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

Delaunay D, Robini MC, Dehay C - Front Cell Neurosci (2015)

Bottom Line: We therefore set out to develop an alternative method for accurately measuring spindle asymmetry.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.We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Brain Research Institute, Institut National de la Santé et de la Recherche Médicale, U846 Bron, France ; Université de Lyon I Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus

2D SSA analysis (Area determination). (A) Optical sections of an E14.5 metaphase cell stained with α-tubulin (to reveal the microtubules, green) and DAPI (blue). The optical sections are taken every 0.5 μm from top to bottom. The magnet sized pictures show the centrosome appearance (red, pericentrin staining). (B) Maximum intensity stack projection showing that the entirety of the spindle apparatus is taken into consideration thanks to the equal sized centrosomes (pericentrin, red). (C) Maximum intensity stack projection of the same cell revealing the tubules only. (D–K) Detailed methods for 2D area determination. (D,H) Schematic representation of a symmetric (D) and an asymmetric metaphase cells (H). When the spindles are symmetric, each part are of equal sizes (Green = Left spindle, Yellow = Right spindle, arbitrarily consider), conversely, when the spindles are asymmetric, the left spindle area is significantly larger than the right one. (E,I) For each cells, a primary reconstruction is made to verify that the centrosome are of equal sizes (E,I, yellow dots). (F,J) The SSA intensity is determined on maximal intensity stack projection reconstructed under the ImageJ software. Each spindle pole is manually drawn and the corresponding area (1 and 2) calculated. Arbitrarily, the bigger area will be defined as the Left spindle and the smallest as the Right spindle. (G,K) The difference between the Left (green) and the Right area (yellow) expressed in percentage will be the unit of measurement, delta (Δ). (L) SSA distribution at different time points during in vitro cortical cells development. Cells were respectively taken at E10, E11.5, E13 and cultured for 1 day to 1.5 day in vitro (DIV). Consistent with previous report (Delaunay et al., 2014), the SSA variation follows a folded normal distribution and parallels the asymmetric cell division kinetics: first an increase with a peak at E14.5 followed by a decrease at E16.5. (M) The change in SSA at E14.5 appears highly significant, as demonstrated by the permutation test (p = 5.10−4). Scale bars: (A) 5 μm E, I 10 μm.
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Figure 3: 2D SSA analysis (Area determination). (A) Optical sections of an E14.5 metaphase cell stained with α-tubulin (to reveal the microtubules, green) and DAPI (blue). The optical sections are taken every 0.5 μm from top to bottom. The magnet sized pictures show the centrosome appearance (red, pericentrin staining). (B) Maximum intensity stack projection showing that the entirety of the spindle apparatus is taken into consideration thanks to the equal sized centrosomes (pericentrin, red). (C) Maximum intensity stack projection of the same cell revealing the tubules only. (D–K) Detailed methods for 2D area determination. (D,H) Schematic representation of a symmetric (D) and an asymmetric metaphase cells (H). When the spindles are symmetric, each part are of equal sizes (Green = Left spindle, Yellow = Right spindle, arbitrarily consider), conversely, when the spindles are asymmetric, the left spindle area is significantly larger than the right one. (E,I) For each cells, a primary reconstruction is made to verify that the centrosome are of equal sizes (E,I, yellow dots). (F,J) The SSA intensity is determined on maximal intensity stack projection reconstructed under the ImageJ software. Each spindle pole is manually drawn and the corresponding area (1 and 2) calculated. Arbitrarily, the bigger area will be defined as the Left spindle and the smallest as the Right spindle. (G,K) The difference between the Left (green) and the Right area (yellow) expressed in percentage will be the unit of measurement, delta (Δ). (L) SSA distribution at different time points during in vitro cortical cells development. Cells were respectively taken at E10, E11.5, E13 and cultured for 1 day to 1.5 day in vitro (DIV). Consistent with previous report (Delaunay et al., 2014), the SSA variation follows a folded normal distribution and parallels the asymmetric cell division kinetics: first an increase with a peak at E14.5 followed by a decrease at E16.5. (M) The change in SSA at E14.5 appears highly significant, as demonstrated by the permutation test (p = 5.10−4). Scale bars: (A) 5 μm E, I 10 μm.

Mentions: To design a reliable 2D SSA quantification method, we analyzed the same data set as for the 3D analysis, that is, dissociated primary cortical precursors from E10.5 to E16.5 for a mean period of 1 day in vitro (DIV) (Figure 3). As for the 3D analysis, cells were fixed and stained for α-tubulin, pericentrin and DAPI to reveal the condensed nuclei. For each metaphase cell, optical sections were acquired from top to bottom with 0.2–0.5 μm intervals using a Leica DM6000 confocal microscope. The same criteria as for the 3D quantification were applied (Figures 3A–C). To quantify the SSA, we measured each spindle pole area and named the larger of the two “Left spindle” (green, Figures 3D,H) and the smaller “Right spindle” (yellow, Figures 3D,H). The difference between the left and the right spindle poles (called the “2D spindle pole difference”), denoted by Δ and expressed as a percentage, reveals the SSA magnitude. Using the ImageJ software, for each cell, optical sections were transformed into maximal intensity stack projections (Figures 3E,I). The resulting left and right spindle pole domains were then manually drawn as ROI (Figures 3F, J) and their respective surface area estimated. Let AL and AR, respectively denote the left and right surface areas, the 2D spindle-pole difference is defined by


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

Delaunay D, Robini MC, Dehay C - Front Cell Neurosci (2015)

2D SSA analysis (Area determination). (A) Optical sections of an E14.5 metaphase cell stained with α-tubulin (to reveal the microtubules, green) and DAPI (blue). The optical sections are taken every 0.5 μm from top to bottom. The magnet sized pictures show the centrosome appearance (red, pericentrin staining). (B) Maximum intensity stack projection showing that the entirety of the spindle apparatus is taken into consideration thanks to the equal sized centrosomes (pericentrin, red). (C) Maximum intensity stack projection of the same cell revealing the tubules only. (D–K) Detailed methods for 2D area determination. (D,H) Schematic representation of a symmetric (D) and an asymmetric metaphase cells (H). When the spindles are symmetric, each part are of equal sizes (Green = Left spindle, Yellow = Right spindle, arbitrarily consider), conversely, when the spindles are asymmetric, the left spindle area is significantly larger than the right one. (E,I) For each cells, a primary reconstruction is made to verify that the centrosome are of equal sizes (E,I, yellow dots). (F,J) The SSA intensity is determined on maximal intensity stack projection reconstructed under the ImageJ software. Each spindle pole is manually drawn and the corresponding area (1 and 2) calculated. Arbitrarily, the bigger area will be defined as the Left spindle and the smallest as the Right spindle. (G,K) The difference between the Left (green) and the Right area (yellow) expressed in percentage will be the unit of measurement, delta (Δ). (L) SSA distribution at different time points during in vitro cortical cells development. Cells were respectively taken at E10, E11.5, E13 and cultured for 1 day to 1.5 day in vitro (DIV). Consistent with previous report (Delaunay et al., 2014), the SSA variation follows a folded normal distribution and parallels the asymmetric cell division kinetics: first an increase with a peak at E14.5 followed by a decrease at E16.5. (M) The change in SSA at E14.5 appears highly significant, as demonstrated by the permutation test (p = 5.10−4). Scale bars: (A) 5 μm E, I 10 μm.
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Related In: Results  -  Collection

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Figure 3: 2D SSA analysis (Area determination). (A) Optical sections of an E14.5 metaphase cell stained with α-tubulin (to reveal the microtubules, green) and DAPI (blue). The optical sections are taken every 0.5 μm from top to bottom. The magnet sized pictures show the centrosome appearance (red, pericentrin staining). (B) Maximum intensity stack projection showing that the entirety of the spindle apparatus is taken into consideration thanks to the equal sized centrosomes (pericentrin, red). (C) Maximum intensity stack projection of the same cell revealing the tubules only. (D–K) Detailed methods for 2D area determination. (D,H) Schematic representation of a symmetric (D) and an asymmetric metaphase cells (H). When the spindles are symmetric, each part are of equal sizes (Green = Left spindle, Yellow = Right spindle, arbitrarily consider), conversely, when the spindles are asymmetric, the left spindle area is significantly larger than the right one. (E,I) For each cells, a primary reconstruction is made to verify that the centrosome are of equal sizes (E,I, yellow dots). (F,J) The SSA intensity is determined on maximal intensity stack projection reconstructed under the ImageJ software. Each spindle pole is manually drawn and the corresponding area (1 and 2) calculated. Arbitrarily, the bigger area will be defined as the Left spindle and the smallest as the Right spindle. (G,K) The difference between the Left (green) and the Right area (yellow) expressed in percentage will be the unit of measurement, delta (Δ). (L) SSA distribution at different time points during in vitro cortical cells development. Cells were respectively taken at E10, E11.5, E13 and cultured for 1 day to 1.5 day in vitro (DIV). Consistent with previous report (Delaunay et al., 2014), the SSA variation follows a folded normal distribution and parallels the asymmetric cell division kinetics: first an increase with a peak at E14.5 followed by a decrease at E16.5. (M) The change in SSA at E14.5 appears highly significant, as demonstrated by the permutation test (p = 5.10−4). Scale bars: (A) 5 μm E, I 10 μm.
Mentions: To design a reliable 2D SSA quantification method, we analyzed the same data set as for the 3D analysis, that is, dissociated primary cortical precursors from E10.5 to E16.5 for a mean period of 1 day in vitro (DIV) (Figure 3). As for the 3D analysis, cells were fixed and stained for α-tubulin, pericentrin and DAPI to reveal the condensed nuclei. For each metaphase cell, optical sections were acquired from top to bottom with 0.2–0.5 μm intervals using a Leica DM6000 confocal microscope. The same criteria as for the 3D quantification were applied (Figures 3A–C). To quantify the SSA, we measured each spindle pole area and named the larger of the two “Left spindle” (green, Figures 3D,H) and the smaller “Right spindle” (yellow, Figures 3D,H). The difference between the left and the right spindle poles (called the “2D spindle pole difference”), denoted by Δ and expressed as a percentage, reveals the SSA magnitude. Using the ImageJ software, for each cell, optical sections were transformed into maximal intensity stack projections (Figures 3E,I). The resulting left and right spindle pole domains were then manually drawn as ROI (Figures 3F, J) and their respective surface area estimated. Let AL and AR, respectively denote the left and right surface areas, the 2D spindle-pole difference is defined by

Bottom Line: We therefore set out to develop an alternative method for accurately measuring spindle asymmetry.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.We therefore provide a reliable and efficient technique to measure SSA in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Brain Research Institute, Institut National de la Santé et de la Recherche Médicale, U846 Bron, France ; Université de Lyon I Lyon, France.

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.

No MeSH data available.


Related in: MedlinePlus