Figure 7
Figure 7. Asymmetric and symmetric AP2A2 during mitosis. Still frames from live-cell videomicroscopy. All cells, adult CD150+48−LSK: DNA (Fitc-green); AP2A2 (Cherry-red/orange). All time points referenced to onset of mitosis (time, 00:00:00 as hours:minutes:seconds). The mean total mitosis times for an ACD was 3:04:17 (n = 6 visualized mitotic cells) and for SCD was 2:56:04 (n = 32 visualized mitotic cells). Differences were not statistically significant. (A) Hematopoietic cell showing AP2A2 asymmetric clustering during and after cell division into daughter cells. (B) Hematopoietic cell (right) with AP2A2-polarized clustering and asymmetric segregation into only one daughter cell (asterisk). Other daughter cell (no asterisk) reexpressed small vesicles of AP2A2 from time 3:58:04 onward (see also supplemental Videos 1-3). (C) HSC with diffuse, symmetric Ap2a2 distribution during and after mitosis with both daughter cells acquiring Ap2a2. (D) HSC with AP2A2 symmetric in division. Telophase midbody concentration is seen at time 2:39:42 (see also supplemental Videos 4-5). Videos were acquired with a DeltaVision video microscope fitted with a 37°C environmental chamber (Applied Precision) using Olympus 60×/1.42 numerical aperture oil-immersion lens and a Photometric CoolSnap HQ2 camera. Video analyses and still frames were performed with softWoRxExplorer Version 2.0 software (Applied Precision). All scale bars indicate 10 μm. (E-F) Influence of different feeder layers (external environment) on AP2A2 localization during mitosis. The terms symmetric and asymmetric were as defined in Figure 6E and I and C and F, respectively. D3-5 and D7-12 refer to days after CD150+48−KLS cell transduction with Ap2a2. NIH 3T3 (ie, GP+E-86) and OP9 are respective feeder layers. CK refers to cytokinesis seen (ie, a successful mitotic division). Failed M (mitosis)–apoptosis was defined as cells seen to be in mitosis for longer than 4 hours, because these cells would eventually abort mitosis to reenter interphase or undergo apoptosis. Analyses based on accumulated total number of dividing cells seen in each time period. (E) D3-5 with NIH 3T3 layer, total cells = 162 from 3 independent experiments; D7-12 with NIH 3T3 layer, total cells = 128 from 2 independent experiments. (F) D3-5 with OP9 layer, total cells = 147 from 2 independent experiments. All values presented are means ± SEM of respective experiment groups.

Asymmetric and symmetric AP2A2 during mitosis. Still frames from live-cell videomicroscopy. All cells, adult CD150+48−LSK: DNA (Fitc-green); AP2A2 (Cherry-red/orange). All time points referenced to onset of mitosis (time, 00:00:00 as hours:minutes:seconds). The mean total mitosis times for an ACD was 3:04:17 (n = 6 visualized mitotic cells) and for SCD was 2:56:04 (n = 32 visualized mitotic cells). Differences were not statistically significant. (A) Hematopoietic cell showing AP2A2 asymmetric clustering during and after cell division into daughter cells. (B) Hematopoietic cell (right) with AP2A2-polarized clustering and asymmetric segregation into only one daughter cell (asterisk). Other daughter cell (no asterisk) reexpressed small vesicles of AP2A2 from time 3:58:04 onward (see also supplemental Videos 1-3). (C) HSC with diffuse, symmetric Ap2a2 distribution during and after mitosis with both daughter cells acquiring Ap2a2. (D) HSC with AP2A2 symmetric in division. Telophase midbody concentration is seen at time 2:39:42 (see also supplemental Videos 4-5). Videos were acquired with a DeltaVision video microscope fitted with a 37°C environmental chamber (Applied Precision) using Olympus 60×/1.42 numerical aperture oil-immersion lens and a Photometric CoolSnap HQ2 camera. Video analyses and still frames were performed with softWoRxExplorer Version 2.0 software (Applied Precision). All scale bars indicate 10 μm. (E-F) Influence of different feeder layers (external environment) on AP2A2 localization during mitosis. The terms symmetric and asymmetric were as defined in Figure 6E and I and C and F, respectively. D3-5 and D7-12 refer to days after CD150+48−KLS cell transduction with Ap2a2. NIH 3T3 (ie, GP+E-86) and OP9 are respective feeder layers. CK refers to cytokinesis seen (ie, a successful mitotic division). Failed M (mitosis)–apoptosis was defined as cells seen to be in mitosis for longer than 4 hours, because these cells would eventually abort mitosis to reenter interphase or undergo apoptosis. Analyses based on accumulated total number of dividing cells seen in each time period. (E) D3-5 with NIH 3T3 layer, total cells = 162 from 3 independent experiments; D7-12 with NIH 3T3 layer, total cells = 128 from 2 independent experiments. (F) D3-5 with OP9 layer, total cells = 147 from 2 independent experiments. All values presented are means ± SEM of respective experiment groups.

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