scholarly journals The N-terminal coiled-coil of Ndel1 is a regulated scaffold that recruits LIS1 to dynein

2011 ◽  
Vol 192 (3) ◽  
pp. 433-445 ◽  
Author(s):  
Eliza Żyłkiewicz ◽  
Monika Kijańska ◽  
Won-Chan Choi ◽  
Urszula Derewenda ◽  
Zygmunt S. Derewenda ◽  
...  
Keyword(s):  
Experimental Approach ◽  
Coiled Coil ◽  
Self Organization ◽  
Binding Partners ◽  
Coiled Coil Domain ◽  
C Terminus ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs

1991 ◽  
Vol 98 (3) ◽  
pp. 271-279
Author(s):  
J. Meier ◽  
K.H. Campbell ◽  
C.C. Ford ◽  
R. Stick ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Membrane Structures ◽  
Chromatin Decondensation ◽  
Immunoblotting Analysis ◽  
Nuclear Assembly ◽  
Contrast Microscopy ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts

Nature ◽  
1996 ◽  
Vol 382 (6590) ◽  
pp. 420-425 ◽  
Author(s):  
Rebecca Heald ◽  
Régis Tournebize ◽  
Thiemo Blank ◽  
Raphael Sandaltzopoulos ◽  
Peter Becker ◽  
...  
Keyword(s):  
Self Organization ◽  
Artificial Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2197
Author(s):  
Zachary M. Geisterfer ◽  
Gabriel Guilloux ◽  
Jesse C. Gatlin ◽  
Romain Gibeaux
Keyword(s):  
Experimental System ◽  
Structure And Function ◽  
Self Organization ◽  
African Clawed Frog ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
The Many ◽  
And Function ◽  
Xenopus Egg Extracts ◽  
Clawed Frog

Self-organization of and by the cytoskeleton is central to the biology of the cell. Since their introduction in the early 1980s, cytoplasmic extracts derived from the eggs of the African clawed-frog, Xenopus laevis, have flourished as a major experimental system to study the various facets of cytoskeleton-dependent self-organization. Over the years, the many investigations that have used these extracts uniquely benefited from their simplified cell cycle, large experimental volumes, biochemical tractability and cell-free nature. Here, we review the contributions of egg extracts to our understanding of the cytoplasmic aspects of self-organization by the microtubule and the actomyosin cytoskeletons as well as the importance of cytoskeletal filaments in organizing nuclear structure and function.

scholarly journals Xenopus HJURP and condensin II are required for CENP-A assembly

2011 ◽  
Vol 192 (4) ◽  
pp. 569-582 ◽  
Author(s):  
Rafael Bernad ◽  
Patricia Sánchez ◽  
Teresa Rivera ◽  
Miriam Rodríguez-Corsino ◽  
Ekaterina Boyarchuk ◽  
...  
Keyword(s):  
Protein A ◽  
Human Cells ◽  
Epigenetic Mark ◽  
Temporal Specificity ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Centromeric Protein ◽  
Xenopus Egg Extracts

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction–recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

scholarly journals Activation of myosin V–based motility and F-actin–dependent network formation of endoplasmic reticulum during mitosis

2002 ◽  
Vol 159 (4) ◽  
pp. 571-577 ◽  
Author(s):  
Torsten Wöllert ◽  
Dieter G. Weiss ◽  
Hans-Hermann Gerdes ◽  
Sergei A. Kuznetsov
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Network Formation ◽  
Active Role ◽  
Myosin V ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

It is widely believed that microtubule- and F-actin–based transport of cytoplasmic organelles and membrane fusion is down-regulated during mitosis. Here we show that during the transition of Xenopus egg extracts from interphase to metaphase myosin V–driven movement of small globular vesicles along F-actin is strongly inhibited. In contrast, the movement of ER and ER network formation on F-actin is up-regulated in metaphase extracts. Our data demonstrate that myosin V–driven motility of distinct organelles is differently controlled during the cell cycle and suggest an active role of F-actin in partitioning, positioning, and membrane fusion of the ER during cell division.

scholarly journals The role of keratin filaments during nuclear envelope reassembly in Xenopus egg extracts 1

FEBS Letters ◽  
1998 ◽  
Vol 428 (1-2) ◽  
pp. 52-56 ◽  
Author(s):  
Bo Zhang ◽  
Ying Chen ◽  
Zhiyang Han ◽  
Hans Ris ◽  
Zhonghe Zhai
Keyword(s):  
Nuclear Envelope ◽  
Keratin Filaments ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

2003 ◽  
Vol 161 (6) ◽  
pp. 1041-1051 ◽  
Author(s):  
Sarah M. Wignall ◽  
Renée Deehan ◽  
Thomas J. Maresca ◽  
Rebecca Heald
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Condensin Complex ◽  
Egg Extracts ◽  
Chromosomal Architecture ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
And Function ◽  
Xenopus Egg Extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.

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