bluuebunny/crossref_metadata_2025_split huggingface dataset:
Association of the γ12 subunit of G proteins with actin filaments
Authors: Ueda Hiroshi , Saga Shinsuke , Shinohara Haruo , Morishita Rika , Kato Kanefusa , Asano Tomiko 
Published: April 2021

ABSTRACT
Recent studies have suggested an association between heterotrimeric G proteins, which play a major role in transmembrane signal transduction, and intracellular components. We therefore examined the subcellular localization of isoforms of G protein γ subunits in Swiss 3T3 and C6 glioma cells, mainly containing the γ5 and γ12 subunits. Immunocytochemical double staining with phalloidin showed co-localization of the γ12 subunit with actin filaments (F-actin), while the γ5 co-localized with vinculin, suggesting an association with focal adhesion. Pretreatment of cells with Triton X-100 eliminated the γ5 but not the γ12 staining. Co-localization of γ12 and F-actin was preserved when F-actin was disorganized with cytochalasin D or reorganized using fetal calf serum. Large amounts of γ12 were recovered in the vimentinand tubulin-free F-actin-rich fraction prepared from crude cytoskeleton preparations by double depolymerization-repolymerization. Co-localization of Gi2α, β and γ12 in the F-actin-rich fraction suggested the existence of γ12 as a βγ or heterotrimeric complex. Furthermore, purified βγ12 was found to associate with F-actin in vitro more tightly than βγ5. These results strongly suggest that the γ12 subunit associates with F-actin in cells. The observed differential distribution of γ12 and γ5 implies functional differences for the two γ subunits.
A programmed oxyradical burst causes hatching of mouse blastocysts
Authors: Thomas M. , Jain S. , Kumar G. P. , Laloraya M. 
Published: April 2021

ABSTRACT
The emergence of the mammalian blastocysts from their thick glycoprotein investment known as the zona pellucida is an important, but poorly understood, event in embryogenesis. In this paper, we demonstrate that peri-hatching blastocysts generate a considerably high quantum of an active oxyradical species for an extremely short period of time when compared to the pre-hatching (unhatched) and post-hatching (hatched) blastocysts. Hatching could be induced in pre-hatching blastocysts by exposing them to superoxide artificially generated to match the observed peri-implantation stage specific levels of superoxide, without impairing their viability. These observations suggest the operation of a superoxide-dependent hatching initiation in developing mammalian embryos.
NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export
Authors: Shan Xiaoyin , Xue Zhixiong , Euskirchen Ghia , Mélèse Teri 
Published: April 2021

ABSTRACT
The nuclear envelope is central to nuclear structure and function. It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body). We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions. Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate. They also have a fragmented nucleolous and accumulate poly(A) * RNA inside the nucleus. A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein. Our results suggest that Nnf1p plays a role in a number of nuclear functions.
Arachidonic acid stimulates protein kinase C-ε redistribution in heart cells
Authors: Huang Xu Pei , Pi YeQing , Lokuta Andrew J. , Greaser Marion L. , Walker Jeffery W. 
Published: April 2021

ABSTRACT
Arachidonic acid is elevated in a variety of cell types in response to extracellular stimuli, and has been hypothesized to exert at least some of its intracellular actions via activation of protein kinase C. Here we show that arachidonic acid stimulates a unique pattern of translocation of the μ-isoform of protein kinase C in isolated adult rat cardiac myocytes. Using western blot analysis, the majority of ε-protein kinase C was found in a cytosolic fraction in unstimulated cells. Treatment with 50 μM arachidonic acid caused a transient increase of δ-protein kinase C in a membrane fraction within 1 minute, then after 5-20 minutes most was found in a filament/nuclear fraction. Immunofluorescence and confocal microscopy of the filament fraction revealed a striated staining pattern with ε-protein kinase C localized near the Z-line where actin filaments are anchored and where transverse tubules are closely apposed to the myofilaments. δ-Protein kinase C, another isoform highly expressed in these cells, did not redistribute significantly in response to arachidonic acid, but in response to phorbol ester displayed a predominantly nuclear localization. Arachidonic acid also stimulated phosphorylation of the thin filament protein, troponin I, consistent with a filament localization for activated PKC. The physiological relevance of these findings was supported by the observation that 50 μM arachidonic acid promoted a 2.3-fold enhancement of myocyte twitch amplitude, an effect that was significantly blocked by the protein kinase C antagonist chelerythrine. Moreover, the onset of this physiological response correlated in time with translocation of ε-protein kinase C to the filaments. The results suggest that arachidonic acid initiates a redistribution of ε-protein kinase C to myofilament structures at or near the Z-line where this isozyme would be strategically located to regulate myofilament function and excitation-contraction coupling.
Cytosolic phospholipase A2 is activated by the hepatocyte growth factor receptor-kinase in Madin Darby canine kidney cells
Authors: Skouteris George G. , Schröder Claus H. 
Published: April 2021

ABSTRACT
The hepatocyte growth factor/scatter factor (HGF/SF) receptor which is a transmembrane protein encoded by the Met oncogene, possesses intrinsic tyrosine kinase activity which transduces the mitogenic, morphogenic and the scattering effect of HGF/SF. The pluripotent signal of HGF/SF is transduced through association of the Met receptor with various intracellular adaptors. Phosphorylation of cytosolic phospholipase A2 (cPLA2) is associated with activation of this molecule which in turn leads to arachidonic acid production followed by release of prostaglandins and related compounds exerting their roles onto cell proliferation, chemotaxis and vascular motility. Arachidonic acid and its metabolites were shown to be involved in processes like liver regeneration where growth factor receptors possessing tyrosine kinase activity are implicated. In this study we examined whether stimulation of the HGF/SF-receptor’s tyrosine kinase activity would involve changes in the phosphorylation state and the activity of cPLA2 in MDCK cells, where HGF/SF is known to induce scattering responses rather than mitogenesis. The activated p145βMET was shown to associate with and to phosphorylate cPLA2on tyrosine residues, this leading to subsequent release of arachidonic acid. cPLA2 was also phosphorylated in serine residues and such a role has been so far assigned to the mitogen activated protein (MAP) kinase. Our data have also shown that MAP kinase is associated and phosphorylated on tyrosine by the activated p145βMET Immunodepletion of MAP kinase via electroporation of an anti-MAP kinase antibody, did not significantly decrease arachidonic acid release in HGF/SF-stimulated MDCK cells. It is therefore emerging that phosphorylation of cPLA2 on tyrosine by the HGF/SF receptor kinase is capable of triggering arachidonic acid release and that MAP kinase is contributing to full, but does not drive, the activity of cPLA2. The release of arachidonic acid by MDCK cells following HGF/SF stimulation is establishing this fatty acid and its metabolites as major components involved in the transduction of MET-driven signals and at the same time in the amplification of such signals.
A specific domain in α-catenin mediates binding to β-catenin or plakoglobin
Authors: Huber Otmar , Krohn Michael , Kemler Rolf 
Published: April 2021

ABSTRACT
The E-cadherin-catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either β-catenin or plakoglobin, which both can assemble α-catenin into the complex. Recently we have identified an α-catenin binding site in β-catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in α-catenin which mediates its interaction with β-catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of α-catenin expressed as recombinant fusion proteins, we found that the Nterminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117-143) which is necessary and sufficient to bind β-catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two α-helices.
Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II
Authors: Grande Marjolein A. , Kraan Ineke van der , Jong Luitzen de , Driel Roel van 
Published: April 2021

ABSTRACT
We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogenously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 μm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.
Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast
Authors: Yamada Hiroshi , Kumada Kazuki , Yanagida Mitsuhiro 
Published: April 2021

ABSTRACT
We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcn1, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pka1) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/cyclosome function through protein-protein interactions and phosphorylation.
Transient increase of free cytosolic calcium during neutrophil motility responses
Authors: Alteraifi Abdullatif M. , Zhelev Doncho V. 
Published: April 2021

ABSTRACT
The release of free cytosolic calcium is a secondary messenger for many cell functions. Here we study the coupling between the release of intracellular calcium and motility responses of the human neutrophil. Two groups of motility responses are studied: motility responses in the presence of adhesion, such as cell crawling and phagocytosis, and motility responses ‘in suspension’, such as pseudopod formation. The motility responses are stimulated by the chemoattractant N-formyl-methionyl-leucylphenylalanine (fMLP) and the release of calcium is monitored by measuring the fluorescence from fluo-3. fMLP induces a single release of free cytosolic calcium both in suspended cells and in crawling cells. Calcium release is a threshold process where the number of cells releasing calcium is dependent on the chemoattractant concentration while the amount of released calcium is not. For suspended cells the threshold fMLP concentration for calcium release is in the order of 10−7 M, while for crawling cells it is in the order of 5×10−9 M. The smaller value of the threshold fMLP concentration for crawling cells compared to that for suspended cells suggests that bound adhesion receptors are involved in the calcium release. The threshold fMLP concentration for suspended cells is also larger than the minimum fMLP concentration (in the order of 10−10 M) for initiating pseudopod formation. So, there is a range of fMLP concentrations where pseudopod formation occurs without calcium release. To explore this relationship further, pseudopod extension and calcium release are stimulated many times in a single cell by using fMLP concentrations above the threshold. The result is that calcium release is desensitized by fMLP while pseudopod extension is not. All the results taken together suggest that the release of free cytosolic calcium and the rearrangement of the F-actin network during motility follow different signaling pathways.
Cell-specific expression and heat-shock induction of Hsps during spermatogenesis in Drosophila melanogaster
Authors: Michaud Sébastien , Marin Raquel , Westwood J. Timothy , Tanguay Robert M. 
Published: April 2021

ABSTRACT
The developmental and heat-shock-induced expression of two small heat-shock proteins (Hsp23 and Hsp27) was investigated during spermatogenesis in Drosophila melanogaster. Both of these Hsps were expressed in unstressed and stressed male gonads as shown by immunoblotting. Immunostaining of whole-mount organs and thin sections of testes showed that an anti-Hsp23 antibody specifically decorated cells of the somatic lineage, such as the cyst cells and the epithelial cells of the testis and of the seminal vesicle. Hsp27 was expressed in some somatic cells (cyst cells and epithelial cells of the accessory glands) and, in addition, was also visible in the maturing spermatocytes of the germline. The same cell-specific pattern of expression was observed after heat shock, and cells which did not express Hsp23 and Hsp27 in the absence of stress were similarly unable to mount a heat shock response for these s-Hsps. However other Hsps such as Hsp70 and Hsp22 were induced under heat-shock conditions in testes. Actinomycin D prevented the heat-induced accumulation of these Hsps indicating that the induction of Hsps was regulated at the transcriptional level. The heat shock transcriptional factor of Drosophila (DmHSF), present in significantly lower amount in testes when compared to other tissues such as the head, was shown to be required for the heat activation of Hsp22 and Hsp70. Immunostaining revealed that HSF expression was restricted to specific cells such as cyst cells, epithelial pigment cells, spermatogonia and spermatids but not the primary spermatocytes. These data show that the expression and induction of the different small Hsps is regulated in a cell-specific manner under both normal and heat shock conditions and suggest that factors other than the DmHSF are involved in this regulation in male gonads.
An actin-related protein in Drosophila colocalizes with heterochromatin protein 1 in pericentric heterochromatin
Authors: Frankel Stewart , Sigel Eric A. , Craig Carolyn , Elgin Sarah C. R. , Mooseker Mark S. , Artavanis-Tsakonas Spyros 
Published: April 2021

ABSTRACT
The actin-related proteins have been identified by virtue of their sequence similarity to actin. While their structures are thought to be closely homologous to actin, they exhibit a far greater range of functional diversity. We have localized the Drosophila actin-related protein, Arp4, to the nucleus. It is most abundant during embryogenesis but is expressed at all developmental stages. Within the nucleus Arp4 is primarily localized to the centric heterochromatin. Polytene chromosome spreads indicate it is also present at much lower levels in numerous euchromatic bands. The only other protein in Drosophila reported to be primarily localized to centric heterochromatin in polytene nuclei is heterochromatin protein 1 (HP1), which genetic evidence has linked to heterochromatin-mediated gene silencing and alterations in chromatin structure. The relationship between Arp4 and heterochromatin protein 1 (HP1) was investigated by labeling embryos and larval tissues with antibodies to Arp4 and HP1. Arp4 and HP1 exhibit almost superimposable heterochromatin localization patterns, remain associated with the heterochromatin throughout prepupal development, and exhibit similar changes in localization during the cell cycle. Polytene chromosome spreads indicate that the set of euchromatic bands labeled by each antibody overlap but are not identical. Arp4 and HP1 in parallel undergo several shifts in their nuclear localization patterns during embryogenesis, shifts that correlate with developmental changes in nuclear functions. The significance of their colocalization was further tested by examining nuclei that express mutant forms of HP1. In these nuclei the localization patterns of HP1 and Arp4 are altered in parallel fashion. The morphological, developmental and genetic data suggest that, like HP1, Arp4 may have a role in heterochromatin functions.
MPM-2 antibody-reactive phosphorylations can be created in detergentextracted cells by kinetochore-bound and soluble kinases
Authors: Renzi L. , Gersch M. S. , Campbell M. S. , Wu L. , Osmani S. A. , Gorbsky G. J. 
Published: April 2021

ABSTRACT
The MPM-2 antibody labels mitosis-specific and cell cycleregulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) α and β? In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosophoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.
Out of balance: consequences of a partial keratin 10 knockout
Authors: Reichelt Julia , Bauer Christoph , Porter Rebecca M. , Lane E. Birgitte , Herzog Volker , Magin Thomas M. 
Published: April 2021

ABSTRACT
Recently we generated keratin 10 knockout mice which provided a valuable model for the dominantly inherited skin disorder epidermolytic hyperkeratosis. Here we investigated the molecular basis for their phenotype. Hetero- and homozygotes expressed a truncated keratin 10 peptide which has been identified directly by microsequencing. Epitope mapping of monoclonal antibodies to keratin 10T enabled us to study its distribution relative to keratin 6, which is highly expressed in keratin 10 knockout mice, by double-immunogold electron microscopy. This revealed that keratin 10T was restricted to complexes with keratin 1 but did not mix with keratin 6. The latter did not form extended filaments with keratins 16/17 but aggregates. Keratins 6/16 were unable to compensate for the lack of normal keratin 1/10 filaments. Remarkably keratin 6 aggregates strictly colocalized with keratohyalin granules. Residual keratin 1/10T clumps were located in the cell periphery and at desmosomes which maintained a normal architecture. Surprisingly keratin 2e, a keratin tailored to sustain mechanical stress, was completely lost in paw sole epidermis of homozygous keratin 10 knockout mice, pointing to keratin 10 as its partner. The selective pairing of keratin 10T and the loss of keratin 2e indicate that in vivo keratins are less promiscuous than in vitro. Skin fragility in keratin 10 knockout mice and in epidermolytic hyperkeratosis is probably the consequence of two complementing mechanisms namely a decrease of normal keratin 1/10 filaments and an increase in keratins 6/16 with a poor filament-forming capacity.