Mechanisms of interkinetic nuclear migration Y Kosodo et al Figure 4 Tpx2 alters microtubule organization in the apical process of G2-phase neural progenitor cells. (A) Images of cell cycle phase marker (CCPM; a) and EB3-tagRFP (a0) in living brain slice cultures. CCPM and EB3-tagRFP were co-electroporated into neural progenitor cells in E12.5 mouse brain, and ﬂuorescent signals from two separate wavelengths were acquired from living tissue slices prepared from E13.5 brain. Note that CCPM localization inside the nucleus indicates that the cell is in G1-phase (ﬁlled arrowhead in a and a00), whereas its localization peripheral to the nucleus indicates that the cell is in G2-phase (open arrowhead in a and a00). Arrows in a0 and a00 indicate organizations of microtubules in the apical process identiﬁed by EB3-tagRFP. (B) Examples of microtubule organization in each cell-cycle phase combined with knockdown of Tpx2 functions. Images were acquired as described in (A). LacZ miR RNAi (control, a, b) or Tpx2 miR RNAi (c, d) were co- electroporated together with CCPM and EB3-tagRFP. Cell-cycle phases identiﬁed by the localization of CCPM are indicated in the panel. Arrowheads and arrows are as described in (A). (C) Quantitation of EB3-tagRFP in the apical process. (a) Scanning of EB3-tagRFP signal in the apical process. The pixel intensity of tagRFP ﬂuorescence in the region of the apical process close to the nucleus (dotted rectangle, 5 mm in length) was measured using MetaMorph software. The double arrow indicates the direction of scanning. (b) Relative intensity of EB3-tagRFP ﬂuorescence in each cell-cycle phase combined with knockdown of Tpx2 functions. The pixel intensity of tagRFP ﬂuorescence in each condition (colour codes are indicated in the panel) was quantiﬁed as described in (a). Signals were normalized as follows: after subtracting the background level from the raw digitized value of pixel intensity, the remaining value was divided by the sum of all the background-corrected values to calculate percentages (y-coordinate). Error bars indicate s.e.m. Bars in (A–C) ¼ 10 mm. (D) Ultrastructural analysis of Tpx2 function on microtubule organization. LacZ miR RNAi (control, a) or Tpx2 miR RNAi (b) were co-electroporated together with CCPM into neural progenitor cells in E13.5 mouse brain, and a slice culture was prepared. After the ﬁxation and permeabilization, pre-embed immunolabelling (Toida et al, 2000) was performed on the brain slice using GFP antibody and 3,30-diaminobenzidine (DAB) reaction to identify the apical process of G2-phase cells. After embedding, 50-nm thick ultrathin sections parallel to the apical surface were prepared to observe the cross- sections of the apical process (10–15 mm away from the apical surface). Asterisk in (b) show DAB-negative cell, its microtubules are seen as small dots. Note that DAB precipitation around microtubules is distinct in DAB-positive cells (arrows in a, b). Bar ¼ 500 nm. (c, d) Number of 2 microtubules in a single (c) or unit area (0.1 mm ) (d) of the cross-sections of DAB-positive apical process in control or Tpx2 knocked-down situation (eight cases in each condition). ***Po0.001, t-test (in d). Error bars indicate s.e.m. For (A–C), apical surface is down. might affect microtubule orientation, the resolution of our assay system could not distinguish differences of EB3 move- ments in the thin process of neural progenitors between TPX2 knocked-down cells and control cells. We further explored the role of Tpx2 in the apical process during G2-phase cells by performing immunoelectron micro- scopy to observe the microtubule organization at ultrastruc- tural resolution in the cross-section of the apical process. 1696 The EMBO Journal VOL 30 | NO 9 | 2011 & 2011 European Molecular Biology Organization