bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate
Authors: Niessen Heiner , Harz Hartmann , Bedner Peter , Krämer Karsten , Willecke Klaus 
Published: April 2021

ABSTRACT
Intercellular propagation of signals through connexin32- containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin- transfected HeLa cells was standardized by measuring the spreading of microinjected Mn2+ that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca2+ wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32- containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.
Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells
Authors: Krasnov Peter A. , Enikolopov Grigori 
Published: April 2021

ABSTRACT
We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.
Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed
Authors: Geese Marcus , Schlüter Kathrin , Rothkegel Martin , Jockusch Brigitte M. , Wehland Jürgen , Sechi Antonio S. 
Published: April 2021

ABSTRACT
The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.
Functional overlap of the Dictyostelium RasG, RasD and RasB proteins
Authors: Khosla Meenal , Spiegelman George B. , Insall Rob , Weeks Gerald 
Published: April 2021

ABSTRACT
Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG− cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects.
Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG−) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG− cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG− cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD.
Introduction of the rasB gene under the control of the rasG promoter into rasG− cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG− cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG− cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG− cells also reduced the level of RasD protein.
In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG− cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.
Regulation of cytokinesis by the Elm1 protein kinase in Saccharomyces cerevisiae
Authors: Bouquin Nicolas , Barral Yves , Courbeyrette Régis , Blondel Marc , Snyder Mike , Mann Carl 
Published: April 2021

ABSTRACT
A Saccharomyces cerevisiae mutant unable to grow in a cdc28-1N background was isolated and shown to be affected in the ELM1 gene. Elm1 is a protein kinase, thought to be a negative regulator of pseudo-hyphal growth. We show that Cdc11, one of the septins, is delocalised in the mutant, indicating that septin localisation is partly controlled by Elm1. Moreover, we show that cytokinesis is delayed in an elm1Δ mutant. Elm1 levels peak at the end of the cell cycle and Elm1 is localised at the bud neck in a septin-dependent fashion from bud emergence until the completion of anaphase, at about the time of cell division. Genetic and biochemical evidence suggest that Elm1 and the three other septin-localised protein kinases, Hsl1, Gin4 and Kcc4, work in parallel pathways to regulate septin behaviour and cytokinesis. In addition, the elm1Δ morphological defects can be suppressed by deletion of the SWE1 gene, but not the cytokinesis defect nor the septin mislocalisation. Our results indicate that cytokinesis in budding yeast is regulated by Elm1.
Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization
Authors: Sun Jing , Paddock Cathy , Shubert Julie , Zhang Hong-Bing , Amin Kunjlata , Newman Peter J. , Albelda Steven M. 
Published: April 2021

ABSTRACT
PECAM-1/CD31, a vascular cell adhesion/signaling molecule that has been implicated in a number of vascular functions (including angiogenesis and the transmigration of leukocytes through endothelium) is highly enriched at the cell-cell borders of adjacent endothelial cells. To identify the mechanisms responsible for this localization, a series of PECAM-1 mutants and chimeric PECAM-1 molecules were transfected into non-PECAM-expressing cells and the ability of the constructs to move to cell-cell borders of adjacent cells was determined using immunohistochemistry and confocal microscopy. Although neither the extracellular domain, by itself, nor the cytoplasmic domain, by itself, was sufficient to direct cell-cell localization, the combination of the extracellular and transmembrane domains with a small group of highly charged amino acids in a membrane proximal region of the cytoplasmic domain was sufficient to direct efficient localization of the molecule to cell-cell borders. Importantly, only constructs that supported PECAM-1 mediated adhesion localized to cell-cell borders. Our data are consistent with a ‘diffusion trapping’ model in which movement of PECAM-1 in the cell membrane occurs relatively freely until the ‘stablized’ extracellular domain of the molecule encounters its ligand on an adjacent cell. When this occurs, the complex is ‘captured’ at the cell-cell interface leading to localization at cell-cell borders.
Nup2p is located on the nuclear side of the nuclear pore complex and coordinates Srp1p/importin-α export
Authors: Hood Jennifer K. , Casolari Jason M. , Silver Pamela A. 
Published: April 2021

ABSTRACT
Proteins bearing canonical nuclear localization sequences are imported into the nucleus by the importin/karyopherin-α/β heterodimer. Recycling of the importin-α subunit to the cytoplasm requires the action of Cas, a member of the importin-β superfamily. In the yeast Saccharomyces ceresivisiae, the essential gene CSE1 encodes a Cas homologue that exports the yeast importin-α protein Srp1p/Kap60p from the nucleus. In this report, we describe a role for the FXFG nucleoporin Nup2p, and possibly the related Nup1p, in the Cse1p-mediated nuclear export pathway. Yeast cells lacking Nup2p or containing a particular temperature-sensitive mutation in NUP1 accumulate Srp1p in the nucleus. Similarly, Cse1p is displaced from the nuclear rim to the nuclear interior in Δnup2 cells. We do not observe any biochemical interaction between Cse1p and Nup2p. Instead, we find that Nup2p binds directly to Srp1p. We have localized Nup2p to the interior face of the nuclear pore complex, and have shown that its N terminus is sufficient for targeting Nup2p to the pore, as well as for binding to Srp1p. Taken together, these data suggest that Nup2p is an important NPC docking site in the Srp1p export pathway.
Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies
Authors: Liu Juinn-Lin , Hebert Michael D. , Ye Ying , Templeton Dennis J. , Kung Hsing-Jien , Matera A. Gregory 
Published: April 2021

ABSTRACT
We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as ‘docking stations’ for CDK2-cyclin E activation by CAKs during the G1/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 μM) or RNA polymerase I (actinomycin D, 0.05 μg/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex-mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well-documented association of CBs, histone gene clusters and RNA 3′ end processing factors are discussed.
Glycocalyx modulation is a physiological means of regulating cell adhesion
Authors: Sabri Siham , Soler Mireille , Foa Colette , Pierres Anne , Benoliel Anne-Marie , Bongrand Pierre 
Published: April 2021

ABSTRACT
Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity.
Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble gamma interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells.
The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells.
Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion.
Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins.
Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins
Authors: Cozzolino Mauro , Giovannone Barbara , Serafino Annalucia , Knudsen Karen , Levi Andrea , Alemà Stefano , Salvatore AnnaMaria 
Published: April 2021

ABSTRACT
Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120ctn and of β-catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases.
Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain
Authors: Bouchard Philippe , Chomilier Jacques , Ravet Viviane , Mornon Jean-Paul , Viguès Bernard 
Published: April 2021

ABSTRACT
Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.
Targeting of cytoskeletal proteins to the flagellum of Trypanosoma brucei
Authors: Ersfeld Klaus , Gull Keith 
Published: April 2021

ABSTRACT
The eukaryotic flagellum represents one of the most complex macromolecular structures found in any organism and contains more than 250 proteins. Due to the relative ease of genetic manipulation the flagellum of Trypanosoma brucei has emerged as an accessible model system to study the morphogenesis and dynamics of this organelle. We have recently started to characterise the mechanisms by which components of the cytoskeletal fraction of the flagellum, such as the axoneme, the paraflagellar rod and the flagellar attachment zone, are targeted by proteins synthesised in the cytoplasm and assembled. Here, we present the identification of a novel actin-related protein as a component of the axoneme. We show that this protein shares the tripeptid motif histidine-leucine-alanine (HLA) with one of the major proteins of the paraflagellar rod, PFRA. Building on previous work from this lab which showed that a deletion comprising this motif abolished targeting of PFRA to the flagellum we demonstrate in this study that the deletion of the tripeptid motif is sufficient to achieve mistargeting both of the PFRA and the actin- related protein. We propose that this motif represents an essential part of a flagellar targeting machinery in trypanosomes and possibly in other flagellated organisms.
Glycosaminoglycan synthesis and secretion by the retinal pigment epithelium: polarized delivery of hyaluronan from the apical surface
Authors: Senanayake Preenie deS , Calabro Anthony , Nishiyama Kazutoshi , Hu Jane G. , Bok Dean , Hollyfield Joe G. 
Published: April 2021

ABSTRACT
Hyaluronan and chondroitin sulfate glycosaminoglycan secretion from retinal pigment epithelial cells was established in confluent cultures with high transepithelial resistance. Cell cultures were maintained on Millicell-PCF culture plates, which allow separation of culture medium exposed to apical and basal epithelial surfaces. Following various times in culture, apical and basal culture media were sampled at three day intervals and the glycosaminoglycan content was quantified. Samples were digested with proteinase K to free the glycosaminoglycans from their core proteins, the glycosaminoglycans were ethanol precipitated, and subjected to hyaluronidase SD and chondroitinase ABC digestion to release hyaluronan and chondroitin sulfate disaccharides. Disaccharides were fluorotagged with 2-aminoacridone, separated on polyacrylamide gels and the molar fluorescence in each disaccharide band quantitated. Hyaluronan in the apical medium was significantly higher than in the basal medium (5-12 times) at all recovery intervals (P&lt;0.0001). In contrast, the distribution of unsulfated chondroitin, 4-sulfated chondroitin and 6-sulfated chondroitin disaccharides in apical and basal media was non-polar. Confocal microscopy of cultures probed with a hyaluronan- specific fluorotag established that the HA evident in these cultures is restricted to the apical border of the RPE cultures. Collectively, these data indicate that hyaluronan synthesized by the retinal pigment epithelium is secreted preferentially from the apical surface, suggesting that this tissue is an important source of hyaluronan present in the interphotoreceptor matrix.
Immunolocalization of cytoplasmic dynein and dynactin subunits in cultured macrophages: enrichment on early endocytic organelles
Authors: Habermann Anja , Schroer Trina A. , Griffiths Gareth , Burkhardt Janis K. 
Published: April 2021

ABSTRACT
Cytoplasmic dyneins and their cofactor, dynactin, work together to mediate the movement of numerous cargo organelles toward the minus-ends of microtubules. In many cases, there is compelling evidence that dynactin functions in part to attach dyneins to cargo organelles, but this may not always be the case. We have localized three dynactin subunits (Arp1, p62 and p150Glued) and two subunits of conventional cytoplasmic dynein (dynein intermediate chain and dynein heavy chain 1) in murine macrophages using immunogold labeling of thawed cryosections. Using stereological techniques, we have quantified the relative distributions of each of these subunits on specific membrane organelles to generate a comprehensive analysis of the distribution of these proteins in a single cell type. Our results show that each of the subunits tested exhibits the same distribution with respect to different membrane organelles, with highest levels present on early endosomes, and lower levels present on later endocytic organelles, the mitochondrial outer membrane, the plasma membrane and vesicles in the Golgi region. An additional pool of punctate dynactin labeling was detected in the cell periphery, in the absence of dynein labeling. Even when examined closely, membrane organelles could not be detected in association with these dynactin-positive sites; however, double labeling with anti-tubulin antibody revealed that at least some of these sites represent the ends of microtubules. The similarities among the labeling profiles with respect to membrane organelles suggest that dynein and dynactin bind to membrane organelles as an obligate unit. In contrast, our results show that dynactin can associate with microtubule ends in the absence of dynein, perhaps providing sites for subsequent organelle and dynein association to form a functional motility complex.