scholarly journals Head and/or CaaX Domain Deletions of Lamin Proteins Disrupt Preformed Lamin A and C But Not Lamin B Structure in Mammalian Cells

2000 ◽  
Vol 11 (12) ◽  
pp. 4323-4337 ◽  
Author(s):  
Masako Izumi ◽  
O. Anthony Vaughan ◽  
Christopher J. Hutchison ◽  
David M. Gilbert
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Form ◽  
Lamin A ◽  
Lamin B1 ◽  
Head Domain ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A “head” domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, “head-less” lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.

scholarly journals Mammalian nuclei become licensed for DNA replication during late telophase

2002 ◽  
Vol 115 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Daniela S. Dimitrova ◽  
Tatyana A. Prokhorova ◽  
J. Julian Blow ◽  
Ivan T. Todorov ◽  
David M. Gilbert
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Significant Degree ◽  
Initiation Of Replication ◽  
Egg Extracts ◽  
Late Telophase ◽  
Xenopus Egg ◽  
Mcm Proteins ◽  
Nuclear Envelope Formation ◽  
Xenopus Egg Extracts

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

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.

scholarly journals The Microtubule-destabilizing Kinesin XKCM1 Regulates Microtubule Dynamic Instability in Cells

2002 ◽  
Vol 13 (8) ◽  
pp. 2718-2731 ◽  
Author(s):  
Susan L. Kline-Smith ◽  
Claire E. Walczak
Keyword(s):  
Mammalian Cells ◽  
Dynamic Instability ◽  
Critical Role ◽  
Dependent Manner ◽  
Related Protein ◽  
Microtubule Dynamic ◽  
Mitotic Delay ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.

scholarly journals The Nup107-160 Nucleoporin Complex Is Required for Correct Bipolar Spindle Assembly

2006 ◽  
Vol 17 (9) ◽  
pp. 3806-3818 ◽  
Author(s):  
Arturo V. Orjalo ◽  
Alexei Arnaoutov ◽  
Zhouxin Shen ◽  
Yekaterina Boyarchuk ◽  
Samantha G. Zeitlin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Sperm Chromatin ◽  
Nuclear Pore ◽  
Checkpoint Proteins ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

scholarly journals Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Songli Zhu ◽  
Mohammadjavad Paydar ◽  
Feifei Wang ◽  
Yanqiu Li ◽  
Ling Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

DNA double strand breaks (DSBs) have detrimental effects on cell survival and genomic stability, and are related to cancer and other human diseases. In this study, we identified microtubule-depolymerizing kinesin Kif2C as a protein associated with DSB-mimicking DNA templates and known DSB repair proteins in Xenopus egg extracts and mammalian cells. The recruitment of Kif2C to DNA damage sites was dependent on both PARP and ATM activities. Kif2C knockdown or knockout led to accumulation of endogenous DNA damage, DNA damage hypersensitivity, and reduced DSB repair via both NHEJ and HR. Interestingly, Kif2C depletion, or inhibition of its microtubule depolymerase activity, reduced the mobility of DSBs, impaired the formation of DNA damage foci, and decreased the occurrence of foci fusion and resolution. Taken together, our study established Kif2C as a new player of the DNA damage response, and presented a new mechanism that governs DSB dynamics and repair.

scholarly journals A Biochemical Screen for Identification of Small-Molecule Regulators of the Wnt Pathway Using Xenopus Egg Extracts

2011 ◽  
Vol 16 (9) ◽  
pp. 995-1006 ◽  
Author(s):  
Curtis A. Thorne ◽  
Bonnie Lafleur ◽  
Michelle Lewis ◽  
Alison J. Hanson ◽  
Kristin K. Jernigan ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Wnt Pathway ◽  
Renilla Luciferase ◽  
Signal Distribution ◽  
Egg Extracts ◽  
Bona Fide ◽  
Xenopus Egg ◽  
The Stability ◽  
Xenopus Egg Extracts

Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.

scholarly journals Apoptotic Cleavage of Cytoplasmic Dynein Intermediate Chain and P150GluedStops Dynein-Dependent Membrane Motility

2001 ◽  
Vol 153 (7) ◽  
pp. 1415-1426 ◽  
Author(s):  
Jon D. Lane ◽  
Maïlys A.S. Vergnolle ◽  
Philip G. Woodman ◽  
Victoria J. Allan
Keyword(s):  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Animal Cells ◽  
Dynein Intermediate Chain ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Dynactin Complex ◽  
Intermediate Chains

Cytoplasmic dynein is the major minus end–directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150Glued. We have found that both CD-IC and p150Glued are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH2-terminal p150Glued binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150Glued in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein–driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein–dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

scholarly journals Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.

1992 ◽  
Vol 116 (6) ◽  
pp. 1431-1442 ◽  
Author(s):  
B Buendia ◽  
G Draetta ◽  
E Karsenti
Keyword(s):  
Critical Concentration ◽  
Cyclin A ◽  
Soluble Form ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Polymer Assembly

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

scholarly journals Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors

2011 ◽  
Vol 108 (42) ◽  
pp. 17331-17336 ◽  
Author(s):  
O. Ganier ◽  
S. Bocquet ◽  
I. Peiffer ◽  
V. Brochard ◽  
P. Arnaud ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Mammalian Cells ◽  
Combined Exposure ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

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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