(A–F) S2 cells were fixed and stained for F-actin (phalloidin/red), nuclear envelope (wheat germ agglutinin/green), and DNA (Hoechst 33258/blue) (main panels) or for tubulin (green), F-actin (red), and DNA (blue) (insets). (A) Control; (B–E) 2–3 day treatment with dsRNA for (B) racGap50C, (C) pavarotti, (D) anillin, (E) citron-kinase, and (F) α-SNAP. RNAi of these genes induced multinucleate cells. The insets show late mitotic/telophase cells demonstrating furrowing in (A and D–F) and no furrowing in (B and C). The scale bar represents 10 μm.

(A) A pair of sister cells connected by a bridge where Pav-GFP had concentrated into a compact structure that persisted throughout the 4 hr duration of the movie. Note that in this and subsequent movies, the cells had already completed furrow ingression at the time that the record was started and that the times do not correspond to the time after cytokinesis. (B) Two days of anillin RNAi. Amid rampant membrane blebbing, bundles of Pav-GFP fluorescence coalesced into a compacted midbody structure (00:10:00), although this gradually fragmented into the individual bundles as they decomposed (00:26:00–00:32:00). (C) Three days of citron-kinase RNAi. Bundles of Pav-GFP fluorescence began to split apart (00:18:00) and progressively separate as the plasma membrane rapidly regressed (00:23:00). However, the bundles remained loosely associated with one another and did not stay attached to the regressing membrane (00:27:00). Times are given as (hr:min:s) from the start of each sequence. The scale bar represents 2 μm. See the Supplemental Data for the movie files.

Cells were fixed and stained with Hoechst 33258 (blue) and antibodies to α-tubulin (red) and Anillin (green). (A) In control cells (no RNAi), Anillin localized to the cleavage furrow during anaphase (left panel), formed rings around the midbody matrix in telophase (center panel; note the gap in tubulin immunoreactivity), and persisted at this location into interphase (right panel). (B) Control cells (no RNAi) showing that Anillin-positive bridges connected pairs of cells throughout much of interphase (arrows). Note that Anillin accumulates in the nucleus as cells progress through interphase [20]. (C) Three days of citron-kinase RNAi. Although Anillin accumulated normally in interphase nuclei, and in telophase bridges (not shown), fewer interphase cells were connected by bridges. In addition, remnants of Anillin rings were rarely seen in binucleate cells after citron-kinase RNAi. (D) Quantification of paired cells in controls and after citron-kinase RNAi. After gentle transfer to conA-coated coverslips, cells were immunostained for Anillin and α-tubulin. Mononucleate cells that were paired (sister cells still connected by an Anillin-positive bridge) or unpaired (cells no longer connected by an Anillin-positive bridge) and binucleate cells were counted in controls (n = 255 pairs) and after 3 days of citron-kinase RNAi (n = 264 pairs). Plot shows the percent of cells in each category. (E) Two days of α-SNAP RNAi. Remnants of Anillin rings persisted in the binucleate cells after cytokinesis failure. These rings often appeared stretched or broken (insets). Note also the pair of cells separated by an Anillin ring (arrow); much of the contents of one sister appear to have been transferred to the other, as seen in the live movies, indicating that the bridge is unsealed. The scale bars represent 3 μm.

(A and B) Control cell illustrating anaphase spindle progression, cleavage furrow ingression, and formation of an intercellular bridge. In a longer record (B), the tubulin content of the bridge gradually declined, but the bridge was detectable for at least 1 hr 30 min (arrow). (C) Example of a control cell treated with the inhibitor of F-Actin assembly Latrunculin A (LatA; 1 μg/ml) added shortly (6 min 30 s) after furrowing. This disrupted the compact bundle of microtubules and resulted in widening of the bridge and gradual cleavage furrow regression. Note that during regression membrane attachment to the bridge did not appear to be compromised (arrow). (D) Example of a control cell where LatA was added slightly later (approximately 10 min) after furrowing. Although the tubulin content of the bridge began to decline more rapidly than usual (00:27:21 and 00:42:53) and the connection appeared to widen slightly, bridge integrity was maintained, and cell fusion was not observed within the 2 hr duration of the movie. (E) Two days of anillin RNAi. Anaphase cell elongation, central spindle formation, and furrow ingression proceeded normally (00:00:00–00:05:30). However, shortly after furrowing, extensive membrane blebbing occurred in the cleavage area, associated with microtubules (arrow, 00:10:00). The blebbing subsided, and the microtubules compacted as an apparently normal bridge formed (00:36:30). Later, the microtubule bundle slowly dissociated, then gradually disintegrated amid renewed blebbing (00:50:30). Ultimately, the furrow slowly regressed to form a binucleate cell (01:10:30). (F) Three days of citron-kinase RNAi. The cell progressed normally through anaphase and telophase (00:00:00-00:12:36). Shortly after furrowing, initial elaboration of the intercellular bridge was marked by reversible membrane blebbing (arrows in 00:12:36 and 00:53:33; transiently—see movie S6—this blebbing was more pronounced than in controls but less than in anillin RNAi). Otherwise, the sister cells appeared normal as the intercellular bridge progressively thinned and matured (01:23:39–01:55:30). Eventually, when the bridge was barely still visible (arrow, 1:55:30), the cells abruptly merged (02:04:57). (G) Two days of α-SNAP RNAi. A faint connection is visible between a pair of sister cells at the start of the movie (00:00:00, arrow). By 03:45:00, the right-hand sister began to shrink as the left-hand one grew. Cell fusion occurred at 05:00:00, but note that the bridge was already mature by the start of the movie. Times are given as (hr:min:s) from the start of each sequence. The scale bar represents 3 μm. See the Supplemental Data for the movie files.