bluuebunny/crossref_metadata_2025_split huggingface dataset:
Latent TGF-β binding protein LTBP-1 contains three potential extracellular matrix interacting domains
Authors: Unsöld Christine , Hyytiäinen Marko , Bruckner-Tuderman Leena , Keski-Oja Jorma 
Published: April 2021

ABSTRACT
Latent TGF-β binding proteins (LTBPs) are components of the extracellular matrix (ECM). They belong to the fibrillin/LTBP-superfamily, and are high molecular weight glycoproteins characterized by EGF-like repeats and 8-Cys repeats. Most LTBPs associate with the small latent forms of TGF-β. Their roles include to facilitate the secretion of latent TGF-β and to target it to the ECM. In order to identify new matrix-binding domains of LTBP-1 and to characterize their association with the extracellular matrix, we have produced (in a mammalian expression system) partly overlapping recombinant fragments of its shorter form, LTBP-1S, and analyzed the binding of the purified fusion proteins to extracellular matrices of cultured human dermal and lung fibroblasts. Recombinant fragments from three different regions of the N- and C-termini showed affinity to the matrix. These interacting regions contain either the first (hybrid), second or fourth 8-Cys domains of the LTBP-1S molecule. They bound independently to the matrix. Each of them had an ability to inhibit the association of native exogenous LTBP-1 with fibroblast extracellular matrix. The interactions of the LTBP-1 fragments with the extracellular matrix resisted treatment with sodium deoxycholate, suggesting strong, possibly covalent binding. The binding occurred in a time- and dose- dependent fashion. The N-terminal fragments bound more readily to the matrices. With all fragments the binding took place both with intact fibroblast matrices and with matrices isolated by sodium deoxycholate. When using CHO cell layers, which form sparse matrices, only the N-terminal fragment of LTBP-1 was efficiently incorporated. The association of the binding fragments with isolated matrices was enhanced by soluble, cell-derived factors. The current data suggest that LTBP-1 contains three different domains with an ability to associate with the extracellular matrix.
Localization of cellubrevin-related peptide, endobrevin, in the early endosome in pancreatic β cells and its physiological function in exo-endocytosis of secretory granules
Authors: Nagamatsu Shinya , Nakamichi Yoko , Watanabe Takashi , Matsushima Satsuki , Yamaguchi Shinya , Ni Jian , Itagaki Eiji , Ishida Hitoshi 
Published: April 2021

ABSTRACT
Cellubrevins are integral membrane proteins expressed in a wide variety of tissues and usually localized in recycling vesicles. Here, we investigated the cellular localization of a cellubrevin-related peptide, endobrevin, in pancreatic β cells and its implication in the exo-endocytosis of insulin and γ-amino butyric acid (GABA). Immunocytochemistry showed that endobrevin is associated with tubulo-vesicular structures, which are colocalized with early endosomes labeled by early endosome antigen (EEA)-1 in insulinoma MIN6 cells. To determine the cellular localization of endobrevin, we appended the green fluorescent protein (GFP) to endobrevin and the fusion protein was introduced into MIN6 cells. The subcellular localization of GFP- endobrevin was visualized by confocal laser microscopy. Colocalization study based on the expressed GFP- endobrevin and endocytosed Texas-Red(Tx-R) labeled transferrin receptor and immunocytochemistry with anti- EEA1 antibody revealed that endobrevin was preferentially localized in the early endosome. Then, we examined the functional role of endobrevin in the exocytosis of insulin and GABA from pancreatic β cells. Endobrevin overexpression increased the amount of GABA released from MIN6 cells; in contrast, it decreased the glucose- stimulated insulin release from rat islets, MIN6 and INS1- D cells to approximately 50% of the control levels. Both in vitro and in vivo binding studies showed that endobrevin binds to syntaxin 1. Finally, using the fluorescent probe FM4-64, it was revealed that endobrevin overexpression accelerates vesicle recycling. We conclude that (1) endobrevin is localized in the early endosome in pancreatic β cells and (2) endobrevin plays a physiological role in the exo-endocytosis of insulin and GABA from pancreatic β cells, probably via an interaction between endocytic vesicles and the endosome.
Mitotic segregation of viral and cellular acentric extrachromosomal molecules by chromosome tethering
Authors: Kanda Teru , Otter Michele , Wahl Geoffrey M. 
Published: April 2021

ABSTRACT
Mitotic chromosome segregation is mediated by spindle microtubules attached to centromeres. Recent studies, however, revealed that acentric DNA molecules, such as viral replicons and double minute chromosomes, can efficiently segregate into daughter cells by associating with mitotic chromosomes. Based on this similarity between viral and cellular acentric molecules, we introduced Epstein-Barr virus vectors into cells harboring double minute chromosomes and compared their mitotic behaviors. We added lac operator repeats to an Epstein- Barr virus vector, which enabled us to readily identify the transgene in cells expressing a fusion protein between the lac repressor and green fluorescent protein. Unexpectedly, we found that Epstein-Barr virus vectors integrated into the acentric double minute chromosomes, but not into normal chromosomes, in all of the six stably transfected clones examined. While transiently transfected Epstein- Barr virus vectors randomly associated with wheel-shaped prometaphase chromosome rosettes, the chimeras of double minute chromosomes and Epstein-Barr virus vectors in stably transfected clones always attached to the periphery of chromosome rosettes. These chimeric acentric molecules faithfully represented the behavior of native double minute chromosomes, providing a tool for analyzing their behavior in living cells throughout the cell cycle. Further detailed analyses, including real-time observations, revealed that double minute chromosomes appeared to be repelled from the spindle poles at the same time that they attached to the chromosome periphery, while centromeric regions were pulled poleward by the attached microtubules. Disrupting microtubule organization eliminated such peripheral localization of double minute chromosomes, but it did not affect their association with chromosomes. The results suggest a model in which double minute chromosomes, but not Epstein-Barr virus vectors, are subject to the microtubule-mediated antipolar force, while they both employ chromosome tethering strategies to increase their segregation to daughter cells.
Transcriptional activation by the human Hsp70-associating protein Hap50
Authors: Niyaz Yilmaz , Zeiner Matthias , Gehring Ulrich 
Published: April 2021

ABSTRACT
We investigated human Hap50, the large isoform of the previously characterized Hsp70/Hsc70-associating protein Hap46, also called BAG-1, for effects on transcriptional activities. Overproduction by transient transfection led to enhanced expression of reporter gene constructs in various cell types using different promoters, suggesting independence of promoter type. Similarly, overexpression of Hap50 resulted in increased levels of poly(A)+ mRNAs in HeLa, COS-7, 3T3 and HTC cells. Concomitantly, the expression of some selected endogenous genes, such as those coding for c-Jun and the glucocorticoid receptor, was enhanced significantly relative to actin. Nuclear runoff transcription assays using HeLa cells showed that the effect is caused by increased transcription rates rather than mRNA stabilization. Activation of transcription by Hap50 occurred at 37°C and did not require prior thermal stress, as is the case for Hap46. In accordance with these biological effects, Hap50 is localized exclusively in the nuclear compartment of different cell types, whereas Hap46 is mostly cytoplasmic in unstressed cells, as revealed by use of fusion constructs with green fluorescent protein. High cellular levels of Hap50 were found to make cells less susceptible to adverse environmental effects such as heat stress. Our data suggest that Hap50 is a nuclear protein that acts in cells to increase the transcription of various genes.
Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress
Authors: Klibanov Sergey A. , O’Hagan Heather M. , Ljungman Mats 
Published: April 2021

ABSTRACT
The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21Waf1 gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.
Rma1, a novel type of RING finger protein conserved from Arabidopsis to human, is a membrane-bound ubiquitin ligase
Authors: Matsuda Noriyuki , Suzuki Toshiaki , Tanaka Keiji , Nakano Akihiko 
Published: April 2021

ABSTRACT
Rma1 is a protein with a RING finger motif and a C-terminal membrane-anchoring domain and is well conserved among higher eukaryotes. We show that fusion proteins between maltose binding protein (MBP) and human or Arabidopsis Rma1 are polyubiquitinated, when incubated with the rabbit reticulocyte or the wheat germ lysate, respectively. The polyubiquitination of MBP-Rma1 has been reconstituted by incubation with purified ubiquitin, the ubiquitin-activating enzyme E1, and one of the two ubiquitin-conjugating E2 enzymes (Ubc4 or UbcH5a). Other E2 enzymes tested, E2-20k, E2-25k, Ubc3 and Ubc8, are not able to confer this modification. Mutational analysis shows that the RING finger motif of Rma1 is necessary for the auto-ubiquitination of MBP-Rma1. Thus, Rma1 represents a novel, membrane-bound type of ubiquitin ligase E3, which probably functions with the Ubc4/5 subfamily of E2. The MBP moiety but not Rma1 itself is ubiquitinated in the auto-ubiquitination reaction of MBP-Rma1. Free MBP in solution is not a substrate of Rma1. These observations indicate that bringing the substrate into its physical vicinity is very important for the action of ubiquitin ligase.
Rabbit M cells and dome enterocytes are distinct cell lineages
Authors: Lelouard Hugues , Sahuquet Alain , Reggio Hubert , Montcourrier Philippe 
Published: April 2021

We have studied the M cell origin and differentiation pathway in rabbit gut-associated lymphoid tissues. Micro-dissected domes and epithelium isolated by ethylene diamine tetra acetic acid detachment allowed us to view the whole epithelial surface from the bottom of crypts to the top of domes. We used monoclonal antibodies specific to the apex of either M cells or dome enterocytes, lectins, and antibodies to vimentin in appendix, distal Peyer’s patches and caecal patches.The earliest vimentin-labeled M cells were observed in the BrdU-positive proliferative zone of dome-associated crypts. Gradual differentiation of the M cell vimentin cytoskeleton started at this site to progressively give rise to the first pocket-forming M cells in the upper dome. Therefore, these mitotic cells of the crypts appear as the direct precursors of M cells. In addition to an early appearance of M cell markers, a regular mosaic-like relative distribution of M cells and dome enterocytes was already detected in the vicinity of crypts, similar to that observed on the lateral surface of domes where functional M cells lie. This constant distribution implies that there is no trans-differentiation of enterocytes to M cells along the crypt-dome axis. Together, these observations provide very strong evidence in favor of an early commitment in crypts of M cell and enterocyte distinct lineages.
Protein phosphatase 2A binds to the cytoplasmic tail of carboxypeptidase D and regulates post-trans-Golgi network trafficking
Authors: Varlamov Oleg , Kalinina Elena , Che Fa-Yun , Fricker Lloyd D. 
Published: April 2021

ABSTRACT
Carboxypeptidase D (CPD) is a transmembrane protein that processes proteins in the trans-Golgi network (TGN). A 20-residue region within the cytoplasmic tail of CPD binds protein phosphatase 2A (PP2A). PP2A also binds to the cytoplasmic tails of other secretory pathway proteins: peptidylglycine-α-amidating mono-oxygenase, the cation-independent mannose-6-phosphate receptor and TGN38. The CPD tail is phosphorylated on Thr residues in the AtT-20 cell line. The CPD tail can also be phosphorylated by purified protein kinase A, protein kinase C and casein kinase II. Both the in vitro and the in vivo phosphorylated CPD tail can be dephosphorylated by purified PP2A. The binding of CPD tail peptide to PP2A does not influence phosphatase activity.
The rate of transport of CPD from the TGN to the cell surface of AtT-20 cells is decreased 45% by okadaic acid, a PP2A inhibitor. Microinjection of the CPD tail into AtT-20 cells inhibits the transition of CPD from endosomal compartments to the TGN. However, okadaic acid does not affect the rate of budding of CPD from the TGN into nascent vesicles or the rate of uptake from the cell surface into endosomal compartments. These results are consistent with the model that PP2A is involved in the trafficking of proteins between a TGN recycling loop and a cell-surface recycling loop, but is not involved in the individual recycling loops.
Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization
Authors: Seipel Katja , O’Brien Stephen P. , Iannotti Elizabeth , Medley Quintus G. , Streuli Michel 
Published: April 2021

ABSTRACT
Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.
Structural models for the self-assembly and microtubule interactions of γ-, δ- and ε-tubulin
Authors: Inclán Yuki F. , Nogales Eva 
Published: April 2021

ABSTRACT
αβ-tubulin heterodimers self-assemble to form microtubules nucleated by γ-tubulin in the cell. γ-tubulin is believed to recruit the αβ-tubulin dimers that form the minus ends of microtubules, but the molecular mechanism of this action remains a matter of heated controversy. Still less is known about the function and molecular interactions of δ-tubulin and ε-tubulin. δ-tubulin may seed the formation of the C triplet tubules in the basal bodies of Chlamydomonas and ε-tubulin is known to localize to the centrosome in a cell cycle-dependent manner. Using the structure of αβ tubulin as a model, we have analyzed the sequences of γ-, δ- and ε-tubulin in regions corresponding to different polymerization interfaces in the tubulin αβ dimer. The sequence comparisons sometimes show clear conservation, pointing to similar types of contacts being functionally important for the new tubulin considered. Conversely, certain surfaces show marked differences that rule out equivalent interactions for non-microtubular tubulins. This sequence/structure analysis has led us to structural models of how these special tubulins may be involved in protein-protein contacts that affect microtubule self-assembly. δ-tubulin most likely interacts longitudinally with α-tubulin at the minus ends of microtubules, while ε-tubulin most likely binds to the plus end of β-tubulin. Conservation of key residues in γ-tubulin suggests that it is capable of longitudinal self-assembly. The implications for the protofilament and template models of nucleation are considered.
The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases
Authors: Davy Deborah A. , Campbell Hugh D. , Fountain Shelley , Jong Danielle de , Crouch Michael F. 
Published: April 2021

ABSTRACT
The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with β-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.
Anomalous apical plasma membrane phenotype in CK8-deficient mice indicates a novel role for intermediate filaments in the polarization of simple epithelia
Authors: Ameen Nadia A. , Figueroa Yolanda , Salas Pedro J. I. 
Published: April 2021

ABSTRACT
Previous results from our laboratory have indicated a requirement for CK intermediate filaments (IF) for the organization of the apical domain in polarized epithelial cells in culture. The results seemed to be challenged by the phenotype of cytokeratin (CK) 8-deficient mice, which comprises only colorectal hyperplasia, female sterility and a weaker hepatocyte integrity. In this work localization with anti-CK antibodies indicated that many Ck8−/− epithelia still form IF in CK8-deficient mice, perhaps because of the expression of the promiscuous CK7. In the small intestine, only villus enterocytes lacked IFs. These cells appeared to lose syntaxin 3, and three apical membrane proteins (alkaline phosphatase, sucrase isomaltase and cystic fibrosis transmembrane conductance regulator) as they progressed along the villus. At the distal third of the villi, γ-tubulin was found scattered within the cytoplasm of enterocytes, in contrast to its normal sub-apical localization, and the microtubules were disorganized. These results could not be attributed to increased numbers of apoptotic or necrotic cells. The only other cell type we found without IFs in CK8 null mice, the hepatocyte, displayed increased basolateral levels of one apical marker (HA4), indicating a correlation between the lack of intermediate filaments and an apical domain phenotype. These data suggest a novel function for intermediate filaments organizing the apical pole of simple polarized epithelial cells.
Where it all starts: eukaryotic origins of DNA replication
Authors: Bielinsky Anja-Katrin , Gerbi Susan A. 
Published: April 2021

ABSTRACT
Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has revealed a single, defined start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.