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First published online 25 September 2007
doi: 10.1242/jcs.016907

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Liver tetraploidization is controlled by a new process of incomplete cytokinesis

¹ Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France
² Inserm, U567, Paris, France
³ Inserm, U785, Université Paris sud, Centre Hépato-Biliaire, Hôpital Paul Brousse, Villejuif F-94804, France

View larger version (51K): [in this window] [in a new window]   Fig. 1. An incomplete mode of cytokinesis takes place in the liver, triggered by weaning. (A) Immunostaining for -catenin (red) and -tubulin (green) of liver sections (10- and 25-day-old rats). Images of telophase were visualized taking into account condensed chromatin staining (Hoechst 33342, blue). Immunostaining allowed us to distinguish between telophase that completed or did not complete cytokinesis. The percentages of cells with complete cytokinesis are indicated. Bars, 3 µm. (B) Events of incomplete cytokinesis before and after weaning (fixed at 19 days, arrow). At each point, four rats were independently analyzed. Percentages of complete and incomplete cytokinesis in telophase cells were calculated using -catenin/-tubulin/Hoechst immunostaining (see A). A total of 50 telophase cells were analyzed per animal. Bars represent s.e., P<0.0001.

View larger version (109K): [in this window] [in a new window]   Fig. 2. Events of incomplete cytokinesis occur both in periportal (PP) and periveneous (PV) hepatocytes. (A) Immunostaining for glutamine synthetase (GS; proximal PV hepatocyte staining, green, right panel), Pepck1 (proximal PP hepatocyte staining, green, left panel) and -catenin (membrane labelling, red, both panels) was performed on rat liver sections (25-day-old rats). -catenin/Hoechst staining allowed us to distinguish between telophase that completed cytokinesis (ingression of the membrane) and telophase that did not complete cytokinesis (no ingression of the membrane) (Hoechst staining in blue). Bars, 40 µm. (B) Percentage of telophase cells that completed cytokinesis or did not were calculated in each region (proximal PP: GS-positive staining; proximal PV: Pepck1-positive staining; distal PP/PV: GS/Pepck1-negative staining). A total of 50 images of telophase were analyzed per animals (n=4). Bars represent s.e., P<0.0001.

View larger version (126K): [in this window] [in a new window]   Fig. 3. Defect in anaphase cell elongation is observed when hepatocytes do not complete cytokinesis. (A) Hepatocytes were isolated from either 15- or 25-day-old rats. Mitosis of mononuclear 2n cells was monitored. Images are shown at selected time points (minutes). Complete cytokinesis: the cell divides successfully (supplementary material Movie 1, 15-day-old-rat, 100% of cytokinesis events). Incomplete cytokinesis: division leads to the genesis of a binucleated hepatocyte (supplementary material Movie 2, 25-day-old-rats, 28±3.6% of cytokinesis events). Movies are representative of 50 cells analyzed in four independent cultures. The outlines show cell shape. (B) Cell elongation from metaphase to telophase was determined using time-lapse sequences. Time 0 correspond to chromosome alignment along the metaphase plate. Data are presented as the mean±s.e. from 25 independent time-lapse sequences. (C) Chromosome-to-membrane distance was measured on the same time-lapse sequences presented in B. The histogram represents the average chromosome-to-membrane distance of complete/incomplete cytokinesis. Data are presented as the mean±s.e. from 25 independent time-lapse sequences, P<0.0001. Images shown are representative of anaphase cells that will complete or not complete cytokinesis. The outlines show cell shape; arrows represent chromosome-to-membrane distance. Bars, 5 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Actin cytoskeleton rearrangement does not occur during incomplete cytokinesis. (A) Hepatocytes were isolated from either 15- or 25-day-old rats. Hepatocytes were stained with Alexa-Fluor-488-phalloidin (Actin) and nuclei with Hoechst (DNA). A total of 200 cells from four independent experiments were analyzed. (B) The Alexa-Fluor-488-phalloidin fluorescence profile was determined along the cortex in early telophase of complete (left) and incomplete (right) cytokinesis. Quantification was measured only on half of the cell, from one pole to the other (see upper scheme, red line). The broken vertical lines surround the equatorial area. A total of 50 early telophase cells were analyzed in four independent cultures. (C) Cell elongation from metaphase to telophase was determined using time-lapse sequences; hepatocytes were treated or not with HA1077 (15-day-old rats). Time 0 correspond to chromosome alignment along the metaphase plate. Data are presented as the mean±s.e. from ten independent time-lapse sequences. (D) Hepatocytes were isolated as in A. Hepatocytes were stained with anti-phospho-Myosin light chain 2 (Phospho-RLC) and nuclei with Hoechst (DNA). Early telophase cells are shown (n=200 from four independent cultures). Bars, 5 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 5. Organization of astral and central spindle microtubules during incomplete cytokinesis. (A) Hepatocytes were isolated from either 15- or 25-day-old rats. Hepatocytes were stained with anti--tubulin and nuclei with Hoechst (DNA). A total of 200 cells from four independent experiments were analyzed. Bars, 5 µm. (B,C) Hepatocytes (25-day-old rats) were stained with anti--tubulin (B, microtubules, green) or anti-EB1 (C, microtubule tips, green) and anti--catenin (B,C, cell cortex, red). A total of 100 cells from four independent experiments were analyzed. (B) Images represent magnified equatorial regions of anaphase hepatocytes. Bars, 1 µm. (C) Nuclei were stained with Hoechst (blue). Boxed areas are shown below: higher-magnification of the equatorial cortical region. Bars, 5 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Incomplete cytokinesis is characterized by an absence of active RhoA localization at the putative cleavage plane. (A,B) Hepatocytes were isolated from 25-day-old rats. Hepatocytes were stained with anti-MgcRacGAP (A, red) and anti--tubulin (A, green) or with anti-RhoA (B, green) antibodies. Nuclei were stained with Hoechst 33342 (A,B, blue). A total of 200 cells from four independent experiments were analyzed. Bars, 5 µm.

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