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Centrosomin (CNN), a core Drosophila centrosome protein, interacts with the newly identified protein Centrocortin to promote cleavage furrow formation in the early embryo. Significantly, this activity is distinct from CNN's well-established role in centrosome-based microtubule organization.
Centrosome-based astral microtubule arrays play a key role in the formation and positioning of the cleavage furrow. The work presented in a recent issue of Current Biology by Kao and Megraw  identify a centrosome-associated protein, Centrocortin (CEN), that does not influence microtubule organization but has a profound effect on furrow formation. These studies have their conceptual origin in an ingenious experiment conducted almost a half century ago . By passing a glass rod through a single-celled sand dollar embryo and allowing it to go through a round of division, Rappaport created a syncytial embryo containing two nuclei. When these nuclei divide, furrows form in the expected position between separated sister chromosomes. In addition, a third, ectopic furrow is formed between neighboring non-sister centrosomes. Significantly, the region of the cytoplasm in which these ectopic furrows formed did not contain chromosomes or a spindle. Follow-up experiments demonstrated that a minimal distance between the two centrosomes and between the centrosomes and cortex were critical factors in inducing formation of these furrows . Equivalent experiments performed in mammalian cells as well as in other systems demonstrated this to be a general phenomenon .
One explanation for the origin of these ectopic furrows, now referred to as Rappaport furrows (Figure 1A), is that activities associated with the centrosomes and their associated astral microtubules are sufficient for induction of the cleavage furrow . Rappaport's experiments were key to the development of the equatorial stimulation model of cytokinesis. This model proposes that positive signals from opposing, overlapping astral microtubules interacting with the cortex at the cell equator provide the initial signals inducing cleavage furrow formation . While it is now clear that additional features of the mitotic spindle, such as the central spindle, also play critical roles in furrow induction and position, recent studies have provided molecular support for the equatorial stimulation model [7,8]. Bringmann and colleagues  have identified cortically localized LET-99 and the interacting heterotrimeric G-proteins GOA-1 and GPA-16 as essential for astral-microtubule-induced furrow formation in Caenorhabditis elegans embryos. Mechanical displacement of the spindle resulted in an equivalent displacement of LET-99 such that it always concentrated at the spindle midpoint and the presumptive site of furrow formation. Although the exact mechanism by which LET-99 is positioned is unknown, the authors suggest LET-99 responds to microtubule-induced cortical tension.
Thus, centrosomes play a key role in cytokinesis through the generation of astral microtubule arrays that interact with the cortex to induce furrow formation. As reported in their recent paper, Kao and Megraw  tackle the less well explored, but equally important, issue of whether centrosome-associated activities, distinct from organizing microtubules, are required for cleavage furrow formation. These studies take advantage of the unique furrows that form during the initial divisions of Drosophila embryogenesis. Following fertilization and nine rounds of rapid synchronous divisions in the interior of the embryo, syncytial nuclei are organized in a monolayer along the actin-rich embryo cortex . During interphase of these cortical divisions, each nucleus and its apically associated centrosome pair organize actin into caps encompassing the centrosomes and their asters. As the nuclei progress into prophase with separated centrosomes, the actin reorganizes into furrows that encompass each maturing spindle (Figure 1B). Although the timing and positioning of these furrows is unusual, they are structurally and compositionally indistinguishable from conventional cytokinesis furrows. In the absence of these furrows, known as pseudocleavage or metaphase furrows, neighboring spindles fuse . Thus, metaphase furrows function as barriers between the highly dynamic, closely packed syncytial spindles. During the late cortical divisions, thousands of metaphase furrows form interlocking rings across the entire embryo cortex. With respect to furrow position, these naturally occurring metaphase furrows are equivalent to the experimentally induced Rappaport furrows . Like Rappaport furrows, metaphase furrows form between neighboring non-sister centrosomes in the absence of chromosomes and spindles. It appears that their position is determined by overlapping astral microtubule arrays during early prophase.
Kao and Megraw  began their studies by characterizing a hypomorphic cnn allele, cnnB4. Drosophila Centrosomin (CNN) is a core centrosomal protein required for normal pericentriolar material organization and astral microtubule assembly [13,14]. In contrast to null alleles, cnnB4 had no discernable effect on microtubule organization yet still produced severe disruptions in furrow formation. Thus, the microtubule- and furrow-organizing functions of CNN are genetically separable. Sequence analysis revealed a point mutation in the conserved carboxy-terminal domain of CNN. Reasoning that proteins interacting with this domain would be essential for CNN's role in furrow formation, Kao and Megraw  identified CEN through two-hybrid analysis. CEN has uncharacterized mammalian orthologs, including the human genes cerebellar degeneration related-2 (Cdr2) and Cdr2-like . CEN localization partially overlaps CNN at the centrosome. During the interphase/prophase transition, CEN localizes between centrosome pairs. Upon centrosome separation, as the nuclei enter prophase, CEN segregates asymmetrically with only one of the two centrosomes. As no other asymmetries have been identified during these divisions, this was unexpected. The functional significance of this asymmetric localization remains unclear. Significantly, CEN also localizes to the metaphase furrows. These localization studies combined with the fact that CEN specifically binds the carboxy-terminal domain of CNN make it an excellent candidate for a molecular link between the centrosomes and cleavage furrow.
Analysis of cen mutants support this interpretation. Strong cen alleles produce phenotypes very similar to the cnnB4 hypomorph described above. Like the cnnB4 mutant, cen mutants had no effect on microtubule organization. In addition, interphase actin-cap organization was normal in these mutants. However, cen mutant embryos displayed a high frequency of broken and weak furrows during prophase and metaphase. These defects readily account for the numerous spindle fusions observed in cen mutant embryos. Taken together, these data support a model in which the conserved carboxy-terminal domain of centrosome-localized CNN is essential for proper cleavage furrow assembly. CNN signaling to the furrow relies on CEN, a protein that localizes at the centrosome and cleavage furrow.
The specific function of CEN at the centrosomes and furrows remains unclear. Previous studies demonstrated that proper organization of the centrosome-associated recycling endosome is required for vesicle-based membrane delivery and proper actin organization at the metaphase furrows [16,17]. Mutants that disrupt recycling endosome organization produce furrow defects strikingly similar to the cen mutant phenotypes . However, cen mutations do not appear to disrupt recycling endosome organization. Thus, in addition to astral microtubule formation and recycling endosome organization, Kao and Megraw  have identified a new centrosome-associated activity required for furrow formation. Fortunately, the identification of CEN provides a means to characterize the components and function of this unexpected signaling pathway between the centrosome and cleavage furrow.