scholarly journals Stable inheritance of CENP-A chromatin: Inner strength versus dynamic control

2020 ◽  
Vol 219 (10) ◽  
Author(s):  
Sreyoshi Mitra ◽  
Bharath Srinivasan ◽  
Lars E.T. Jansen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Dynamic Control ◽  
Centromeric Chromatin ◽  
Long Term Stability ◽  
Binding Partners ◽  
Histone H3 Variant ◽  
Long Lifetime

Chromosome segregation during cell division is driven by mitotic spindle attachment to the centromere region on each chromosome. Centromeres form a protein scaffold defined by chromatin featuring CENP-A, a conserved histone H3 variant, in a manner largely independent of local DNA cis elements. CENP-A nucleosomes fulfill two essential criteria to epigenetically identify the centromere. They undergo self-templated duplication to reestablish centromeric chromatin following DNA replication. More importantly, CENP-A incorporated into centromeric chromatin is stably transmitted through consecutive cell division cycles. CENP-A nucleosomes have unique structural properties and binding partners that potentially explain their long lifetime in vivo. However, rather than a static building block, centromeric chromatin is dynamically regulated throughout the cell cycle, indicating that CENP-A stability is also controlled by external factors. We discuss recent insights and identify the outstanding questions on how dynamic control of the long-term stability of CENP-A ensures epigenetic centromere inheritance.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

scholarly journals Phosphorylation of Drosophila CENP-A on serine 20 regulates protein turn-over and centromere-specific loading

2019 ◽  
Vol 47 (20) ◽  
pp. 10754-10770 ◽  
Author(s):  
Anming Huang ◽  
Leopold Kremser ◽  
Fabian Schuler ◽  
Doris Wilflingseder ◽  
Herbert Lindner ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Posttranslational Modifications ◽  
Centromeric Chromatin ◽  
Phosphorylated Form ◽  
Specific Loading ◽  
Histone H3 Variant ◽  
Proteasome Pathway ◽  
Turn Over ◽  
Fine Tune

Abstract Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites.

scholarly journals Intrinsic elasticity of nucleosomes is encoded by histone variants and calibrated by their binding partners

2019 ◽  
Vol 116 (48) ◽  
pp. 24066-24074 ◽  
Author(s):  
Daniël P. Melters ◽  
Mary Pitman ◽  
Tatini Rakshit ◽  
Emilios K. Dimitriadis ◽  
Minh Bui ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Chromosome Segregation ◽  
Binding Proteins ◽  
Histone Variants ◽  
Histone Variant ◽  
Binding Partners ◽  
Damage Repair ◽  
Fine Tune

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins, which discriminate between histone variant nucleosomes, suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Taken together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.

scholarly journals Development and test for long-term stability of a synthetic standard for a quantitative Cryptosporidium parvum LightCycler™ PCR assay

2005 ◽  
Vol 3 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Renata Filkorn-Kaiser ◽  
Konrad Botzenhart ◽  
Albrecht Wiedenmann
Keyword(s):  
Cryptosporidium Parvum ◽  
Surrogate Marker ◽  
Target Sequence ◽  
Positive Trend ◽  
Long Term Stability ◽  
Synthetic Standard ◽  
One Year

A recently described quantitative rapid cycle real time PCR (LightCycler™) assay detects Cryptosporidium parvum after in vitro excystation, which is a surrogate marker for the viability of the organisms. In the original assay the quantification standard is a dilution series of C. parvum oocysts with a microscopically determined excystation rate. The need to keep suspensions of viable oocysts in stock and to continuously monitor their excystation rate, however, renders the assay impracticable for routine application. A synthetic standard was developed to replace the in vivo standard and was calibrated using oocysts with known excystation rates. The standard consists of a 486 bp DNA segment ranging from 229 bp upstream to 79 bp downstream of the actual PCR target site. Aliquots of the standard were frozen and stored at −20 °C and at −70 °C or lyophilised and stored at room temperature in the dark. For a period of one year samples preserved with each of the three methods were restored every four or five weeks. They were amplified in the LightCycler™ and the crossing points (CP) were monitored. No significant trend in the raw CP values could be observed for any of the three storage methods. However, when the methods were compared to each other by calculating the CP ratios (−20 °C/−70 °C; −20 °C/lyophilised; −70 °C/lyophilised) at the 10 monitoring dates, the CP ratios −20 °C/−70 °C and −20 °C/lyophilised showed a highly significant positive trend (p<0.0001) while the CP ratio −70 °C/lyophilised did not differ from the null hypothesis (p=0.53). It can be concluded that the latter two preservation methods are both appropriate, while storage at −20 °C is less advisable. Calculations based on the molecular weight of the standard and on the assumption of an average yield of three sporozoites per oocyst led to the conclusion that the target sequence is probably located on a double copy gene

Modifying Brushite Cement Degradation Using Calcium Alginate Beads

2007 ◽  
Vol 361-363 ◽  
pp. 311-314 ◽  
Author(s):  
Liam M. Grover ◽  
Sarika Patel ◽  
Y. Hu ◽  
Uwe Gbureck ◽  
J.E. Barralet
Keyword(s):  
Calcium Alginate ◽  
Alginate Beads ◽  
The Body ◽  
Cement Matrix ◽  
Calcium Alginate Beads ◽  
Long Term Stability ◽  
The Matrix ◽  
Implant Degradation

The hydrolysis of brushite in calcium phosphate cements to form hydroxyapatite is known to result in the long term stability of the material in the body. It has previously been established that this hydrolysis reaction can be influenced by implant volume, media refreshment rate and media composition. In this study, the effect of macroporosity on the rate of degradation of the material is investigated. Macroporosity was incorporated into the material using calcium alginate beads mixed into the cement paste. The inclusion of the calcium alginate beads did not influence the degree of conversion of the material and allowed the incorporation of porosity at up to maximum of 57%. The macroporosity weakened the cement matrix (from 46.5 to 3.2 MPa). When aged the brushite in the macroporous cement dissolved completely from the matrix resulting in a weight loss of 60wt% in a period of 28 days. This suggests that the controlled incorporation of calcium alginate beads into brushite cements in vivo can be used to control implant degradation rate.

Nontoxic Silicon Nanocrystals and Nanodiamonds as Substitution for Harmful Quantum Dots

2009 ◽  
Vol 1241 ◽  
Author(s):  
Anna Fucikova ◽  
Jan Valenta ◽  
Ivan Pelant ◽  
Vitezslav Brezina
Keyword(s):  
Quantum Dots ◽  
Chemical Composition ◽  
Silicon Nanocrystals ◽  
Nanocrystalline Silicon ◽  
Semiconductor Quantum Dots ◽  
In Vivo Studies ◽  
Long Term Stability

AbstractThe commercially available semiconductor quantum dots have been proven to be slightly to significantly toxic by recent publications depending on the chemical composition. We are developing new non-toxic fluorescent labels based on (i) nanocrystalline silicon, suitable for in vivo studies due to their biodegrability, and on (ii) nanodiamonds, intended mainly for in vitro use due to their long-term stability and nondegradilibity.

scholarly journals CENP-A nucleosome—a chromatin-embedded pedestal for the centromere: lessons learned from structural biology

2020 ◽  
Vol 64 (2) ◽  
pp. 205-221
Author(s):  
Ahmad Ali-Ahmad ◽  
Nikolina Sekulić
Keyword(s):  
Cell Division ◽  
Structural Biology ◽  
Histone H3 ◽  
Lessons Learned ◽  
Centromeric Chromatin ◽  
Centromere Proteins ◽  
Molecular Determinants ◽  
Histone H3 Variant ◽  
Segregation Of Chromosomes

Abstract The centromere is a chromosome locus that directs equal segregation of chromosomes during cell division. A nucleosome containing the histone H3 variant CENP-A epigenetically defines the centromere. Here, we summarize findings from recent structural biology studies, including several CryoEM structures, that contributed to elucidate specific features of the CENP-A nucleosome and molecular determinants of its interactions with CENP-C and CENP-N, the only two centromere proteins that directly bind to it. Based on those findings, we propose a role of the CENP-A nucleosome in the organization of centromeric chromatin beyond binding centromeric proteins.

scholarly journals ZapA tetramerization is required for midcell localization and ZapB interaction in Escherichia coli

2019 ◽  
Author(s):  
Nils Y. Meiresonne ◽  
Tanneke den Blaauwen
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bacterial Cell Division ◽  
Cellular Processes ◽  
Interaction Partners ◽  
Associated Proteins

AbstractBacterial cell division is guided by FtsZ treadmilling precisely at midcell. FtsZ itself is regulated by FtsZ associated proteins (Zaps) that couple it to different cellular processes. ZapA is known to enhance FtsZ bundling but also forms the synchronizing link with chromosome segregation through ZapB and matS bound MatP. ZapA exists as dimers and tetramers in the cell. Using the ZapAI83E mutant that only forms dimers, this paper investigates the effects of ZapA multimerization state on its interaction partners and cell division. By employing (fluorescence) microscopy and Förster Resonance Energy Transfer in vivo it is shown that; dimeric ZapA is unable to complement a zapA deletion strain and localizes diffusely through the cell but still interacts with FtsZ that is not part of the cell division machinery. Dimeric ZapA is unable to recruit ZapB, which localizes in its presence unipolarly in the cell. Interestingly, the localization profiles of the chromosome and unipolar ZapB anticorrelate. The work presented here confirms previously reported in vitro effects of ZapA multimerization in vivo and further places it in a broader context by revealing the strong implications for ZapB localization and ter linkage.

scholarly journals N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

2017 ◽  
Author(s):  
Kentaro Ohkuni ◽  
Reuben Levy-Myers ◽  
Jack Warren ◽  
Wei-Chun Au ◽  
Yoshimitsu Takahashi ◽  
...  
Keyword(s):  
Genome Stability ◽  
Histone H3 ◽  
Physiological Role ◽  
Centromeric Chromatin ◽  
E3 Ligases ◽  
Physiological Conditions ◽  
Evolutionarily Conserved ◽  
Histone H3 Variant

AbstractStringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

scholarly journals CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

2019 ◽  
Author(s):  
N Cortes-Silva ◽  
J Ulmer ◽  
T Kiuchi ◽  
E Hsieh ◽  
G Cornilleau ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Gene Editing ◽  
Mitotic Progression ◽  
Critical Function ◽  
Kinetochore Assembly ◽  
Histone H3 Variant ◽  
Essential Function ◽  
Necessary And Sufficient ◽  
Assembly Pathways

AbstractAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-deficient kinetochores of Lepidoptera (butterflies and moths). We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.

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