bluuebunny/crossref_metadata_2025_split huggingface dataset:
Up-regulation of p27Kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation
Authors: Harvat Beth L. , Wang Amy , Seth Prem , Jetten A. M. 
Published: April 2021

ABSTRACT
Irreversible growth arrest is an early and integral part of squamous cell differentiation in normal human epidermal keratinocytes (NHEKs) and is assumed to be linked to the control of expression of differentiation-specific genes. In this study, we examine the link between the molecular events associated with growth arrest and the expression of differentiation genes. NHEKs that have been induced to undergo growth arrest and differentiation by suspension culture contain populations in both G1 and G2/M of the cell cycle. The irreversible growth arrest state in NHEKs is characterized by an accumulation of the hypophosphorylated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-regulated genes such as cyclin A. These events correlate with an inhibition of G1 cdk activity, mediated in part by an increase in the cdk inhibitors p21WAF1/Cip1, p27Kip1 and p16Ink4a. Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the squamous-specific genes cornifin α (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differentiation. However, growth arrest induced by over-expression of p27, p21 or p16 by recombinant adenovirus is not sufficient to induce expression of the differentiation genes, or to invoke the pattern of cell cycle regulatory protein expression characteristic of the differentiation-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the complete differentiation program.
Identification of laminin-10/11 as a strong cell adhesive complex for a normal and a malignant human epithelial cell line
Authors: Ferletta Maria , Ekblom Peter 
Published: April 2021

ABSTRACT
Laminins are heterotrimeric proteins of basement membranes. More than 50 different trimers may exist. Laminin-10 (α5β1γ1) rather than laminin-1 (α1β1γ1) could be the most abundant isoform in the adult stage, and laminin-10 is made by several developing epithelial sheets. We show here that a much used commercial human preparation contains laminin-10 (α5β1γ1), some laminin-11 (α5β2γ1), but no laminin-1. Moreover, the laminin-10/11 mixture was found to be a strong adhesive for two human cell lines derived from epithelia. Antibodies against integrin β1, α6 or α3 (at 50 µg/ml) or dystroglycan did not inhibit cell attachment to laminin-10/11, although lower concentrations of anti-dystroglycan and integrin α6 antibodies inhibited cell binding to laminin-1.
Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells
Authors: Toomre Derek , Keller Patrick , Olivo Jamie White, Jean-Christophe , Simons Kai 
Published: April 2021

ABSTRACT
The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (&lt;250 nm) and larger vesicular-tubular structures (&gt;1.5 µm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of ∼0.7 µm/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.
Key words: VSVG, GFP, Golgi, TGN, Cytoskeleton, Microtubule, Exocytosis
The tyrosinase tail mediates sorting to the lysosomal compartment in MDCK cells via a di-leucine and a tyrosine-based signal
Authors: Simmen Thomas , Schmidt Andrea , Hunziker Walter , Beermann Friedrich 
Published: April 2021

ABSTRACT
Tyrosinase is a type I membrane protein found in melanosomes, which are lysosomal-like organelles and specific for pigment cells. A mutation of mouse tyrosinase, platinum (cp), leads to truncation of tyrosinase’s cytosolic tail, and results in misrouting to the cell periphery. In this study, we expressed chimeras of wild-type and mutant cytosolic tails of mouse tyrosinase fused to rat lysosome-associated membrane protein-1 luminal and transmembrane domain to study sorting of tyrosinase in Madin-Darby canine kidney cells. The study shows that the mouse tyrosinase cytosolic tail is necessary and sufficient to mediate sorting of a heterologous type I membrane protein to compartments of the lysosomal lineage. Whereas deletions of 7 or 10 C-terminal amino acids of the tail still result in sorting to lysosomes, a deletion mutant corresponding to platinum (cp) tail fails to sort correctly and corroborates the in situ findings in cp homozygous mutant mice. Correct sorting of tyrosinase-lysosome-associated membrane protein-1 chimeras is mediated by the interplay of a di-leucine signal and a tyrosine motif of the Y-X-X-Ø type.
The ubiquitin-proteasome system and endocytosis
Authors: Strous Ger J. , Govers Roland 
Published: April 2021

ABSTRACT
Internalization of membrane proteins has been studied for more than three decades without solving all the underlying mechanisms. Our knowledge of clathrin-mediated endocytosis is certainly sufficient to understand the basic principles. However, more detailed insight is required to recognize why different proteins enter clathrin-coated pits with different rates and affinities. In addition to clathrin coat components, at least two adaptor systems and even more accessory proteins have been described to preselect membrane proteins before they can enter cells. Recent experimental data have identified the ubiquitin-proteasome Nonetheless, mean peeling velocity (µm/second) generally increased with detachmentsystem as a regulatory system for endocytosis. This system is well-known for its basic regulatory function in protein degradation, and controls a magnitude of key events. The ubiquitin-proteasome system is now identified as a regulator of the endocytosis of selected membrane proteins. In this review, we will discuss the complexity and implications of this mechanism for receptor-mediated endocytosis.
Muscle cell peeling from micropatterned collagen: direct probing of focal and molecular properties of matrix adhesion
Authors: Ra Hyun J. , Picart Catherine , Feng Huisheng , Sweeney H. Lee , Discher Dennis E. 
Published: April 2021

ABSTRACT
To quantitatively elucidate attributes of myocyte-matrix adhesion, muscle cells were controllably peeled from narrow strips of collagen-coated glass. Initial growth of primary quail myoblasts on collagen strips was followed by cell alignment, elongation and end-on fusion between neighbors. This geometric influence on differentiation minimized lateral cell contact and cell branching, enabling detailed study of myocyte-matrix adhesion. A micropipette was used to pull back one end of a quasi-cylindrical cell while observing in detail the non-equilibrium detachment process. Peeling velocities fluctuated as focal roughness, µm in scale, was encountered along the detachment front. Nonetheless, mean peeling velocity ( µm/second) generally increased with detachment force (nN), consistent with forced disruption of adhesion bonds. Immunofluorescence of β1-integrins correlated with the focal roughness and appeared to be clustered in axially extended focal contacts. In addition, the peeling forces and rates were found to be moderately well described by a dynamical peeling model for receptor-based adhesion (Dembo, M., Torney, D. C., Saxman, K. and Hammer, D. (1988). Proc. R. Soc. Lond. B 234, 55-83). Estimates were thereby obtained for the spontaneous, molecular off-rate and the receptor complex stiffness (approx.10 -5 -10 -6N/m) of adherent myocytes. Interestingly, the local stiffness is within the range of flexible proteins of the spectrin superfamily. The overall approach lends itself to elucidating the developing function of other structural and adhesive components of cells, particularly skeletal muscle cells with specialized components, such as the spectrinhomolog dystrophin and its membrane-linked receptor dystroglycan.
A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock
Authors: Weighardt Florian , Cobianchi Fabio , Cartegni Luca , Chiodi Ilaria , Villa Antonello , Riva Silvano , Biamonti Giuseppe 
Published: April 2021

ABSTRACT
A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells.
Heat-shock treatment at 42°C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37°C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.
Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth
Authors: Boleti Haralabia , Benmerah Alexandre , Ojcius David M. , Cerf-Bensussan Nadine , Dautry-Varsat Alice 
Published: April 2021

ABSTRACT
Chlamydiae enter epithelial cells via a mechanism that still remains to be fully elucidated. In this study we investigated the pathway of entry of C. psittaci GPIC and C. trachomatis LGV/L2 into HeLa cells and demonstrated that it does not depend on clathrin coated vesicle formation. We used mutant cell lines defective in clathrin-mediated endocytosis due to overexpression of dominant negative mutants of either dynamin I or Eps15 proteins. When clathrindependent endocytosis was inhibited by overexpression of the dynK44A mutant of dynamin I (defective in GTPase activity), Chlamydia entry was not affected. However, in these cells there was a dramatic inhibition in the proliferation of Chlamydia and the growth of the chlamydia vacuole (inclusion). When clathrin-dependent endocytosis was inhibited by overexpression of an Eps15 dominant negative mutant, the entry and growth of Chlamydia was unaltered. These results indicate that the effect on the growth of Chlamydia in the dynK44A cells was not simply due to a deprivation of nutrients taken up by endocytosis. Instead, the dominant-negative mutant of dynamin most likely affects the vesicular traffic between the Chlamydia inclusion and intracellular membrane compartments. In addition, cytochalasin D inhibited Chlamydia entry by more than 90%, indicating that chlamydiae enter epithelial cells by an actin-dependent mechanism resembling phagocytosis. Finally, dynamin is apparently not involved in the formation of phagocytic vesicles containing Chlamydia.
Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments
Authors: Ehler Elisabeth , Rothen Barbara M. , Hämmerle Sibylle P. , Komiyama Masatoshi , Perriard Jean-Claude 
Published: April 2021

ABSTRACT
Myofibrillogenesis in situ was investigated by confocal microscopy of immunofluorescently labelled whole mount preparations of early embryonic chicken heart rudiments. The time-course of incorporation of several components into myofibrils was compared in triple-stained specimens, taken around the time when beating starts. All sarcomeric proteins investigated so far were already expressed before the first contractions and myofibril assembly happened within a few hours. No typical stress fibre-like structures or premyofibrils, structures observed in cultured cardiomyocytes, could be detected during myofibrillogenesis in the heart. Sarcomeric proteins like α-actinin, titin and actin were found in a defined localisation pattern even in cardiomyocytes that did not yet contain myofibrils, making up dense body-like structures. As soon as the heart started to beat, all myofibrillar proteins were already located at their exact position in the sarcomere. The maturation of the sarcomeres was characterised by a short delay in the establishment of the pattern for M-line epitopes of titin with respect to Z-disk epitopes and the incorporation of the M-line component myomesin, which preceded that of myosin binding protein-C. Thus dense body-like structures, made up of titin, α-actinin and actin filaments serve as the first organised complexes also during myofibrillogenesis in situ and titin functions as a ruler for sarcomere assembly as soon as its C termini have become localised. We suggest that assembly of thin and thick filament occurs independently during myofibrillogenesis in situ and that myomesin might be important for integrating thick filaments with the M-line end of titin.
The putative nuclear receptor mediator TIF1α is tightly associated with euchromatin
Authors: Remboutsika Eumorphia , Lutz Yves , Gansmuller Anne , Vonesch Jean-Luc , Losson Régine , Chambon Pierre 
Published: April 2021

ABSTRACT
Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1α is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1α is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1α exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1α, most of TIF1α is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1α. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1α is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1α could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.
Polarised expression pattern of focal contact proteins in highly motile antigen presenting dendritic cells
Authors: Madruga Jaime , Koritschoner Nicolás P. , Diebold Sandra S. , Kurz Steffen M. , Zenke Martin 
Published: April 2021

ABSTRACT
Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues to elicit specific T cell responses. Here we used an in vitro differentiation system for generating highly motile dendritic cells from chicken bone marrow progenitors by employing the conditional v-Rel estrogen receptor (ER) fusion protein v-RelER. Molecular mechanisms of dendritic cell motility were investigated. Differentiation of v-relER progenitors into dendritic cells is associated with a reduction in cell-cell and cell-extracellular matrix interactions as cells acquire motility. We demonstrate that v-relER progenitors and dendritic cells express several adhesion receptors and components of adhesion complexes. Differentiation of v-relER cells was accompanied by downregulation of focal adhesion kinase (FAK), a key molecule of adhesion complexes, but ectopic FAK expression did not affect cell adhesion and motility. Interestingly, v-relER dendritic cells exhibit a polarised expression pattern of actin and vimentin, with actin being highly concentrated at the leading edge of the cells where lamellipodia are formed. FAK, paxillin and tyrosine phosphorylated proteins are found at both poles of the cell and colocalise with actin at the leading edge, while surface β1 integrin is confined to the uropod at the rear. CD34+ stem cell-derived human dendritic cells also exhibited an elongated bipolar morphology, mode of migration and a polarised pattern of actin-vimentin expression similar to v-relER dendritic cells.
Cytoplasmic acidification is not an effector mechanism of VP16 or DEX-induced apoptosis in CEM T leukaemia cells
Authors: Benson R. S. P. , Dive C. , Watson A. J. M. 
Published: April 2021

ABSTRACT
The role of intracellular acidification in the execution phase of apoptosis is not well understood. Here we examine the effect of Bcl-2 over-expression on intracellular acidification occurring during apoptosis. We found, that in CEM cells, neither DEX nor VP16-induced apoptosis lead to a significant change in intracellular pH (pHi). Furthermore, we found that shifting pHi away from physiological values was unable to induce chromatin condensation or poly(ADP-ribose) polymerase (PARP) cleavage in the presence of Bcl-2 over-expression. However, it was found that maximum chromatin condensation and PARP cleavage occurred at near physiological pHi values. Taken together these data suggest that intracellular acidification does not trigger the effector phase of CEM apoptosis.
Homologous chromosome pairing in wheat
Authors: Martínez-Pérez Enrique , Shaw Peter , Reader Steve , Aragón-Alcaide Luis , Miller Terry , Moore Graham 
Published: April 2021

ABSTRACT
Bread wheat is a hexaploid (AABBDD, 2n=6x=42) containing three related ancestral genomes, each having 7 chromosomes, giving 42 chromosomes in diploid cells. During meiosis true homologues are correctly associated in wild-type wheat, but a degree of association of related chromosomes (homoeologues) occurs in a mutant (ph1b). We show that the centromeres are associated in non-homologous pairs in all floral tissues studied, both in wild-type wheat and the ph1b mutant. The non-homologous centromere associations then become homologous premeiotically in wild-type wheat in both meiocytes and the tapetal cells, but not in the mutant. In wild-type wheat, the homologues are colocalised along their length at this stage, but the telomeres remain distinct. A single telomere cluster (bouquet) is formed in the meiocytes only by the onset of leptotene. The sub-telomeric regions of the homologues associate as the telomere cluster forms. The homologous associations at the telomeres and centromeres are maintained through meiotic prophase, although, during leptotene, the two homologues and also the sister chromatids within each homologue are separate along the rest of their length. As meiosis progresses, first the sister chromatids and then the homologues associate intimately. In wild-type wheat, first the centromere grouping, then the bouquet disperse by the end of zygotene.
Tenascin-C modulates tumor stroma and monocyte/macrophage recruitment but not tumor growth or metastasis in a mouse strain with spontaneous mammary cancer
Authors: Talts Jan F. , Wirl Gerhard , Dictor Michael , Muller William J. , Fässler Reinhard 
Published: April 2021

ABSTRACT
The local growth of tumors and their ability to metastasize are crucially dependent on their interactions with the surrounding extracellular matrix. Tenascin-C (TNC) is an extracellular matrix protein which is highly expressed during development, tissue repair and cancer. Despite the high levels of TNC in the stroma of primary and metastatic tumors, the function of TNC is not known. In the present study we have crossed TNC-null mice with a mouse strain where both female and male mice spontaneously develop mammary tumors followed by metastatic disease in the lungs. We report that the absence of TNC had no effect on the temporal occurrence of mammary tumors and their metastatic dissemination in lungs. Furthermore, the number and size of tumors, the number and size of metastatic foci in the lungs, the proliferation rate and apoptosis of tumor cells and tumor angiogenesis were not altered in the absence of TNC. Histological examination revealed that the tumor organisation, however, was modulated by TNC. In the presence of TNC both primary as well as metastatic tumors were organised in large tumor cell nests surrounded by thick layers of extracellular matrix proteins. In the absence of TNC these tumor cell nests were smaller but still separated from each other by extracellular matrix proteins. In addition, the TNC-null stromal compartment contained significantly more monocytes/macrophages than tumor stroma from TNC wild-type mice. Using in vitro coculture experiments we show that TNC-null tumor cells were still able to activate the TNC gene in fibroblasts which express low basal levels of TNC. Altogether these data indicate that TNC has a very limited role during the spontaneous development and growth of mamary tumors and their metastasis to the lungs.