bluuebunny/crossref_metadata_2025_split huggingface dataset:
Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation
Authors: Perrot-Applanat Martine , Gualillo Oreste , Buteau Helene , Edery Marc , Kelly Paul A. 
Published: April 2021

ABSTRACT
Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells : they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4×10−10M) and have the same capacity to stimulate the transcriptional activity of a milk protein (β-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-local-ization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.
Sphingolipid organization in biomembranes: what physical studies of model membranes reveal
Authors: Brown Rhoderick E. 
Published: April 2021

ABSTRACT
Recent cell biological studies suggest that sphingolipids and cholesterol may cluster in biomembranes to form raft-like microdomains. Such lipid domains are postulated to function as platforms involved in the lateral sorting of certain proteins during their trafficking within cells as well as during signal transduction events. Here, the physical interactions that occur between cholesterol and sphingolipids in model membrane systems are discussed within the context of microdomain formation. A model is presented in which the role of cholesterol is refined compared to earlier models.
RagA is a functional homologue of S. cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway
Authors: Hirose Eiji , Nakashima Nobutaka , Sekiguchi Takeshi , Nishimoto Takeharu 
Published: April 2021

ABSTRACT
Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p. According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR. Both cDNAs rescued a cold sensitivity of S. cerevisiae, gtr1-11. Furthermore, we introduced into the cloned human RagA cDNA, the mutation ‘T21L’ corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1−, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein. The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1− and rna1-1 mutations. These results indicated that RagA and RagBs are functional homologues of S. cervisiae Gtr1p. Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S. cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex. In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm. Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.
Platelet-derived growth factor (PDGF)-induced actin rearrangement is deregulated in cells expressing a mutant Y778F PDGF β-receptor
Authors: Ruusala Aino , Sundberg Christian , Arvidsson Ann-Kristin , Rupp-Thuresson Eva , Heldin Carl-Henrik , Claesson-Welsh Lena 
Published: April 2021

ABSTRACT
Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor β-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type β-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase Cγ, and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor β-receptor, potentially through binding of a novel signaling molecule to this site.
In vivo association of lamins with nucleic acids in Drosophila melanogaster
Authors: Rzepecki Ryszard , Bogachev Sergei S. , Kokoza Elena , Stuurman Nico , Fisher Paul A. 
Published: April 2021

ABSTRACT
A 32P-labeling strategy was developed to study the interaction(s) in tissue culture cells between proteins and nucleic acids. Interphase and mitotic nuclear lamins were studied in Drosophila Kc cells. After bromodeoxyuridine incorporation and in vivo photo-crosslinking with 366 nm light, it was found that interphase lamins were associated with nucleic acid. Interactions with DNA as well as RNA were detected. In contrast, interaction of nucleic acids with mitotic lamin was not observed. Photo-crosslinking in the presence of antibiotics distamycin and/or chromomycin suggested that interphase lamins interacted with both A-T-rich DNA and G-C-rich DNA; interactions with G-C-rich DNA predominated. These results have implications for understanding the interphase organization of the higher eukaryotic cell nucleus as well as the transition of cells from interphase to mitosis. A model of nuclear organization, consistent with our results, is proposed.
Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus
Authors: Röttger Sabine , White Jamie , Wandall Hans H. , Olivo Jean-Christophe , Stark Annika , Bennett Eric P. , Whitehouse Caroline , Berger Eric G. , Clausen Henrik , Nilsson Tommy 
Published: April 2021

ABSTRACT
O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.
T cell syncytia induced by HIV release T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber
Authors: Shutt Damon C. , Jenkins Leslie M. , Carolan Edward J. , Stapleton Jack , Daniels Karla J. , Kennedy Ronald C. , Soll David R. 
Published: April 2021

ABSTRACT
A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.
Ret finger protein is a normal component of PML nuclear bodies and interacts directly with PML
Authors: Cao Tongyu , Duprez Estelle , Borden Katherine L. B. , Freemont Paul S. , Etkin Laurence D. 
Published: April 2021

ABSTRACT
The ret finger protein (rfp) is a member of the B-box zinc finger gene family many of which may function in growth regulation and in the appropriate context become oncogenic. Members of this family are nuclear proteins that possess a characteristic tripartite motif consisting of the RING and B-box zinc binding domains and a coiled-coil domain. The promyelocytic leukemia gene (PML), another B-box family member, produces a protein product that is detected within punctate nuclear structures called PML nuclear bodies (NBs) or PML oncogenic domains (PODs). These NBs are complex structures that consist of a number of different proteins many of which have yet to be identified. In the disease acute promyelocytic leukemia (APL) a fusion protein, PML-RARA, is produced through the t(15:17) translocation. In APL the morphology of the NBs is altered. We report that rfp co-localizes with PML in a subset of the PML NBs and that it interacts directly with PML. This interaction is mediated through the rfp B-box and the distal two coils. In contrast, homomultimerization of rfp preferentially involves the B-box and the proximal coil. The association of rfp with the PML NBs is altered by mutations that affect rfp/PML interaction and in NB4 cells that are derived from APL patients. When treated with retinoic acid, rfp reassociates with the NBs in a pattern similar to non APL cells. Additionally, we found that rfp colocalizes with PML-RARA protein produced in APL patients. These results suggest that rfp, along with the other known/unknown components of PML NBs, have an important role in regulating cellular growth and differentiation.
Clustering of pericentromeres initiates in step 9 of spermiogenesis of the rat (Rattus norvegicus) and contributes to a well defined genome architecture in the sperm nucleus
Authors: Meyer-Ficca Mirella , Müller-Navia Jutta , Scherthan Harry 
Published: April 2021

ABSTRACT
Fluorescence in situ hybridization with centromeric, telomeric and whole chromosome paint probes was used to study nuclear topology in epididymal sperm as well as spermatids from testis tissue sections of the rat. Pericentromeric regions of 9 chromosomes of the rat (n=21) were labeled with a satellite I specific DNA probe. Pericentromeres showed few tandem associations in spermatids of steps 1-8 of spermiogenesis. At step 9, pericentromeric regions associated to form an elongated cluster in the spermatid nucleus. This arrangement was also seen in the sperm nucleus. FISH with telomere probes revealed numerous, variably arranged signals in round and elongated spermatids as well as sperm nuclei. Telomere signals showed a tendency for pairwise association which was more pronounced in elongated spermatid and epididymal sperm nuclei. FISH to DTT treated sperm suggested that telomeres reside at the periphery and that pericentromeres are located in the nuclear interior. Chromosome painting with rat chromosome 2 and 12 specific microdissection library probes showed that these chromosomes predominantly occupy compact and variably shaped territories during spermatid maturation. In elongated epididymal sperm nuclei chromosome 2 and 12 territories took up specific positions. We suppose that the associations of pericentromeres during step 9 render a well defined nuclear topology which facilitates the ordered compaction of the genome at subsequent stages.
The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215
Authors: Charrasse Sophie , Schroeder Marianne , Gauthier-Rouviere Cécile , Ango Fabrice , Cassimeris Lynne , Gard David. L. , Larroque Christian 
Published: April 2021

ABSTRACT
We have recently identified a 6,449 bp cDNA, termed colonic, hepatic tumor over-expressed gene (ch-TOG), that is highly expressed in human tumors and brain. Its single open reading frame encodes a putative 218,000 Da polypeptide, TOGp. Antibodies generated against a bacterially expressed TOGp fragment specifically recognize a 218,000 Da polypeptide in two human cell lines and in brain. Immunofluorescence microscopy using affinity-purified TOGp antibodies revealed that the distribution of TOGp was dependent upon the cell cycle. During interphase, TOGp was found concentrated in the perinuclear cytoplasm, where it co-localized with ER markers. In contrast anti-TOGp antibodies stained centrosomes and spindles in mitotic cells. TOGp co-sedimented with taxol-stabilized microtubules in vitro. Moreover, a TOGp enriched fraction promotes microtubule assembly both in solution and from nucleation centers. Finally, sequence comparison and immunologic cross-reaction suggest that TOGp is homologous to XMAP215, a previously described microtubule associated protein (MAP) from Xenopus eggs. These results suggest that TOGp is a MAP and that TOGp/XMAP215 may be necessary for microtubules rearrangements and spindle assembly in rapidly dividing cells.
LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis
Authors: Duband-Goulet Isabelle , Courvalin Jean-Claude , Buendia Brigitte 
Published: April 2021

ABSTRACT
Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the aminoterminal domain of LBR is specifically cleaved during apoptosis to generate an ∼20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.
BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function
Authors: Lies C. Mark , Cheng Jijun , James Steven W. , Morris N. Ronald , O’Connell Matthew J. , Mirabito P. M. 
Published: April 2021

ABSTRACT
Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a &gt;25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECydin B. Our results indicate that B?MAAPC?, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.
Ligand-independent role of the β4 integrin subunit in the formation of hemidesmosomes
Authors: Nievers Mirjam G. , Schaapveld Roel Q. J. , Oomen Lauran C. J. M. , Fontao Lionel , Geerts Dirk , Sonnenberg Arnoud 
Published: April 2021

ABSTRACT
Recently, we have shown that a region within the β4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of α6β4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the β4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the β4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of β4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/β4 chimeric constructs in COS-7 and CHO cells, which do not express α6β4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/β4 chimeras in a keratinocyte cell line derived from a patient lacking β4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of α6β4-expressing cells to fibronectin. These findings establish that the β4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of α6β4 to ligand, and heterodimerization of α6 with β4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.
The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells
Authors: Assmann Volker , Marshall John F. , Fieber Christina , Hofmann Martin , Hart Ian R. 
Published: April 2021

ABSTRACT
The receptor for hyaluronan mediated motility (RHAMM) has been reported to mediate migration, transformation, and metastatic spread of murine fibroblasts. Here we describe the expression of two human RHAMM isoforms, which are generated by alternative splicing of the primary gene transcript, by a series of human breast carcinoma cell lines. A polyclonal antibody, raised against a bacterially expressed RHAMM fusion protein, detected an 85-90 kDa protein by western blot analysis. No correlation between the level of RHAMM mRNA and protein expression with known metastatic/malignant potential of the tumour cell lines was observed. Interestingly, we found that the antibody did not stain the cell surface but the cytoplasm of breast cancer cells. The intracellular localisation of RHAMM was confirmed by subcellular fractionation studies. RHAMM proteins were capable of binding to hyaluronan, but not to heparin or chondroitin sulphate, in an vitro binding assay. We also provide evidence that a potential hyaluronan-binding motif in the N terminus of the protein is not involved in the interaction of RHAMM with hyaluronan. Our findings lead us to conclude that RHAMM does not function as a conventional motility receptor for HA in human breast cancer cells and we suggest the term RHAMM be substituted by ‘intracellular hyaluronic acid binding protein’ (IHABP).