siRNA treatment and rescue HeLa cells were grown to 50C60% confluency and transfected with 40 nM or 120 nM double-stranded siRNA (anillin and SEPT9, respectively) using lipofectamine 2000 (Invitrogen). action of the anillinCseptin dBET1 complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis. S2 cells can be bypassed, and the cells consequently show defects in midbody ring assembly [20C22]. Likewise, in mammalian cells, depletion of SEPT9 prolongs cytokinesis with an eventual failure to abscise . These combined observations suggest that anillin and septins may have specific functions during the later stages of mammalian cytokinesis. Although recent studies have shed light on the final stages of abscission and its regulation [14C16,24,25], little is known about the mechanism of ICB formation that provides the platform for subsequent cell abscission. Here, we investigate the role of anillin in ICB formation and maturation through its function in recruiting the septin cytoskeleton. Using novel tools that allow initial furrow ingression to progress in the absence of the septin cytoskeleton combined with live imaging and subdiffraction three-dimensional structured illumination microscopy (3D-SIM), we define new dBET1 stages and structures required for ICB assembly and abscission in mammalian cells. 3.?Results 3.1. Anillin dynamically associates with late cytokinetic structures To assess anillin’s role in the late stages of cytokinesis, we analysed anillin dynamics during cytokinesis in a HeLa cell line stably expressing inducible green fluorescent protein (GFP)-anillin at levels similar to endogenous anillin (see electronic supplementary material, figure S1). Time-lapse analysis of GFP-anillin revealed previously undescribed phases of anillin organization, suggesting new distinct stages of cytokinesis where anillin may function (figure 1and the electronic supplementary material, video S1). Prior to chromosome segregation, anillin is distributed along the plasma membrane before it concentrates in the furrow upon ingression. As the opposing membranes of the furrow converge, anillin localizes to a distinct collar that defines the initial ICB (1.89 0.08 m diameter, and 1.27 0.03 m long, as measured along the axis of the ICB, = 18). Next, the anillin collar elongates almost Mapkap1 threefold to 3.61 0.25 m in length and narrows by 25% to a diameter of 1 1.44 0.06 m, = 18 (see electronic supplementary material, video S2). The anillin collar then retracts and reorganizes to form three distinct rings (figure 1and the electronic supplementary material, figure S2and the electronic supplementary material, video S3). By contrast, GFP-anillin localized to the ICB later in cytokinesis showed lower levels of recovery dBET1 after photobleaching (figure 1and electronic supplementary material, video S4). Later in cytokinesis at the three-ring stage, GFP-anillin was bleached at the central stem body and one constriction site. Now, only 33.2 1.9% of GFP-anillin signal at the constriction site was recovered. However, this was significantly more than the 26.5 2.2% of the GFP-anillin signal recovered at the stem body (= 0.03, figure 1and the dBET1 electronic supplementary material, video S5). The microtubules of the spindle midzone become increasingly bundled as the furrow ingresses. As the microtubule bundles become denser, two regions of lower GFP-tubulin intensity form on either side of the central bulge that marks the stem body. We refer to these regions of decreased microtubule staining as constriction sites because they are of a narrower diameter than the dBET1 microtubule bundles elsewhere in the ICB. Next, the microtubule bundle narrows further to the diameter of the two constriction sites, resulting in a bundle of microtubules with a uniform diameter, except for a less pronounced bulge of GFP-tubulin intensity at the stem body. Following this, we observed the oscillation of the microtubule bundle from side to side within the ICB. Finally, asymmetric cleavage occurs, termed abscission, and the ICB remnant is consumed by one of the daughter cells. Open in a separate window Figure?2. The ICB forms through a series of defined organizational states. (= 17, collar, = 28, three-ring,.