Centrosome-Dependent Exit of Cytokinesis in Animal Cells Matthieu Piel, Joshua Nordberg, Ursula Euteneuer, and Michel Bornens

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• Movie 1
  (analyzed in Fig. 1A) A post-anaphase HeLa cell from a clone stably expressing centrin-GFP, a centriole marker, was recorded both in phase contrast and in epifluorescence, during 4 hours (30 images/hour). Red arrows point to the midbody (MB), white arrows indicate the attachment points of the intercellular bridge (APs), and green arrows point to the centrosomes (CTR) or to the mother centriole (MC) of the upper daughter cell. The first part of the movie shows the separation of the centrioles from both centrosomes. The GFP signal alone is displayed, first as the whole field recorded and then as a zoom on both pole regions. The second part shows the repositioning of the mother centriole of the upper cell toward the midbody. Phase contrast and fluorescence images were overlaid. The sequence is replayed with a blowup of the midbody region and replayed for a third time with pauses at important steps. First, the mother centriole and the midbody come close to one another; second, the bridge narrows on both sides of the midbody; and third, the mother centriole leaves the midbody region and the bridge cuts. The third part of the movie shows again the whole sequence of the movement of the mother centriole, until it goes back to its former position near the nucleus.

• Movie 2
  A post-anaphase U2OS cell from a clone stably expressing tubulin-GFP and centrin-RFP was recorded in both colors during 2 hours (one image/minute). The movie shows cartoons at various key points to illustrate the working model that we draw from these observations (Fig. 4). Centrioles are also indicated at key points by a large white star for the mother centriole and a small white star for the daughter centriole. The important point in this movie is the release of microtubule bundles from both sides of the midbody, which follows the repositioning of a mother centriole near the tip of the bundle in the upper cell. Microtubule bundles then seem to be sucked inside the cell toward the main aster anchored on the mother centriole, rather than disassembled (their length remains constant).

• Movie 3
  This movie illustrates the variations in the abscission process observed in various cell lines under various culture conditions. The cell lines used stably expressed centrin-GFP, a centriole marker. Cells were recorded both in phase contrast and in epifluorescence. Only key images are shown. The midbody (MB) is indicated by a red arrowhead, and centrioles are indicated by green arrowheads. The first example is a HeLa cell in which both mother centrioles repositioned toward the intercellular bridge: the midbody moved toward the upper cell, and the bridge cut on that side. The second example is a L929 cell seeded on a fibronectin-coated coverslip (5 mg/ml solution, which induce a strong anchorage but does not inhibit cytokinesis): the two daughter cells are very motile, centrioles are widely split, and the bridge elongates. Eventually, the mother centriole in the upper cell moved toward the bridge, entered deeply inside, and reached the midbody. The bridge then cuts, and the centriole goes back inside the cell body. The third example is a 3T3 cell grown on a fibronectin-coated coverslip (5 mg/ml): the two daughter cells become rapidly flat and migrate away from one another; no centriole ever repositions toward the bridge, which elongates and is eventually torn apart.

Supplemental Figure 1. HeLa cells stably expressing centrin-GFP were seeded on a CELLocate (Eppendorf) coverslip and synchronized by a double thymidine block. They were then fixed (2% glutaraldehyde in PBS for 20 min) 14 hours after the release from the block, in order to obtain cells that had passed through mitosis 1 or 2 hours before. Coverslips were then examined under the light microscope to localize cells with centrioles near the intercellular bridge. They were prepared for EM, and cells were localized and imaged with an EM microscope. MC stands for mother centriole, which can be identified by its subdistal appendages (small black arrowhead); DC stands for daughter centriole; and MB stands for midbody. The black arrow indicates the pinched region of the intercellular bridge (see also Fig. 1B). Magnification, ×25,000.

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Supplemental Figure 2. A pair of L929 daughter-cells from a clone stably expressing centrin-GFP, which concentrates into the two centrioles (green arrows), stained with an antibody to tubulin revealed with a secondary antibody coupled to AMCA (shown in black and white), and an antibody to ninein, which strongly stains the microtubule-anchoring subdistal appendages of the mother centriole. Images were acquired and reconstructed as described [M. Piel et al., J. Cell Biol. 149, 317 (2000)]. In both cells, the mother centriole seems to be at the center of diverging rectilinear bundles of microtubules. The lower figure is a ×2 magnification of the mother centriole region of the right daughter cell, showing bundles of microtubules almost homogeneously distributed around the mother centriole. This could reflect the overall tension exerted by the cell periphery on the mother centriole, through an angular sectoring roughly corresponding to that of the microtubule-anchoring subdistal appendages on the mother centriole. One of the bundles ends into the intercellular bridge. Bar, 5 mm

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