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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

3D SSA analysis (Volume determination). (A) 3D representation of the spindle apparatus in metaphase cell. The larger spindle is colored in green and the right spindle in yellow. The intersection between both sides is represented by the red dashed line and both spindle poles rotate along the same revolution axis (gray). (B–H) Spindle volume determination using the VolumeJ program. (B,C) Stack projection of an in vitro metaphase cell stained with α-tubulin. (C) ROI extraction of the spindle apparatus. (D) Signal tresholding for each optical section. (E) Signal transformation in pixels. (F) Selection of one spindle pole using the Wand tool. (G) Creation of a mask displaying the selected Left and right spindle pole. (H) 3D volume rendering and calculation. The volume is independently calculated for each spindle pole. Here, the difference in volume (Δv) is 20.7, typical of an asymmetric cell.
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Figure 2: 3D SSA analysis (Volume determination). (A) 3D representation of the spindle apparatus in metaphase cell. The larger spindle is colored in green and the right spindle in yellow. The intersection between both sides is represented by the red dashed line and both spindle poles rotate along the same revolution axis (gray). (B–H) Spindle volume determination using the VolumeJ program. (B,C) Stack projection of an in vitro metaphase cell stained with α-tubulin. (C) ROI extraction of the spindle apparatus. (D) Signal tresholding for each optical section. (E) Signal transformation in pixels. (F) Selection of one spindle pole using the Wand tool. (G) Creation of a mask displaying the selected Left and right spindle pole. (H) 3D volume rendering and calculation. The volume is independently calculated for each spindle pole. Here, the difference in volume (Δv) is 20.7, typical of an asymmetric cell.

Mentions: In dividing apical cortical progenitors, spindle size is correlated with daughter cell identity. The daughter cell inheriting the large spindle gives rise to a neuron, and the daughter cell inheriting the small spindle a self-renewing apical progenitor (Delaunay et al., 2014; Figure 1). 3D reconstruction of the two spindle poles, which allows calculation of the volume of each pole, appears as the method of choice for the accurate determination of SSA (Delaunay et al., 2014). Dissociated mouse apical precursors were fixed and stained for α-tubulin, pericentrin and DAPI to reveal the condensed nuclei. For each metaphase cell, optical sections were acquired every 0.2 to 0.5 μm from top to bottom, using confocal microscopy. To avoid problems with tilted or incomplete spindles, we only considered the metaphases displaying centrosomes of equal size (based on the pericentrin staining). We measured each spindle pole volume and named the larger of the two spindles “Left spindle” (green, Figure 2A) and the smaller “Right spindle” (yellow, Figure 2A). The difference between the left and right spindle poles, called the “3D spindle pole difference,” denoted by ΔV and expressed as a percentage, revealed the SSA magnitude:△V = (VL−VRVL+VR )×100,where VL and VR denote the volumes of the left and right spindle poles, respectively.


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

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

3D SSA analysis (Volume determination). (A) 3D representation of the spindle apparatus in metaphase cell. The larger spindle is colored in green and the right spindle in yellow. The intersection between both sides is represented by the red dashed line and both spindle poles rotate along the same revolution axis (gray). (B–H) Spindle volume determination using the VolumeJ program. (B,C) Stack projection of an in vitro metaphase cell stained with α-tubulin. (C) ROI extraction of the spindle apparatus. (D) Signal tresholding for each optical section. (E) Signal transformation in pixels. (F) Selection of one spindle pole using the Wand tool. (G) Creation of a mask displaying the selected Left and right spindle pole. (H) 3D volume rendering and calculation. The volume is independently calculated for each spindle pole. Here, the difference in volume (Δv) is 20.7, typical of an asymmetric cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321609&req=5

Figure 2: 3D SSA analysis (Volume determination). (A) 3D representation of the spindle apparatus in metaphase cell. The larger spindle is colored in green and the right spindle in yellow. The intersection between both sides is represented by the red dashed line and both spindle poles rotate along the same revolution axis (gray). (B–H) Spindle volume determination using the VolumeJ program. (B,C) Stack projection of an in vitro metaphase cell stained with α-tubulin. (C) ROI extraction of the spindle apparatus. (D) Signal tresholding for each optical section. (E) Signal transformation in pixels. (F) Selection of one spindle pole using the Wand tool. (G) Creation of a mask displaying the selected Left and right spindle pole. (H) 3D volume rendering and calculation. The volume is independently calculated for each spindle pole. Here, the difference in volume (Δv) is 20.7, typical of an asymmetric cell.
Mentions: In dividing apical cortical progenitors, spindle size is correlated with daughter cell identity. The daughter cell inheriting the large spindle gives rise to a neuron, and the daughter cell inheriting the small spindle a self-renewing apical progenitor (Delaunay et al., 2014; Figure 1). 3D reconstruction of the two spindle poles, which allows calculation of the volume of each pole, appears as the method of choice for the accurate determination of SSA (Delaunay et al., 2014). Dissociated mouse apical precursors were fixed and stained for α-tubulin, pericentrin and DAPI to reveal the condensed nuclei. For each metaphase cell, optical sections were acquired every 0.2 to 0.5 μm from top to bottom, using confocal microscopy. To avoid problems with tilted or incomplete spindles, we only considered the metaphases displaying centrosomes of equal size (based on the pericentrin staining). We measured each spindle pole volume and named the larger of the two spindles “Left spindle” (green, Figure 2A) and the smaller “Right spindle” (yellow, Figure 2A). The difference between the left and right spindle poles, called the “3D spindle pole difference,” denoted by ΔV and expressed as a percentage, revealed the SSA magnitude:△V = (VL−VRVL+VR )×100,where VL and VR denote the volumes of the left and right spindle poles, respectively.

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