65 NUCLEAR REPROGRAMMING OF PORCINE CELLS AND THEIR USE AS DONOR CELL FOR NUCLEAR TRANSFER AFTER TREATMENT IN XENOPUS EGG EXTRACTS

2007 ◽  
Vol 19 (1) ◽  
pp. 150 ◽  
Author(s):  
K. Miyamoto ◽  
M. Ohnuki ◽  
N. Minami ◽  
M. Yamada ◽  
H. Imai
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Revealing an adequate cell state for nuclear reprogramming is essential to achieve efficient production of cloned embryos and animals. Previous reports suggest that nuclei from undifferentiated cells such as blastomeres or embryonic stem cells can support efficient development of cloned embryos to term. In recent years, differentiated somatic cells are frequently used for donor cells because of ease of preparation and application for genetic modification. The efficiency of the somatic cell nuclear transfer (SCNT) is still extremely low. We hypothesized that somatic cells that had been reprogrammed to dedifferentiated states before SCNT might support higher developmental ability of SCNT embryos. To test this hypothesis, porcine fibroblast cells were treated with Xenopus egg extracts, and the extract-treated cells (ETCs) were used as donor cell for SCNT to examine their ability to support early embryonic development. Xenopus egg extracts were prepared from activated S-phase eggs. Porcine fibroblast cells (106/mL) were permeabilized by 500 ng mL-1 of Streptolysin O and were incubated in the egg extracts with the energy-regenerating system for 2 hours at 23�C. After the extract treatment, permeabilized membranes were resealed in DMEM containing 2 mM CaCl2. The ETCs were fused with porcine enucleated oocytes and simultaneously activated. The reconstructed embryos were cultured in PZM-3 medium for 7 days. All statistical differences were analyzed by ANOVA. Reprogramming of ETCs was evaluated on changes of chromatin states and gene expression. Chromatin-binding proteins of ETCs were separated and analyzed on SDS-PAGE. Some proteins were incorporated onto and/or released from chromatins after the extract treatment. Especially, Xenopus egg-specific linker histone B4 was assembled on chromatins. Non-permeabilized control cells did not show these protein exchanges. Deacetylation of histone H3 lysine9 was detected in half number of ETCs in an ATP-dependent manner. In contrast, a high population of histone H3-acetylated cells was observed in buffer-treated cells as well as cells before the extract treatment. The pluripotent marker gene expression, such as OCT4 and SOX2, was also observed in ETCs after culture. The gene expression of these genes was not detected in non-treated cells. These results indicate that the extract treatment induces or triggers a part of dedifferentiation of somatic cells. These ETCs were used as donor cell for SCNT, and reconstructed cloned embryos were cultured. SCNT embryos showed no significant difference in cleavage rates and developmental rates to the blastocyst stage (25%) compared with non-treated control cells (26%). However, the total cell number of embryos at the blastocyst stage was significantly higher in SCNT embryos from ETCs compared with those of control cells (62 � 7 vs. 43 � 2, respectively; P < 0.05). These results indicate that the extract treatment before nuclear transfer may stimulate cell proliferation of SCNT embryos but not improve early development. More studies, however, are needed to investigate their developmental ability to term.

3 NUCLEAR REPROGRAMMING OF PORCINE FIBROBLAST CELLS BY XENOPUS EGG EXTRACTS

2006 ◽  
Vol 18 (2) ◽  
pp. 110 ◽  
Author(s):  
K. Miyamoto ◽  
Y. Nagao ◽  
N. Minami ◽  
M. Yamada ◽  
K. Ohsumi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Marker Gene ◽  
Calcium Ionophore ◽  
Pcr Analysis ◽  
Ionophore A23187 ◽  
Fibroblast Cells ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Much evidence indicates that somatic cells can be reprogrammed in an oocyte cytoplasm. The nuclear reprogramming consists of many unknown processes, and mechanisms underlying these processes still remain to be elucidated. Recently some reports noted that Xenopus oocytes or eggs can induce some of the reprogramming events in mammalian cells. We investigated the processes of nuclear reprogramming of porcine fibroblast cells by Xenopus egg extracts to understand how egg extracts trigger the reprogramming and/or dedifferentiation of cells. Unfertilized Xenopus eggs were collected from mature females. After removal of the jelly coat, activation was routinely achieved by calcium ionophore A23187. The eggs were immediately centrifuged and the cytoplasmic fraction was used as egg extracts. Porcine fibroblast cells were permeabilized by streptolysin O and incubated in the egg extracts under the ATP-generating system (1 mM ATP, 5 mM phosphocreatine, and 20 U/mL creatine kinase) for 30 min at 37�C or 2 h at 23�C. The incorporation of Xenopus-specific linker histone B4 into porcine fibroblasts was examined by immunofluorescence and immunobloting analysis. After collection of cells from the extracts, permeabilized membranes of the cells were resealed in culture medium containing 2 mM CaCl2 for 2 h. The cells were then incubated in DMEM with 10% fetal bovine serum (FBS) or porcine zygote medium-3 (PZM-3: Yoshioka et al. 2002 Biol. Reprod. 66, 112-119) containing 5.55 mM glucose and 5% FBS. RNAs were extracted from the cells in each culture dish and Oct-4 expression was examined by RT-PCR analysis every day until Day 8. The primers were designed to span the 99 base-pair intron region of porcine Oct-4 gene for recognizing both spliced and unspliced transcripts. The incorporation of histone B4 from Xenopus egg extracts was observed at the nuclear region of the porcine fibroblasts under both the 37�C and the 23�C conditions. Because the histone B4 incorporation was inhibited by addition of Apyrase, an ATPase, a part of reprogramming might be an ATP-dependent process. When treated cells were incubated in DMEM or PZM-3, Oct-4 expression was detected in the cells cultured in DMEM, but not in PZM-3. However, the transcripts of Oct-4 were mainly obtained in unspliced form at the earlier stage of culture (after Day 1 to Day 4 of culture), suggesting that a part of reprogramming processes by the egg extracts involves induction of dedifferention of cells or activation of a pluripotent marker gene such as Oct-4. Xenopus egg extract may provide a system to investigate the processes involving nuclear reprogramming and the pluripotent state of mammalian cells in vitro.

121 DEMETHYLATION OF MAMMALIAN SOMATIC DNA BY XENOPUS EGG AND OOCYTE EXTRACTS

2006 ◽  
Vol 18 (2) ◽  
pp. 169
Author(s):  
Y. Bian ◽  
R. Alberio ◽  
A. Johnson ◽  
K. Campbell
Keyword(s):  
Dna Replication ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Donor Cell ◽  
Dna Demethylation ◽  
Xenopus Laevis Oocyte ◽  
Dependent Manner ◽  
Embryonic Cells ◽  
Egg Extracts ◽  
Xenopus Egg

In mammals, the successful development of live offspring by somatic cell nuclear transfer (SCNT) has demonstrated the ability of oocyte or egg cytoplasm to reprogram the differentiated status of somatic DNA. However, the efficiency of development is low, and this has been attributed to incomplete or inappropriate reprogramming of epigenetic status. One such epigenetic marker is methylation of genomic DNA at CpG islands. In SCNT, derived embryo abnormal DNA methylation patterns have been reported by a number of groups; in particular, it has been observed that the methylation pattern of embryonic cells resembles that of the donor cell (Santos et al. 2003 Curr. Biol. 13, 1116-1121). One strategy to improve reprogramming and, hence, development is to erase or reprogram the epigenetic status of the donor cell prior to nuclear transfer. We have previously reported that Xenopus egg and oocyte extracts show a differential effect on transcription. In oocyte extracts Pol I and II transcripts are maintained in the somatic cells; in egg extracts, these are abolished (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). To extend these studies, we have investigated the ability of oocyte and egg extracts to demethylate bovine somatic DNA. Preparation of Xenopus oocyte and egg extracts, culture, permeabilization of donor cells, and incubation conditions were all as previously described (Alberio et al. 2005 Exp. Cell. Res. 307, 131-141). Cells were incubated in extracts for 1 and 3 h at 21�C, centrifuged onto glass slides fixed in 4% Para formaldehyde for 15 min, followed by 4 M HCL for 1 h at 39�C, and blocked for 1 h. Cells were stained with mouse monoclonal anti-1MeC (1:50) overnight at 4�C followed by FITC-conjugated goat anti-mouse antibody (1:20) for 1 h at room temperature and mounted in Vectashied containing 10 �g of propidium iodide/mL. Nuclei were scored as positive or negative for 5MeC staining. In control cells, 90% of nuclei stained positively for 5MeC. In both oocyte and egg extracts the number of positive nuclei decreased with time showing demethylation of the somatic DNA 68 and 58% and 38 and 42% positive, respectively, after 1 and 3 h of incubation. Addition of apyrase (2%) to hydrolyze ATP inhibited demethylation in both extracts (90% nuclei positive). High rates of DNA replication were observed in somatic cells in egg extracts in contrast to no replication in oocyte extracts. Aphidicolin (1 �g/20 �L) was added to egg extracts to inhibit DNA replication, and under these conditions, DNA demethylation was abolished, suggesting a passive DNA demethylation mechanism as a result of DNA replication. In conclusion, Xenopus laevis oocyte and egg extracts can demethylate mammalian somatic DNA in an energy-dependent manner. In oocyte extracts, demethylation is independent of DNA replication, suggesting an active mechanism. In egg extracts, DNA replication is required, suggesting a passive mechanism. These studies further demonstrate the differences in reprogramming activities between oocyte and egg cytoplasm and suggest that interspecies extracts may provide a tool for nuclear reprogramming.

273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS

2009 ◽  
Vol 21 (1) ◽  
pp. 234 ◽  
Author(s):  
C.-Y. Chiang ◽  
P.-C. Tang
Keyword(s):  
Somatic Cells ◽  
Egfp Expression ◽  
3T3 Cells ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.

65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 138
Author(s):  
K. M. Whitworth ◽  
J. Zhao ◽  
L. D. Spate ◽  
R. S. Prather
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Real Time ◽  
Histone Deacetylase ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Transcriptional Profiling ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Hdac Activity

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.

Long-term effect on in vitro cloning efficiency after treatment of somatic cells with Xenopus egg extract in the pig

2014 ◽  
Vol 26 (7) ◽  
pp. 1017 ◽  
Author(s):  
Ying Liu ◽  
Olga Østrup ◽  
Rong Li ◽  
Juan Li ◽  
Gábor Vajta ◽  
...  
Keyword(s):  
Donor Cell ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Long Term Effect ◽  
Donor Cells ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7–22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.

scholarly journals Cats cloned from fetal and adult somatic cells by nuclear transfer

Reproduction ◽  
2005 ◽  
Vol 129 (2) ◽  
pp. 245-249 ◽  
Author(s):  
X J Yin ◽  
H S Lee ◽  
Y H Lee ◽  
Y I Seo ◽  
S J Jeon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Somatic Cells ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Skin Cells ◽  
Fetal Fibroblasts ◽  
Adult Skin

This work was undertaken in order to study the developmental competence of nuclear transfer (NT ) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.

scholarly journals 23REPROGRAMMING MAMMALIAN CELLS IN XENOPUS EGG EXTRACTS: ROLE OF EMBRYONIC LAMIN B3

2004 ◽  
Vol 16 (2) ◽  
pp. 134
Author(s):  
R. Alberio ◽  
K.H.S. Campbell
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Somatic Cells ◽  
Nuclear Transplantation ◽  
Heterologous System ◽  
Fetal Fibroblasts ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The generation of animals by nuclear transplantation has demonstrated that a fully differentiated cell can be reversed into totipotency when transferred into an oocyte. Identification of oocyte specific molecules responsible for the reprogramming of somatic cells may contribute to the understanding of cell differentiation and embryo development. We have developed a heterologous system to investigate the effect of lamin B3, a major component of Xenopus laevis egg cytoplasm, on DNA replication of mammalian somatic cells. Bovine fetal fibroblasts were arrested at G1/S by incubation in aphidicolin for 18h. After permeabilization with digitonin, the cells were incubated in either (1) lamin B3 depleted, or (2) whole Xenopus egg extracts (1000 cells μL−1 extract) supplemented with an energy regenerating system for a period of 3h at 21°C. Xenopus lamin B3-depleted egg extracts were prepared by three rounds of incubation with Dynabeads coated with a mouse monoclonal lamin B3 antibody (mAbLB3). Immunodepletion was confirmed by western blotting. Purified lamin B3 was obtained by dialysis of the beads after immunodepletion, and the purified lamin B3 was used for rescue experiments. DNA replication of cells incubated in the extracts was assessed by adding 25μM Biotin-11-dUTP for 3h. After treatment cells were fixed in 70% methanol at −20°C and incubated in mAbLB3 for 30min at 37°C. This was followed by incubation in FITC-conjugated sheep anti-mouse antibody and in 5mgmL−1 Texas Red-conjugated Streptavidin for 40min at 37°C. After three hours’ incubation in egg extracts, DNA replication was detected in 60% of cells and more than 95% of cells were lamin B3 positive. In contrast, DNA replication in immunodepleted extracts was significantly lower (P≤0.01, by one-way ANOVA) than in cells incubated in whole extracts and was coincident with the few lamin B3-positive cells observed. More than 95% of cells were lamin B3-negative and did not replicate DNA. When purified lamin B3 was re-added to depleted extracts, DNA replication was detected in 60% of cells. DNA synthesis resumed in 93% of control cells 3h after release from aphidicolin into culture medium at 39°C. These experiments show that somatic nuclei, which possess a nuclear envelope with somatic variants of lamins, are able to synthesize DNA in egg extracts only when Xenopus lamin B3 is incorporated into the nuclear envelope. This heterologous system provides new information on the role of an embryonic molecule, namely Xenopus lamin B3, in the reprogramming of DNA replication of somatic cells incubated in egg environment. These results open new questions as to whether embryonic lamins also exist in mammals, and whether failure in development of cloned animals is in part due to abnormal or incomplete replacement of somatic variants of proteins with their embryonic counterparts.

scholarly journals Stathmin/Op18 Phosphorylation Is Regulated by Microtubule Assembly

2001 ◽  
Vol 12 (2) ◽  
pp. 437-448 ◽  
Author(s):  
Thomas Küntziger ◽  
Olivier Gavet ◽  
Valérie Manceau ◽  
André Sobel ◽  
Michel Bornens
Keyword(s):  
Feedback Loop ◽  
Somatic Cells ◽  
Network Control ◽  
Microtubule Assembly ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

Stathmin/Op 18 is a microtubule (MT) dynamics-regulating protein that has been shown to have both catastrophe-promoting and tubulin-sequestering activities. The level of stathmin/Op18 phosphorylation was proved both in vitro and in vivo to be important in modulating its MT-destabilizing activity. To understand the in vivo regulation of stathmin/Op18 activity, we investigated whether MT assembly itself could control phosphorylation of stathmin/Op18 and thus its MT-destabilizing activity. We found that MT nucleation by centrosomes from Xenopus sperm or somatic cells and MT assembly promoted by dimethyl sulfoxide or paclitaxel induced stathmin/Op18 hyperphosphorylation in Xenopus egg extracts, leading to new stathmin/Op18 isoforms phosphorylated on Ser 16. The MT-dependent phosphorylation of stathmin/Op18 took place in interphase extracts as well, and was also observed in somatic cells. We show that the MT-dependent phosphorylation of stathmin/Op18 on Ser 16 is mediated by an activity associated to the MTs, and that it is responsible for the stathmin/Op18 hyperphosphorylation reported to be induced by the addition of “mitotic chromatin.” Our results suggest the existence of a positive feedback loop, which could represent a novel mechanism contributing to MT network control.

scholarly journals Anaphase A Chromosome Movement and Poleward Spindle Microtubule Flux Occur At Similar Rates in Xenopus Extract Spindles

1998 ◽  
Vol 141 (3) ◽  
pp. 703-713 ◽  
Author(s):  
Arshad Desai ◽  
Paul S. Maddox ◽  
Timothy J. Mitchison ◽  
E.D. Salmon
Keyword(s):  
Somatic Cells ◽  
Cell Types ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Spindle Microtubule ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
The Difference ◽  
Xenopus Egg Extracts ◽  
Different Cell Types

We have used local fluorescence photoactivation to mark the lattice of spindle microtubules during anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and anaphase A chromosome movement occur at similar rates (∼2 μm/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymerization near the spindle poles, is the predominant mechanism for anaphase A in Xenopus egg extracts. In contrast, in vertebrate somatic cells a “Pacman” kinetochore mechanism, coupled to microtubule depolymerization near the kinetochore, predominates during anaphase A. Consistent with the conclusion from fluorescence photoactivation analysis, both anaphase A chromosome movement and poleward spindle microtubule flux respond similarly to pharmacological perturbations in Xenopus extracts. Furthermore, the pharmacological profile of anaphase A in Xenopus extracts differs from the previously established profile for anaphase A in vertebrate somatic cells. The difference between these profiles is consistent with poleward microtubule flux playing the predominant role in anaphase chromosome movement in Xenopus extracts, but not in vertebrate somatic cells. We discuss the possible biological implications of the existence of two distinct anaphase A mechanisms and their differential contributions to poleward chromosome movement in different cell types.

Reversible Membrane Permeabilization of Mammalian Cells Treated with Digitonin and Its Use for Inducing Nuclear Reprogramming by Xenopus Egg Extracts

2008 ◽  
Vol 10 (4) ◽  
pp. 535-542 ◽  
Author(s):  
Kei Miyamoto ◽  
Teruyoshi Yamashita ◽  
Tomoyuki Tsukiyama ◽  
Naoya Kitamura ◽  
Naojiro Minami ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Membrane Permeabilization ◽  
Nuclear Reprogramming ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
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