scholarly journals Directly probing the mechanical properties of the spindle and its matrix

2010 ◽  
Vol 188 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Jesse C. Gatlin ◽  
Alexandre Matov ◽  
Gaudenz Danuser ◽  
Timothy J. Mitchison ◽  
Edward D. Salmon
Keyword(s):  
Mechanical Properties ◽  
Xenopus Laevis ◽  
Metaphase Plate ◽  
Constant Length ◽  
Spindle Matrix ◽  
Microtubule Motors ◽  
Egg Extracts ◽  
Length Regulation ◽  
Length Determination ◽  
Poleward Flux

Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.

scholarly journals Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts

2005 ◽  
Vol 169 (6) ◽  
pp. 859-869 ◽  
Author(s):  
Thomas J. Maresca ◽  
Benjamin S. Freedman ◽  
Rebecca Heald
Keyword(s):  
Xenopus Laevis ◽  
Metaphase Plate ◽  
Histone H1 ◽  
Linker Histone ◽  
Chromosome Architecture ◽  
Linker Histone H1 ◽  
Egg Extracts ◽  
Chromatin Proteins ◽  
And Function

During cell division, condensation and resolution of chromosome arms and the assembly of a functional kinetochore at the centromere of each sister chromatid are essential steps for accurate segregation of the genome by the mitotic spindle, yet the contribution of individual chromatin proteins to these processes is poorly understood. We have investigated the role of embryonic linker histone H1 during mitosis in Xenopus laevis egg extracts. Immunodepletion of histone H1 caused the assembly of aberrant elongated chromosomes that extended off the metaphase plate and outside the perimeter of the spindle. Although functional kinetochores assembled, aligned, and exhibited poleward movement, long and tangled chromosome arms could not be segregated in anaphase. Histone H1 depletion did not significantly affect the recruitment of known structural or functional chromosomal components such as condensins or chromokinesins, suggesting that the loss of H1 affects chromosome architecture directly. Thus, our results indicate that linker histone H1 plays an important role in the structure and function of vertebrate chromosomes in mitosis.

scholarly journals Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

2006 ◽  
Vol 103 (32) ◽  
pp. 11904-11909 ◽  
Author(s):  
P. Selenko ◽  
Z. Serber ◽  
B. Gadea ◽  
J. Ruderman ◽  
G. Wagner
Keyword(s):  
Xenopus Laevis ◽  
Protein G ◽  
Nmr Analysis ◽  
Quantitative Nmr ◽  
B1 Domain ◽  
Egg Extracts

scholarly journals Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

2015 ◽  
Vol 35 (15) ◽  
pp. 2626-2640 ◽  
Author(s):  
Lingjun Meng ◽  
Jung-Eun Park ◽  
Tae-Sung Kim ◽  
Eun Hye Lee ◽  
Suk-Youl Park ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Spindle Assembly ◽  
Human Cells ◽  
Mitotic Progression ◽  
Spindle Formation ◽  
Content Type ◽  
Bipolar Spindle ◽  
Egg Extracts ◽  
Cultured Human Cells ◽  
Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

The NIMA-related kinase X-Nek2B is required for efficient assembly of the zygotic centrosome in Xenopus laevis

2000 ◽  
Vol 113 (11) ◽  
pp. 1973-1984 ◽  
Author(s):  
A.M. Fry ◽  
P. Descombes ◽  
C. Twomey ◽  
R. Bacchieri ◽  
E.A. Nigg
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Basal Body ◽  
Chromatin Condensation ◽  
Embryonic Cells ◽  
Serine Threonine Kinase ◽  
Cell Cycles ◽  
Functional Studies ◽  
Mammalian Cell Cycle ◽  
Egg Extracts

Nek2 is a mammalian cell cycle-regulated serine/threonine kinase that belongs to the family of proteins related to NIMA of Aspergillus nidulans. Functional studies in diverse species have implicated NIMA-related kinases in G(2)/M progression, chromatin condensation and centrosome regulation. To directly address the requirements for vertebrate Nek2 kinases in these cell cycle processes, we have turned to the biochemically-tractable system provided by Xenopus laevis egg extracts. Following isolation of a Xenopus homologue of Nek2, called X-Nek2B, we found that X-Nek2B abundance and activity remained constant through the first mitotic cycle implying a fundamental difference in Nek2 regulation between embryonic and somatic cell cycles. Removal of X-Nek2B from extracts did not disturb either entry into mitosis or the accompanying condensation of chromosomes providing no support for a requirement for Nek2 in these processes at least in embryonic cells. In contrast, X-Nek2B localized to centrosomes of adult Xenopus cells and was rapidly recruited to the basal body of Xenopus sperm following incubation in egg extracts. Recruitment led to phosphorylation of the X-Nek2B kinase. Most importantly, depletion of X-Nek2B from extracts significantly delayed both the assembly of microtubule asters and the recruitment of gamma-tubulin to the basal body. Hence, these studies demonstrate that X-Nek2B is required for efficient assembly of a functional zygotic centrosome and highlight the possibility of multiple roles for vertebrate Nek2 kinases in the centrosome cycle.

Fatigue responses of cracked Ti/APC-2 nanocomposite laminates at elevated temperature

2019 ◽  
Vol 54 (13) ◽  
pp. 1705-1715
Author(s):  
MHR Jen ◽  
GT Kuo ◽  
YH Wu ◽  
YJ Chen
Keyword(s):  
Mechanical Properties ◽  
Elevated Temperature ◽  
Composite Laminates ◽  
Elevated Temperatures ◽  
Tensile Tests ◽  
Cyclic Tests ◽  
Constant Length ◽  
Nano Powder ◽  
Strength And Stiffness ◽  
Inclined Angle

The mechanical properties and fatigue responses of Ti/APC-2 neat and nanocomposites with inclined single-edged cracks due to tensile and cyclic tests at elevated temperature were investigated. Two types of composite laminates [Ti/(0/90)s/Ti] were fabricated with and without (W/WO) nanoparticles SiO2 of optimal 1 wt.%. The geometry and dimensions of specimens were L × W × t = 240 × 25 × 1.55 mm3. The cracks were of constant length 3 mm and width 0.3 mm. The inclined angles were 0°, 45°, and 60°. Both the tensile and cyclic tests were conducted at elevated temperatures 25℃ (RT), 100℃, 125℃, and 150℃. From the tensile tests we obtained the load vs. displacement curves for both types of laminates with varied inclinations at elevated temperatures. Next, we received the applied load vs. cycles curves for the same laminates with inclined cracks at the corresponding temperature due to cyclic tests. According to the experimental data of both tensile and cyclic tests the mechanical properties, such as strength, stiffness, and life, decreased as the temperature rises. The greater the inclined angles were, the greater the strength and stiffness were. Similarly, the fatigue life was in the same trend. However, the effect of inclined angle on mechanical properties was more strong than those of temperature. The mechanical properties of nanocomposite laminates were higher than those of neat composite laminates, but not significant. The main reason was that the enhancement of spreading nano-powder silica on the laminate interfaces did not effectively eliminate the stress intensity at the crack tip locally.

scholarly journals Inhibition of protein synthesis in frog (Xenopus laevis) egg extracts by an antibody against phosphatidylinositol 4,5-bisphosphate

1996 ◽  
Vol 317 (3) ◽  
pp. 643-646
Author(s):  
Thomas J. KEATING ◽  
Kiyoko FUKAMI ◽  
Kenneth R. ROBINSON
Keyword(s):  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Similar Reduction ◽  
Inhibition Of Protein Synthesis ◽  
Egg Extracts ◽  
Hydrolysis Of

The antibody kt10, which is directed against the phospholipid PtdIns(4,5)P2, inhibits protein synthesis when added to cytosolic extracts prepared from frog eggs. Addition of stable analogues of diacylglycerol and Ins(1,4,5)P3 failed to rescue the inhibition of translation, suggesting that the effect of the antibody was not to block hydrolysis of PtdIns(4,5)P2. Neomycin, which also binds PtdIns(4,5)P2, produced a similar reduction in protein-synthesis levels in the extract system, supporting the idea that it is the interaction of the antibody with PtdIns(4,5)P2 that is producing the effect.

scholarly journals Micromanipulation studies of chromosome movement. I. Chromosome-spindle attachment and the mechanical properties of chromosomal spindle fibers.

1979 ◽  
Vol 82 (2) ◽  
pp. 528-541 ◽  
Author(s):  
D A Begg ◽  
G W Ellis
Keyword(s):  
Mechanical Properties ◽  
Tensile Force ◽  
Metaphase Plate ◽  
Compressive Force ◽  
Preliminary Evidence ◽  
Spindle Pole ◽  
Polarization Microscopy ◽  
Similar Distance ◽  
Mechanical Attachment ◽  
Spindle Fibers

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers. Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure of the spindle or the positions of the nonmanipulated chromosomes. From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery. In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle; however, the chromosome resists displacement away from the spindle pole. When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole. The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate. No evidence was found for mechanical attachments between separating half-bivalents in anaphase. Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force. Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy.

scholarly journals Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

2008 ◽  
Vol 19 (11) ◽  
pp. 4900-4908 ◽  
Author(s):  
Claudia M. Casanova ◽  
Sofia Rybina ◽  
Hideki Yokoyama ◽  
Eric Karsenti ◽  
Iain W. Mattaj
Keyword(s):  
Xenopus Laevis ◽  
Detailed Analysis ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
Spindle Midzone ◽  
Microtubule Growth ◽  
Spindle Microtubules

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

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