Rho-kinase Is Required for Anaphase Cell Elongation (A-B) Selected frames from time-lapse sequences of Drosophila S2 cells, stably expressing histone H2B-GFP, progressing through anaphase (time from the metaphase/anaphase transition indicated in hr:min:s). (A) A typical control cell undergoing anaphase cell elongation. Note the appearance of a polar bleb (arrows: 00:01:52, 00:02:08), allowing for the establishment of a new cell boundary to accommodate the segregating DNA masses (00:02:24; see also Movie 1). (B) A cell following 72 hr rok RNAi failed to elongate in anaphase. No polar blebs appeared, and the spindle poles and segregating DNA masses smashed up against the cell cortex, which bulged slightly outward (arrow). Although furrow ingression was delayed and did not initiate within the 6 min of the sequence depicted here, the cell ultimately divided successfully (see Movie 2). (C) Fixed images of S2 cells following 72 hr rok RNAi, stained for DNA (blue), MTs (green), and F-actin (red). Whereas the cell in late anaphase A (left) appears normal, the anaphase B cell (middle) and telophase cell (right) have failed to elongate. Note the buckled appearance of the MTs in the middle panel. (D-F) Selected frames from fluorescent time-lapse sequences of GFP-tubulin expressing S2 cells in anaphase. (D) A typical control cell. As the cell and spindle coordinately elongated in anaphase, polar blebs appeared (arrows), and the centrally localized interpolar MTs elongated parallel to the spindle axis (00:03:30-00:04:54, see Movie 3). (E) A cell following 48 hr rok RNAi. As the separating spindle poles reached the cell cortex, no polar blebs were observed, cell elongation did not keep pace with spindle extension, and the interpolar MTs buckled outwards (arrow, 00:04:08, see Movie 4). (F) A cell following 120 hr rok RNAi. Again, the interpolar MTs buckled outward in the absence of cell elongation (arrow, 00:03:51, see Movie 5). Scale bars represent 3 μm.

Anaphase Cell Elongation Requires Both rok and Myosin II Function, but Is Less Dependent on Other Cytokinesis Regulators (A-B) Selected frames from time-lapse sequences of S2 cells expressing Spaghetti squash-GFP (in green and in [Á] and [B́]) and mRFP-tubulin (in red) and progressing through anaphase. (A) A control cell shows the rapid recruitment of Sqh-GFP to the equatorial cortex (see Movie 8). (B) Eighty-four hour rok RNAi caused a dramatic delay and reduction of cortical Sqh-GFP recruitment (Movie 9). (C) Quantification of Sqh-GFP recruitment to the equatorial cortex in individual control and rok RNAi cells, performed as detailed in the Experimental Procedures. All of the rok RNAi cells ultimately furrowed, as in (B). Data points corresponding to the selected frames in Á and B́ are indicated (^*, #). (D) Anaphase cell elongation (mean increase in cell length from metaphase ± SE, n = 3-9) was determined from time-lapse sequences of individual cells following the indicated RNAi treatments. (E-I) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (E) Seventy-two hour spaghetti squash RNAi. The spindle buckled when the poles encountered the cortex, and cell elongation and cytokinesis failed (see Movie 10). (F) Seventy-two hour zipper (zip; myosin heavy chain) RNAi. Similarly, the spindle buckled as it tried to elongate in anaphase; cell elongation and cytokinesis both failed (00:13:52-00:20:48, see Movie 11). (G) Seventy-two hour pebble RNAi. Cell elongation occurred and a furrow initiated but then regressed (see Movie 12). (H) Seventy-two hour RacGAP50C RNAi. Some cell elongation occurred, but furrow ingression did not (see Movie 13). (I) Ninety-six hour diaphanous RNAi. Cell elongation occurred normally; a furrow initiated but then the cortex contracted wildly from side to side for several minutes before subsiding (see Movie 14). Times from the metaphase/anaphase transition are indicated in hr:min:s. Scale bars represent 3 μm.

Other Potential MRLC Kinases Do Not Appear to Contribute Significantly to Furrowing (A) Quantification of the incidence of bi/multinucleate cells induced by rok dsRNA in combination with a control dsRNA against E. coli LacI, or with further additions of Mbs dsRNA, which partially suppresses the rok RNAi phenotype, together with dsRNAs of other putative myosin RLC regulators: bent (Projectin myosin light chain kinase, CG32019); Mlck (stretchin-MLCK, CG18255); Pak (PAK-kinase, p21-activated kinase, CG10296); gek (genghis khan, Myotonic dystrophy kinase related, CG4012); and cit (citron, CG10522). S2 cells were incubated with equal amounts of dsRNA for 4 days, fixed, and stained, and the proportion of multinucleate cells counted. LacI dsRNA was used to standardize the total amounts of RNA added. Mbs RNAi greatly reduced the number of multinucleate cells induced by rok RNAi (compare first two bars). Except for citron, which is itself required for cytokinesis (see below), dsRNAs to other putative MRLC kinases had no effect on the frequency of failed cytokinesis in cells depleted of Rok and Mbs.(B-C) Selected frames from time-lapse sequences of GFP-tubulin S2 cells. (B) A cell following 7 days rok + Mbs RNAi. Cell elongation was perturbed (note the buckled spindle 00:04:39), and furrowing was delayed and slowed, but cytokinesis was successful (see Movie 16). (C) A cell following 4 days rok + Mbs + cit RNAi. As with rok + Mbs RNAi, furrowing was delayed and slowed but nonetheless successful. However, cytokinesis still failed through instability of the intercellular bridge, the phenotype characteristic of citron RNAi alone (see Movie 17).

The Buckling of the Anaphase Spindle Following rok RNAi Depends on an Intact Actin Cortex (A) Interpolar MT lengths (d) were determined from time-lapse sequences of individual GFP-tubulin-expressing cells with or without rok RNAi, as indicated in the cartoon. The fold increase in d over time from anaphase onset is plotted (mean ± standard deviation [SD], n = 5). (B) Anaphase cell elongation (mean increase in cell length from metaphase ± standard error [SE], n = 5-12) was determined from time-lapse sequences of individual cells with or without rok RNAi in the presence or absence of Latrunculin A (LatA, 1 μg/ml, added 1-2 hr before imaging). (C and D) Selected frames from representative time-lapse sequences in the presence of LatA (time from the metaphase/anaphase transition indicated in hr:min:s). (C) A control cell (see Movie 6). (D) A cell following 72 hr rok RNAi. Note how the morphology of the elongating spindle following rok RNAi closely resembles that of the control (see Movie 7). Scale bars represent 3 μm.

Contributions of Rok to Furrowing (A and B) Fixed S2 cells stained for F-actin (phalloidin/red), nuclear envelope (wheat germ agglutinin/green), and DNA (Hoechst 33258/blue). (A) Control cells. (B) One hundred forty-four hour rok RNAi showing bi/multinucleate phenotype. (C) Quantification of the incidence of bi/multinucleate cells as a function of duration of rok RNAi (mean ± SE, from three independent experiments). (D-G) Selected frames from time-lapse sequences of GFP-tubulin S2 cells (time from the metaphase/anaphase transition indicated in hr:min:s). (D) Control cell undergoing cell elongation and cytokinesis (see Movie 3). (E) Cell following 48 hr rok RNAi. Cell elongation was delayed and reduced relative to mitotic progress, the spindle buckled, and the cell formed a robust cleavage furrow that was nonetheless delayed (see Movie 4). (F) Cell following 120 hr rok RNAi. Little or no cell elongation occurred, the spindle buckled, and after a large delay an asymmetric furrow formed and ingressed very slowly (arrow). Note the unusual shape of the furrow, which initiates as a narrow cleft rather than a smooth valley. Despite these abnormalities, cytokinesis succeeded (see Movie 5). (G) Cell following 120 hr rok RNAi. The buckling spindle was severely disrupted, and furrowing did not occur (see Movie 15). Note the cells depicted in (D)-(F) are the same as in Figures 1D-1F, but emphasizing later events. Scale bars represent 3 mm.

Model for Anaphase Cell Elongation and Initiation of Cytokinesis in Drosophila S2 Cells We propose that both polar relaxation and equatorial contraction contribute to anaphase cell elongation and the initiation of cytokinesis in S2 cells. In our model, the cell undergoes the transition to anaphase with the rigid cortex of a metaphase cell (in black), but this rigidity must be modulated during anaphase. As the chromosomes (in blue) segregate and the anaphase spindle (MTs in green) extends, the polar cortices relax (dotted lines) and a broad contraction begins at the cell equator (in red). Both of these processes, which normally occur around 2-3 min from anaphase onset, require Rok (and Myosin II): As a result, elongation is effectively blocked by depletion of Rok. In contrast, Pebble and RacGAP50C only have an input into the broad equatorial contraction so that depletion does not remove the polar relaxation input into cell elongation. Later, around 5 min after anaphase onset, an actin ring (dotted red line) drives furrow ingression from the center of the earlier broad zone of contractility. Actin ring contraction is sensitive to Pebble, RacGAP50C, and Diaphanous depletion, but Rok depletion, unless severe or prolonged, leads only to a delay in furrowing and modification of the shape of the ingressing furrow. We propose that all three processes of polar relaxation, broad equatorial contraction, and actin ring contraction are promoted by Rok and myosin II, whereas other cytokinesis regulators such as Pebble are more specialized in the promotion of equatorial contraction and actin ring assembly. Accordingly, Rok depletion is particularly effective at blocking/delaying the earliest events in cytokinesis.