Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Using micromanipulation to analyze control of vertebrate meiotic spindle size.

ABSTRACT: The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles that self-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the validity of the equation, we cut the spindle into two fragments along the pole-to-pole axis by micromanipulation techniques that rapidly decrease the amount of MTs. The spheroidal shape spontaneously recovered within 5 min, but the size of each fragment remained small. The equation we obtained quantitatively describes how the spindle size correlates with the amount of MTs while maintaining the shape and the MT density.

SUBMITTER: Takagi J

PROVIDER: S-EPMC4155493 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Using micromanipulation to analyze control of vertebrate meiotic spindle size.

Takagi Jun J Itabashi Takeshi T Suzuki Kazuya K Kapoor Tarun M TM Shimamoto Yuta Y Ishiwata Shin'ichi S

Cell reports 20131010 1

The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles that self-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the vali ...[more]

PMID: 24120869

Similar Datasets

Probing the mechanical architecture of the vertebrate meiotic spindle.

Project description:Accurate chromosome segregation during meiosis depends on the assembly of a microtubule-based spindle of proper shape and size. Current models for spindle-size control focus on reaction diffusion-based chemical regulation and balance in activities of motor proteins. Although several molecular perturbations have been used to test these models, controlled mechanical perturbations have not been possible. Here we report a piezoresistive dual cantilever-based system to test models for spindle-size control and examine the mechanical features, such as deformability and stiffness, of the vertebrate meiotic spindle. We found that meiotic spindles prepared in Xenopus laevis egg extracts were viscoelastic and recovered their original shape in response to small compression. Larger compression resulted in plastic deformation, but the spindle adapted to this change, establishing a stable mechanical architecture at different sizes. The technique we describe here may also be useful for examining the micromechanics of other cellular organelles.

| S-EPMC2766809 | biostudies-literature

Kif2a Scales Meiotic Spindle Size in Hymenochirus boettgeri.

Project description:Size is a fundamental feature of biological systems that affects physiology at all levels. For example, the dynamic, microtubule-based spindle that mediates chromosome segregation scales to a wide range of cell sizes across different organisms and cell types. Xenopus frog species possess a variety of egg and meiotic spindle sizes, and differences in activities or levels of microtubule-associated proteins in the egg cytoplasm between Xenopus laevis and Xenopus tropicalis have been shown to account for spindle scaling [1]. Increased activity of the microtubule severing protein katanin scales the X. tropicalis spindle smaller compared to X. laevis [2], as do elevated levels of TPX2, a protein that enriches the cross-linking kinesin-5 motor Eg5 at spindle poles [3]. To examine the conservation of spindle scaling mechanisms more broadly across frog species, we have utilized the tiny, distantly related Pipid frog Hymenochirus boettgeri. We find that egg extracts from H. boettgeri form meiotic spindles similar in size to X. tropicalis but that TPX2 and katanin-mediated scaling is not conserved. Instead, the microtubule depolymerizing motor protein kif2a functions to modulate spindle size. H. boettgeri kif2a possesses an activating phosphorylation site that is absent from X. laevis. Comparison of katanin and kif2a phosphorylation sites across a variety of species revealed strong evolutionary conservation, with X. laevis and X. tropicalis possessing distinct and unique alterations. Our study highlights the diversity and complexity of spindle assembly and scaling mechanisms, indicating that there is more than one way to assemble a spindle of a particular size.

| S-EPMC6832855 | biostudies-literature

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

Project description: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.

| S-EPMC4121975 | biostudies-literature

Micromechanics of the vertebrate meiotic spindle examined by stretching along the pole-to-pole axis.

Project description:The meiotic spindle is a bipolar molecular machine that is designed to segregate duplicated chromosomes toward the opposite poles of the cell. The size and shape of the spindle are considered to be maintained by a balance of forces produced by molecular motors and microtubule assembly dynamics. Several studies have probed how mechanical perturbations of the force balance affect the spindle structure. However, the spindle's response to a stretching force acting at the spindle pole and along its long axis, i.e., the direction in which chromosomes are segregated, has not been examined. Here, we describe a method to apply a stretching force to the metaphase spindle assembled in Xenopus egg extracts and measure the relationship between the force and the three-dimensional deformation of the spindle. We found that the spindle behaves as a Zener-type viscoelastic body when forces are applied at the spindle pole, generating a restoring force for several minutes. In addition, both the volume of the spindle and the tubulin density are conserved under the stretching force. These results provide insight into how the spindle size is maintained at metaphase.

| S-EPMC3944988 | biostudies-literature

Op18 reveals the contribution of nonkinetochore microtubules to the dynamic organization of the vertebrate meiotic spindle.

Project description:Accuracy in chromosome segregation depends on the assembly of a bipolar spindle. Unlike mitotic spindles, which have roughly equal amounts of kinetochore microtubules (kMTs) and nonkinetochore microtubules (non-kMTs), vertebrate meiotic spindles are predominantly comprised of non-kMTs, a large subset of which forms an antiparallel "barrel" array at the spindle equator. Though kMTs are needed to drive chromosome segregation, the contributions of non-kMTs are more mysterious. Here, we show that increasing the concentration of Op18/stathmin, a component of the chromosome-mediated microtubule formation pathway that directly controls microtubule dynamics, can be used to deplete non-kMTs in the vertebrate meiotic spindle assembled in Xenopus egg extracts. Under these conditions, kMTs and the spindle pole-associated non-kMT arrays persist in smaller spindles. In excess Op18, distances between sister kinetochores, an indicator of tension across centromeres, remain unchanged, even though kMTs flux poleward with a approximately 30% slower velocity, and chromosomes oscillate more than in control metaphase spindles. Remarkably, kinesin-5, a conserved motor protein that can push microtubules apart and is required for the assembly and maintenance of bipolar meiotic spindles, is not needed to maintain spindle bipolarity in the presence of excess Op18. Our data suggest that non-kMTs in meiotic spindles contribute to normal kMT dynamics, stable chromosome positioning, and the establishment of proper spindle size. We propose that without non-kMTs, metaphase meiotic spindles are similar to mammalian mitotic spindles, which balance forces to maintain metaphase spindle organization in the absence of extensive antiparallel microtubule overlap at the spindle equator or a key mitotic kinesin.

| S-EPMC2741252 | biostudies-other

Cytoplasmic Determination of Meiotic Spindle Size Revealed by a Unique Inter-Species Germinal Vesicle Transfer Model.

Project description:Spindle sizes are different in diverse species and cell types. In frogs, the meiotic spindle size is positively correlated with the egg cell volume. Across species, relatively small mouse oocytes (70-80 μm) have a relatively large spindle while larger pig oocytes (about 120 μm) have a considerably smaller spindle. In this study we investigated whether species-specific oocyte spindle size was determined by cytoplasmic or nuclear factors. By exchanging the germinal vesicle between mouse and pig oocytes, we obtained two kinds of reconstructed oocytes: one with mouse ooplasm and pig GV (mCy-pGV oocyte), and the other with pig ooplasm and mouse GV (pCy-mGV oocyte). We show that the MII spindle size of the mCy-pGV oocyte is similar to that of the mouse meiotic spindle and significantly larger than that of the pig meiotic spindle. The timing of oocyte maturation also followed that of the species from which the oocyte cytoplasm arose, although some impact of the origin of the GV was observed. These data suggest that spindle size and the timing of meiotic progression are governed by cytoplasmic components rather than cytoplasmic volume and GV materials.

| S-EPMC4728387 | biostudies-literature

Katanin controls mitotic and meiotic spindle length.

Project description:Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that gamma-tubulin-dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.

| S-EPMC2064698 | biostudies-literature

OsMTOPVIB is required for meiotic bipolar spindle assembly.

Project description:The organization of microtubules into a bipolar spindle is essential for chromosome segregation. Both centrosome and chromatin-dependent spindle assembly mechanisms are well studied in mouse, Drosophila melanogaster, and Xenopus oocytes; however, the mechanism of bipolar spindle assembly in plant meiosis remains elusive. According to our observations of microtubule assembly in Oryza sativa, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum, we propose that a key step of plant bipolar spindle assembly is the correction of the multipolar spindle into a bipolar spindle at metaphase I. The multipolar spindles failed to transition into bipolar ones in OsmtopVIB with the defect in double-strand break (DSB) formation. However, bipolar spindles were normally assembled in several other mutants lacking DSB formation, such as Osspo11-1, pair2, and crc1, indicating that bipolar spindle assembly is independent of DSB formation. We further revealed that the mono-orientation of sister kinetochores was prevalent in OsmtopVIB, whereas biorientation of sister kinetochores was frequently observed in Osspo11-1, pair2, and crc1 In addition, mutations of the cohesion subunit OsREC8 resulted in biorientation of sister kinetochores as well as bipolar spindles even in the background of OsmtopVIB Therefore, we propose that biorientation of the kinetochore is required for bipolar spindle assembly in the absence of homologous recombination.

| S-EPMC6689953 | biostudies-literature

Transcriptome profiling of maize anthers using genetic ablation to analyze pre-meiotic and tapetal cell types

Project description:Agilent oligonucleotide arrays were used to profile gene expression in dissected maize anthers of 3 types of male-sterile plants and their fertile siblings at four stages of development: after anther initiation, at the rapid mitotic proliferation stage, pre-meiosis, and meiotic prophase I. The male-sterile mutants (ms23, msca, and mac1) lack a range of normal cell types resulting from a temporal progression of anther failure. By combining the data sets from the comparisons between individual sterile and fertile anthers, candidate genes predicted to play important roles during maize anther development were assigned to stages and to likely cell types. Keywords: anther development, maize, male sterility

2008-09-16 | GSE12756 | GEO

Axin-1 Regulates Meiotic Spindle Organization in Mouse Oocytes.

Project description:Axin-1, a negative regulator of Wnt signaling, is a versatile scaffold protein involved in centrosome separation and spindle assembly in mitosis, but its function in mammalian oogenesis remains unknown. Here we examined the localization and function of Axin-1 during meiotic maturation in mouse oocytes. Immunofluorescence analysis showed that Axin-1 was localized around the spindle. Knockdown of the Axin1 gene by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in severely defective spindles, misaligned chromosomes, failure of first polar body (PB1) extrusion, and impaired pronuclear formation. However, supplementing the culture medium with the Wnt pathway activator LiCl improved spindle morphology and pronuclear formation. Downregulation of Axin1 gene expression also impaired the spindle pole localization of γ-tubulin/Nek9 and resulted in retention of the spindle assembly checkpoint protein BubR1 at kinetochores after 8.5 h of culture. Our results suggest that Axin-1 is critical for spindle organization and cell cycle progression during meiotic maturation in mouse oocytes.

| S-EPMC4902301 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data