scholarly journals CaM kinase II initiates meiotic spindle depolymerization independently of APC/C activation

2008 ◽  
Vol 183 (6) ◽  
pp. 1007-1017 ◽  
Author(s):  
Simone Reber ◽  
Sabine Over ◽  
Iva Kronja ◽  
Oliver J. Gruss
Keyword(s):  
Meiotic Spindle ◽  
Free Calcium ◽  
Microtubule Depolymerization ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Anaphase Onset ◽  
Egg Extracts ◽  
Dependent Signal ◽  
Chromosome Separation ◽  
Xenopus Egg Extracts

Altered spindle microtubule dynamics at anaphase onset are the basis for chromosome segregation. In Xenopus laevis egg extracts, increasing free calcium levels and subsequently rising calcium-calmodulin–dependent kinase II (CaMKII) activity promote a release from meiosis II arrest and reentry into anaphase. CaMKII induces the activation of the anaphase-promoting complex/cyclosome (APC/C), which destines securin and cyclin B for degradation to allow chromosome separation and mitotic exit. In this study, we investigated the calcium-dependent signal responsible for microtubule depolymerization at anaphase onset after release from meiotic arrest in Xenopus egg extracts. Using Ran–guanosine triphosphate–mediated microtubule assemblies and quantitative analysis of complete spindles, we demonstrate that CaMKII triggers anaphase microtubule depolymerization. A CaMKII-induced twofold increase in microtubule catastrophe rates can explain reduced microtubule stability. However, calcium or constitutively active CaMKII promotes microtubule destabilization even upon APC/C inhibition and in the presence of high cyclin-dependent kinase 1 activity. Therefore, our data demonstrate that CaMKII turns on parallel pathways to activate the APC/C and to induce microtubule depolymerization at meiotic anaphase onset.

scholarly journals Probing the Force-Balancing Mechanism of the Meiotic Spindle in Xenopus Egg Extracts

2010 ◽  
Vol 98 (3) ◽  
pp. 365a
Author(s):  
Jun Takagi ◽  
Takeshi Itabashi ◽  
Yuta Shimamoto ◽  
Tarun M. Kapoor ◽  
Shin'ichi Ishiwata
Keyword(s):  
Meiotic Spindle ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Augmin promotes meiotic spindle formation and bipolarity in Xenopus egg extracts

2011 ◽  
Vol 108 (35) ◽  
pp. 14473-14478 ◽  
Author(s):  
S. Petry ◽  
C. Pugieux ◽  
F. J. Nedelec ◽  
R. D. Vale
Keyword(s):  
Meiotic Spindle ◽  
Spindle Formation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts

1995 ◽  
Vol 108 (11) ◽  
pp. 3557-3568 ◽  
Author(s):  
H.D. Lindsay ◽  
M.J. Whitaker ◽  
C.C. Ford
Keyword(s):  
Mitotic Cell ◽  
Free Calcium ◽  
Cdc2 Kinase ◽  
Kinase Activation ◽  
Cell Cycles ◽  
Calcium Buffer ◽  
Egg Extracts ◽  
Calcium Buffers ◽  
Xenopus Egg Extracts ◽  
Activation Phase

Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translation modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500 nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2A) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.

Alteration of Acylphosphate Formation of Cardiac Sarcoplasmic Reticulum ATPase by Calmodulin-Dependent Phosphorylation

1984 ◽  
Vol 39 (3-4) ◽  
pp. 289-292 ◽  
Author(s):  
Christian Pifl ◽  
Brigitte Plank ◽  
Gertrude Hellmann ◽  
Wolfgang Wyskovsky ◽  
Josef Suko
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Transport ◽  
State Level ◽  
Hill Coefficient ◽  
Free Calcium ◽  
Sr Protein ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Transport Atpase ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent

The calcium-dependent acylphosphate formed by the calcium transport ATPase of cardiac sarcoplasmic reticulum and the calcium-, calm odulin-dependent phosphoester(s) of sarcoplasmic reticulum fractions formed by a calcium-, calmodulin-dependent membrane-bound protein kinase can be distinguished by removal of calcium and/or magnesium by EDTA or hydroxylamine treatment of the acid denaturated membranes. Both procedures decompose the acylphosphate with little effect on the phosphoester(s). Calmodulin-dependent phosphorylation (2.44 nmol/mg SR protein) reduces the apparent K(Ca) of the acylphosphate steady state level of the calcium transport ATPase from 0.56 to 0.34 μM free calcium, without affecting the maximum phosphoenzyme level (0.93 versus 0.89 nmol/mg protein), and has little, if any, effect on the Hill-coefficient (1.32 versus 1.54)

scholarly journals TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts

2014 ◽  
Vol 206 (3) ◽  
pp. 385-393 ◽  
Author(s):  
Kara J. Helmke ◽  
Rebecca Heald
Keyword(s):  
Parallel Architecture ◽  
Cell Types ◽  
Meiotic Spindle ◽  
Spindle Poles ◽  
Frog Species ◽  
Two Factors ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Xenopus Egg Extracts

The spindle segregates chromosomes in dividing eukaryotic cells, and its assembly pathway and morphology vary across organisms and cell types. We investigated mechanisms underlying differences between meiotic spindles formed in egg extracts of two frog species. Small Xenopus tropicalis spindles resisted inhibition of two factors essential for assembly of the larger Xenopus laevis spindles: RanGTP, which functions in chromatin-driven spindle assembly, and the kinesin-5 motor Eg5, which drives antiparallel microtubule (MT) sliding. This suggested a role for the MT-associated protein TPX2 (targeting factor for Xenopus kinesin-like protein 2), which is regulated by Ran and binds Eg5. Indeed, TPX2 was threefold more abundant in X. tropicalis extracts, and elevated TPX2 levels in X. laevis extracts reduced spindle length and sensitivity to Ran and Eg5 inhibition. Higher TPX2 levels recruited Eg5 to the poles, where MT density increased. We propose that TPX2 levels modulate spindle architecture through Eg5, partitioning MTs between a tiled, antiparallel array that promotes spindle expansion and a cross-linked, parallel architecture that concentrates MTs at spindle poles.

scholarly journals A computational model predicts Xenopus meiotic spindle organization

2010 ◽  
Vol 191 (7) ◽  
pp. 1239-1249 ◽  
Author(s):  
Rose Loughlin ◽  
Rebecca Heald ◽  
François Nédélec
Keyword(s):  
Dynamic Instability ◽  
Length Distribution ◽  
Spindle Pole ◽  
Two Dimensions ◽  
Meiotic Spindle ◽  
Bipolar Structure ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Xenopus Egg Extracts

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5–like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.

scholarly journals BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

2002 ◽  
Vol 158 (3) ◽  
pp. 487-496 ◽  
Author(s):  
Rey-Huei Chen
Keyword(s):  
Spindle Checkpoint ◽  
Kinase Domain ◽  
Wild Type ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Spindle Microtubules ◽  
Xenopus Egg Extracts ◽  
Checkpoint Signal

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.

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.

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