bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of nucleation in patterning microtubule networks
Authors: Hyman A. , Karsenti E. 
Published: April 2021

ABSTRACT
Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.
Localization and processing from a polycistronic precursor of novel snoRNAs in maize
Authors: Shaw Peter J. , Beven Alison F. , Leader David J. , Brown John W. S. 
Published: April 2021

ABSTRACT
We have shown previously that groups of U14 snoRNA genes are clustered with other, novel snoRNAs in maize. These genes are transcribed polycistronically from an upstream promoter to give a precursor snoRNA, which is processed by a splicing-independent mechanism. The clusters contain both box C/D snoRNAs, thought to guide rRNA O-ribose methylations, and the first plant box H/ACA snoRNA so far identified, thought to guide an rRNA pseudo-uridylation. Here we show that four novel snoRNAs identified as members of U14-containing gene clusters each show distinct sub-nucleolar localizations. Two of the snoRNAs (snoR2, a box H/ACA snoRNA, and snoR3, a box C/D snoRNA) colocalise closely with nucleolar rDNA transcription sites. A third box C/D snoRNA, U49, is localised to a more extended region which includes the transcription sites. On the other hand snoR1, another box C/D snoRNA, is located in a quite different region of the nucleolus, and shows a similar distribution to that of 7–2/MRP, a snoRNA involved in the later pre-rRNA cleavage reactions. This may indicate that this snoRNA is involved at later stages of processing, whereas the other snoRNAs are involved early or cotranscriptionally. Probes to intergenic spacer regions of the precursor snoRNA have been used to determine the location of the precursor. This shows a clear labelling of both the dense fibrillar component of the nucleolus, and of coiled bodies. This distribution implies that the polycistronic precursor is imported into the nucleolus for processing to the mature snoRNAs, and that the import or processing pathway involves coiled bodies.
Advillin (p92): a new member of the gelsolin/villin family of actin regulatory proteins
Authors: Marks Peter W. , Arai Maya , Bandura Jennifer L. , Kwiatkowski David J. 
Published: April 2021

ABSTRACT
A new member of the gelsolin/villin family of actin regulatory proteins was initially identified by screening an adult murine brain cDNA library with a probe for bovine adseverin. The predicted amino acid sequence of the 92 kDa murine protein p92 (advillin) is 75% homologous to villin and 65% homologous to gelsolin and adseverin. It shares a six domain structure with other gelsolin family members and has a carboxy-terminal headpiece, similar to, yet distinct from, villin. Northern blot analysis shows a high level of mRNA expression in murine uterus and human intestine. In situ mRNA analysis of adult murine tissues demonstrates that the message is most highly expressed in the endometrium of the uterus, the intestinal lining, and at the surface of the tongue. In murine embryonic development, strong expression of the message is observed by day 14.5 in dorsal root ganglia and trigeminal ganglia. Expression is also noted at day 16.5 in cerebral cortex. We propose that p92 (advillin) has unique functions in the morphogenesis of neuronal cells which form ganglia, and that it may compensate to explain the near normal phenotype observed in villin-deficient mice.
Antisense oligonucleotides against ‘cardiac’ and ‘skeletal’ DHP-receptors reveal a dual role for the ‘skeletal’ isoform in EC coupling of skeletal muscle cells in primary culture
Authors: Bulteau Laurence , Raymond Guy , Cognard Christian 
Published: April 2021

ABSTRACT
Two dihydropyridine receptor mRNA isoforms (cardiac and skeletal) are expressed in rat skeletal muscle cells in primary culture. The progressive changes in excitationcontraction coupling mode from dual mode (‘skeletal’ and ‘cardiac’) to predominant ‘skeletal’ one during in vitro myogenesis are thought to be linked to the developmental changes in the relative expression of the two types of molecular entity previously observed in this preparation. In order to test this hypothesis, myotube cultures (5-to 7-day-old) were treated with antisense phosphorothioated oligodeoxynucleotides against cardiac or skeletal α1 subunit of L-type calcium channel. The oligodeoxynucleotide uptake by cells was checked by means of imaging of fluorescent oligodeoxynucleotide derivatives within the cells. Optimum concentration used (10 μM in the extracellular medium) and incubation time (70 hours) were empirically determined. Antisense directed against the cardiac type led to a 54% decrease in the averaged L-type calcium current peak density at −10 mV. The same type of experiment was performed with antisense against the skeletal isoform and led to a same order of inihibition (46%). This result clearly shows that the two isoforms can work as a calcium channel. Conversely, analysis of the shape of T–V (relative contractile amplitude versus membrane potential) curves shows that the treatment with ‘skeletal’ antisense depressed the contractile response in the medium membrane potential range whereas treatment with ‘cardiac’ antisense had no effect. This and other results taken together suggest that the skeletal isoform of dihydropyridine receptor is involved in both ‘cardiac’ and ‘skeletal’ types of EC coupling mechanisms at work in early stages of myotubes in vitro development. The type of coupling probably depends on the proximity of the skeletal dihydropyridine receptor and the ryanodine receptor.
Altered stem cell regeneration in irradiated intestinal crypts of senescent mice
Authors: Martin K. , Potten C. S. , Roberts S. A. , Kirkwood T. B. L. 
Published: April 2021

ABSTRACT
Ageing is associated with a progressive deterioration in the functions of many organs within the body. In tissue with high cell turnover, the maintenance of the stem cells is of particular importance. Any accumulation of damage in stem cells may affect their function and hence threaten the homeostasis and regenerative capacity of the tissue. The small intestine represents a good model for the study of stem cells because of its spatial and hierarchical organisation. We have examined the effect of age on stem cell regenerative capacity after irradiation, using the microcolony assay. Crypt survival levels, the growth rate of surviving crypts, and the number of cells able to repopulate a crypt have been investigated by irradiating groups of 67 month old and 28-30 month old ICRFa male mice. After high doses of irradiation, the surviving crypts in old mice were both smaller and fewer in number than in young mice. The growth rate of surviving crypts was determined by measuring the crypt area and the number of cells/crypt at various times after 14 Gy irradiation. There was a growth delay of between about one half and one day in the older mice. Surprisingly, the number of clonogenic cells per crypt was estimated to be greater in the older mice. These studies indicate important age-related alterations in the capacity to regenerate the crypts after radiation damage.
Splenic T lymphocytes die preferentially during heat-induced apoptosis: NuMA reorganization as a marker
Authors: Sodja Caroline , Brown David L. , Walker P. R. , Chaly Nathalie 
Published: April 2021

ABSTRACT
We are investigating nuclear events during apoptosis in mouse splenic lymphocytes cultured immediately after isolation (controls) or after heat treatment (42°C, 30 minutes), and have found that hyperthermia increased the level of apoptosis to double that of spontaneous apoptosis in controls within 6 hours. Immunolabelling for Nuclear Mitotic Apparatus Protein (NuMA) suggested that splenocytes were responding heterogeneously to the heat treatment. Whereas all nuclei in controls and about half of nuclei in heat-treated samples showed the usual diffuse nucleoplasmic labelling, 40-60% of nuclei in heated samples also contained numerous bright spots. We then examined whether the heterogeneity in NuMA organization might be an indication of a differential response of B and T lymphocytes to hyperthermia, and whether the presence of NuMA spots is related to the apoptotic process. NuMA labelling of heated fractionated splenocyte populations showed that 90% of nuclei in T-enriched cultures (≤4% IgG+ cells), but only 25% of nuclei in B-enriched samples (≤80% IgG+ cells), contained spots. As well, 2 hours after heat treatment of unfractionated cultures, ≥90% of nuclei that were accumulating DNA strand breaks, as detected by TUNEL, exhibited NuMA spots. These data indicate that cells with NuMA spots are targetted for, or have initiated, the death program. Since most T cells, but few or no B cells, were spotty after heating, we conclude further that hyperthermia induces apoptosis preferentially in splenic T lymphocytes. The observation that the proportion of T cells was, on average, threefold greater in control than in heated samples after 24 hours in culture reinforces this conclusion.
Regulation of macropinocytosis in v-Src-transformed fibroblasts: cyclic AMP selectively promotes regurgitation of macropinosomes
Authors: Veithen Alex , Amyere Mustapha , Smissen Patrick Van Der , Cupers Philippe , Courtoy Pierre J. 
Published: April 2021

ABSTRACT
Stable transformation of Rat-1 fibroblasts by the v-Src oncoprotein results into the constitutive formation of macropinosomes. In the present report, we found that macropinosomes do not fuse with transferrin-containing endosomes and investigated the effects of cyclic AMP as a regulator of macropinocytosis in this cell system. The permeant analogs dibutyryl cyclic AMP and 8-bromo-cyclic AMP, as well as the pharmacological activator of adenylate cyclase forskolin, similarly decreased by about 35% the net endocytic accumulation of the fluid-phase tracer horseradish peroxidase at intervals &gt;5 minutes in v-Src-transformed cells but not in the non-transformed parental Rat-1 cell line. However, and in contrast to the phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate or the phosphatidylinositol 3-kinase inhibitor wortmannin, dibutyryl cyclic AMP neither returned the peroxidase accumulation rate of v-Src-transformed cells to that of parental Rat-1/control cells, nor prevented macropinosome formation, as shown by confocal microscopy. Detailed analysis of the kinetics of tracer entry and efflux in transformed cells revealed that dibutyryl cyclic AMP inhibited peroxidase accumulation only after intervals &gt;5 minutes, due to accelerated peroxidase regurgitation, but did not alter the rate of transferrin recycling. Taken together, these data indicate that, in v-Src-transformed fibroblasts, macropinocytosis and micropinocytosis serve different pathways and that cyclic AMP affects neither micropinocytosis nor the formation of macropinosomes, but selectively promotes regurgitation therefrom.
Characterization of novel nuclear targeting and apoptosis-inducing domains in FAS associated factor 1
Authors: Fröhlich Thomas , Risau Werner , Flamme Ingo 
Published: April 2021

ABSTRACT
FAS associated factor 1 (FAF1) has been described as an unusual protein that binds to the intracellular portion of the apoptosis signal transducing receptor FAS/Apo-1 and potentiates apoptosis in L-cells. By means of mRNA differential display we have identified the avian homologue (qFAF) as a fibroblast growth factor-inducible gene in pluripotent cells from E0 quail embryos during mesoderm induction in vitro. Later during embryonic development, qFAF expression is ubiquitous. We confirm that qFAF is associated with FAS, and show that it is phosphorylated on serine residues and localized to the nucleus. By in vitro mutagenesis we have delimited a novel nuclear targeting domain to a short 35 amino acid alpha-helical region in the amino-terminal half of the protein. The nuclear function of qFAF remains unclear. However, a probably dominant negative deletion mutant of qFAF causes apoptosis of transfected cells. This function resides in the carboxyterminal domain of qFAF which shares remarkable sequence homologies with a putative ubiquitin conjugating enzyme from Caenorhabditis elegans. Our data indicate a complex function for FAF, which may be executed during FAS signalling and/or in the ubiquitination pathway, and may be essential for cell differentiation and survival.
Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes
Authors: Weil Miguel , Jacobson Michael D. , Raff Martin C. 
Published: April 2021

ABSTRACT
We show that mouse sperm die spontaneously within 1-2 days in culture and that treatment with either staurosporine (STS) and cycloheximide (CHX) or a peptide caspase inhibitor does not accelerate or delay the cell death. Chicken erythrocytes, by contrast, are induced to die by either serum deprivation or treatment with STS and CHX, and embryonic erythrocytes are more sensitive than adult erythrocytes to both treatments. Although these erythrocyte deaths display a number of features that are characteristic of apoptosis, they are not blocked, or even delayed, by peptide caspase inhibitors, and most of the cells die without apparently activating caspases. A small proportion of the dying erythrocytes do activate caspase-3, but even these cells, which seem to be the least mature erythrocytes, die just as quickly in the presence of caspase inhibitors. Our findings raise the possibility that both mouse sperm and chicken erythrocytes have a death programme that may not depend on caspases and that chicken erythrocytes lose caspases as they mature. Chicken erythrocytes may provide a useful ‘stripped down’ cell system to try to identify the protein components of such a death programme, which may serve to back-up the conventional caspase-dependent suicide mechanism in many cell types.
Endothelial cells assemble two distinct α6β4-containing vimentin-associated structures: roles for ligand binding and the β4 cytoplasmic tail
Authors: Homan Suzanne M. , Mercurio Arthur M. , LaFlamme Susan E. 
Published: April 2021

ABSTRACT
The α6β4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the α6β4 integrin. Because endothelial cells lose expression of α6β4 in culture, we expressed recombinant α6β4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing α6β4. Using immunofluorescence microscopy, we found that recombinant α6β4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the β4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant α6β4. In addition, this IL-2R-β4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant α6β4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the β4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant α6β4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The β4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant α6β4. Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct α6β4-containing and vimentin-associated structures on their basal surface and that the β4 cytoplasmic tail and the availability of specific α6β4 ligands regulate receptor localization to these structures.
The vitronectin receptor associates with clathrin-coated membrane domains via the cytoplasmic domain of its β5 subunit
Authors: Deyne Patrick G. De , O’Neill Andrea , Resneck Wendy G. , Dmytrenko George M. , Pumplin David W. , Bloch Robert J. 
Published: April 2021

ABSTRACT
Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing β3- and β5-integrins) and fibronectin receptors (FnR, containing β1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the β5 subunit of the VnR at both domains.
We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the β5 subunit of the VnR.
Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human β5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous β1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the β5 but not the β1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the β5 but not the β1 peptide. Thus, a sequence including the NPXY motif in the integrin β5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.
Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly
Authors: Chernousov Michael A. , Stahl Richard C. , Carey David J. 
Published: April 2021

ABSTRACT
Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. α5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.
Targeting of the myosin-I myr 3 to intercellular adherens type junctions induced by dominant active Cdc42 in HeLa cells
Authors: Stöffler Hanns-Eugen , Honnert Ulrike , Bauer Christian A. , Höfer Dirk , Schwarz Heinz , Müller Rainer T. , Drenckhahn Detlev , Bähler Martin 
Published: April 2021

ABSTRACT
Myr 3, a member of the myosin-I family from rat, is shown in this study to be localized at adherens-type intercellular junctions in epithelial and nonepithelial tissues. Formation of intercellular junctions and the accompanying recruitment of myr 3 to these junctions involves signaling by the Rho subfamily of small GTP-binding proteins. This conclusion is based on studies with HtTA-1 HeLa cells that were induced by overexpression of constitutively active Cdc42Hs to form typical adherens-type intercellular junctions enriched in cadherins (N-cadherin), β-catenin, filamentous actin and myr 3. Recruitement of myr 3 to Cdc42-induced adherens junctions in HeLa cells was dependent on a short region of the tail domain and a functional myosin motor domain, but was independent of its myosin-I tail homology and SH3 regions. Overexpression of constitutively active Rac1 induced a distinct type of adherens junction in HeLa cells that was characterized by elaborate intercellular interdigitations enriched in N-cadherin, β-catenin and F-actin. Myr 3 was often present, but not specifically enriched in the intercellular junctions induced by constitutively active Rac1.
Strategies of epithelial repair: Modulation of stem cell and transit amplifying cell proliferation
Authors: Lehrer Michael S. , Sun Tung-Tien , Lavker Robert M. 
Published: April 2021

ABSTRACT
Using double labeling techniques, we studied the replication of corneal epithelial stem cells that reside exclusively in the limbal zone, and their progeny transit amplifying cells. We show that corneal epithelial stem cells can be induced to enter DNA synthesis by wounding and by TPA. We demonstrate the existence of a hierarchy of TA cells; those of peripheral cornea undergo at least two rounds of DNA synthesis before they become post-mitotic, whereas those of central cornea are capable of only one round of division. However, the cell cycle time of these TA cells can be shortened and the number of times these TA cells can replicate is increased in response to wounding. These results thus demonstrate three strategies of epithelial repair: (i) stem cell replication, (ii) the unleashing of additional rounds of cell proliferation that remain as an untapped reserve under normal circumstances, and (iii) enhancement of TA cell proliferation via a shortening of the cycling time.