bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA damage inhibits proteolysis of the B-type cyclin Clb5 in S. cerevisiae
Authors: Germain Doris , Hendley Joy , Futcher Bruce 
Published: April 2021

ABSTRACT
Cell cycle progression is mediated by waves of specific cyclin dependent kinases (CDKs) in all eukaryotes. Cyclins are degraded by the ubiquitin pathway of proteolysis. The recent identification of several components of the cyclin proteolysis machinery has highlighted both the importance of proteolysis at multiple transition points in the cell cycle and the involvement of other substrates degraded by the same machinery. In this study, we have investigated the effects of DNA damage on the cyclin proteolytic machinery in Saccharomyces cerevisiae. We find that the half-life of the B-type cyclin Clb5 is markedly increased following DNA damage while that of G1 cyclins is not. This effect is independent of cell cycle phase. Clb5 turnover requires p34CDC28 activity. Stabilisation of Clb5 correlates with an increase in tyrosine phosphorylation of p34CDC28, but stabilisation does not require this tyrosine phosphorylation. The stabilisation is independent of the checkpoint genes Mec1 and Rad53. These observations establish a new link between the regulation of proteolysis and DNA damage.
Aggregation reroutes molecules from a recycling to a vesicle-mediated secretion pathway during reticulocyte maturation
Authors: Vidal Michel , Mangeat Paul , Hoekstra Dick 
Published: April 2021

ABSTRACT
Endocytosis of the Tf/TfR complex is essentially the only pathway active in maturing reticulocytes, while exosomes, formed by invagination of the endosomal membrane, provide a mechanism to eliminate seemingly obsolescent proteins, including the TfR, when their function is completed. In this study, we examined molecular trafficking in the recycling and exosome-directed pathways during endocytosis in maturing reticulocytes. To this end, the flow of two exogenously inserted fluorescent lipid analogs, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]) sphingomyelin (C6-NBD-SM) and N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) was monitored and compared to that of the transferrin (Tf)/Tf receptor (TfR) complex. Prior to elimination via exosomes, the TfR actively recycles with a half-time of approx. 2 minutes. The recycling kinetics of C6-NBD-SM, as bulk plasma membrane marker, are identical to those of the apoTf/TfR complex, as shown by fluorescence microscopy and biochemical analysis. By contrast, although efficiently internalized along the same pathway, N-Rh-PE does not return to the cell surface. More specifically, sucrose gradient analysis and immunoisolation experiments demonstrated that N-Rh-PE accumulates in exosomes, which are eventually released into the extracellular medium. Fluorometric measurements showed that exogenously inserted N-Rh-PE is present in the reticulocyte plasma membrane as small molecular clusters. Moreover, a close correlation was observed between the fate of crosslinked proteins, including the TfR and acetylcholinesterase (AChE), and the fate of the clustered lipid N-Rh-PE. Thus antibody-induced aggregation of specific proteins like the TfR and AChE, which are normally sorted into exosomes during reticulocyte maturation, enhances their shedding by the exosomal pathway. Taken together, the results support the hypothesis that aggregation of either proteins or lipids act as a general sorting signal for exosomal processing, thereby inhibiting reentry in a recycling pathway and providing an effective means for clearing molecules from the cell surface and their eventual elimination from the cells.
Microtubules suppress actomyosin-based cortical flow in Xenopus oocytes
Authors: Canman Julie C. , Bement William M. 
Published: April 2021

ABSTRACT
Several cell motility processes including cytokinesis and cell locomotion are dependent on the interplay of the microtubule and actomyosin cytoskeletons. However, because such processes are essentially visual phenomena, interactions between the two cytoskeletal systems have been difficult to study quantitatively. To overcome this difficulty, we have developed the Xenopus oocyte as an inducible, quantitative model system for actomyosin-based cortical flow and then exploited the strengths of this system to assess the relationship between microtubules and cortical flow. As in other systems, oocyte cortical flow entails: (1) redistribution of cortical filamentous actin (f-actin); (2) a requirement for actomyosin; (3) redistribution of cell surface proteins; (4) a requirement for cell surface protein mobility; and (5) directed movement of cortical organelles. Cortical flow rate in the oocyte system is inversely proportional to the level of polymeric tubulin and microinjection of free tubulin has no effect on the rate of cortical flow. Enhancement of microtubule polymerization inhibits cortical f-actin cable formation during cortical flow. The effects of microtubule depolymerization on cortical flow are rapid, independent of transcription or translation, independent of effects on the oocyte intermediate filament system, and independent of the upstream stimulus for cortical flow. The results show that the microtubules themselves, or a factor associated with them, suppress cortical flow, either by mechanically resisting flow, or by modulating the actomyosin cytoskeleton.
Hydra regeneration from recombined ectodermal and endodermal tissueII. Differential stability in the ectodermal and endodermal epithelial organization
Authors: Murate Motohide , Kishimoto Yasuyuki , Sugiyama Tsutomu , Fujisawa Toshitaka , Takahashi-Iwanaga Hiromi , Iwanaga Toshihiko 
Published: April 2021

ABSTRACT
Hydra tissue consists of the ectodermal and the endodermal layers. When the two layers were separated by procaine treatment and then recombined, the ectodermal epithelial cells spread as a single cell layer over the endoderm as in epiboly in vertebrate embryogenesis, and the resultant spherical structure subsequently regenerated into a complete hydra. In this study, light and electron microscopy were used to examine the structural changes which took place in the cells and tissue during this epibolic ectodermal spreading process. Within a few hours after tissue recombination, the endoderm underwent dramatic changes; it lost its epithelial sheet organization, and turned into a mass of irregularly shaped cells without the apicalbasal cell polarity initially present. In contrast, the ectoderm maintained its basic epithelial sheet organization as it spread over the endoderm. Later, the endodermal epithelial cells reorganized themselves into a single-layered epithelial sheet underneath the spreading ectodermal layer. The resultant spherical structure consisted of a single layer of ectodermal epithelial cells outside, a single layer of endodermal epithelial cells inside, and an empty cavity in the center as in normal hydra tissue. This structure regenerated into hydra in the following days. These and other observations demonstrate that the two-layered epithelial sheet organization is highly dynamic, and that its stability is maintained by strong interactions between the two layers in normal hydra. It is suggested that this dynamic nature of the hydra tissue, particularly the high plasticity of the endodermal epithelial sheet organization, may be an important element for the high regenerative capacity of this organism.
Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice
Authors: Holwell Tricia A. , Schweitzer Susan C. , Evans Robert M. 
Published: April 2021

ABSTRACT
Fibroblast cell lines were derived from vim−/− mice that express a mouse vimentin transgene in a tetracycline regulatable manner. Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim− cell lines were isolated. A vim− cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences. Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline. The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts. The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion. Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus. Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force. Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound. Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture. These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.
Integrated interactions between chondroitin sulphate proteoglycans and weak dc electric fields regulate nerve growth cone guidance in vitro
Authors: Erskine L. , McCaig C. D. 
Published: April 2021

ABSTRACT
During development and regenerative growth, neuronal pathways are defined in part by several endogenous cues that collectively determine directed growth. The interactions between such cues largely are unknown. To address potential interactions, we have examined in vitro the combined effect on nerve growth of two endogenous growth cone guidance cues: chondroitin sulphate proteoglycans and weak dc electric fields. Addition to the culture medium of a chondroitin 6-sulphate/keratan sulphate containing PG (BNC-PG) markedly enhanced the cathodal re-orientation of embryonic Xenopus neurites in an electric field, whereas a proteoglycan containing chondroitin 4-sulphate (RC-PG) was inhibitory. These effects of BNC-PG and RC-PG were reproduced by their chondroitin sulphate glycosaminoglycan side chains alone. Chondroitin 6-sulphate or chondroitin 4-sulphate, respectively, enhanced and inhibited cathodally-directed nerve re-orientation. This was dependent on the integrity of the glycosaminoglycan chain structure; when digested into their disaccharide subunits both molecules became inactive. Keratan sulphate, a minor component of BNC-PG, was found to be inhibitory, whereas dermatan sulphate, an epimer of chondroitin 4-sulphate, had no effect. We conclude that in vitro specific interactions between these two nerve guidance cues do occur and that the specificity of the response is critically dependent on the charge pattern of the proteoglycans chondroitin sulphate side chains. The expression of a host of proteoglycans with differing glycosaminoglycan side chains varies in both time and place in the developing nervous system, thus the scope is vast for spatial and temporal modulation of nerve guidance by interacting cues.
Association of the nucleolar transcription factor UBF with the transcriptionally inactive rRNA genes of pronuclei and early Xenopus embryos
Authors: Bell Peter , Mais Christine , McStay Brian , Scheer Ulrich 
Published: April 2021

ABSTRACT
When nuclei (pronuclei) were assembled from sperm chromatin in Xenopus egg extract and examined by immunofluorescence microscopy, UBF was concentrated at a single intranuclear dot-like or more extended necklacelike structure. These UBF-foci contained rDNA as demonstrated by in situ hybridization and hence represent the chromosomal nucleolus organizing regions (NORs). Besides UBF, other components of the transcription machinery such as the TATA-box binding protein (TBP) and RNA polymerase I (pol I) as well as several nucleolar proteins could not be detected at the NORs. Immunodepletion experiments indicated that UBF is maternally provided and taken up by the pronuclei. Essentially the same results were obtained when we examined the NORs of early Xenopus embryos up to the midblastula stage. After this stage, when transcription of the rRNA genes has begun, nucleoli developed and the NORs acquired TBP and pol I. Our results support the hypothesis that UBF is an architectural element which converts the rDNA chromatin into a transcriptionally competent form.
Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome
Authors: Dehio Christoph , Meyer Marlene , Berger Jürgen , Schwarz Heinz , Lanz Christa 
Published: April 2021

ABSTRACT
Vascular colonisation by Bartonella henselae may cause vaso-proliferative tumour growth with clumps of bacteria found in close association with proliferating endothelial cells. By using B. henselae-infected human umbilical vein endothelial cells as an in vitro model for endothelial colonisation, we report here on a novel mechanism of cellular invasion by bacteria. First, the leading lamella of endothelial cells establishes cellular contact to sedimented bacteria and mediates bacterial aggregation by rearward transport on the cell surface. Subsequently, the formed bacterial aggregate is engulfed and internalised by a unique host cellular structure, the invasome. Completion of this sequence of events requires 24 hours. Cortical F-actin, intercellular adhesion molecule-1 and phosphotyrosine are highly enriched in the membrane protrusions entrapping the bacterial aggregate. Actin stress fibres, which are anchored to the numerous focal adhesion plaques associated with the invasome structure, are typically found to be twisted around its basal part. The formation of invasomes was found to be inhibited by cytochalasin D but virtually unaffected by nocodazole, colchicine or taxol, indicating that invasome-mediated invasion is an actin-dependent and microtubuli-independent process. Bacterial internalisation via the invasome was consistently observed with several clinical isolates of B. henselae, while a spontaneous mutant obtained from one of these isolates was impaired in invasome-mediated invasion. Instead, this mutant showed increased uptake of bacteria into perinuclear localising phagosomes, suggesting that invasome-formation may interfere with this alternative mechanism of bacterial internalisation. Internalisation via the invasome represents a novel paradigm for the invasion of bacteria into host cells which may serve as a cellular colonisation mechanism in vivo, e.g. on proliferating and migrating endothelial cells during Bartonella-induced vaso-proliferative tumour growth.
Involvement of caspases in sympathetic neuron apoptosis
Authors: McCarthy Mary Jane , Rubin Lee L. , Philpott Karen L. 
Published: April 2021

ABSTRACT
In order to study the involvement of caspases in neuronal cell death, we have examined the effects of the viral caspase inhibitor p35 and peptide caspase inhibitors on sympathetic neurons isolated from the superior cervical ganglion (SCG). In these neurons, apoptosis can be induced by the withdrawal of nerve growth factor (NGF) and also by the addition of the kinase inhibitor staurosporine. p35 has been shown to be a broad spectrum inhibitor of the caspase family and promotes the survival of SCG neurons withdrawn from NGF. We show that p35 is also protective when apoptosis is induced by staurosporine. In addition, p35 inhibits a number of the morphological features associated with apoptosis, such as nuclear condensation, TUNEL labelling, and externalisation of phosphatidylserine. The tri-peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethylketone (zVAD-fmk) was effective at inhibiting NGF withdrawal-induced and staurosporine-induced apoptosis of SCG neurons. Two other peptide inhibitors, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) and acetyl-Asp-Glu-Ala-Asp-aldehyde (Ac-DEVD-CHO), also inhibited apoptosis induced by both means when microinjected into SCG neurons but peptides derived from the caspase cleavage site in p35 were not protective. We present data to suggest that apoptosis induced by separate death stimuli can result either in the activation of distinct caspases or in differences in the time of activation of the family members.
Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro
Authors: Moursi Amr M. , Globus Ruth K. , Damsky Caroline H. 
Published: April 2021

ABSTRACT
We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cellbinding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin’s effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor α5β1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, α3β1, α8β1 and αvβ3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that α5β1 levels are constant throughout osteoblast differentiation whereas levels of α3β1 and α8β1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing antiintegrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-α5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against α3β1 and α8β1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to αvβ3 and αvβ5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes α5β1, α3β1 and α8β1. We further conclude that the specific α5β1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.
Surface residue mutations of the PML RING finger domain alter the formation of nuclear matrix-associated PML bodies
Authors: Boddy Michael N. , Duprez Estelle , Borden Katherine L. B. , Freemont Paul S. 
Published: April 2021

ABSTRACT
The human protein PML, was first identified as part of a fusion protein with retinoic acid receptor alpha as found in the chromosomal translocation which gives rise to acute promyelocytic leukaemia. PML is normally localised to large matrix-associated nuclear domains (known as ND10, Kr bodies, PODS or PML NBs) which comprise several multi-protein complexes. Within the PML protein, there are a number of identified zinc-binding domains, one of which called the RING finger is found in a large family of diverse and unrelated proteins. Here, we report the effect of site-directed mutations within the context of the whole PML protein, of amino acids found on the surface of the PML RING finger domain and PML NB formation in vivo. Mutations of a small region of the RING finger domain surface affect the size and numbers of PML NBs in a mouse fibroblast expression assay, resulting in fewer but larger exogenous PML NBs. Mutations of other surface RING residues, however, do not affect exogenous PML NB formation. Furthermore, all of the PML RING mutants co-localise to both endogenous and exogenous wild-type PML NBs. These data identify a specific region of the PML RING finger domain which is directly involved in correct PML NB formation. They also provide evidence to suggest that the PML RING finger is involved in mediating PML-PML oligomeric interactions, as part of a mechanism leading to the assembly of the PML NB complex.
Homotypic fusion between aggregated lysosomes triggered by elevated [Ca2+]i in fibroblasts
Authors: Bakker Annette C. , Webster Paul , Jacob Wim A. , Andrews Norma W. 
Published: April 2021

ABSTRACT
Previous studies demonstrated that microinjection of antibodies to the cytoplasmic domain of the lysosomal glycoprotein lgp120 induces aggregation of lysosomes in NRK cells. Here we show that the antibody-clustered vesicles do not co-localize with MPR and β-COP-containing organelles, confirming their lysosomal nature. Observations by transmission and high voltage electron microscopy indicated that, although tightly apposed to each other, aggregated lysosomes remained as separate vesicles, with an average diameter of 0.3-0.4 μm. However, when cells microinjected with antibody were exposed to the Ca2+ ionophore ionomycin, large vesicles were formed within the lysosome clusters, suggesting the occurrence of lysosomelysosome fusion. Stereological measurements of lysosome diameters on confocal and transmission electron microscopy indicated that the large lgp120-positive vesicles could have originated from the fusion of 3 up to 15 individual lysosomes. To verify if agents that mobilize Ca2+ from intracellular stores had the same effect, anti-lgp120-microinjected cells were treated with thapsigargin, and with the receptor-mediated agonists bombesin and thrombin. Thapsigargin also induced the formation of large lgp120-containing vesicles, detected by both confocal and transmission electron microscopy. Analysis of antibody-clustered lysosomes in streptolysin O-permeabilized cells indicated that an intracellular free Ca2+ concentration of 1 μM was sufficient to trigger formation of large lysosomes.
Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts
Authors: Battistoni Alessandra , Guarguaglini Giulia , Degrassi Francesca , Pittoggi Carmine , Palena Antonella , Matteo Gigliola Di , Pisano Claudio , Cundari Enrico , Lavia Patrizia 
Published: April 2021

ABSTRACT
RanBPI is a molecular partner of the Ran GTPase, which is implicated in the control of several processes, including DNA replication, mitotic entry and exit, cell cycle progression, nuclear structure, protein import and RNA export. While most genes encoding Ran-interacting partners are constitutively active, transcription of the RanBPI mRNA is repressed in non proliferating cells, is activated at the G1/S transition in cycling cells and peaks during S phase. We report here that forced expression of the RanBPI gene disrupts the orderly execution of the cell division cycle at several stages, causing inhibition of DNA replication, defective mitotic exit and failure of chromatin decondensation during the telophase-to-interphase transition in cells that achieve nuclear duplication and chromosome segregation. These results suggest that deregulated RanBP? activity interferes with the Ran GTPase cycle and prevents the functioning of the Ran signalling system during the cell cycle.