bluuebunny/crossref_metadata_2025_split huggingface dataset:
p53-independent apoptosis induced by muscle differentiation stimuli in polyomavirus large T-expressing myoblasts
Authors: Gottifredi Vanesa , Peschiaroli Angelo , Fimia Gian Maria , Maione Rossella 
Published: April 2021

ABSTRACT
Abnormal proliferation signals, driven by cellular or viral oncogenes, can result in the induction of apoptosis under sub-optimal cell growth conditions. The tumor suppressor p53 plays a central role in mediating oncogene-induced apoptosis, therefore transformed cells lacking p53 are generally resistant to apoptosis-promoting treatments. In a previous work we have reported that the expression of polyomavirus large T antigen causes apoptosis in differentiating myoblasts and that this phenomenon is dependent on the onset of muscle differentiation in the absence of a correct cell cycle arrest. Here we report that polyomavirus large T increases the levels and activity of p53, but these alterations are not involved in the apoptotic mechanism. Apoptosis in polyomavirus large T-expressing myoblasts is not prevented by the expression of a p53 dominant-negative mutant nor it is increased by p53 over-expression. Moreover, forced differentiation induced through the over-expression of the muscle regulatory factor MyoD, leads to apoptosis without altering p53 function and, more significantly, even in a p53-null background. Our results indicate that apoptosis induced by the activation of muscle differentiation pathways in oncogene-expressing cells can occur in a p53-independent manner.
Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts
Authors: Zhang Chuanmao , Hughes Mike , Clarke Paul R. 
Published: April 2021

ABSTRACT
Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.
LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction
Authors: Furukawa K. 
Published: April 2021

ABSTRACT
Lamina-associated polypeptide (LAP) 2, which directly interacts with B-type lamins and chromosomes, is an integral membrane protein specifically distributed along the inner nuclear membrane of the nuclear envelope. The chromatin- and lamin-binding activity of LAP2 suggests that LAP2 plays an important role in targeting mitotic vesicles to chromosomes and reorganizing the nuclear structure at the end of mitosis. Here I identified a LAP2 interacting protein, termed L2BP1 (LAP2 binding protein 1). The rat L2BP1 cDNA sequence is predicted to encode a protein of 89 amino acids which turns out to be a rat homolog of mouse and human BAF (Barrier-to-Autointegration Factor). L2BP1 is distributed diffusely throughout the nucleus in interphase cells. It is, however, highly concentrated at the chromosomes during the M-phase. Further, the L2BP1 binding domain of LAP2 overlaps its chromosome-binding region. These findings suggest that L2BP1 is a candidate mediator of LAP2-chromosome interaction at the end of mitosis.
A hyperphosphorylated form of RNA polymerase II is the major interphase antigen of the phosphoprotein antibody MPM-2 and interacts with the peptidyl-prolyl isomerase Pin1
Authors: Albert Alexandra , Lavoie Sébastien , Vincent Michel 
Published: April 2021

ABSTRACT
The monoclonal antibody MPM-2 recognizes a subset of M phase phosphoproteins in a phosphorylation-dependent manner. It is believed that phosphorylation at MPM-2 antigenic sites could regulate mitotic events since most of the MPM-2 antigens identified to date have M phase functions. In addition, many of these proteins are substrates of the mitotic regulator Pin1, a peptidyl-prolyl isomerase which is present throughout the cell cycle and which is thought to alter its mitotic targets by changing their conformation. In interphase cells, most MPM-2 reactivity is confined to nuclear speckles. We report here that a hyperphosphorylated form of the RNA polymerase II largest subunit is the major MPM-2 interphase antigen. These findings were made possible by the availability of another monoclonal antibody, CC-3, that was previously used to identify a 255 kDa nuclear matrix protein associated with spliceosomal components as a hyperphosphorylated form of the RNA polymerase II largest subunit. MPM-2 recognizes a phosphoepitope of the large subunit that becomes hyperphosphorylated upon heat shock in contrast to the phosphoepitope defined by CC-3, whose reactivity is diminished by the heat treatment. Therefore, these two antibodies may discriminate between distinct functional forms of RNA polymerase II. We also show that RNA polymerase II large subunit interacts with Pin1 in HeLa cells. Pin1 may thus regulate transcriptional and post-transcriptional events by catalyzing phosphorylation-dependent conformational changes of the large RNA polymerase II subunit.
The major olive pollen allergen (Ole e I) shows both gametophytic and sporophytic expression during anther development, and its synthesis and storage takes place in the RER
Authors: Alché Juan de Dios , Jesús Castro Antonio , Olmedilla Adela , Fernández María del Carmen , Rodríguez Rosalía , Villalba Mayte , Rodríguez-García María Isabel 
Published: April 2021

ABSTRACT
The distribution of Ole e I (the major olive pollen allergen) and its transcripts was investigated in the anther from premeiotic stages until the dehiscent pollen stage. Crude protein extracts were analyzed by immunoblotting and probed with a monoclonal antibody to Ole e I. The protein, with three variants, was found to accumulate from the early microspore stage onwards. In addition to the previously reported localization of the protein, Ole e I has been immunolocalized for the first time within the pollen wall and in the tapetum. Reverse transcription-polymerase chain reaction analysis using specific oligonucleotides and RNA extracted from whole anthers revealed that the Ole e I gene is expressed from the late tetrad stage onwards. No expression was found in control tissues such as petals, roots or leaves. Light microscopy in situ hybridization on developing flower buds and dehiscent pollen confirmed the transcripts to be present in both the microspores and the sporophytic tissue (tapetum). Labeling was found primarily in the tapetum, reaching the highest concentration in the cytoplasm of the developing and mature pollen, once tapetum started to degenerate. In situ hybridization at the transmission electron microscope level showed the transcripts to accumulate on ribosomes of the rough endoplasmic reticulum. These studies, together with others carried out previously by us, indicated that both synthesis and storage of Ole e I take place in the endoplasmic reticulum, coincidentally with the conspicuous changes suffered by this membrane system during pollen development. This process is most likely controlled at the transcriptional level. The localization of the protein in the pollen ectexine bring new insights into the function of the allergen, which are discussed.
The SFL activity secreted by metastatic carcinoma cells is related to laminin 5 and mediates cell scattering in an integrin-independent manner
Authors: Grassi M. , Moens G. , Rousselle P. , Thiéry J. P. , Jouanneau J. 
Published: April 2021

ABSTRACT
We have previously reported that an in vivo-selected metastatic variant of NBT-II rat carcinoma cells, M-NBT-II, produces and secretes a factor with cell-scattering activity, SFL, that is potentially involved in tumor progression. This biological activity was purified and characterized as a laminin 5 (LN5) -related protein. This SFL/LN5 protein consists of the α3, β3 and γ2 chains of expected sizes. Laminin 5 is a multifunctional secreted glycoprotein thought to be involved in cell adhesion and migration, mainly via its interaction with α3β1 and α6β4 integrins. SFL/LN5, and purified human laminin 5, induced the scattering and motility of MDCK cells and the formation of actin stress fibers and focal contacts in A549 cells. These events were dependent on activation of the small GTP-binding protein Rho. αv colocalized with vinculin in the focal contacts of activated cells whereas α3 and α6 integrins did not. Blocking antibodies directed against α3 and α6 integrins or the laminin 5 integrin-binding site did not abolish SFL/LN5 biological activity, which, in contrast, was completely inhibited by heparin. Thus, SFL/LN5 activity in epithelial cell scattering and cytoskeletal reorganization is probably independent of integrin receptors.
Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches
Authors: Balguerie Axelle , Sivadon Pierre , Bonneu Marc , Aigle Michel 
Published: April 2021

ABSTRACT
In this report, we have shown that the yeast amphiphysin-like protein Rvs167p was localized mainly in small cortical patches throughout the cell in unbudding cells. During budding, the patches were polarized at bud emergence site. During mating, Rvs167p was concentrated at the tip of the shmoo. Rvs167p colocalized with actin patches during yeast vegetative growth and mating. Complete disruption of the actin cytoskeleton using Latrunculin-A did not affect Rvs167p localization in patches throughout the cell. In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected. These observations suggest that Rvs167p may localize the actin cortical complex properly. Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, was required but not sufficient for the correct localization of the protein.
Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs
Authors: Gajewski Andreas , Krohne Georg 
Published: April 2021

ABSTRACT
The p58/lamin B receptor of vertebrates is localized in the inner nuclear membrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast majority of this membrane protein is localized in cytoplasmic membranes. Only very small amounts of p58 not detectable by immunofluorescence microscopy were contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nuclei reconstituted in vitro in Xenopus egg extracts contained p58, and the nucleoplasmic domain of Xp58 could be specifically bound to sperm chromatin in vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane whereas the complement of lamins is identical in both cell types. To gain insight into the properties of oocyte p58 we microinjected isolated nuclei of cultured rat cells into the cytoplasm of Xenopus oocytes. The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nuclear membrane of rat nuclei. Our data indicate that the peripheral chromatin but not lamins are required for the retention of p58 in the inner nuclear membrane.
Sucrose step gradient centrifugation of total oocyte membranes revealed that the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertilized eggs were found in the same fractions. By electron microscopical immunolocalizations one major population of meiotic p58 vesicles was identified that contained exclusively p58 and a second minor population (ca. 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins. Egg vesicles containing pore membrane proteins were predominantly recovered in gradient fractions that did not contain p58 and B-type lamins. Our data indicate that the targeting of p58 to chromatin at the end of mitosis in the early Xenopus embryo is a process independent from that of lamin targeting.
Comparable to the situation in oocytes and eggs, a significant proportion of p58 of interphase cells could be recovered in fractions that did not contain lamins. This population of p58 molecules could be extracted from A6-cells with buffers containing 1% Triton X-100/0.15 M NaCl and could be pelleted by a 50,000 g centrifuation. A- and B-type lamins were not detectable in the p58 containing pellet.
Three actin cross-linking proteins, the 34 kDa actin-bundling protein, α-actinin and gelation factor (ABP-120), have both unique and redundant roles in the growth and development of Dictyostelium
Authors: Rivero Francisco , Furukawa Ruth , Fechheimer Marcus , Noegel Angelika A. 
Published: April 2021

ABSTRACT
The contribution of three actin cross-linking proteins, α-actinin (αA), gelation factor (ABP-120), and the 34 kDa actin-bundling protein to cellular functions has been studied in three single mutant (αA−, 120−, and 34−) and three double mutant (αA−/120−, 34−/αA−, 34−/120−) strains of Dictyostelium generated by homologous recombination. Strains αA−/120− and 34−/αA− exhibited a reduced rate of pinocytosis, grew to lower saturation densities, and produced small cells in shaking cultures. All strains grew normally in bacterial suspensions and on agar plates with a bacterial lawn. Slow growth under conditions of reduced temperature and increased osmolarity was observed in single mutants 34− and αA−, respectively, as well as in some of the double mutant strains. Motility, chemotaxis, and development were largely unaltered in 34−/αA− and 34−/120− cells. However, 34−/αA− cells showed enhanced aggregation when starved in suspension. Moreover, morphogenesis was impaired in both double mutant strains and fruiting bodies of aberrant morphology were observed. These defects were reverted by re-expression of one of the lacking cross-linking proteins. The additive and synthetic phenotypes of these mutations indicate that actin cross-linking proteins serve both unique and overlapping functions in the actin cytoskeleton.
In vitro reconstitution of calreticulin-substrate interactions
Authors: Peterson Jeffrey R. , Helenius Ari 
Published: April 2021

ABSTRACT
Calreticulin is a soluble, endoplasmic reticulum-resident protein and a molecular chaperone for glycoproteins. We have reconstituted the binding of recombinant calreticulin to two glycoprotein substrates, vesicular stomatitis virus G protein and influenza hemagglutinin, in vitro. The binding was found to be direct and to require monoglucosylated, asparagine-linked oligosaccharides on the substrate glycoprotein but no other cellular factors. The binding could be modulated in vitro by incubation of substrate with purified preparations of the glycan modifying enzymes glucosidase II and the UDP-glucose:glycoprotein glucosyltransferase, thus recapitulating the regulation of calreticulin-binding by glycan modification that occurs in vivo. Using the purified ER enzymes and the recombinant calreticulin, an assay was established for reconstituting a complex, multicomponent chaperone binding cycle in vitro. We demonstrated, moreover, that the acidic C-terminal 62 residues of calreticulin are dispensable for substrate binding whereas further deletions inhibit substrate binding.
VIP36 localisation to the early secretory pathway
Authors: Füllekrug Joachim , Scheiffele Peter , Simons Kai 
Published: April 2021

ABSTRACT
VIP36, an integral membrane protein previously isolated from epithelial MDCK cells, is an intracellular lectin of the secretory pathway. Overexpressed VIP36 had been localised to the Golgi complex, plasma membrane and endocytic structures suggesting post-Golgi trafficking of this molecule (Fiedler et al., 1994). Here we provide evidence that endogenous VIP36 is localised to the Golgi apparatus and the early secretory pathway of MDCK and Vero cells and propose that retention is easily saturated. High resolution confocal microscopy shows partial overlap of VIP36 with Golgi marker proteins. Punctate cytoplasmic structures colocalise with coatomer and ERGIC-53, labeling ER-Golgi intermediate membrane structures. Cycling of VIP36 is suggested by colocalisation with anterograde cargo trapped in pre-Golgi structures and modification of its N-linked carbohydrate by glycosylation enzymes of medial Golgi cisternae. Furthermore, after brefeldin A treatment VIP36 is segregated from resident Golgi proteins and codistributes with ER-Golgi recycling proteins.
Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells
Authors: Mishima Masanori , Nishida Eisuke 
Published: April 2021

ABSTRACT
Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64-299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64-299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.
Orientation of chemotactic cells and growth cones: models and mechanisms
Authors: Meinhardt Hans 
Published: April 2021

ABSTRACT
A model is proposed for an amplification step in chemotactically sensitive cells or growth cones that accounts for their extraordinary directional sensitivity. It is assumed that cells have an intrinsic pattern forming system that generates the signals for extension of filopods and lamellipods. An external signal such as a graded cue is assumed to impose some directional preference onto the pattern formed. According to the model, a saturating, self-enhancing reaction is coupled with two antagonistic reactions. One antagonist equilibrates rapidly over the whole cell, causing competition between different surface elements of the cell cortex for activation. It will be won by those cortical regions of the cell that are exposed to the highest concentrations of the external graded cues. The second antagonistic reaction is assumed to act more locally and has a longer time constant. It causes a destabilization of peaks after they have formed. While the total activated area on the cell surface is maintained, the disappearance of some hot spots allows the formation of new ones, preferentially at positions specified by the actual external guiding signal. Computer simulations show that the model accounts for the highly dynamic behaviour of chemotactic cells and growth cones. In the absence of external signals, maxima of the internal signals emerge at random positions and disappear after some time. Travelling waves or oscillations in counter phase can emerge on the cell cortex, in agreement with observations reported in the literature. In other ranges of parameters, the model accounts for the generation of a stable cell polarity.
The Drosophila centrosomal protein Nuf is required for recruiting Dah, a membrane associated protein, to furrows in the early embryo
Authors: Rothwell Wendy F. , Zhang Claire X. , Zelano Christina , Hsieh Tao-shih , Sullivan William 
Published: April 2021

ABSTRACT
During mitosis of the Drosophila cortical syncytial divisions, actin-based membrane furrows separate adjacent spindles. Our genetic analysis indicates that the centrosomal protein Nuf is specifically required for recruitment of components to the furrows and the membrane-associated protein Dah is primarily required for the inward invagination of the furrow membrane. Recruitment of actin, anillin and peanut to the furrows occurs normally in dah-derived embryos. However, subsequent invagination of the furrows fails in dah-derived embryos and the septins become dispersed throughout the cytoplasm. This indicates that stable septin localization requires Dah-mediated furrow invagination. Close examination of actin and Dah localization in wild-type embryos reveals that they associate in adjacent particles during interphase and co-localize in the invaginating furrows during prophase and metaphase. We show that the Nuf centrosomal protein is required for recruiting the membrane-associated protein Dah to the furrows. In nuf-mutant embryos, much of the Dah does not reach the furrows and remains in a punctate distribution. This suggests that Dah is recruited to the furrows in vesicles and that the recruiting step is disrupted in nuf mutants. These studies lead to a model in which the centrosomes play an important role in the transport of membrane-associated proteins and other components to the developing furrows.