InteractiveFly: GeneBrief

scraps: Biological Overview | References

Anillin is a conserved component of the contractile ring that is essential for cytokinesis, and physically interacts with three conserved cleavage furrow proteins, F-actin, myosin II and septins in biochemical assays. The Drosophila scraps gene, identified as a gene involved in cellularization, encodes Anillin. Defects have been characterized in cellularization, pole cell formation and cytokinesis in a series of maternal effect and zygotic anillin alleles. Mutations that result in amino acid changes in the C-terminal PH domain of Anillin cause defects in septin recruitment to the furrow canal and contractile ring. These mutations also strongly perturb cellularization, altering the timing and rate of furrow ingression. They cause dramatic vesiculation of new plasma membranes, and destabilize the stalk of cytoplasm that normally connects gastrulating cells to the yolk mass. A mutation closer to the N terminus blocks separation of pole cells with less effect on cellularization, highlighting mechanistic differences between contractile processes. Cumulatively, these data point to an important role for Anillin in scaffolding cleavage furrow components, directly stabilizing intracellular bridges, and indirectly stabilizing newly deposited plasma membrane during cellularization (Field, 2005).

Cytokinesis in animal cells occurs by ingression of a cleavage furrow, driven by constriction of an actomyosin-based contractile ring coupled to insertion of new plasma membrane. An important unanswered question is how are furrow ingression and new membrane deposition coordinated? The Drosophila embryo provides an interesting model system with which to address this question and the general cell biology of furrowing. During the syncytial blastoderm stage, typical cytokinesis does not occur, but transient furrows ingress to keep mitotic spindles separate. At this stage, pole cells separate from the yolk mass by a type of cytokinesis that has been little studied. Later, during a process called cellularization, interconnected furrows ingress between each nucleus and synchronously partition the embryo into ~6000 individual cells. After cellularization, conventional cytokinesis starts in the mitotic domains of the gastrulating embryo (Field, 2005 and references therein).

Cellularization is more complex than conventional cytokinesis. It requires an estimated 25-fold increase in surface area during a single cell cycle, and is thus useful for exploring the coupling between actomyosin contraction and insertion of new plasma membrane. It also requires ingression in two different planes. Initially, furrows ingress perpendicularly to the embryo surface. The tips of cellularization furrows are called furrow canals, and they interconnect as an almost hexagonal network surrounding each nucleus. Ingression of the plasma membrane occurs in at least two stages that differ in rate (initially slow, then fast) and mechanism, with certain mutations selectively affecting one stage. Later, once the furrow canals pass the nuclei, ingression also occurs parallel to the embryo surface. The furrow canals broaden and become almost triangular-shaped in cross-section, and the network transforms into an almost hexagonal array of contractile rings. These constrict around the base of each nucleus, individualizing the cells. Constriction is incomplete. The newly formed cells remain connected to the yolk mass by a thin neck of cytoplasm or 'stalk' as the embryo initiates gastrulation movements (Field, 2005 and references therein).

Given their rich contractile biology, Drosophila embryos have been useful for investigating the mechanism of furrowing. As expected, embryonic furrows contain the contractile proteins F-actin and cytoplasmic Myosin II. They also contain septins and Anillin, conserved furrow components whose function is less clear. Septins were discovered as CDC mutants in budding yeast and were implicated in animal cytokinesis by analysis of mutations in the Drosophila septin Peanut. Peanut was later shown to be involved in cellularization. Biochemical investigation in Drosophila embryos showed that septins bind GTP and assemble into heteromeric complexes and filaments. Their molecular function is unknown, although septins have been implicated in vesicle trafficking (Field, 2005).

Anillin was originally isolated from Drosophila embryos by affinity chromatography on F-actin (Field, 1995) and homologs were later found in vertebrates (Oegema, 2000; Straight, 2005) and C. elegans (see Maddox, 2005). Anillin is required for cytokinesis in Drosophila and vertebrate tissue culture cells (Oegema, 2000; Somma, 2002; Kiger, 2003; Echard, 2004; Straight, 2005), but its function during furrowing remains unclear. Mid1 and Mid2, two proteins with more limited homology to Anillin, play central roles in cytokinesis in S. pombe (Berlin, 2003; Paoletti, 2000; Tasto, 2003; Field, 2005 and references therein).

Anillin is a multi-domain protein that physically interacts with several other cleavage furrow components in vitro. Its N terminus contains a region that binds and bundles F-actin (Field, 1995; Oegema, 2000), and a second region that binds phosphorylated cytoplasmic Myosin II (Straight, 2005). Its C-terminus comprises a predicted PH domain, an ~100 amino acid module often implicated in binding to membranes via inositol lipids (Lemmon, 2004; Lemmon, 2002). The C-terminal region of vertebrate Anillin was implicated in septin binding by expression of truncated protein and biochemical assays (Oegema, 2000; Kinoshita, 2002), but the physiological relevance of that proposed interaction was not tested. To explore the function of Anillin, in particular its potential role in coupling cytoskeletal and membrane dynamics, the effects were analyzed of a series of mutations in Anillin on the diverse, cell cycle regulated furrows in the early Drosophila embryo (Field, 2005).

To find mutations in Anillin, P-element insertions were sought near its map location, 43DE (Field, 1995). The genomic region around the insertion site in line P3427, was sequenced, revealing a P-element insertion 53 bp upstream of a potential anillin start codon. The scraps gene also maps in this region. A screen for recessive female sterile mutations (Schupbach, 1989) isolated six maternal effect alleles in this gene, designated scraps^RS, scraps^PQ, scraps^HP, scraps^PE, scraps^RV and scraps^PB. Females homozygous for any of these alleles, or trans-heterozygous for any allelic combination, lay morphologically normal eggs that fail to hatch. Observation of mutant embryos using bright-field microscopy showed cellularization defects. Complementation tests over a deficiency of the region revealed a zygotic function for scraps (Schupach, 1989) and a later study identified two zygotic alleles, scraps⁷ and scraps⁸ (Heitzler, 1993). Two additional maternal effect alleles, scraps^B26-35 and scraps^C82-45, have been identified. In complementation tests, it was found that that the P3427 insertion line is allelic to scraps (Field, 2005).

To confirm that Anillin is the product of the scraps gene, rescue experiments were performed by injecting anillin cDNA. All the embryos from scraps^RV/RV mothers that received the transgene were rescued to hatching, and ~90% of these developed into fertile adults. It is concluded that Anillin is essential for embryonic viability and is the product of the scraps gene. (Field, 2005).

The strongest molecular conclusion from this work is that Anillin is required to target and maintain septins in cortical structures. Strong maternal anillin alleles map to the N terminus of the PH domain, the region of Anillin previously predicted to interact with septins on the basis of fragment expression (Oegema, 2000) and in vitro binding assays (Kinoshita, 2002). These alleles cause severe mis-localization of the septin Peanut and also result in a significant reduction in the amount of Anillin in furrow canals late in cellularization. Thus, the genetic and biochemical data together suggest that a physical interaction between the PH domain of Anillin and septin complexes is required to stably recruit both proteins to ingressing furrow canals. This interdependence is also supported by analysis of Peanut mutants. In embryos that lack Peanut, F-actin rings failed to form late in cellularization, similar to strong anillin alleles. Anillin localization is normal in slow phase, but the protein is progressively lost from furrow canals during fast phase, showing that Peanut is necessary for stable localization of Anillin. A role for Anillin in targeting and stabilizing septins is also supported by studies in C. elegans, where the Anillin homolog ANI-1 is required to target the septins to the contractile ring (Maddox, 2005), and in S. pombe (Berlin, 2003; Tasto, 2003), where the Anillin-related protein Mid2 stabilizes cortical septins in the medial ring (Field, 2005).

PH domains in many proteins have been implicated in lipid binding, and the amino acid changes in strong anillin alleles fall in the region of this domain known to interact with lipids. However, the PH domain of Anillin lacks the positively charged amino acids that mediate specific binding to phosphoinositides, and GFP fusions to the C terminus of human Anillin transiently expressed in mammalian cells did not localize to the plasma membrane, but instead assembled into small septin-containing foci (Oegema, 2000). It therefore seems possible that the PH domain of Anillin mediates a protein-protein interaction with septins and not a protein-lipid interaction. However, Drosophila Anillin can target to the cellularization front in the absence of Peanut (Adam, 2000) and the C. elegans ortholog ANI-1 can target to the furrow normally in the absence of the septins (Maddox, 2005). Many PH domains do not bind inositol lipids with high affinity and require oligomerization or additional motifs within the same protein to impart membrane localization (Lemmon, 2002). It is therefore possible that the PH domain of Anillin mediates association with membranes by mechanisms other than septin binding (Field, 2005).

Morphologically, the most dramatic phenotype of strong anillin alleles was the appearance of sheets of vesicles (that appear as lines in thin sections) between nuclei during cellularization, in place of the intact, apposed plasma membranes deposited behind the cellularization front in wild-type embryos. The presence of some intact membranes in mutant embryos fixed early in cellularization is interpreted as evidence that lateral plasma membranes are initially deposited in anillin mutant embryos, but they are unstable and subsequently vesiculate. Other membranes in the mutant embryos, including pole cell and apical plasma membranes, are unaffected, arguing that vesiculation is not a fixation artifact, and that the new lateral plasma membranes have a specific requirement for Anillin and septins for stability. These membranes are special in at least three ways that might account for their fragility: they assemble very rapidly, by highly dynamic exo- and endocytosis; they are probably under tension from the ingressing furrow canals; and they are closely apposed to each other. Anillin might regulate vesicle trafficking dynamics; for example, decreasing exocytosis could lead to a build up of tension and membrane fragmentation. Septins have been argued to regulate exocytosis in mammalian cells but, in this case, inactivation of septins leads to increased, rather than decreased, exocytosis. Alternatively, Anillin might directly regulate physical stability of membranes. Plasma membranes are physically stabilized in most situations by attachment to a cortical actin cytoskeleton, and loss of Anillin might destabilize them by weakening the cortex or its attachment to the membrane. Destabilization of membranes under tension might lead to the fusion of closely apposed membranes and the lines of vesicles observed are reminiscent of some stages of programmed cell fusion, e.g. myoblast fusion. Anillin itself does not localize to the cortex of the apposed plasma membranes, but it might function to recruit and leave behind other proteins required for stability under tension. Septins are normally present and localized ectopically in anillin mutants; although targeting of F-actin is less affected, its organization in mutants is unknown. Loss of septins or F-actin bundling could result in a more fragile, or more weakly attached, cortical cytoskeleton, causing the membranes to fragment as tension builds up during cellularization. A role for septins in stabilizing membranes could be tested by TEM of peanut mutant embryos, which are known to exhibit defects in nuclear positioning similar to those observed in anillin mutants (Adam, 2000). Roles for Anillin in regulating membrane trafficking compared with physical stability might be distinguished by live imaging of membrane markers to measure exo- and endocytosis, and by imaging thermal fluctuations of the new plasma membranes to estimate their stiffness, as a function of anillin genotype (Field, 2005).

An interesting aspect of the vesiculation phenotype is the tendency of plasma membrane-derived vesicles to remain localized in sheets behind the cellularization front, rather than diffusing away. It is thought that they may be adhering to the baskets of microtubules that surround each nucleus, that are unaffected in anillin mutant embryos. Remarkably, the physical organization of vesicles is sufficient to allow gastrulation movements, even though cellularization has failed completely in terms of generating cells bounded by plasma membranes (Field, 2005).

Although defects in septin recruitment mirrored the allelic series, septin recruitment is clearly not the only function of Anillin. Similar defects are observed in the timing and rate of cellularization front ingression in all alleles examined, including weak alleles that had no obvious defects in septin recruitment. Defects were also observed in F-actin and Myosin II localization/organization that are consistent with previously identified biochemical interactions (Field, 1995; Straight, 2005). Since the alleles did not alter the N terminus of Anillin, where it interacts with F-actin and Myosin II, these defects probably result from reduced localization of functional Anillin. A role for Anillin in scaffolding contractile structures has been demonstrated in C. elegans, where the Anillin homolog ANI-1 is required to organize foci containing Myosin II that pull on the plasma membrane during polarity establishment (Maddox, 2005). Focusing of actomyosin contraction by Anillin is also suggested by excessive membrane blebbing and mislocalization of myosin II seen during cytokinesis after knocking down Anillin by RNAi (Echard, 2004; Somma, 2002; Straight, 2005; Field, 2005 and references therein).

A more structural role for Anillin may be important late in cytokinesis and cellularization. Myosin II, and most F-actin, typically leave furrows before cytokinesis is complete, and it is important that something prevents the furrow from opening back up when it is no longer actively contractile. Anillin and septins may assemble into a structure under the plasma membrane to stabilize the neck of cytoplasm late in cytokinesis. One allele, anillin^PE/PE, exhibited a severe effect on pole cell formation because of re-opening of the neck of cytoplasm connecting the pole cell to the yolk mass. Interestingly, this allele exhibited milder defects in cellularization, was viable over zygotic mutations and thus was termed 'weak'. The mutation maps to a region in the middle of Anillin not implicated in any protein interactions. In other systems, removal of Anillin also leads to defects late in cytokinesis, with furrows reopening (Echard, 2004; Somma, 2002; Rogers, 2003; Straight, 2005). Anillin and septins are strongly enriched as rings or short tubes in stable intracellular bridges, including male ring canals during spermatogenesis (Hime, 1996; Robinson, 1996), and the stalks that connect cells to the yolk mass after cellularization. They also remain in mid-bodies after the contractile proteins leave during conventional cytokinesis (Field, 1995). Cumulatively, these data suggests that ring- or tube-shaped Anillin-septin assemblies [rings are the preferred assembly state of mammalian septins (Kinoshita, 2002)] may stabilize intracellular bridges to facilitate the completion of normal cytokinesis, and to allow communication between sister cells following incomplete cytokinesis (Field, 2005).

Given its interaction with multiple conserved furrow proteins, and its functional involvement in both contractility and membrane stability, further study of Anillin is likely to reveal detailed aspects of how the cytoskeleton and membrane systems work together during cytokinesis, and related furrowing processes in embryos (Field, 2005).

Anillin binds nonmuscle myosin II and regulates the contractile ring

The contractile ring protein anillin interacts directly with nonmuscle myosin II and this interaction is regulated by myosin light chain phosphorylation. Despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. A role is proposed for anillin in spatially regulating the contractile activity of myosin II during cytokinesis (Straight, 2005).

The anillin protein is a multifunctional component of the cytoskeleton that is recruited to the furrow early in cytokinesis but functions primarily late in cytokinesis to focus contractility at the furrow. Anillin is known to directly interact with actin and contribute to the organization of the septin complex along actin filaments (Field, 1995; Kinoshita, 2002). This study shows that anillin also directly interacts with nonmuscle myosin II. This interaction with myosin II depends upon phosphorylation of myosin II regulatory light chain by MLCK, suggesting that anillin only associates with active myosin II (Straight, 2005).

Whether anillin functions in cytokinesis to recruit activated myosin II to the cleavage furrow was tested. Anillin depletion data in vivo rule out this simple model because myosin II is able to localize to the division site and promote furrow contraction with normal timing in the absence of anillin. Oegema (2000) observed reduction of the initial rate of furrow contraction after inhibiting anillin by antibody injections, but the current depletion data suggest this may have been due to the presence of antibody in the furrow rather than anillin removal. This study also found that anillin targets to the furrow normally when myosin II is depleted, although in this case contraction is completely inhibited. Those data are consistent with previous pharmacological studies where it was shown that inhibition of kinases that regulate cytokinesis interfere with targeting of myosin II, but not of anillin, to the furrow (Straight, 2003). It will be interesting to test in the future where the pathways that target myosin II and anillin diverge. Both require the continual presence of microtubules (Straight, 2003) and probably also activated Rho (Somma, 2002) to target normally (Straight, 2005).

The primary defect observed in cells that lack anillin is a delocalization of contraction at the end of cytokinesis. Observation of myosin II dynamics in anillin-depleted cells revealed that myosin II is no longer constrained to the contractile ring as it is in control cells and instead is found in the cell cortex concomitant with aberrant cell contraction. This aberrant contraction often results in both mispositioning of the cleavage furrow to yield binucleate cells or to furrow regression and thus binucleation. It was not possible to deplete all of the anillin by RNAi in human cells, thus complete depletion or inhibition of the anillin protein may result in an even more severe cytokinesis phenotype. It is not clear whether the phenotype observed represents extra contraction, for example, due to hyperactivation of myosin II, or relocalization of contraction due to mislocalization of active myosin II to ectopic sites. Distinguishing these hypotheses will require measuring contractile properties of the cortex at different positions. Because anillin is restricted to the contractile ring in unperturbed cells, it is unlikely that anillin outside the furrow inhibits myosin II. Overall, these data point to a model whereby anillin binding to activated myosin II restricts its activity to the furrow until cytokinesis can complete. Anillin is retained in the fully contracted furrow much longer than myosin II, and it is also present in intracellular bridges that are no longer contracting (Field, 1995). An extension of this model proposes that loss of myosin II from the fully contracted furrow is promoted by cell cycle-dependent modification of anillin and/or myosin, such as dephosphorylation of myosin regulatory light chain (Straight, 2005).

Several lines of evidence suggest that anillin controls, or at least coordinates multiple aspects of cytokinesis. Two anillin-related proteins in yeast, Mid1 and Mid2, organize distinct steps during cytokinesis. The Mid1 protein, like anillin, relocalizes from the nucleus to the contractile ring early in cytokinesis (Sohrmann, 1996; Wu, 2003). Mid1 mutants are defective in septum placement and formation (Sohrmann, 1996) and overexpression of Mid1 disrupts cytokinesis (Bahler, 1998; Paoletti, 2000). Several important differences exist between Mid1 and anillin. Mid1 is not essential, does not require actin filaments or microtubules to be maintained at the division site, and does not contract with the actomyosin contractile ring (Paoletti, 2000; Wu, 2003). However, Mid1 is important for the initial organization of myosin II at the contractile ring and can interact with myosin II (Wu, 2003; Motegi, 2004). A second anillin-like protein in fission yeast, Mid2, performs other functions that depend on anillin in metazoan cells. In particular, Mid2 organizes septins in fission yeast and is necessary for proper cell separation (Berlin, 2003; Tasto, 2003), whereas metazoan anillin binds directly to septins (Kinoshita, 2002) and participates in targeting septins to the cortex (Oegema, 2000). Mid2 mutant cells have no defect in myosin II localization or contraction at the end of cytokinesis (Berlin, 2003). Thus, metazoan anillin may encompass the activities of both Mid1 and Mid2. It is speculated that the functions of anillin may be split in fission yeast because of the different mechanical requirements for cytokinesis. In yeast, remodeling of the cell wall may be the primary requirement for cytokinesis, whereas cytokinesis in metazoan animals is dominated by the need to physically constrict the equator of the dividing cell. In budding yeast, the mechanical requirements are different again, because the cell division site is predetermined at a narrow constriction. In that system, myosin II targets very early and no anillin-like proteins have been identified (Straight, 2005).

Anillin is known to be essential for the completion of cytokinesis in vertebrate cells and in Drosophila (Somma, 2002). Anillin's interaction with both the septin complex and with filamentous actin may be required for cell abscission. Myosin II leaves the contractile ring late in cytokinesis, but anillin persists at these contracted furrows, suggesting that anillin's role in the completion of cytokinesis may only be partially explained by its interaction with myosin II. The current results suggest an early role for anillin in cytokinesis to properly organize the contractile ring and a late function for anillin in restricting myosin II contraction to the furrow. In Drosophila embryos expressing mutant anillin, actin, and myosin II are disorganized during cellularization. This may reflect an analogous role for anillin in organizing myosin II at the cellularization front as well as at the contractile ring during cytokinesis (Straight, 2005).

The events of mitosis are temporally coupled by the activities of protein kinases that drive the cell cycle and the proteasome that inactivates these kinases and degrades other proteins involved in mitosis. A role has been demonstrated for proteolysis in the disassembly of the contractile ring (Straight, 2003) in vertebrate cells. Possible substrates for this proteolysis are anillin and the cell cycle kinase Polo. In yeast, Mid2 is degraded by ubiquitin-mediated proteolysis (Tasto, 2003), it will be interesting to determine whether in somatic cells anillin is degraded upon mitotic exit, although no change was observed in anillin levels during the metaphase-to-interphase transition in Xenopus egg extracts. Mid1 is controlled by the activity of Polo kinase in fission yeast. In Xenopus extracts, anillin is rapidly dephosphorylated as cells exit mitosis and is efficiently phosphorylated by Polo kinase in vitro. Regulation of anillin by phosphorylation may provide another effective means of coupling the early and late events of cytokinesis to the cell cycle (Straight, 2005).

The results demonstrate a role for anillin in localizing the contractile activity of myosin in addition to anillin's previously identified functions in binding actin and organizing the septins. Thus, anillin seems to be a central factor for coupling the filament systems that interact during cytokinesis. Understanding how proteins such as anillin dynamically organize the cytoskeletal and regulatory networks that are integrated to accomplish cytokinesis will be key to understanding the process of cell division (Straight, 2005).

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA, which subsequently recruits contractile-ring components such as actin, myosin, and Anillin. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. This study reports the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. The spindle-associated component, RacGAP50C, which specifies the site of cleavage, interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex (Gregory, 2008).

Previous work has shown that initiation of contractile-ring formation is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed microtubule protein Pav-KLP, which accumulates at the equatorial cortex during anaphase. This study reports another crucial interaction involving RacGAP, this time relating to the stability of the contractile ring once its position has been set by equatorial microtubules. It was expected that there must be some link between the microtubule-bound positioning complex and the cortical ring, made of cytoskeletal polymers like actin, myosin, and septins. Previous models proposed that this link is through activated Rho: equatorial RacGAP bringing Pebble to activate Rho, which then recruits the actomyosin contractile apparatus via Diaphanous and Rho-dependent kinases. However, this model does not explain why a newly formed ring remains where it began unless there is something structurally connecting the nascent ring to the spindle. In fact, the relationship between the position of microtubules and the ring is extremely robust, as demonstrated by classic manipulation experiments. Furthermore, inhibitor studies have shown that an ongoing interaction between microtubules and the cortex is critical for cytokinesis to proceed. This study presents the first structural connection between the actin-based ring and the microtubule based positioning mechanism that can explain their relationship. RacGAP, localized to the equatorial microtubules by Pav-KLP, induces contractile-ring formation via Pebble activation of Rho and then anchors the forming contractile ring by binding Anillin, which binds both myosin and actin. It is anticipated that this link ensures not only the stable localization of the ring but also the continued delivery of Rho-activating signals by the RacGAP-linked RhoGEF Pebble (Gregory, 2008).

In the absence of Anillin, phenotypes such as ring slippage have been observed (Straight, 2005) that are strikingly similar to those seen when microtubules are removed, consistent with the model for its role in attaching the ring to cortical microtubules, in order that the cleavage position is dictated by the spindle. The loss of Anillin phenotype described, in which RacGAP is found only on interpolar microtubules, can also be seen in human cells (Zhao, 2005). RacGAP and Pav-KLP are normally found on equatorial microtubules, and this localization is critical for cytokinesis, at least in Drosophila. Evidence from vertebrate cells indicates that contractile-ring formation is similarly dependant on cortical RacGAP (Yuce, 2005). Recent evidence for the role for cytokinesis failure in the generation of aneuploid cells and the promotion of tumorigenesis and the strong conservation of the Anillin sequence across phyla suggests that this newly discovered link between centralspindlin and the contractile ring is likely to be broadly significant (Gregory, 2008).

Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site

Anillin, one of the first factors recruited to the cleavage site during cytokinesis, interacts with actin, myosin II and septins, and is essential for proper organization of the actomyosin contractile ring. Affinity-purification methodology coupled with mass spectrometry was used to identify Anillin-interacting molecules in Drosophila cells. Several actin and myosin proteins, three of the five Drosophila septins and RacGAP50C (Tum), a component of the centralspindlin complex, were isolated. Using drug and RNA interference (RNAi) treatments it was established that F-actin is essential for Anillin cortical localization in prometaphase but not for its accumulation at the cleavage furrow after anaphase onset. Moreover, septins are not recruited to the cleavage site in cells in which Anillin os knocked down by RNAi, but localizes to central-spindle microtubules, suggesting that septins travel along microtubules to interact with Anillin at the furrow. Finally, it was demonstrated that RacGAP50C is necessary for Anillin accumulation at the furrow and that the two proteins colocalize in vivo and interact in vitro. Thus, in addition to its role in activating RhoA signalling, RacGAP50C also controls the proper assembly of the actomyosin ring by interacting with Anillin at the cleavage furrow (D'Avino, 2008).

It is surmised that Anillin initially localizes to the cortex after mitotic entry because of its strong affinity for F-actin. Then, after anaphase onset, at least two distinct mechanisms co-operate to localize Anillin at the furrow. The first, mediated by Rho but independent of RacGAP50C, excludes Anillin from the polar regions, probably as a result of cell elongation, as already shown for myosin. The second involves a direct interaction with RacGAP50C, which restricts and maintains the localization of Anillin to the cleavage site during furrow ingression (D'Avino, 2008).

Three of the five Drosophila septins -- Pnut, Sep2 and Septin 5 (Sep5) -- were identified in pull-down assays. A fourth septin, Septin 1 (Sep1), was detected only in one of the two separate purifications and with a very low score, making its identification more uncertain. Although the interaction between Anillin and septins has already been described in another system (Kinoshita, 2002), it was previously unclear which of the five Drosophila septins could complex with Anillin. Interestingly, the two showing the highest scores, Pnut and Sep2, are also the only two septins that were identified in a genome-wide screen for genes required for cytokinesis in Drosophila cultured cells (Echard, 2004). The localization of Pnut and Sep2 were analyzed after Anillin RNAi. Sep5 localization could not be analyzed because no antibodies are currently available. Moreover, its high homology with Sep2 (~70%), and the low score and number of peptides obtained from mass-spectrometry analysis, made its identification as an interacting protein dubious. Western blot analysis indicated that Anillin could not be detected after a 48-hour incubation with dsRNA. Both Pnut and Sep2 failed to localize to the cleavage site in these Anillin-depleted cells, and instead localized to the central-spindle microtubules in a significant percentage (30%-40%) of telophase cells. Because these two septins, unlike Anillin, did not accumulate to the cortex after nuclear envelope break-down, the results suggest that Pnut and Sep2 travel along the central-spindle microtubules to be delivered to the cortex, in which they then interact with Anillin. A similar failure to recruit Pnut to the cleavage furrow was also observed in scraps (Anillin) mutant embryos, although mislocalization to the central spindle was not reported in that study (Field, 2005). Conversely, Anillin localization during cytokinesis was unaffected in cells incubated for 96 hours with dsRNAs directed against either pnut or Sep2, or against both septins simultaneously. Western blot analysis confirmed that the expression of both septins was severely reduced after RNAi treatment. Interestingly, the expression of Pnut was reduced after Sep2 RNAi and vice-versa, suggesting that depletion of one septin can affect the stability of the other, consistent with the observation that these two septins are found in a three-protein complex along with Sep1 (Field, 1996). These results indicated that severe depletion of Pnut and Sep2 did not compromise Anillin localization during cytokinesis. No significant increase in the number of multinucleate cells, however, was detected in septin-depleted cells and telophase figures appeared normal. This is in accord with two previous reports showing that septin RNAi treatments for either 3 or 4 days did not cause cytokinesis failure (Eggert, 2004; Somma, 2002). However, Echard (2004) reported an increase in cytokinesis defects when cells were exposed to dsRNAs against pnut or Sep2 for a longer period (6 days). The difference in incubation time might very well explain the discrepancy between these two conflicting sets of results (D'Avino, 2008).

In summary, it is concluded that, after anaphase onset, Anillin directly binds RacGAP50C to establish a connection between the actomyosin filaments responsible for furrow ingression and a sub-population of spindle microtubule that contact the equatorial cortex, the peripheral microtubules. This interaction appears necessary to restrict and/or maintain the localization of Anillin to the cleavage site during furrowing. In turn, Anillin recruits at least two septins, Pnut and Sep2. These proteins then form a scaffold necessary for proper organization of the actomyosin filaments and their interaction with the plasma membrane (Eggert, 2006). These data also provide a molecular mechanism for the previous observation that a membrane-tethered version of RacGAP50C could generate ectopic furrows that contained both Anillin and Pnut (D'Avino, 2006). Thus, RacGAP50C not only activates RhoA during cytokinesis through its interaction with the RhoGEF Pbl, but also binds Anillin at the furrow to control the proper organization of the actomyosin contractile ring. These results further support a model in which the RacGAP component of the centralspindlin complex acts as a key regulator of furrow formation and ingression, and so represents a major furrow-inducing signal (D'Avino, 2005; Saint, 2003; D'Avino, 2008 and references therein).

Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization

Cortical compartments partition proteins and membrane at the cell surface to define regions of specialized function. This study asks how cortical compartments arise along the plasma membrane furrows that cellularize the early Drosophila embryo, and investigates the influence that this compartmentalization has on furrow ingression. Zygotic gene product Nullo aids the establishment of discrete cortical compartments, called furrow canals, which form at the tip of incipient furrows. Upon nullo loss-of-function, proteins that are normally restricted to adjacent lateral regions of the furrow, such as Neurotactin and Discs large, spread into the furrow canals. At the same time, cortical components that should concentrate in furrow canals, such as Myosin 2 (Zipper) and Anillin (Scraps), are missing from some furrows. Depletion of these cortical components from the furrow canal compartments precipitates furrow regression. Contrary to previous models, it was found that furrow compartmentalization does not require cell-cell junctions that border the furrow canals. Instead, compartmentalization is disrupted by treatments that reduce levels of cortical F-actin. Because the earliest uniform phenotype detected in nullo mutants is reduced levels of F-actin at furrow canals, it is proposed that Nullo compartmentalizes furrows via its regulation of F-actin, thus stabilizing furrows and insuring their ingression to complete cellularization (Sokac, 2008).

The establishment of compartments around the cortical F-actin/ Myosin 2 array appears to be a conserved feature of furrowing during conventional cytokinesis, common from yeast, to sea urchin and Xenopus embryos, to cultured mammalian cells. Previousanalyses in cellularizing Drosophila embryos similarly suggested that the F-actin/Myosin 2 furrow canals are discrete compartments that form at the tips of incipient furrows and are maintained as the furrows ingress. According to the data in this study, compartmentalization of the cellularization furrow emerges as an essential cellular mechanism to concentrate cortical components at the furrow canal and so ensure sustained furrow ingression. Nullo has been identified as a developmental regulator that aids compartment establishment and maintenance. Nullo activity serves to partition proteins along the furrow from the beginning of cellularization, retaining cortical components such as Myosin 2 at the furrow canal while excluding the lateral proteins Dlg and Nrt. In this way, Nullo stabilizes furrows such that they ingress to convert the syncytial embryo into a primary epithelial sheet (Sokac, 2008).

The data show that Nullo is unlikely to compartmentalize the cellularization furrow via basal junctions or the cortical scaffold proteins Anillin or Septin, and instead supports a model whereby Nullo regulates cortical F-actin to establish and then maintain furrow canal compartments. In nulloX embryos, furrow canal breaks and regression occur at only a fraction of furrows. This is in contrast to the global expression profile of Nullo protein. Discontinuous furrow canal phenotypes have similarly been reported following alternative perturbations of cortical F-actin in cellularizing embryos. For example, Cyto-B injection does not halt furrowing, but rather induces breaks in the Myosin 2 furrow canal network. RhoGEF2 and diaphanous (dia) mutants, which also have reduced F-actin levels at furrow canals, are also missing some furrow canals. Taken together, it is now suggested that reduced F-actin compromises furrow canal compartments at all furrows. In support of this, it was found that furrow canal morphology is altered at all furrows and lateral PM components spread into every furrow canal in nulloX mutants. However, the spreading of lateral components is not sufficient to precipitate furrow regression. Instead, it is the stochastic loss of cortical components from the furrow canal that destabilizes furrows to the extreme that they may regress. In the case of nulloX embryos, it was found that even in the absence of furrow canal components such as Myosin 2, as long as the furrow canal contains some F-actin, the associated furrow continues to ingress. But these furrows appear to be sensitized and perhaps the continued dilution of furrow canal actin by the spreading lateral PM components eventually precipitates their regression (Sokac, 2008).

Several published results now suggest that, in addition to Nullo, the Rho1 GTPase may contribute to furrow compartmentalization. Rho1 and its activator, RhoGEF2, localize to furrow canals and the lateral furrow membrane during cellularization, suggesting that Rho1 is specifically activated there. RhoGEF2 localization is independent of F-actin, and in RhoGEF2 mutants Dia fails to accumulate at the furrow canal and the embryos have reduced levels of cortical F-actin. Interestingly, RhoGEF2 and dia mutants show phenotypes strikingly similar to nulloX mutants in that some furrow canals are missing. This supports the assertion that cortical F-actin helps to maintain furrow canal integrity. Furthermore, following RNAi depletion of nullo in RhoGEF2 mutants, F-actin does not assemble at furrow canals, and so the two proteins may function in separate but parallel pathways. Thus a model is favored whereby the combined activities of Nullo and Rho1 provide the full complement of F-actin at and around the furrow canal, which is in turn required to establish and/or maintain the furrow canal compartment (Sokac, 2008).

Why would reduced cortical F-actin compromise furrow compartmentalization? One possibility is that F-actin recruits and/or retains particular cytoskeletal or scaffold proteins at the furrow canal that are required for compartment establishment and/or maintenance. In fact, in nulloX and Cyto-D-treated embryos, Myosin 2, Anillin and Septin are missing from some furrow canal compartments. This mechanism is consistent with that of the axon initial segment, where PM compartments develop by the progressive recruitment of the scaffold protein Ankyrin G that tethers cortical F-actin/Spectrin to the PM. The resulting meshwork traps and concentrates additional proteins in the compartment, including transmembrane receptors and ion channels. Alternatively, F-actin levels might control membrane trafficking events that occur at the furrow canal compartment. In support of this, cytoplasmic Myosin 2 punctae are seen following F-actin perturbation in cellularizing embryos, which might represent some form of trafficking intermediate. Since cortical F-actin modulates both endocytosis and exocytosis, changes in actin level might change the rates of membrane and/or protein uptake and delivery within PM compartments. Lastly, F-actin levels at the furrow canal might promote lipid heterogeneities at the PM in the form of lipid micro-domains or rafts, as has been reported in a spectrum of mammalian cell types and in sea urchin embryos. Lipid rafts may then define compartments by virtue of their intrinsic chemical properties or by recruiting additional signaling or scaffolding complexes. Of course, during cellularization these mechanisms may not be mutually exclusive, but should converge on the formation and maintenance of discrete furrow canal compartments that stabilize furrows and ensure their sustained ingression (Sokac, 2008).

Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)^Pbl-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF^Pbl-dependent control themselves. Microscopy of Drosophila S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, a separate RhoGEFPbl-dependent pathway was uncovered that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF^Pbl-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. It is proposed that such interactions occur dynamically and transiently to promote furrow stability (Hickson, 2008).

Drosophila S2 cell lines expressing anillin-GFP were generatated. The anillin-GFP fusion rescued loss of endogenous anillin and its localization paralleled that of endogenous anillin. In interphase it was nuclear, at metaphase it was uniformly cortical, and in anaphase it accumulated at the equator while being lost from the poles. In some highly expressing cells, nuclear anillin-GFP formed filaments not ordinarily seen with anillin immunofluorescence, but these disassembled upon nuclear envelope breakdown and the overexpression had no appreciable effect on the progress or success of cytokinesis (Hickson, 2008).

Tests were performed to see whether RhoGEF^Pbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEF^Pbl RNAi or Rho1 RNAi, anillin-GFP was found to be localized to the cortex in metaphase but does not relocalize to the equator during anaphase, indicating a requirement for RhoGEF^Pbl, consistent with prior analysis of fixed RhoGEF^Pbl mutant embryos. Because anillin can bind F-actin (Field, 1995) and phosphorylated myosin regulatory light chain (MRLC; Straight, 2005), RhoGEF^Pbl might regulate anillin indirectly through its control of F-actin and myosin II (Hickson, 2008).

Latrunculin A (LatA) was used to test whether F-actin was required for anillin-GFP localization. A 30-60-min incubation of 1 µg/ml LatA abolished cortical anillin-GFP localization in metaphase, indicating an F-actin requirement at this phase. However, when anillin normally relocalizes to the equator (~3-4 min after anaphase onset), anillin-GFP formed punctate structures that became progressively more filamentous over the next few minutes, reaching up to several micrometers in length and having a thickness of ~0.3 µm. These linear anillin-containing structures contained barely detectable levels of F-actin and formed specifically at the plasma membrane and preferentially at the equator, although subsequent lateral movement often led to a more random distribution. Thus, anillin responds to spatiotemporal cytokinetic cues even after major disruption of the F-actin cytoskeleton. A substantial (albeit incomplete) reacquisition of cortical phalloidin staining was observed in cells fixed after washing out the drug for a few minutes. In live cells, LatA washout immediately after formation of the anillin structures allowed the preformed structures to migrate from a broad to a compact equatorial zone as the cells attempted to complete cytokinesis. This movement indicates that an F-actin–dependent process can contribute to the equatorial focusing of anillin (Hickson, 2008).

The influence of RhoGEF^Pbl on anillin behavior was tested in LatA. After RNAi of RhoGEF^Pbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEF^Pbl and Rho1 are required for anaphase anillin behavior, whether the cortex is intact or disrupted by LatA treatment (Hickson, 2008).

Tests were performed to see whether myosin II impacts anillin-GFP localization. Compared with controls, RNAi of the gene encoding MRLC spaghetti squash (MRLC^Sqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEF^Pbl RNAi, equatorial accumulation of anillin-GFP was not altogether blocked. It was still recruited but in a broad zone. Furthermore, in the presence of LatA, MRLC^Sqh RNAi did not affect the formation of the anillin-GFP structures. It is concluded that myosin II contributes to the equatorial focusing of anillin when the F-actin cortex is unperturbed but that myosin II is dispensable for anillin behavior in LatA (Hickson, 2008).

Collectively, these data suggest that multiple RhoGEF^Pbl-dependent inputs control anillin localization. The slowed equatorial accumulation of anillin when myosin II function was impaired indicates a myosin II-dependent input. That reassembly of the cortical F-actin network (after washout of LatA) allowed preformed anillin structures to move toward the cell equator indicates an F-actin-dependent input. This is consistent with the concerted actions of myosin II and F-actin driving cortical flow, as observed in other cells, and is reminiscent of the coalescence of cortical nodes during contractile ring assembly in Schizosaccharomyces pombe. However, the F-actin- and myosin II-independent behavior of anillin in LatA indicates an additional RhoGEF^Pbl-dependent input. Thus, RhoGEF^Pbl can control anillin behavior in anaphase via a previously unrecognized route. Immunofluorescence analysis revealed extensive colocalization between endogenous Rho1 and anillin-GFP in LatA, indicating that Rho1 was itself a component of these structures. These findings are consistent with the idea that Rho1 and anillin directly interact (Hickson, 2008).

Myosin II localization was studied, since it can bind anillin and is controlled by RhoGEF^Pbl. MRLC^Sqh-GFP is able to localize to the equatorial membrane independently of F-actin, and in doing so forms filamentous structures resembling those observed with anillin-GFP. Indeed, anillin and MRLC^Sqh (detected as either MRLC^Sqh-GFP or with an antibody to serine 21-phosphorylated pMRLC^Sqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLC^Sqh-GFP localization were tested. MRLC^Sqh-GFP recruitment and furrow initiation appeared normal, but within a few minutes of initiation, furrows became laterally unstable and oscillated back and forth across the cell cortex, parallel to the spindle axis, in repeated cycles, each lasting ~1-2 min and eventually subsiding to yield binucleate cells after ~20 min. The phenotype was very similar to that reported for anillin RNAi in HeLa cells (Straight, 2005; Zhao, 2005) and represents a requirement for anillin at an earlier stage than previously noted in Drosophila (Somma, 2002; Echard, 2004). Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008). <{>In LatA, anillin RNAi did not prevent equatorial MRLC^Sqh-GFP recruitment, but instead of appearing as persistent linear structures distorting the cell surface, a more reticular and dynamic structure lacking cell surface protrusions was observed. Thus myosin II can localize independently of both anillin and F-actin but the filamentous appearance of myosin II in the presence of LatA requires anillin, indicating that anillin can influence myosin II behavior in the absence of F-actin, whereas myosin II appeared capable of influencing anillin behavior only in the presence of F-actin (Hickson, 2008).

Septins are multimeric filament-forming proteins that can bind anillin in vitro and function with anillin in vivo (Kinoshita, 2002; Field, 2005; Maddox, 2005; Maddox, 2007). Using an antibody to the septin Peanut, it was found that in nontransfected S2 cells, septin^Pnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septin^Pnut antibody also strongly labeled bundles of cytoplasmic ordered cylindrical structures, each ~0.6 µm in diameter and of variable length (up to several micrometers). These staining patterns could be greatly reduced by septin^Pnut RNAi and were thus specific. The cylindrical structures did not appear to be cell cycle regulated, as they were apparent in interphase, mitotic, and postmitotic cells. They also did not colocalize with anillin, nor did their stability rely on anillin. Incubation with 1 µg/ml LatA before fixation inevitably led to disassembly of most of these large structures; however, the resulting distribution of septin^Pnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septin^Pnut formed cytoplasmic rings, ~0.6 µm in diameter, which are similar to the Septin2 rings seen in interphase mammalian cells treated with F-actin drugs or in the cell body of unperturbed ruffling cells (Kinoshita, 2002; Schmidt, 2004). In LatA-treated mitotic cells, septin^Pnut was diffusely cytoplasmic (or barely detectable) in early mitosis, whereas in anaphase/telophase, it localized to the same plasma membrane-associated anillin-containing filamentous structures (Hickson, 2008).

Anillin behavior was analyzed after septin^Pnut RNAi. Although unable to fully deplete septin^Pnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septin^Pnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septin^Pnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septin^Pnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septin^Pnut. However, in this case the plasma membrane to which anillin-GFP localized subsequently exhibited unusual behavior. It was internalized in large vesicular structures, apparently in association with midzone MTs. Although this phenomenon is not understood, it may be related to events induced by point mutations in the septin-interacting region of anillin that give rise to abnormal vesicularized plasma membranes during Drosophila cellularization (Hickson, 2008).

The effects were tested of anillin RNAi on the localization of septin^Pnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septin^Pnut. In LatA-treated cells, anillin RNAi did not affect the formation of septin^Pnut rings in interphase cells, but it greatly reduced the formation of septin^Pnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septin^Pnut and for the formation of septin^Pnut-containing structures in 1 µg/ml LatA. In contrast, Dia could still localize to the equatorial plasma membrane after combined anillin RNAi and LatA treatment, indicating that it can localize independently of both anillin and F-actin. Thus, although Dia partially colocalized with anillin in LatA, this likely reflected independent targeting to the same location rather than an association between anillin and Dia (Hickson, 2008).

These data argue that Rho1, anillin, septins, and the plasma membrane participate independently of F-actin in the formation of a complex. However, anillin, septins and F-actin can also form a different complex in vitro, independently of Rho (Kinoshita, 2002). Perhaps two such complexes dynamically interchange in vivo (Hickson, 2008).

The involvement of MTs in anillin behavior was tested in LatA. Overnight incubation with 25 µM colchicine effectively depolymerized all MTs in mitotic cells and promoted mitotic arrest, as expected. Using Mad2 RNAi to bypass the arrest, anillin-GFP was observed during mitotic exit in the absence of MTs and in the presence of LatA. Under such conditions, anillin-GFP formed filamentous structures very similar to those formed when MTs were present, indicating that MTs were dispensable for their formation. However, the structures appeared uniformly around the plasma membrane rather than restricted to the equatorial region, which is consistent with the role MTs play in the spatial control of Rho activation (Hickson, 2008).

The LatA-induced anillin structures localize to the ends of nonoverlapping astral MTs directed toward the equator. Live imaging of cells coexpressing cherry-tubulin and anillin-GFP revealed bundles of MTs associating with the filamentous anillin-GFP structures as they formed. Colocalization between anillin-GFP structures and MT ends persisted over many minutes, even after considerable lateral movement at the membrane. Thus, although the anillin structures formed independently of MTs, they stably associated with MTs. These findings support prior biochemical evidence for interactions of MTs with both anillin and septins (Sisson, 2000) and reveal a potential positive-feedback loop in which MTs directed where Rho1-anillin-septin formed linear structures at the plasma membrane, whereas the structures in turn associated with the MT ends. An MT plus end-binding ability of anillin-septin could explain the furrow instability phenotype elicited by anillin RNAi. Accordingly, anillin may physically link Rho1 to MT plus ends during furrow ingression, thereby promoting the focusing and retention of active Rho1, thus stabilizing the furrow at the equator (Hickson, 2008).

These live-cell analyses highlight an unusual behavior of the Rho-dependent anillin-containing structures at the plasma membrane. Initially forming beneath and parallel to the plasma membrane, the structures then often lifted on one side to appear perpendicular to the cell surface while remaining anchored at their base by MTs. This reorientation is interpreted as reflecting avid binding to and subsequent envelopment by the plasma membrane. Although intrinsically stable, the structures exhibited dynamic movement within the plane of the plasma membrane and were capable of sticking to one another, via their ends, giving rise to branched structures that were also capable of breaking apart. Anillin has a pleckstrin homology domain within its septin-interacting region and a membrane-anchoring role of anillin has long been postulated (Field, 1995). These data support such a role and suggest that it is controlled by Rho (Hickson, 2008).

The data highlight the complexity of RhoGEF^Pbl signaling and lead to a model in which multiple Rho-dependent inputs synergize to control anillin behavior during cytokinesis (Hickson, 2008). At the appropriate time and location of normal cytokinesis, several proteins, including Rho1, MRLC^Sqh, Dia, anillin, and Septin^Pnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and Septin^Pnut were uniquely and specifically required for the formation of the linear filamentous structures describe in this study. The behaviors of these structures are consistent with prior studies of ordered assemblies of septins and anillin (Oegema, 2000; Kinoshita, 2002) and of interaction between anillin and MTs (Sisson, 2000). Although such structures are not normally seen in furrowing cells, anillin localizes to remarkably similar filamentous structures in the cleavage furrows of HeLa cells (Straight, 2005) arrested with the myosin II inhibitor blebbistatin (Hickson, 2008).

It seems unlikely that LatA induces the described structures through nonspecific aggregation of proteins. Rather, it is proposed that LatA blocks a normally dynamic disassembly of Rho1-anillin-septin complexes (by blocking an F-actin-dependent process required for the event) and that continued assembly promotes formation of the linear structures. Because blebbistatin slows F-actin turnover, blebbistatin and LatA may have induced filamentous anillin-containing structures via a common mechanism. A dynamic assembly/disassembly cycle involving anillin could promote transient associations between the plasma membrane and elements of the contractile ring and MTs, properties that could contribute to furrow stability and plasticity. Finally, because local loss of F-actin accompanies and may indeed trigger midbody formation, LatA treatment may have stabilized events in a manner analogous to midbody biogenesis and could therefore be useful in understanding this enigmatic process (Hickson, 2008).

Anillin is unequally required during asymmetric cell divisions of the PNS

During Drosophila embryogenesis, timely and orderly asymmetric cell divisions ensure the correct number of each cell type that make up the sensory organs of the larval peripheral nervous system. A role is reported for scraps, Drosophila Anillin, during these divisions. Anillin, a constitutive member of the contractile ring is essential for cytokinesis in Drosophila and vertebrates. During embryogenesis it was found that zygotically transcribed scraps is required specifically for the unequal cell divisions, those in which cytokinesis occurs in an 'off-centred' manner, of the pIIb and pIIIb neuronal precursor cells, but not the equal cell divisions of the lineage related precursor cells. Complementation and genetic rescue studies demonstrate this effect results from zygotic scraps and leads to polyploidy, ectopic mitosis, and loss of the neuronal precursor daughter cells. The net result of which is the formation of incomplete sense organs and embryonic lethality (OíFarrell, 2008).

scraps mutation is the source of both the embryonic lethality and PNS phenotype (firmly linking the two phenotypes) associated with the l(2)k08255 chromosome. Zygotic alleles escape the early cellularisation defects via maternally contributed wildtype scraps and instead later suffer severe PNS (and CNS) phenotypes. Notably, neuronal specific expression of scraps was capable of rescuing embryonic lethality. While these larvae likely do not have a completely restored PNS or CNS, the level of restoration achieved is sufficient for survival of the animals to pupal stages. Thus demonstrating that the lack of Anillin within nervous tissues accounted for these phenotypes. High levels of Anillin at critical time-points are likely required in multiple tissues during formation of adult structures, possibly inadequately supplied via our transgenic set-up (OíFarrell, 2008).

Anillin is a key component of the actomyosin contractile ring and one of the earliest detectable contractile ring proteins, potentially mediating contact with the cellular membrane and targeting other proteins. As such Anillin represents a candidate for contractile ring nucleation and hence positioning. Anillin has been shown to fulfil such a role in both fission yeast and C. elegans, although no such role has been demonstrated in Drosophila or higher eukaryotes. Thus Anillin is involved in cytokinesis in several systems although the nature of that role may vary. The consequence of lacking zygotic scraps/Anillin during PNS asymmetric cell divisions was examined in this study (OíFarrell, 2008).

While some observations were in line with those made in other systems, e.g. the enlargement of cells resulting from failed cytokinesis and the associated multi-nucleate phenotypes, other findings were more surprising. A loss of cells was observed stemming specifically from the pIIb neuronal lineage of the Ch- and ES-organs, while pIIa cell divisions were unaffected. A potential caveat to this observation is the maternal contribution of Anillin, which potentially persists during the PNS divisions. However, since the pIIb divides prior to the pIIa and since the pIIa cell division is unperturbed, this explanation was found to be inadequate to account for the differences observed. A perdurance of maternally contributed protein (or RNA) would be ubiquitous, rather than cell specific. The alternate side of this argument then begs the question; do the SOP and pIIa then not require Anillin? This is also unlikely. Possible explanations to reconcile these two arguments include the differential inheritance of maternally contributed scraps mRNA or protein to the pIIa cell, such mechanisms exist. Alternatively, differential degradation of the Anillin protein within pIIb cells specifically, could account for the differences observed. Finally, as the pIIb cell undergoes unequal cell division and hence contractile ring positioning 'off-center', there could be a requirement for additional, or a specifically modified version of the Anillin protein, supplied via zygotic transcription (OíFarrell, 2008).

Interestingly, the resulting polyploid pIIb and pIIIb cells that have failed to divide are capable of differentiating into neurons while simultaneously progressing through the cell cycle. This is likely a re-entry into the cell cycle following a failed cell division event rather than a cell cycle regulatory role for Anillin. Direct links between polyploidy and uncontrolled proliferation have been previously described. A biased requirement for Anillin in unequal pIIb cell divisions is a novel finding, the nature and purpose of which is open to speculation. Further study of the role of Anillin in other models of asymmetric divisions or nervous system development could prove enlightening (OíFarrell, 2008).

Search PubMed for articles about Drosophila Anillin

Adam, J. C., Pringle, J. R. and Peifer, M. (2000). Evidence for functional differentiation among Drosophila septins in cytokinesis and cellularization. Mol. Biol. Cell 11: 3123-3135. PubMed citation: 10982405

Bahler, J., Steever, A. B., Wheatley, S., Wang, Y., Pringle, J. R., Gould, K. L. and McCollum, D. (1998). Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J. Cell Biol. 143: 1603-1616. PubMed citation: 9852154

Berlin, A., Paoletti, A. and Chang, F. (2003). Mid2p stabilizes septin rings during cytokinesis in fission yeast. J. Cell Biol. 160: 1083-1092. PubMed citation: 12654901

D'Avino, P. P., Savoian, M. S. and Glover, D. M. (2005). Cleavage furrow formation and ingression during animal cytokinesis: a microtubule legacy. J. Cell Sci. 118: 1549-1558. PubMed citation: 15811947

D'Avino, P. P., Savoian, M. S., Capalbo, L. and Glover, D. M. (2006). RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis. J. Cell Sci. 119: 4402-4408. PubMed citation: 17032738

D'Avino, P. P., et al. (2008). Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site. J. Cell Sci. 121(Pt 8): 1151-8. PubMed citation: 18349071

Echard, A., Hickson, G. R., Foley, E. and O'Farrell, P. H. (2004). Terminal cytokinesis events uncovered after an RNAi screen. Curr. Biol. 14(18): 1685-93. PubMed citation: 15380073

Eggert, U. S., Mitchison, T. J. and Field, C. M. (2006). Animal cytokinesis: from parts list to mechanisms. Annu. Rev. Biochem. 75: 543-566. PubMed citation: 16756502

Field, C. M. and Alberts, B. M. (1995). Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J. Cell Biol. 131: 165-178. PubMed citation: 7559773

Field, C. M., al-Awar, O., Rosenblatt, J., Wong, M. L., Alberts, B. and Mitchison, T. J. (1996). A purified Drosophila septin complex forms filaments and exhibits GTPase activity. J. Cell Biol. 133: 605-616. PubMed citation: 8636235

Field, C. M., Coughlin, M., Doberstein, S., Marty, T. and Sullivan, W. (2005). Characterization of anillin mutants reveals essential roles in septin localization and plasma membrane integrity. Development 132(12): 2849-60. PubMed citation: 15930114

Gregory, S. L., et al. (2008). Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring. Curr. Biol. 18(1): 25-9. PubMed citation: 18158242

Heitzler, P., Coulson, D., Saenz-Robles, M., Ashburner, M., Roote, J., Simpson, P. and Gubb, D. (1993). Genetic and cytogenetic analysis of the 43 A-E region containing the segment polarity gene costa and the cellular polarity genes prickle and spiny-legs in Drosophila melanogaster. Genetics 135: 105-115. PubMed citation: 8224812

Hickson, G. R. and O'Farrell, P. H. (2008). Rho-dependent control of anillin behavior during cytokinesis. J. Cell Biol. 180(2): 285-94. PubMed citation: 18209105

Hime, G. R., Brill, J. A. and Fuller, M. T. (1996). Assembly of ring canals in the male germ line from structural components of the contractile ring. J. Cell Sci. 109: 2779-2788. PubMed citation: 9013326

Kiger, A. A., Baum, A., Jones, S., Jones, M. R., Coulson, A., Escheverri, C. and Perrimon, N. (2003). A functional genomic analysis of cell morphology using RNAi interference. J. Biol. 2: 1-15. PubMed citation: 14527345

Kinoshita, M., Field, C. M., Coughlin, M. L., Straight, A. F. and Mitchison, T. J. (2002). Self- and actin-templated assembly of mammalian septins. Dev. Cell 3: 791-802. PubMed citation: 12479805

Lemmon, M. A., Ferguson, K. M. and Abrams, C. S. (2002). Pleckstrin homology domains and the cytoskeleton. FEBS Lett. 513: 71-76. PubMed citation: 11911883

Lemmon, M. A. (2004). Pleckstrin homology domains: not just for phosphoinositides. Biochem. Soc. Trans. 32: 707-711. PubMed citation: 15493994

Maddox, A. S., Habermann, B., Desai, A. and Oegema, K. (2005). Distinct roles for two C. elegans anillins in the gonad and early embryo. Development 132: 2837-2848. PubMed citation: 15930113

Maddox, A. S., Lewellyn, L., Desai, A. and Oegema, K. (2007). Anillin and the septins promote asymmetric ingression of the cytokinetic furrow. Dev. Cell 12(5): 827-35. PubMed citation: 17488632

Motegi, F., Mishra, M., Balasubramanian, M. K. and Mabuchi, I. (2004). Myosin-II reorganization during mitosis is controlled temporally by its dephosphorylation and spatially by Mid1 in fission yeast. J. Cell Biol. 165: 685-695. PubMed citation: 15184401

Oegema, K., Savoian, M. S., Mitchison, T. J. and Field, C. M. (2000). Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis. J. Cell Biol. 150: 539-552. PubMed citation: 10931866

O'Farrell, F. and Kylsten, P. (2008). Drosophila Anillin is unequally required during asymmetric cell divisions of the PNS. Biochem. Biophys. Res. Commun. 369(2): 407-13. PubMed citation: 18295597

Paoletti, A. and Chang, F. (2000). Analysis of mid1p, a protein required for placement of the cell dividion site reveals a link between the nucleus and the cell surface in fission yeast. Mol. Biol. Cell 8: 2751-2773. PubMed citation: 10930468

Robinson, D. N. and Cooley, L. (1996). Stable intercellular bridges in development: the cytoskeleton lining the tunnel. Trends Cell Biol. 6: 474-479. PubMed citation: 15157506

Rogers, S. L., Wiedmann, U., Stuurmann, N. and Vale, R. D. (2003). Molecular requirements for actin-based lamella formation in Drosophila S2 Cells. J. Cell Biol. 162: 1079-1088. PubMed Citation: 12975351

Saint, R. and Somers, W. G. (2003). Animal cell division: a fellowship of the double ring? J. Cell Sci. 116: 4277-4281. PubMed citation: 14514883

Schmidt, K. and Nichols, B. J. (2004). Functional interdependence between septin and actin cytoskeleton. BMC Cell Biol. 5: 43. PubMed citation: 15541171

Schupbach, T. and Wieschaus, E. (1989). Female sterile mutations on the second chromosome of Drosophila melanogaster. I. Maternal effect mutations. Genetics 121: 101-117. PubMed citation: 2492966

Sisson, J. C., et al. (2000). Lava lamp, a novel peripheral Golgi protein, is required for Drosophila melanogaster cellularization. J. Cell Biol. 151: 905-918. PubMed citation: 11076973

Sohrmann, M., Fankhauser, C., Brodbeck, C., and Simanis, V. (1996). The dmf1/mid1 gene is essential for correct positioning of the division septum in fission yeast. Genes Dev. 10: 2707-2719. PubMed citation: 8946912

Sokac, A. M. and Wieschaus, E. (2008). Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization. J. Cell Sci. [Epub ahead of print]. PubMed citation: 18460582

Somma, M. P., Fasulo, B., Cenci, G., Cundari, E. and Gatti, M. (2002). Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell 13: 2448-2460. PubMed citation: 12134082

Straight, A. F., Cheung, A., Limouze, J., Chen, I., Westwood, N. J., Sellers, J. R. and Mitchison, T. J. (2003). Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299: 1743-1747. PubMed citation: 12637748

Straight, A. F., Field, C. M. and Mitchison, T. J. (2005). Anillin binds nonmuscle Myosin II and regulates the contractile ring. Mol. Biol. Cell 1: 193-201. PubMed citation; Online text

Tasto, J. J., Morrell, J. L. and Gould, K. L. (2003). An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation. J. Cell Biol. 160: 1093-1103. PubMed citation: 12668659

Wu, J. Q., Kuhn, J. R., Kovar, D. R., and Pollard, T. D. (2003). Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev. Cell 5: 723-734. PubMed citation: 14602073

Yuce, O., Piekny, A. and Glotzer, M. (2005). An ECT2-centralspindlin complex regulates the localization and function of RhoA, J. Cell Biol. 170: 571-582. PubMed citation: 16103226

Zhao, W. M. and Fang, G. (2005). Anillin is a substrate of anaphase-promoting complex/cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis, J. Biol. Chem. 280: 33516-33524. PubMed citation: 16040610

date revised: 20 May 2008

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.