) with that of green fluorescent protein (GFP) and expressed it in HeLa cells. The GFP-CaM protein was found to have similar biochemical properties as the wildtype CaM, and its distribution was also similar to that of the endogenous CaM. Using this GFP-tagged CaM as a probe, we have conducted a detailed examination of the spatialand temporal-dependent redistribution of calmodulin in living mammalian cells during cell division. Our major findings are: (1) high density of CaM was found to distribute in two sub-cellular locations during mitosis; one fraction was concentrated in the spindle poles, while the other was concentrated in the sub-membrane region around the cell. (2) The sub-membrane fraction of CaM became aggregated at the equatorial region where the cleavage furrow was about to form. The timing of this localized aggregation of CaM was closely associated with the onset of cytokinesis. (3) Using a TA-CaM probe, we found that the sub-membrane fraction of CaM near the cleavage furrow was selectively activated during cell division. (4) When we injected a CaM-specific inhibitory peptide into early anaphase cells, cytokinesis was either blocked or severely delayed. These findings suggest that, in addition to Ca2+ ion, CaM may represent a second signal that can also play an active role in determining the positioning and timing of the cleavage furrow formation.