Dataset Information

Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

ABSTRACT: Recent data have uncovered that spindle size asymmetry (SSA) is a key component of asymmetric cell division (ACD) in the mouse cerebral cortex (Delaunay et al., 2014). In the present study we show that SSA is independent of spindle orientation and also occurs during cortical progenitor divisions in the ventricular zone (VZ) of the macaque cerebral cortex, pointing to a conserved mechanism in the mammalian lineage. Because SSA magnitude is smaller in cortical precursors than in invertebrate neuroblasts, the unambiguous demonstration of volume differences between the two half spindles is considered to require 3D reconstruction of the mitotic spindle (Delaunay et al., 2014). Although straightforward, the 3D analysis of SSA is time consuming, which is likely to hinder SSA identification and prevent further explorations of SSA related mechanisms in generating ACD. We therefore set out to develop an alternative method for accurately measuring spindle asymmetry. Based on the mathematically demonstrated linear relationship between 2D and 3D analysis, we show that 2D assessment of spindle size in metaphase cells is as accurate and reliable as 3D reconstruction provided a specific procedure is applied. We have examined the experimental accuracy of the two methods by applying them to different sets of in vivo and in vitro biological data, including mouse and primate cortical precursors. Linear regression analysis demonstrates that the results from 2D and 3D reconstructions are equally powerful. We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

SUBMITTER: Delaunay D

PROVIDER: S-EPMC4321609 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

Delaunay Delphine D Robini Marc C MC Dehay Colette C

Frontiers in cellular neuroscience 20150209

Recent data have uncovered that spindle size asymmetry (SSA) is a key component of asymmetric cell division (ACD) in the mouse cerebral cortex (Delaunay et al., 2014). In the present study we show that SSA is independent of spindle orientation and also occurs during cortical progenitor divisions in the ventricular zone (VZ) of the macaque cerebral cortex, pointing to a conserved mechanism in the mammalian lineage. Because SSA magnitude is smaller in cortical precursors than in invertebrate neuro ...[more]

PMID: 25709568

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Project description:BackgroundThe yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined.ResultsWe present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page) (Continued from previous page).Conclusions"RodCellJ" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells.AvailabilityRodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.

Project description:The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.

Project description:Predicting motion is essential for many everyday life activities, e.g., in road traffic. Previous studies on motion prediction failed to find consistent results, which might be due to the use of very different stimulus material and behavioural tasks. Here, we directly tested the influence of task (detection, extrapolation) and stimulus features (visual vs. audiovisual and three-dimensional vs. non-three-dimensional) on temporal motion prediction in two psychophysical experiments. In both experiments a ball followed a trajectory toward the observer and temporarily disappeared behind an occluder. In audiovisual conditions a moving white noise (congruent or non-congruent to visual motion direction) was presented concurrently. In experiment 1 the ball reappeared on a predictable or a non-predictable trajectory and participants detected when the ball reappeared. In experiment 2 the ball did not reappear after occlusion and participants judged when the ball would reach a specified position at two possible distances from the occluder (extrapolation task). Both experiments were conducted in three-dimensional space (using stereoscopic screen and polarised glasses) and also without stereoscopic presentation. Participants benefitted from visually predictable trajectories and concurrent sounds during detection. Additionally, visual facilitation was more pronounced for non-3D stimulation during detection task. In contrast, for a more complex extrapolation task group mean results indicated that auditory information impaired motion prediction. However, a post hoc cross-validation procedure (split-half) revealed that participants varied in their ability to use sounds during motion extrapolation. Most participants selectively profited from either near or far extrapolation distances but were impaired for the other one. We propose that interindividual differences in extrapolation efficiency might be the mechanism governing this effect. Together, our results indicate that both a realistic experimental environment and subject-specific differences modulate the ability of audiovisual motion prediction and need to be considered in future research.

Dataset Information

Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

Publications

Mitotic spindle asymmetry in rodents and primates: 2D vs. 3D measurement methodologies.

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