scholarly journals NuSAP, a Mitotic RanGTP Target That Stabilizes and Cross-links Microtubules

2006 ◽  
Vol 17 (6) ◽  
pp. 2646-2660 ◽  
Author(s):  
Katharina Ribbeck ◽  
Aaron C. Groen ◽  
Rachel Santarella ◽  
Markus T. Bohnsack ◽  
Tim Raemaekers ◽  
...  
Keyword(s):  
Cross Linking ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Large Numbers ◽  
In Vitro Reconstitution ◽  
Cross Links ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Interfering Rna

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) α, Impβ, and Imp7. While Impα and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impβ specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.

scholarly journals Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

PLoS ONE ◽  
2008 ◽  
Vol 3 (12) ◽  
pp. e3936 ◽  
Author(s):  
Julie Cahu ◽  
Aurelien Olichon ◽  
Christian Hentrich ◽  
Henry Schek ◽  
Jovana Drinjakovic ◽  
...  
Keyword(s):  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

scholarly journals In vitro assembly of coiled bodies in Xenopus egg extract.

1994 ◽  
Vol 5 (6) ◽  
pp. 633-644 ◽  
Author(s):  
D W Bauer ◽  
C Murphy ◽  
Z Wu ◽  
C H Wu ◽  
J G Gall
Keyword(s):  
Rna Processing ◽  
Mrna Splicing ◽  
Immunofluorescent Staining ◽  
Rrna Processing ◽  
Xenopus Egg Extract ◽  
Coiled Bodies ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Sperm Nuclei

When demembranated sperm nuclei are placed in a Xenopus egg extract, they become surrounded by a nuclear envelope and then swell to form morphologically typical pronuclei. Granules ranging from < 1.0 to approximately 3.0 microns in diameter appear within such nuclei. Bell et al. identified four nucleolar proteins in these "prenucleolar bodies" by immunofluorescent staining (fibrillarin, nucleolin, B23/NO38, 180-kDa nucleolar protein). By in situ hybridization we show that these bodies also contain U3 and U8 small nuclear RNAs (snRNAs), known to be involved in pre-rRNA processing. Moreover, they contain all the snRNAs involved in pre-mRNA splicing (U1, U2, U4, U5, and U6), as well as U7, which is required for histone pre-mRNA 3' end formation. In addition to the nucleolar antigens previously identified, we demonstrated staining with antibodies against the Sm epitope, trimethylguanosine, and coilin. Because the composition of these prenucleolar bodies is closer to that of coiled bodies than to nucleoli, we propose that they be referred to as coiled bodies. The existence of large coiled bodies in transcriptionally inactive pronuclei suggests that they may play a role in the import, assembly, and storage of RNA processing components but are not themselves sites of processing. In transcriptionally active nuclei coiled bodies could serve as sites for initial preassembly and distribution of snRNP complexes for the three major RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3' end formation.

scholarly journals In vitro assembly of prenucleolar bodies in Xenopus egg extract.

1992 ◽  
Vol 118 (6) ◽  
pp. 1297-1304 ◽  
Author(s):  
P Bell ◽  
M C Dabauvalle ◽  
U Scheer
Keyword(s):  
Experimental System ◽  
Nucleolus Organizer ◽  
Structural Element ◽  
Interphase Nuclei ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg ◽  
Functional Features ◽  
Nucleolar Components

Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.

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.

23 IMPROVING EFFICIENCY IN WORK WITH TRANSFER OF CLONED PIG EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 126 ◽  
Author(s):  
H. Callesen ◽  
Y. Liu ◽  
R. Li ◽  
M. Schmidt
Keyword(s):  
Large White ◽  
Mean Values ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Donor Cells ◽  
Chi Squared ◽  
Xenopus Egg ◽  
Working Day ◽  
One Way Anova

Cloning is a quite inefficient procedure with only around 10% of offspring born based on number of cloned embryos transferred. Every step to increase this level is therefore welcomed. Our group has worked with cloning of pig embryos since 2006, with the main purpose to establish a well-functioning cloning system to have transgenic piglets born as animal models for important human diseases. Here we report results from our attempts to improve efficiencies in several steps in the whole cloning procedure. Over 7.5 years, donor cells from 3 breeds were nontransgenic (50%, 4 types) or transgenic with 1 of 6 different types of gene. Oocytes from Large White (LW) sows or gilts were handmade cloned, so the zona-free cloned embryos were in vitro cultured until Day 5 to 6 to select 13 311 embryos (morulae or blastocysts) for transfer to 171 LW recipient sows or gilts. Of these, 126 were pregnant (74%; Day 35), but 20 aborted before term. A total of 704 offspring were delivered; half of the piglets were alive after 4 weeks and developed normally after that. Frequencies were compared using Chi-squared test; mean values by one-way ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Specific improvements were tested in 4 areas: (1) donor cells: stimulating reprogramming using Xenopus egg extract (Liu et al. 2013 Reprod. Fertil. Dev. http://dx.doi.org/10.1071/RD13147); (2) oocytes: preferably from sows, but also using larger gilt oocytes (Li et al. 2013 Zygote http://dx.doi.org/10.1017/S0967199412000676); (3) transfer: using both uterine sides (Theriogenology 74 : 1233); (4) number of embryos transferred: after cloning with same nontransgenic cells, embryo numbers per recipient were reduced from 90 to 30 (see Table 1). As a consequence of these different activities, overall results improved over the 7.5-year period [first 3.5 years v. last 4 years: 48% (32/67) v. 90% (94/104) recipients pregnant after transfer (P < 0.05); 5.6 ± 0.6 (n = 22) v. 6.9 ± 0.5 (n = 84) piglets/litter]. In our system, one good cloning person can now produce all embryos needed for one recipient in one good working day. Transfer of fewer cloned embryos results in fewer piglets, but it reduces the workload to produce cloned embryos and does not reduce efficiency. Further work is still needed to better understand the biological and technical challenges in work with cloning; 2 important areas are quality evaluation of the donor cells used for cloning and the recipient's reaction to transfer of many embryos. In conclusion, a reasonable increase in the overall efficiency in pig cloning work was achieved, which reduces the need for personnel, time, and material when working with this technology. Table 1.Results of improving efficiency of cloning

Prenucleolar bodies contain coilin and are assembled in Xenopus egg extract depleted of specific nucleolar proteins and U3 RNA

1997 ◽  
Vol 110 (1) ◽  
pp. 43-54 ◽  
Author(s):  
P. Bell ◽  
U. Scheer
Keyword(s):  
Cultured Cells ◽  
Ultrastructural Level ◽  
Nuclear Bodies ◽  
Xenopus Egg Extract ◽  
Nucleolar Proteins ◽  
Coiled Bodies ◽  
Egg Extract ◽  
Structural Association ◽  
Xenopus Egg

Nuclei assembled in Xenopus egg extract contain numerous spherical aggregations or nuclear bodies. Previously we have shown that they closely resemble prenucleolar bodies (PNBs), both at the compositional and ultrastructural level. Subsequently, coilin was also identified and for this reason they were called coiled bodies. Here we present morphological and immunocytochemical evidence that the in vitro nuclear bodies resemble authentic PNBs and are different from coiled bodies. In particular we show that coilin, previously considered as the defining protein constituent of coiled bodies, is also present in PNBs of cultured cells. In contrast, the PNB-associated nucleolar proteins nucleolin and B23/NO38 are not detectable in coiled bodies and may thus serve as suitable markers for PNBs. Our results suggest that PNBs are primary assembly structures which contribute to the formation of both nucleoli and coiled bodies and thus offer an explanation for the frequently observed structural association of coiled bodies with nucleoli. To gain some insight into the assembly process of PNBs in vitro, specific nucleolar proteins were removed from Xenopus egg extract. Quite surprisingly, the immuno-depleted extracts still promoted the assembly of nuclear bodies which lacked either fibrillarin, nucleolin, xNopp180 or B23/NO38. Only after fibrillarin-depletion fewer PNBs were seen as compared to controls. Digestion of the extract with RNase followed by northern blot analysis revealed that U3 small nucleolar RNA is not required for the formation and structural maintenance of PNBs in vitro.

scholarly journals Coiled bodies without coilin.

1997 ◽  
Vol 8 (1) ◽  
pp. 73-82 ◽  
Author(s):  
D W Bauer ◽  
J G Gall
Keyword(s):  
Rna Processing ◽  
Coiled Body ◽  
Sm Proteins ◽  
Light Microscope Level ◽  
Xenopus Egg Extract ◽  
Coiled Bodies ◽  
Egg Extract ◽  
Processing Pathways ◽  
Xenopus Egg

Nuclei assembled in vitro in Xenopus egg extract contain coiled bodies that have components from three different RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. In addition, they contain SPH-1, the Xenopus homologue of p80-coilin, a protein characteristic of coiled bodies. To determine whether coilin is an essential structural component of the coiled body, we removed it from the egg extract by immunoprecipitation. We showed that nuclei with bodies morphologically identical to coiled bodies (at the light microscope level) formed in such coilin-depleted extract. As expected, these bodies did not stain with antibodies against coilin. Moreover, they failed to stain with an antibody against the Sm proteins, although Sm proteins associated with snRNAs were still present in the extract. Staining of the coilin- and Sm-depleted coiled bodies was normal with antibodies against two nucleolar proteins, fibrillarin and nucleolin. Similar results were observed when Sm proteins were depleted from egg extract: staining of the coiled bodies with antibodies against the Sm proteins and coilin was markedly reduced but bright nucleolin and fibrillarin staining remained. These immunodepletion experiments demonstrate an interdependence between coilin and Sm snRNPs and suggest that neither is essential for assembly of nucleolar components in coiled bodies. We propose that coiled bodies are structurally heterogeneous organelles in which the components of the three RNA processing pathways may occur in separate compartments.

Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei

2000 ◽  
Vol 113 (5) ◽  
pp. 887-898 ◽  
Author(s):  
C.J. Li ◽  
J.A. Bogan ◽  
D.A. Natale ◽  
M.L. DePamphilis
Keyword(s):  
Prolonged Exposure ◽  
Selective Activation ◽  
Initiation Factors ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Dna Substrate

As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the cell division cycle, and their appearance did not depend on the presence of Xenopus Orc proteins. Therefore, hamster pre-replication complexes appear to be assembled at specific chromosomal sites during G(1)-phase. Third, selective activation of ori-(beta) in late G(1)-nuclei depended on the ratio of Xenopus egg extract to nuclei, revealing that epigenetic parameters such as the ratio of initiation factors to DNA substrate could determine the number of origins activated.

Studying nuclear disassembly in vitro using Xenopus egg extract

Methods ◽  
2006 ◽  
Vol 39 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Meda M. Higa ◽  
Katharine S. Ullman ◽  
Amy J. Prunuske
Keyword(s):  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg
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