Protein Interactions

Immunoaffinity studies using antiseptin antibodies reveal that three Drosophia septins, Pnut, Sep1 and Sep2 coimmunoprecipitate and cofractionate as a protein complex. Visualized by electron microcoscopy, the septin complex contains filaments of variable lengths (up to 350 nm) meauring 7-9 nm in diameter. The filaments reveal a periodicity of about 26 nm leading to the belief that filaments are constructed from repeats of a 26-nm subunit. The septin complex binds and hydrolyzes GTP. Exogenous GTP slowly exchanges with the tightly bound guanine nucleotide. The majority of bound guanine nucleotide (added as GTP) is recovered as GDP, and only GDP accumulates over time. Since the unbound nucleotide in reactions remains predominantly GTP, it is thought that hydrolysis is fast, when compared to exchange under the assay conditions. The data for complex formation between the three septins best fit a model in which the complex is a linear heterotrimer of septin homodimers, with a final trimer length of 26 nm. Filaments are distributed in lengths between 1 and 14 subunits long, suggesting a simple linear polymerization in which the subunits associate end-to-end with an association constant that is independent of polymer length. None of the conditions tested so far have resulted in an obvious increase in the polymerization state of the isolated complex. The isolated septin complex copurifies with one molecule of guanine nucleotide per polypeptide. Assuming the trimer of dimers structure, the complex contains four to five molecules of GDP and one to two molecules of GTP. The GTP that copurifies with the complex is most likely bound to the Sep2 polypeptide (Field, 1996).

Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. The septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV-KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. Polo kinase associates with PAV-KLP, with which it shows an overlapping pattern of subcellular localization during the mitotic cycle. This distribution is disrupted in pav mutants. It is suggested that PAV-KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins. PAV-KLP may be responsible for transporting Polo kinase from one set of centrosome-associated substrates to a second set of substrates in the midzone of the spindle as mitosis progresses (Adams, 1998).

Interaction of Peanut with ORC6 reveals a cytokinetic function of Drosophila ORC6 protein

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. The smallest of Drosophila origin recognition complex subunits, Orc6, is found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA results in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-Peanut interaction domain and subsequent overexpression of the Orc6 mutant protein results in the formation of multinucleated cells that have replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis (Chesnokov, 2003).

Septins are polymerizing proteins with a common GTPase activity and were first discovered in S. cerevisiae but now seem to be ubiquitous in fungi and animals. Pnut is one of the five Drosophila septins identified to the date. Genetic data showed that the proteins play diverse roles in organization of the cell cortex and in cytokinesis. At the molecular level, the role of GTP binding and hydrolysis by septins is unclear and is probably not required for filament formation. Thus, formation of filaments at a cleavage furrow or in the cytoplasm during interphase may be an activity of septins independent of other roles for the proteins, perhaps in intracellular signaling, because they seem to be more homologous to the ras superfamily members than to other GTPases. In many cell types examined to date, the septins form rings at the site of the cleavage furrow and septin mutants in S. cerevisiae and Drosophila are defective in cytokinesis. In Drosophila, Pnut is an essential protein. In pnut mutants, cells of the imaginal disk tissues fail to proliferate and instead develop clusters of large multinucleated cells, consistent with an important role in cytokinesis. In S. cerevisiae, septins are required for proper localization of bud site-selection markers Bud3p and Bud4p and of the subunits of the chitin synthase III complex. These and other data led to the hypothesis that the septins function as a scaffold on which other proteins assemble along the cytoplasmic side of the cleavage furrow. How septins themselves localize is unknown; adaptor proteins likely recruit them to specific targets (Chesnokov, 2003 and references therein).

Whole-cell extracts from Drosophila embryos (0-12 h of development) and L2 tissue culture cells were subjected to immunoprecipitation with polyclonal antibody raised against the Drosophila Orc6 subunit. Coprecipitated material was analyzed by Western blotting for the presence of Pnut protein. Polyclonal anti-Orc6 serum coimmunoprecipitates Pnut together with Orc6 protein from both Drosophila embryonic and L2 tissue culture extracts. No Pnut signal was detected in control reactions with polyclonal antibody against the Drosophila Orc2 subunit. The negative results with the Orc2 sera imply that it is the pool of Orc6 unassociated with the other ORC proteins that interacts with Pnut (Chesnokov, 2003).

Although Pnut could be coimmunoprecipitated with anti-Orc6 antibodies, the reciprocal experiment showed that only a very minor fraction of Orc6 was precipitated by monoclonal antibodies raised against Pnut protein. This result was perhaps due to epitope masking in the Orc6-Pnut complex, coupled with the fact that this particular Pnut monoclonal antibody worked best in immunostaining and rather poorly in immunoprecipitations (Chesnokov, 2003).

Both Drosophila embryos at different stages of development and L2 cells were used to determine whether Orc6 and Pnut proteins colocalize. At the stage of cellularization that occurs after the 13th nuclear division, the Pnut signal became apparent at the advancing membrane front and especially at the cytoplasmic connections between cells and yolk. These yolk plugs maintain actin, myosin, anillin, and the septins in a surrounding ring. Orc6 is indeed found with Pnut at these locations. Later, as cell membranes grow and eventually reach a full depth, Orc6 is also localized at the membrane locations together with Pnut protein. Orc6 also colocalizes with Pnut in 3.5- to 4-h-old embryos (stages 8 and 9). At these stages, shortly after gastrulation, cells enter cell cycle 15 when neuroblasts delaminate from the ectoderm. Both Drosophila Orc6 and Pnut proteins are found at the cleavage furrows between dividing cells in the presumptive neurogenic ectoderm (Chesnokov, 2003).

Double staining of Drosophila L2 tissue culture cells with Orc6 and Pnut antibodies also showed a colocalization in dividing cells. In addition to its anticipated nuclear localization, endogenous Orc6 is localized to the cell membranes together with Pnut protein. Moreover, in mitotic cells Orc6 and Pnut colocalize at the cleavage furrow of the dividing cells. Transient ectopic expression of the GFP-Orc6 fusion protein again showed distinct nuclear and membrane localization of the protein. Similar GFP fusions with ORC1 and two genes elicited only nuclear signals, and cytology with ORC2 antibodies detected only a nuclear stain in embryos (Chesnokov, 2003).

Because pnut mutations in Drosophila result in a cytokinesis defect, the role of Orc6 in this process was explored by depletion with RNAi. After transfection of the appropriate double-stranded RNA into an asynchronous population of Drosophila cells, immunoblot analysis of treated L2 cells revealed that the level of Orc6 protein was greatly reduced by 24 h and almost completely lost by 72 h. The level of Orc2 protein was not significantly decreased in cells treated with Orc6 dsRNA, and the cells transfected with luciferase dsRNA as a control showed normal levels of Orc2 and Orc6 proteins during the time course of this experiment. Immunostaining of transfected cells with anti-Orc6 antibody showed a concomitant disappearance of the Orc6 signal. DNA replication in cells treated with Orc6 dsRNA also decreased over time. BrdUrd incorporation was detected in 70%-80% of these cells after the first 24 h of incubation, similar to what was observed with untreated cells. However, the fraction of cells incorporating the precursor nucleotide dropped continuously (40%-50% at 48 h and 10%-15% at 72 h). The most striking phenotype observed after the transfection of L2 cells with Orc6 dsRNA was the rapid appearance of binucleated cells. The number of multinucleated cells observed increased from a background of <0.2% in the population to 5% after the first 24 h and reached =30% after 72 h of transfection. Prolonged periods of the Orc6 depletion by RNAi resulted in a decrease of cell proliferation and increased cell death. This multinucleated phenotype was not observed in vivo for lethal mutations of ORC subunits 2, 3, or 5. Moreover, dsRNA for ORC2, although effective for decreasing BrdUrd incorporation, showed no efficacy for elevating the number of binucleated cells above the background. However, such defects were observed in RNAi-based studies of Drosophila passenger proteins INCENP and Aurora B. From these data, it is speculated that Orc6 participates in some aspects of the cell division cycle that influences cytokinesis. Furthermore, the kinetics of cytokinesis defects suggest that the cytokinetic function is more sensitive to small changes in the Orc6 pool than is DNA replication (Chesnokov, 2003).

The C-terminal 25 aa of Drosophila Orc6 contains a leucine-rich region that may mediate protein-protein interactions through an amphipathic helix. A similar motif, thought to be important for protein-protein interaction, is also found in the Pnut protein and is present in most of the septins described to date. The notion that this region of Orc6 is important for Pnut interaction was tested with a series of Orc6 C-terminal deletion mutants. Purified WT and mutant proteins expressed in E. coli as His-tagged fusions were tested for their ability to precipitate Pnut protein from the Drosophila cell culture extracts. Proteins were precipitated by using cobalt beads (Qiagen) that can bind selectively His-tagged proteins. Precipitated material was analyzed by employing a Western immunoblotting assay and the anti-Pnut antibody for detection. In contrast to the WT Orc6 His-tagged protein, Orc6 mutant protein lacking the terminal 57 aa (Orc6-200) failed to precipitate Pnut from the Drosophila extracts. Orc6 protein truncated at the terminal 33 aa (Orc6-224) was able to precipitate Pnut protein but less efficiently than the intact protein. Thus, the terminal leucine-rich section of Orc6 is important for Pnut interaction but not likely the sole mediator. Equivalent results were obtained with Drosophila embryonic extracts (Chesnokov, 2003).

It was asked whether these deletion mutants might have dominant-negative effects if expressed in cultured cells. Overexpression of WT Orc6 protein does not produce any noticeable effect on either cell morphology or the ability of the cells to replicate DNA in side-by-side comparison to nontransfected cells. However, the overexpression of C-terminal Orc6 mutants resulted in an elevated number of cells with multiple nuclei (5%-7% of transfected cells for Orc6-224 and up to 30% for Orc6-200). Overexpression of the Orc6-200 allele also results in a loss of membrane localization. Cells carrying GFP-Orc6-224 or GFP-Orc6-200 were able to incorporate BrdUrd during a 20-h labeling period at the level of nontransfected cells or cells transfected with GFP-Orc6 WT, as judged by intensity of staining and the fraction of cells with signal. Further, by using a recombinant ORC system, the ORC6-200 allele, when expressed with the other subunits, incorporates effectively into the complex and thus can compete with the WT gene for complex formation when it is overexpressed. This result suggests that the core replication domain of Orc6 is not affected by these mutations, although the cytokinetic function as measured by the presence of binucleated cells is lost. The antimorphic nature of alleles such as GFP-ORC6-200 indicates that the replication domain may also contain critical functions for cytokinesis, but that in the absence of the C terminus the defective protein interferes with the process. For example, the ORC6-200 may bind and sequester a protein important for releasing ORC6 from chromatin to transport the protein to the membrane locations. Overexpression of Orc6-163 mutant protein, in contrast, results in large cells that do not incorporate BrdUrd. Moreover, transfection of L2 cells with Orc6-163 apparently has a toxic effect on cells and results in decreased proliferation and increased cell death (Chesnokov, 2003).

A reasonable extrapolation from these data is that a C-terminal domain of Orc6, perhaps beginning around amino acid 200 and progressing from there toward the C terminus, defines a Pnut interaction domain and a membrane-proximal localization function critical for cytokinesis. To probe the domain organization of Orc6 in silico, a web-based method for protein fold prediction was used that employs 1D and 3D sequence profiles coupled with secondary structure and solvation potential information. With this program, Drosophila and human Orc6 sequences were compared with known protein structures. Unexpectedly, it was found that the predicted Orc6 structure over much of its length was homologous to the structure of the human TFIIB transcription factor bound to the DNA in a complex with TBP. The E value, an inverse measure of the program's reliability for this alignment, was commensurate with a certainty of >99.9% for the human Orc6 and 99.2% for the Drosophila Orc6 homologue. Two points aside from the hypothetical nature of this modeling are emphasized here. (1) The break in the predicted TFIIB homology domain is at amino acid 203, and the C-terminal amino acids of Orc6 do not fit into this fold, in rather good agreement with biochemical and cell-based genetic assays. Deletion alleles map an approximate break in functional activities to this region. (2) A recent report also presented data consistent with a cleavage furrow localization of human Orc6 and function in cytokinesis. Ablation of human ORC6 expression in cultured cells via the RNAi method also leads to a rapid appearance of binucleated cells and a decrease in DNA replication. The domain structure homology predictions are noted as even higher for the human protein (Chesnokov, 2003).

It was asked whether the C-terminal region of Orc6 was both necessary and sufficient for Pnut interaction as anticipated from the hypothesis that this region defines a discrete domain of the protein. A peptide corresponding to the last 71 aa of Drosophila Orc6 protein was synthesized, linked by a spacer to biotin, and used to investigate this point. The biotin-labeled peptide was incubated with Drosophila embryonic whole-cell extract and then reisolated by using paramagnetic beads coupled with streptavidin (Promega). The bound material was subjected to SDS/PAGE and analyzed by Western blotting for the presence of Drosophila Pnut protein. The Orc6 C-terminal peptide was able to precipitate Pnut from the extract with a concentration as low as 5-10 micromol. It is concluded that this C-terminal region is a distinct domain of the protein. The putative structural homology between ORC6 and TFIIB can be tested, for example, by physical methods, and if borne out it would bring forth the notion that certain proteins involved in the initiation of replication coevolved with proteins important for transcription. In this context, it is intriguing that archael organisms have a single gene encoding an Orc1 family member and a TFB (TFIIB homolog); perhaps the respective encoded proteins interact (Chesnokov, 2003).

The primary question raised by these findings might be posed as follows: Does the role of Orc6 in cytokinesis actually link the regulation of DNA replication to this late step in cell division? A priori, it might be envisioned that the first steps toward building a prereplication complex in early G1 or late telophase might be tied to the successful completion of cytokinesis. Orc6 molecules at the cleavage furrow might participate in some event during cytokinesis and then after execution shuttle to chromosomes perhaps with other proteins. This shuttling might make dependent the completion of a cytokinetic function to the start of a new round of replication. This model posits a late step in cytokinesis for ORC6 that might couple the cytokinetic and DNA replication pathways. Alternatively, Orc6 may participate in some early role in cytokinesis assisting in a targeting function for septins in metazoans, thus potentially linking assembly of such septin rings at the cleavage furrow to the completion of DNA replication (Chesnokov, 2003).

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. Mid1 mutants are defective in septum placement and formation and overexpression of Mid1 disrupts cytokinesis. 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 However, Mid1 is important for the initial organization of myosin II at the contractile ring and can interact with myosin II. 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, whereas metazoan anillin binds directly to septins 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. 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, 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).

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 RhoGEFPbl-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 RhoGEFPbl-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 RhoGEFPbl contributes to anillin localization during cytokinesis. After 3 d of RhoGEFPbl 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 RhoGEFPbl, consistent with prior analysis of fixed RhoGEFPbl mutant embryos. Because anillin can bind F-actin and phosphorylated myosin regulatory light chain (MRLC), RhoGEFPbl 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 RhoGEFPbl on anillin behavior was tested in LatA. After RNAi of RhoGEFPbl or Rho1, anillin-GFP remained cytoplasmic through anaphase. Thus RhoGEFPbl 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 (MRLCSqh) inhibited cell elongation during anaphase, slowed furrow formation, and delayed and diminished the equatorial localization of anillin-GFP. However, unlike after RhoGEFPbl 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, MRLCSqh 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 RhoGEFPbl-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 RhoGEFPbl-dependent input. Thus, RhoGEFPbl 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 RhoGEFPbl. MRLCSqh-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 MRLCSqh (detected as either MRLCSqh-GFP or with an antibody to serine 21-phosphorylated pMRLCSqh) colocalize (, although they were often offset as if labeling different regions of the same structures (Hickson, 2008).

The effects of anillin RNAi on MRLCSqh-GFP localization were tested. MRLCSqh-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 and represents a requirement for anillin at an earlier stage than previously noted in Drosophila. Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008).

In LatA, anillin RNAi did not prevent equatorial MRLCSqh-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. Using an antibody to the septin Peanut, it was found that in nontransfected S2 cells, septinPnut localized to the cleavage furrow and midbody where it colocalized with anillin. Unexpectedly, the septinPnut 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 septinPnut 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 septinPnut depended on the cell cycle phase. In LatA-treated interphase cells, when anillin is nuclear, septinPnut 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, septinPnut 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 septinPnut RNAi. Although unable to fully deplete septinPnut, it was found that anillin could localize to the equatorial cortex in regions devoid of detectable septinPnut, which is consistent with findings in C. elegans (Maddox, 2005). Importantly, in septinPnut-depleted cells, anillin-GFP still localized to the plasma membrane in LatA but no longer appeared filamentous, indicating that septinPnut is essential for the filamentous nature of the structures and that Rho1 can promote the association of anillin with the plasma membrane independently of septinPnut. 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 septinPnut. Using Dia as a furrow marker, 3 d of anillin RNAi prevented the furrow recruitment of septinPnut. In LatA-treated cells, anillin RNAi did not affect the formation of septinPnut rings in interphase cells, but it greatly reduced the formation of septinPnut-containing structures during anaphase/telophase. Thus, anillin is required for the furrow recruitment of septinPnut and for the formation of septinPnut-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 RhoGEFPbl 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, MRLCSqh, Dia, anillin, and SeptinPnut, localized to the equatorial membrane in the presence of LatA. Of these (and apart from Rho1 itself), anillin and SeptinPnut 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 and of interaction between anillin and MTs. Although such structures are not normally seen in furrowing cells, anillin localizes to remarkably similar filamentous structures in the cleavage furrows of HeLa cells 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).

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. 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. 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. 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 failureowever, 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. 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. 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, 2008 and references therein).

Anillin-mediated targeting of Peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. This study demonstrates that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevents pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that form. Ran is required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin α and importin β to anillin prevents the binding of Peanut to anillin. Furthermore, RanGTP reverses the inhibitory action of importin α and β. On expression of a mutant form of anillin that lacks an importin α and β binding site, inhibition of Ran no longer restricts the depth and duration of furrow ingression in those pseudocleavage furrows that form. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran (Silverman-Gavrila, 2008).

During cytokinesis, the ingressing plasma membrane physically divides the mother cell into two daughter cells. Membrane ingression during cell division is both temporally and spatially regulated, ensuring that membrane scission occurs (1) only after the chromosomes have fully segregated and (2) between the two chromosomal masses. The signals within the cell that determine cytokinetic furrow positioning are complex, reflecting the strict control needed to ensure that cytokinesis is successful. Signals from astral microtubules, the spindle midbody, the nucleus, and the membrane itself direct the assembly of the contractile ring to the equatorial cortex of the plasma membrane. The contractile ring is an actomyosin-based structure that constricts and generates the force needed to drive membrane ingression. As the membrane ingresses, it is remodeled and stabilized (Silverman-Gavrila, 2008).

Other membrane ingression events share many of the same features and involve many of the same proteins as cytokinetic furrows. In the syncytial Drosophila embryo before cellularization, up to 6000 closely packed nuclei exist in a common cytosol close to the cortex. To ensure faithful chromosome segregation during the rapid nuclear divisions, nuclei are isolated from one another to prevent neighboring spindles from contacting and fusing. To achieve this, plasma membrane ingressions form transiently between nuclei during the rapid nuclear cycles before cellularization. These membrane ingressions, termed pseudocleavage or metaphase furrows, are organized by the actin cytoskeleton and bear a close resemblance to cytokinetic cleavage furrows. First actin caps form at the plasma membrane above each nucleus. Then during interphase, as the centrosomes migrate to either side of the nucleus, the actin caps expand correspondingly. In prophase the cap reorganizes to drive membrane ingression into the embryo such that nuclei and newly forming spindles are separated from one another. Toward the end of metaphase, the furrows begin to retract and dissipate by anaphase. This process is repeated from the tenth through the thirteenth nuclear cycles. During the fourteenth nuclear cycle, the syncytial embryo cellularizes to form 6000 columnar epithelial cells. In this instance the cleavage furrows extend down into the embryo, before growing transversally and fusing to form a single layer of nucleated cells (Silverman-Gavrila, 2008).

Most components required for furrow ingression are conserved between cytokinetic furrows (during conventional mitosis) and pseudocleavage furrows. However, there are some differences. Notably pseudocleavage furrows are membrane ingressions that do not meet and therefore do not lead to membrane fusion. Instead they extend into the embryo, perpendicular to the cortex, and then retract back toward the embryo cortex after the chromosomes have begun to segregate. In addition, there is a difference in the stage of the cell cycle when the furrow components assemble. Although the cytokinetic furrow begins to assemble during anaphase and is required to divide a cell in two, the syncytial embryo pseudocleavage furrows begin to assemble in prophase and serve to prevent neighboring spindles from contacting one another (Silverman-Gavrila, 2008).

A key protein involved in cytokinetic furrow function is anillin, which has multiple domains allowing it to bind and bundle actin filaments, target septins to the plasma membrane, and interact with components of the microtubule-bound centralspindilin complex. Consequently anillin is thought to act as a scaffold for the correct assembly of the contractile ring. It is not fully understood how the role of anillin in cytokinesis is regulated. However, its role in remodeling the actomyosin contractile ring in somatic cells is in part regulated by its differential spatial positioning in the cell during the cell cycle. In interphase anillin localizes to the nucleus where it cannot interact with actin and myosin at the plasma membrane. However, in mitosis upon nuclear envelope breakdown, anillin is released from the nucleus and is targeted to the cortex of the plasma membrane and later to the equatorial cortex of the plasma membrane in a RhoGTP-dependent manner. The spatial regulation of anillin during the cell cycle contributes to the restriction of its function to mitosis. However, in Drosophila syncytial embryos anillin is cytosolic, localizing to pseudocleavage furrows throughout the nuclear cycle, suggesting that it may be regulated by other mechanisms (Silverman-Gavrila, 2008).

One function of anillin is to target septins to the contractile ring. Septins are a family of GTP-binding proteins that can assemble into filaments. Septins have been attributed multiple roles: as membrane diffusion barriers, as stabilizers of the furrow, in membrane trafficking, and as a scaffold. In Drosophila there are five septins: Peanut, Sep1, Sep2, Sep4, and Sep5. Peanut, Sep1 and Sep2 have been isolated as a stoichiometric complex that in vitro can polymerize into filaments. In contrast, Xenopus laevis Sept2 can self assemble into filaments, suggesting that septins may function independently (Silverman-Gavrila, 2008 and references therein).

The GTPase Ran is a key positive regulator of mitosis (Ciciarello, 2007). RanGTP regulates a number of mitotic factors that are sequestered in the nucleus by nuclear transport receptors during interphase. In mitosis RanGTP antagonizes the binding of nuclear transport receptors to these proteins and thereby promotes their activity. RanGTP is at its highest concentration around the chromosomes, where RCC1 the nucleotide exchange factor for Ran is localized. Consequently, RanGTP has been proposed to act as a spatial cue by only activating these mitotic proteins close to the chromosomes (Caudron, 2005; Kalab, 2006). In so doing RanGTP is thought to specify where certain mitotic processes occur in the cell. For example, it could specify that spindle assembly only occurs around chromosomes. The full extent to which this mechanism regulates the mitotic cell is not known and continues to expand (Silverman-Gavrila, 2008).

This study demonstrates a new role for Ran in regulating pseudocleavage furrow ingression, a membrane invagination process in early Drosophila embryos. The Ran pathway regulates the interaction between anillin and the septin Peanut, thereby regulating furrow stability (Silverman-Gavrila, 2008).

A cytological screen was carried out to identify mitotic processes regulated by the Ran pathway. Inhibitors of the Ran pathway were injected into GFP-α-tubulin-expressing embryos just before mitotic entry, and then microtubule organization was monitored by time-lapse microscopy. One phenotype, the fusion of neighboring spindles, occurred more frequently upon the injection of inhibitors of the Ran pathway compared with control injections. In control injected embryos 0.2% of observed spindles fused to a neighboring spindle. In contrast, inhibition of the Ran pathway by injecting either the dominant negative allele of Ran, RanT24N, or importin α resulted in 8.4 and 7.8% of observed spindles fusing to neighboring spindles, respectively (Silverman-Gavrila, 2008).

Peanut is recruited to ingressing furrows by anillin, a multifunctional protein required for cytokinesis that interacts with myosin II, actin, and septins. Septins bind to the carboxy-terminus of anillin, which includes a pleckstrin homology (PH) domain. Drosophila anillin has three potential nuclear localization signals (NLS) that could bind to the nuclear transport receptors importin α and β. Two of the NLS motifs are located in or directly adjacent to the PH domain (Silverman-Gavrila, 2008).

To determine if the carboxy-terminus of anillin could bind to importin α and β, a fusion was constructed between GST and the carboxy-terminus of anillin (amino acids 815-1201, anillin-CT, and its ability to bind to recombinant importin α and β was analyzed. Both importin α and β bound to anillin-CT, and this binding was reversed in the presence of RanQ69L, a point mutant of Ran locked in the GTP-bound state. Of the two potential NLS motifs, the one located between amino acid residues 989 and 999, bares the closest resemblance to an archetypal bipartite NLS and is found in the same region of human anillin (amino acids 887-898). Mutation of lysines 997-999 to alanine (3A-anillin-CT) abrogate both importin α and β binding to this region of anillin, suggesting that amino acids 989-999 constitute a nuclear transport receptor-binding site (Silverman-Gavrila, 2008).

It was next asked if the anillin-CT could interact with Peanut. GST-anillin-CT was incubated with 0-3-h Drosophila embryo extract and then isolated using glutathione agarose beads. Anillin-CT copurified with Peanut and another septin, Sep2. However, the addition of exogenous importin α and importin β inhibited the binding of Peanut to anillin-CT in a concentration- and NLS-dependent manner. This inhibition was specific to Peanut, because Sep2 binding to anillin-CT was not inhibited by importins (Silverman-Gavrila, 2008).

To determine if the in vivo targeting of Peanut and Sep2 to the pseudocleavage furrows is differentially regulated, importin α was injected into syncytial embryos and GFP-Sep2 localization was determined by time-lapse microscopy. Consistent with in vitro results, GFP-Sep2 localization was not perturbed upon interfering with the Ran pathway. Furthermore, in fixed GFP-Sep2-expressing embryos in which the Ran pathway has been perturbed, Peanut fails to localize to nascent furrows, whereas GFP-Sep2 does localize to nascent furrows. These data suggest that Peanut and Sep2 are differentially regulated by Ran and that Sep2 can localize to pseudocleavage furrows independently of Peanut (Silverman-Gavrila, 2008).

This study has identified RanGTP as a regulator of the interaction between Peanut and anillin. This mechanism operates directly and independently of Ran's well-characterized role in regulating the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Studies suggest that anillin is required for the recruitment of septins to the furrow. By perturbing the Ran pathway, this study has demonstrated that the recruitment of the septins Peanut and Sep2 is differentially regulated, consistent with previous observations that Sep1 recruitment to furrows is dependent on Peanut but Sep2 is not. Anillin lacking the importin binding site between residues 997 and 999 can bind to Peanut in the presence of importins, suggesting that importins directly block the anillin-Peanut interaction rather than disrupting the Peanut, Sep1, and Sep2 complex. These data suggest that although Peanut, Sep1, and Sep2 can exist in a single complex, they may be able to function independently of one another as has been demonstrated in vitro for a Xenopus septin (Silverman-Gavrila, 2008).

Perturbing the Ran pathway destabilizes pseudocleavage furrows. One mechanism for this is through the regulation of the anillin-Peanut interaction. In embryos that expressed an anillin mutant lacking the importin-binding site, Peanut recruitment to pseudocleavage furrows occurs even in the presence of exogenous importins, and furrows demonstrate wild-type dynamics. These data suggest that Peanut is required for pseudocleavage furrow stability. This role for anillin and Peanut is consistent with the observed role for these proteins in stabilizing the cellularization furrow later in Drosophila development. These findings may at first appear to contradict those studies, in which embryos lacking Peanut protein progressed through the syncytial nuclear divisions only showing the first defects during cellularization. However, these studies only analyzed syncytial furrows from the top, apical view and not from the lateral view to observe ingression dynamics. Therefore, these studies would not have detected changes in furrow ingression dynamics that were observed upon inhibition of Ran, which correlated with a failure to recruit Peanut to the furrow (Silverman-Gavrila, 2008).

The Ran pathway regulates pseudocleavage furrow ingression directly by regulating importin binding to anillin. It was previously shown that in Drosophila syncytial embryos the importin β, whose injection causes similar effects as importin α, is released from the nucleus upon nuclear envelope breakdown and becomes diffuse throughout the cytosol during the rest of mitosis. During this period pseudocleavage furrows begin to retract. Therefore, as importin β is cytosolic during metaphase and anaphase it could act to prevent the interaction of Peanut and anillin. In turn this would lead to furrow instability and retraction (Silverman-Gavrila, 2008).

It cannot unequivocally be ruled out that some of the defects caused by perturbing the Ran pathway are due to a disruption of microtubule cytoskeleton. Indeed, one microtubule-dependent furrow phenotype, the formation of pseudocleavage furrows that encompassed a small area of cytosol around a nucleus, was observed. This phenotype has also seen in another study upon depolymerization of microtubules in embryos. However, microtubule depolymerization when instigated in interphase does not cause a failure in pseudocleavage furrow formation, a finding consistent with a previous study (Silverman-Gavrila, 2008).

Another mechanism through which Ran could affect pseudocleavage furrows is by disrupting nuclear trafficking. Indeed it was observed that nuclear trafficking can be reduced by up to 50% upon disruption of the Ran pathway. However, it seems unlikely that the changes in nuclear import kinetics in these experiments disrupted the function of anillin because anillin is a cytosolic protein in the syncytial embryo and localizes to the leading edge of the ingressing furrow during interphase. It is not understood how anillin is retained in the cytoplasm of syncytial embryos because it is imported into nuclei in other developmental stages. However, this phenomenon is not unique to anillin and is also exhibited by the kinesin Pavarotti, another protein involved in pseudocleavage furrow organization (Silverman-Gavrila, 2008).

These studies suggest that Ran regulates multiple factors involved in pseudocleavage furrow ingression, because embryos expressing the mutant anillin still exhibit a failure to form all the expected pseudocleavage furrows. Failure to fully suppress the phenotype could be due to the continued presence of endogenous anillin or reflect that other Ran pathway-sensitive factors are involved in pseudocleavage furrow formation. Regulation through the Ran pathway could define a spatial cue concentrated around chromosomes and extending to the cortex. Such a spatiotemporal regulatory mechanism could be involved in promoting cytokinetic furrows in other cells. A recent study in oocytes finds that Ran regulates myosin II, whose activity is required for cytokinetic cleavage furrows. In addition importin α is required for ring canal organization during oogenesis. Ring canals form as a result of incomplete cytokinesis, and many proteins involved in cytokinesis both localize to and are required for their formation, including anillin and septins (Silverman-Gavrila, 2008).

The data suggest that the anillin-Peanut interaction, which is inhibited by importins must occur in regions of the cell where there are low levels of importins or high levels of RanGTP. Recent studies have visualized a RanGTP-importin β gradient and found that it persists from the chromosomes to the centrosomes, a distance similar to that between the metaphase plate and the cortex. Thus, RanGTP could play an important role in positioning the plane of cleavage by defining on the cell cortex where furrow proteins interact (Silverman-Gavrila, 2008).

Although there are clear differences between cytokinetic and pseudocleavage furrows, anillin and septins are involved in both. Therefore, this study suggests that Ran could also have a role in regulating cytokinetic furrows. Whether chromosomes play a significant role in cytokinesis remains controversial. However, studies where nuclei or chromosomes are asymmetrically positioned within a cell show that furrow ingression coincided with the region of the cell that contained the chromosomes, suggesting that signals from the nucleus and in particular the chromosomes had a role in specifying furrow ingression. Similarly, enucleated sea urchin eggs are able to duplicate their centrosomes and generate astral arrays of microtubules, but fail to form stable cleavage furrows. The current study proposes a molecular mechanism to explain, at least in part, these observations, suggesting that RanGTP generated around the chromosomes is a diffusible signal that facilitates multiple processes required for furrow formation. Whether RanGTP is required early in cytokinesis to 'prime' the cortex for a future ingression or acts directly later during the ingression process is unclear. Testing these hypotheses is not straightforward, since Ran is also required for organizing the mitotic microtubule cytoskeleton, which is required for cytokinesis. Taken together these findings suggest an additional mechanism involved in regulating cytokinesis that is dependent on signals from chromosomes in addition to those stemming from the different organizational states of the mitotic microtubule cytoskeleton (Silverman-Gavrila, 2008).

Drosophila Orc6 facilitates GTPase activity and filament formation of the septin complex

This study describes the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis (Huijbregts, 2009).

Both in Drosophila, a considerable pool of Orc6 is cytoplasmic, and the protein is either associated with or proximal to the plasma membrane and cleavage furrows of dividing cells. In Drosophila, Orc6 and Pnut colocalize in vivo at cell membranes and cleavage furrows of dividing cells, and during cellularization in Drosophila early embryos. The C-terminal domain of Orc6 is necessary for this colocalization with Pnut. Moreover, Orc6 RNAi results in cytokinesis defects in Drosophila tissue culture cells (Chesnokov, 2003), whereas Pnut RNAi disrupts the localization of Orc6 to the plasma membrane (Huijbregts, 2009).

Analysis of the cells treated with Pnut dsRNA revealed an elevated number of binucleated cells (5- to 30-fold increase, depending on the experiment). This is in contrast with previously reported data in which no elevated numbers of binucleated cells were detected in cultures treated with Pnut dsRNA. Differences in culture conditions, amount of dsRNA used, as well as cell preparation protocols for analysis might have contributed to the discrepancies between the two studies (Huijbregts, 2009).

Deletion of part of the predicted coiled-coil domain of Pnut impaired its ability to form a complex with both Sep1 and Sep2 together, but it was still able to interact with Sep1. A previous study proposed that different septin complexes may exist within Drosophila. The current data suggests that Pnut and Sep1 might form a precomplex that joins with Sep2 to form the complete septin complex. The interaction of Orc6 with Pnut is also disrupted in C-terminal deletion mutants of Pnut, suggesting that the coiled-coil domain of Pnut is important for both binding with Orc6 and for the formation of the septin complex. However, leucine to alanine substitutions within the coiled-coil domain of Pnut prevent Orc6 binding but do not inhibit complex assembly, indicating that these protein interactions are based on different structural moieties within the C terminus. Furthermore, direct interaction studies revealed that the coil-coil domain of Pnut is not sufficient for the interaction of this septin with Orc6 and that other structural features may also be important for the interaction between the two proteins (Huijbregts, 2009).

To study the effect of Pnut mutations in vivo in Drosophila tissue culture cells, various expression systems were used, including heat shock and metallothionein promoters. In all cases, expression of GFP-Pnut in L2 cells resulted in rod- and spiral-like structures present throughout the cytosol. Expression of either N-terminal or C-terminal GFP fusions to Pnut, or a FLAG-Pnut protein also resulted in the same aberrant structures, compromising the in vivo analysis of Pnut mutants. However, when under control of the native Pnut promoter, proteins could be expressed in L2 cells at lower levels. This figure further shows that the coiled-coil domain of Pnut, which is important for Drosophila septin complex assembly, also is essential for the in vivo localization of Pnut, because FLAG-Pnut(1-427), lacking the coiled-coil domain, had the tendency to accumulate into crescent shaped aggregates. The FLAG-tagged triple leucine mutants of Pnut exhibited mainly diffuse cytoplasmic staining when expressed in L2 cells, although some plasma membrane staining was observed. It is possible that due to the mutations in the coiled-coil domain these Pnut mutants do not interact properly with other proteins (as shown for Orc6), resulting in a release from the plasma membrane at specific cell stages (Huijbregts, 2009).

Native septin complex as well as reconstituted septin complexes exhibit the characteristic properties of filament formation and GTPase activity, indicating that they are functional complexes. Because insect cell lines are closely related to Drosophila, baculovirus-derived reconstituted septin complex was used for further biochemical studies (Huijbregts, 2009).

The human SEPT2-SEPT6-SEPT7 complex can be formed from recombinant proteins all lacking their predicted coiled-coil domains, suggesting that their C termini are dispensable for complex formation (Sirajuddin, 2007). Structural analysis of crystals of the human septin complex revealed that the filaments consist of an assembly of GTP binding domains. However, the coiled-coil domains of SEPT6 and SEPT7 do interact directly with each other, suggesting that although not required for the human septin complex, coiled-coils may further stabilize filament formation (Sirajuddin, 2007). The GTP binding domains of human septin proteins can also interact with coiled-coil structures within the multiple subunit complex. This might also occur with the Drosophila septins when they assemble into complex. However, the interaction of Orc6 with the septin complex seems strongly dependent on the coiled-coil domain of Pnut (Huijbregts, 2009).

One possible role of the interaction of Orc6 with the septin complex could be the regulation of the GTPase activity of the complex during cytokinesis. Orc6 reproducibly increased the GDP-to-GTP ratio of bound nucleotide of the whole septin complex, but no significant increase of total nucleotide bound to complex was detected. It has been hypothesized that GTP hydrolysis might promote disassembly of the septin complex. However, purified recombinant septin complex was retrieved intact with bound Orc6 after 2-h incubation in the presence of GTP, although potentially the disassembly of a small amount of complex might have occurred. It was observed that many larger filaments present in concentrated recombinant septin complex samples were not detected under GTPase assay conditions, most likely due to the dilution of concentrated sample. No differences in filament size were observed for septin complexes incubated either in the presence or absence of GTP. However, due to limitations of the EM setup, subtle changes in small filament size could not be detected (Huijbregts, 2009).

Although GTP hydrolysis by septin complex was accelerated by Orc6 binding (because the presence of a nonbinding mutant of Orc6 had no additional affect on hydrolysis), no significant changes could be detected in turnover rate. The higher turnover rates reported for individual Xenopus, mouse, and human recombinant septin proteins do not exclude a regulatory function for these subunits in vivo when not assembled in complex. A structural rather than regulatory role for septin complex-bound GTP and GDP was proposed from the results obtained with yeast septins. No turnover of yeast septin-bound GTP and GDP could be detected during a cell cycle in vivo. Furthermore, in vitro experiments revealed that GTP hydrolysis of yeast septin complex was limited by its slow binding or exchange activity, similar to the properties described initially for the Drosophila septin complex. The role of Orc6 in GTP hydrolysis and filament disassembly of septin complex also suggests that in the case of Drosophila the guanine nucleotides bound to septins may contribute to the structural properties of the complex. Additionally, the importance of the GTP binding domains for the assembly of the human septin complex and potentially filament formation (Sirajuddin, 2007) also indicates a role for guanine nucleotide in septin complex structure (Huijbregts, 2009).

The addition of Orc6 to septin complex in the absence of GTP, in contrast, greatly induced filament formation, whereas in the presence of GTP the effect was not observed. This indicates that Orc6 exhibits two opposite effects in its interactions with the septin complex. Based on the sequence homologies between human or Drosophila septins, hexamers can be depicted as a linear protein similar to the crystal structure of the human septin complex (Sirajuddin, 2007) with Pnut at either end of the complex (see Model for the interaction of Orc6 with the septin complex). In the absence (or low concentration) of GTP Orc6 binding to Pnut enhances linear filament assembly, potentially due to conformational changes in either Pnut or other septin subunits. Orc6 stabilizes the formation of the filaments by protein-protein interactions. It is interesting to note that purified recombinant Orc6 protein behaves as a dimer in biochemical assays. In the presence of GTP, Orc6 increases the GTPase activity of the septin complex, at the same time resulting in filament disassembly. Increased GTPase activity may lead to conformational changes in the septin complex, causing disassembly of septin filaments. These results suggest that Orc6 may regulate either assembly or disassembly of septin filaments. Whether Orc6 actively induces filament disassembly in the presence of GTP or this process is a result of the increased GTPase activity of the septin complex remains to be investigated (Huijbregts, 2009).

The septins are important for cytokinesis but molecular mechanisms of their functions in this process are not completely understood. These data on interactions between Drosophila Orc6 and the septin complex reveal some new aspects for these proteins. Orc6 has an effect on both GTPase activity and filament formation of the septin complex, suggesting that Orc6 might have a direct role in septin complex functions during the last stage of mitosis (Huijbregts, 2009).



Intense staining for Pnut protein is seen on the cell surfaces of the embryonic central nervous system and on the apical membranes of the developing photoreceptor cells in the eye imaginal disc. Punctate cytoplasmic staining is seen in a subset of eye disc and larval central nervous system cells (Neufeld, 1994).

Peanut and Sep1, a second Drosophila septin identified based on its homolog to yeast septins, colocalize to the leading edge in cellularizing embryos. During the interphase between nuclear divisions 13 and 14, actin first reorganizes from the set of caps over the nuclei to form a hexagonal actin-myosin network at the embryo cortex. During the process of cellularization, the cleavage furrows move into the embryo around each nucleus, with actin and myosin concentrate at their leading edges, and spectrin concentrates slightly behind the leading edges. At the beginning of cellularization, Sep1 also assumes a hexagonal pattern similar to (but apparently less uniform than) that of actin and myosin. The nonuniformity of the Sep1 staining is maintained until the end of cellularization. As cellularization proceeds, Sep1 is concentrated at the leading edges of the advancing cleavage furrows, although some diffuse staining is still observed at the embryo cortex and in the underlying cytoplasm. Both Sep1 and Pnut co-localize at least at the resolution of the light microscope. Examination of double-stained embryos reveal that the septins co-localize with actin and myosin at the very leading edge of the cleavage furrows, in a position distinctly ahead of spectrin (Fares, 1995).

Study of Sep1 localization suggests septin roles that are not related to cytokinesis. Sep1 is concentrated at the leading edge in cells at the front of the extending epithelia during dorsal closure. The staining of Sep1 at the tips of the migrating cells sems to extend further back into their cytoplasms than does the staining of actin. In the developing central and peripheral nervous systems the localizations of Sep1 and Pnut are indistinguishable (Fares, 1995).

To explore the nature of the defects seen in the absence of diaphanous function, wild-type and dia mutant embryos were stained at nuclear cycles 11-13 with the DNA dye DAPI and with an antibody directed against F-actin. In the wild type, nuclei are positioned at the embryo cortex at interphase of nuclear cycles 11-13; a structure referred to as the actin cap is situated between each nucleus and the plasma membrane. During the transition to prophase, filament reorganization results in a concentration of actin at the edge of the caps. At metaphase, the resulting rings of cortical actin, together with associated plasma membrane, invaginate to form metaphase furrows. As viewed from above, actin staining at these furrows appears as a hexagonal array over the embryonic surface. In the sagittal view, actin staining at the metaphase furrow appears as a line between the metaphase nuclei. In dia-deficient embryos, severe structural changes in the actin cytoskeleton are manifested after nuclear cycle 11. Formation of the hexagonal actin arrays is disrupted during prophase and metaphase and there is an absence of actin staining between the metaphase nuclei. Similar patterns of staining are obtained when dia embryos are stained with antibodies directed against anillin (Drosophila gene: Scraps) and Peanut, other components of the metaphase furrow. There is thus a failure in the formation of the metaphase furrow. Consistent with the known role of metaphase furrows in maintaining nuclear organization, the nuclei in dia mutant embryos frequently exhibit abnormal spacing and, in some cases, fuse in subsequent nuclear cycles. These irregularities are readily apparent in contrast to the uniform pattern observed in the wild type. In regions in which cortical actin staining is weak or absent, nuclei are frequently found displaced into the interior of the embryo, although the centrosomes remain at the surface (Afshar, 2000).

To investigate whether the absence of metaphase furrows results from a failure in membrane invagination, dia embryos were stained with antibodies directed against myosin (also known as Zipper). In wild-type embryos, myosin localizes to the embryonic cortex between the actin caps at each interphase, appears at the tip of the invaginating membrane at prophase and disappears at metaphase. In dia embryos, myosin staining, albeit very weak and irregular, is detected between the actin caps at the cortex during interphase. At prophase, myosin, where detectable, remains at the cortex, with no detectable membrane pinching or invagination. Therefore, despite the presence of myosin at the cortex between actin caps, the membrane invagination that precedes metaphase furrowing is absent in dia embryos (Afshar, 2000).

Immunolocalization was used to determine whether Diaphanous plays a role in the recruitment of anillin and Peanut, a Drosophila septin. In wild-type embryos both anillin and Peanut localize to the embryonic cortex, between the actin caps at interphase. During prophase and metaphase, they localize to the metaphase furrow and their pattern of staining is similar to that of actin. In dia embryos, the staining patterns of both anillin and Peanut are very weak during interphase. Similarly, in dia embryos the localization of both of these proteins is disrupted during prophase and metaphase, when the metaphase furrow is being formed in wild-type embryos. Diaphanous is thus required for recruitment and proper localization of anillin and Peanut as well as myosin to the regions of membrane invagination (Afshar, 2000).

Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).

To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).

This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).

Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P2, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).

The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).

The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).

A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).

A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).

In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).

The BAR domain of amphiphysin is required for cleavage furrow tip-tubule formation during cellularization in Drosophila embryos

De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. This study identified the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, it was determined that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression (Su, 2013).

Cellularization in the Drosophila embryo involves de novo generation of 6000 columnar epithelial cells, which are generated by the ingression of plasma membrane furrows (cleavage furrows) that enclose each nucleus. At the tip of ingressing cleavage furrows, CFT-tubules form. This study demonstrated the existence of three populations of CFT-tubules, which can de defined by different septin family members. The different populations of CFT-tubules are differentially regulated, and their presence or absence correlates with changes in cleavage furrow ingression kinetics (Su, 2013).

Septins were identified as additional factors localizing to the CFT-tubules. Of interest, not all septins localize to the same CFT-tubules or the same domain within a single CFT-tubule. This suggests that although the CFT-tubules are formed by an endocytic pathway (Sokac, 2008), the tubules are not homogeneous. Instead, tubules can contain different domains that may have different functions. Three distinct types of tubules were identified: those that contain only amphiphysin and the septins Sep1 and Sep2, those that contain only the septins Peanut, Sep4, and Sep5, and those that possess heterogeneous subdomains each defined by a distinct composition of these various components. Of importance, localization studies suggest that distinct septin complexes localize to different structures. Because Peanut, Sep4, and Sep5 do not colocalize with Sep1 and Sep2 on CFT-tubules, it is predicted that Peanut, Sep4, and Sep5 form a novel septin complex. This new septin complex may resemble the previously isolated complex of Peanut, Sep1, and Sep2, as Sep2 is most closely related to Sep5 (72% identity) and Sep1 is most closely related to Sep4 (47% identity). It was not possible to isolate individual septin complexes by immunoprecipitation, as all septins coimmunoprecipitated. This finding is consistent with studies in mammalian cells and reflects either the heterogeneous nature of septin complexes within the entire embryo or that, in part, partial septin filaments were being immunoprecipitated. Unexpectedly, Peanut did not colocalize with Sep1 and Sep2 on CFT-tubules. This observation raises the possibility that Sep1 and Sep2 alone form a complex. Septin filaments in yeast and mammalian systems are generated from octamers containing two copies of four different septins arranged in an inverted repeat; however, this may not be true for all systems. In the case of Drosophila a hexamer of Peanut, Sep1, and Sep2 has been isolated, and in Caenorhabditis elegans there are only two septin genes (Su, 2013).

Septins have predominantly been implicated in modulating events at the plasma membrane in conjunction with the actin cytoskeleton. In mammalian cells, septins have also been linked to potential roles in membrane trafficking, especially in the exocytic pathway, possibly by regulating vesicle fusion. It seems unlikely that the septins on CFT-tubules are regulating exocytosis, as all evidence suggests that exocytosis occurs at distinct apical sites in the syncytial embryo). In contrast, one study suggests a role for septins in the endocytic pathway by regulating recruitment of the coat protein complex AP-3 to lysosomal membranes (Baust, 2008). The precise roles for septins in this process are unclear. In CFT-tubules, it is possible that septins exert an effect directly on the membrane. Septins can tubulate membranes containing phosphatidylinositol (4,5)-bisphosphate, a lipid that has a key role in cytokinesis. However, the current data demonstrate that CFT-tubule formation is dependent on amphiphysin. Septins have been proposed to stabilize membranes. Therefore septins could stabilize the CFT-tubules once formed. Indeed, reduced recruitment of septins to cleavage furrows destabilizes the entire cleavage cleavage furrow. Furthermore, embryos depleted of Peanut form unstable yolk channels at the end of cellularization, further supporting the model that septins can stabilize membrane structures to which they localize. These findings also suggest that mutations that deplete septins will not allow examination of the role of septins in CFT-tubule organization and function (Su, 2013).

This study found that CFT-tubule formation requires the BAR domain of amphiphysin. The N-BAR subfamily, to which amphiphysin belongs, can bind to membranes and promote their curvature. Amphiphysin is also involved in t-tubule formation in Drosophila indirect flight muscles and mouse heart muscle. These findings suggest a conserved role for amphiphysin in promoting tubule formation and organization (Su, 2013).

Loss of amphiphysin and the prevention of CFT-tubule formation did not inhibit furrow ingression, suggesting that amphiphysin is not required for remodeling of the membrane to drive furrow ingression. Instead, loss of amphiphysin increased the rate of furrow ingression. Because amphiphysin localizes to the tip of the furrow, it is possible that amphiphysin acts as a negative regulator of furrow ingression. Alternatively, by preventing CFT-tubule formation, amphiphysin may render more plasma membrane accessible for furrow ingression, and therefore the rate of furrow ingression increases. Consistent with this model, when CFT-tubules become longer upon disruption of dynamin, the rate of cleavage furrow ingression is reduced. One potential consequence of inhibiting endocytosis at the furrow tip would be to reduce the amount of membrane available for the expansion of the plasma membrane and the ingression of the furrow. In such a scenario membrane derived from endocytosis at the tip of the furrow would be recycled back to the plasma membrane through the exocytic pathway, thereby providing sufficient membrane for the expansion and ingression of the furrow. This reduced availability of membrane could manifest itself as a reduced rate of furrow ingression seen in shibirets embryos at the nonpermissive temperature, where CFT-tubules elongate due to a failure to pinch off. The additional membrane may be especially important for the rapid increase in furrow ingression that is seen once the furrow has ingressed ∼10 μm, a depth of ingression where CFT-tubules normally become shorter and disappear (Su, 2013).

Changes in tubule parameters correlate with changes in cleavage furrow ingression kinetics, especially in the fast phase of ingression; longer, more persistent tubules correlate with slower ingression kinetics, and the absence of tubules correlates with faster ingression kinetics. If the fast phase of cleavage furrow ingression were dependent upon new membrane being inserted into the plasma membrane, then restricting membrane insertion would suppress the fast phase. If membrane was recycled by endocytosis at the cleavage furrow tips through an endocytic compartment back to the plasma membrane, then changes in CFT-tubule parameters might be expected to affect cleavage furrow ingression kinetics (Su, 2013).

In the models outlined in this study CFT-tubules would function to buffer the amount of available membrane that is accessible for efficient cleavage furrow ingression. However, no comparable measurements have been made with respect to t-tubules in muscles. Therefore it remains unclear whether the tubules in these different systems have a common function, whether they are examples of specialized endocytosis, or whether the creation of extra membrane surface area facilitates specialized functions in these different systems (Su, 2013).

Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues

Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. This study uncovers intrinsic and extrinsic forces necessary for cytokinesis and presents a framework for understanding how tissue cohesion is preserved during epithelial division (Guillot, 2013).

Epithelial cells divide in the plane of the tissue, allowing the equal partitioning of polarity proteins. This study delineated two major events during epithelial cytokinesis that shed light on how this is controlled. Cleavage progresses along the apicobasal axis and is polarized, as it is faster from basal to apical. This is not due to polarized contraction of the ring but to apical anchoring of the ring to E-cad complexes. Second, cleavage occurs in the plane of junction and involves local adhesion disengagement. In contrast to standard cytokinesis, this study delineated intrinsic and extrinsic mechanical processes operating during epithelial cytokinesis. Contractility of the ring itself is dependent on septins and Anillin. Ring contraction is resisted by intercellular adhesion mediated by E-cadherin complexes and by tension from neighboring cells transmitted by adhesion. Thus, E-cad-based adhesion plays a pivotal role in epithelial cytokinesis by anchoring the contractile ring, while its disengagement uncouples intrinsic and extrinsic tensile activity (Guillot, 2013).

In Drosophila embryos, epithelial cells exhibit polarized cleavage furrow ingression. This is likely to be general in epithelial cells, albeit at different magnitudes. MDCK cells too divide from the basal side toward the apex, and neuroepithelial cells in vertebrates partition the basal body first before the more apical part of the cell. Polarized cleavage is not a property unique to epithelial cells, however. Embryonic cleavage in several species exhibit a range of patterns, from completely unilateral cleavage, as reported in jellyfish (Clytia and Beroe) and Ctenophores (Pleurobrachia), to partly asymmetric cleavage in the one-cell-stage C. elegans embryos). In the latter case, polarized ingression of the cleavage furrow is stochastic and correlates with heterogeneities in the recruitment of the actin crosslinker Anillin and of septins. In anillin and septin knockdowns, cleavage becomes symmetric. This contrasts with activators of MyoII, such as Rho kinase, which affects the speed of contraction but not its polarity. Thus, in nonepithelial cells, polarized cleavage is a purely autonomous process governed by heterogeneities in regulators of contractility. This study found, however, that in Drosophila embryos, polarized cleavage is not determined by polarized distribution of Anillin and septins or by differential biomechanical properties of the ring. Septins display a marginal yet significant enrichment basally, and Anillin is slightly enriched apically. However, invagination was still normally polarized along the apicobasal axis in both peanut mutants and anillin RNAi embryos, despite strong reduction in constriction rat. Moreover, no significant difference between apical and basal relaxation kinetics was detected following ablation in wild-types. The ablation kinetics reflects the relative effect of stiffness in the ring and friction internal to the ring and with the cytoplasm. With the caveat that the latter cannot be directly measured and and is assumed to be uniform, these ablation experiments indicate the relative stiffness in the ring. The fact that relaxation is faster (<5 s) than turnover of the internal components of the ring, such as MyoII, substantiates the idea that mostly the elastic relaxation of the ring was measured and not a quasi-static relaxation associated with turnover/movements of ring components (Guillot, 2013).

The rate of constriction was monotonic such that big rings and small rings contracted at a constant rate in wild-types but also in anillin or septin mutants, although it was strongly reduced in the latter cases. This contrasts with reports in C. elegans, where constriction was scaling with ring size, suggesting a mechanism based on disassembly of contractile units whose number scales with ring size. This difference may stem from the fact that cytokinesis is especially rapid in Drosophila embryos (about 150 s). Alternatively, it could reflect the epithelial nature of the divisions reported in this study (Guillot, 2013).

The evidence argues instead that polarized ingression depends largely on apical anchoring of the ring to E-cad complexes. First, E-cad complexes colocalize with the contractile ring for the most part of invagination. Second, ingression is symmetric in either e-cad or α-cat RNAi embryos. Although E-cad complexes, in particular α-cat, can recruit regulators of MyoII, this cannot explain polarized invagination of the ring, since apical and basal relaxations are not significantly different in wild-types and in α-cat RNAi embryos. E-cad complexes transmit actomyosin tension in epithelia. Two sets of observation support the idea that junctions exert pulling forces on the ring due to anchoring. The ring is stretched laterally as it constricts, and this requires apical junctions via e-cad and α-cat. The relative deformation of the ring following ablation is larger apically than basally, and this also requires cell junctions. It is striking that extrinsic and intrinsic regulators of the ring contraction have very different effects on ring dynamics. In the absence of Pnut or Anillin, the ring constriction is reduced but it is still polarized. However, following e-cad or α-cat depletion, ring constriction is normal but symmetric. It is concluded that the mechanical connection of E-cad complexes to the contractile ring causes polarized invagination. It is possible that, in other systems, both intrinsic and extrinsic regulation will operate in parallel to increase the cleavage asymmetry. This may be important in highly columnar epithelial cells or when adhesion is lower and unable to resist the ring tension (Guillot, 2013).

Polarized cleavage effectively separates apical and basal cleavage, adhesion complexes being a barrier separating the apical and lateral domains. The central problem becomes: How does cleavage occur at adherens junctions? This study delineated two critical phases in junctional cleavage. First, the adherens junctions invaginate with the actomyosin ring, consistent with the fact that the ring is anchored to the junctions. During this phase, E-cad intercellular adhesion is stable in the face of the tension exerted by the ring, and E-cad colocalizes with the ring at the point of coupling. Invagination of junctions then stops as E-cad levels decrease in this area. However, ring constriction continues and appears to detach from junctions. This is interpreted as a point of adhesion disengagement. Adhesion disengagement marks the formation of the new vertices and of the new junction between daughter cells. Electron microscopy images show this membrane disengagement. Consistent with this, the membrane still invaginates with the actomyosin ring), although E-cad is still not detected. Closer examination shows that E-cad monomers are present at this late stage of cytokinesis but that adhesion complexes form gradually from this stage onward. It is striking that adhesion is very locally (<1 μm out of ∼40 μm of junction perimeter) and transiently (∼200 s) perturbed during division. In the first 150 s, E-cad clusters immediately adjacent to the cleavage furrow remain in position as the junction invaginates. This suggests that the cortex can be extensively remodeled locally. It likely reflects the fact that tension induces membrane flows with respect to the actin-rich cortex and argues that E-cad-mediated adhesion does not prevent membrane flow during disengagement. Interestingly, local disengagement allows local cell deformation without affecting the overall shape of cell contacts. Consistent with the idea that adhesion is locally disengaged, the amount of E-cad has a strong impact on the timing and depth of junctional cleavage. Increasing E-cad delays disengagement (i.e., the formation of the new junction, inducing strong cell deformations. More generally, this implies that increasing adhesion may provide an efficient mechanism to prevent local cell-cell disengagement when internal tension is used to remodel junctions during morphogenesis. In apical constriction in the Drosophila mesoderm, actomyosin cables pull on the junctional cortex and reduce junction lengths. If adhesion was not strong enough, local disengagement would occur and junctions could not remodel. The fact that adhesion disengagement is local and transient during cytokinesis is also probably key to the overall maintenance of cell polarity and adhesion during epithelial division (Guillot, 2013).

It is proposed that adhesion disengagement is mechanically induced by tension in the cytokinetic ring and by tension from neighboring cells. When the cumulated tension is higher that the adhesive force, disengagement occurs. Consistent with this, disengagement and formation of the new junction is strongly delayed in mutants that reduce the constriction of the cytokinetic ring, namely, in septin mutants and in Anillin knockdown embryos. Likewise, ablation of neighboring cells delays disengagement. It is, however, possible that adhesion is also locally disrupted by either E-cad endocytosis or phosphorylation of β-cat/Arm (Guillot, 2013).

Adhesion complexes transmit cell tension exerted by neighboring cells. Surrounding junctions and, more specifically, MyoII cables oriented toward or near the cleavage furrow strongly affect furrow invagination when E-cad is present at high levels. The invagination in this case is very shallow, suggesting a tug of war between intrinsic (ring contraction) and extrinsic tension (MyoII cables in neighbors). This results in asymmetric furrows in the plane of junctions due to the asymmetric distribution of MyoII cables around the cell. When E-cad is expressed at lower levels, even if surrounding junctions are oriented toward the cleavage furrow, invagination is unaffected and symmetric. It is proposed that E-cad complexes sensitize cells to their mechanical environment. This may provide a mechanism for cells to integrate stress coming from the environment. It will be important to explore how E-cad levels may affect cells responsiveness to extrinsic stress during division by affecting the timing of the formation of the new junction by local disengagement and the resulting cell shape and topology (Guillot, 2013).

Larval and Adult

In larval imaginal discs, Sep1 is concentrated mainly at the apical and basal surfaces of cells and is apparently less abundant at the lateral surfaces. Double-staining for Sep1 and D4.1 and for Sep1 and Armadillo reveals that the localization of Sep1 to the apical membranes extends laterally at least as far basally as the septate junctions, although there may be a partial exclusion from the region of the adherens junctions. Sep1 is found predominantly at the lateral and basal cells surfaces of salivary glands in partial overlap with the pattern of actin.

In ovaries, Sep1 staining is weak or undetectable at the nurse-cell plasma membranes and the ring canals but is evident in the cytoplasm of these cells. In addition, although Sep1 is clearly associated with the plasma membranes of the follicle cells, it seems to be more concentrated at their baso-lateral surfaces. Some Sep1 may also be present in the cytoplasm of the follicle cells. This pattern of Sep1 staining of follicle cells is seen in all egg chambers of the ovariole, including in the stalk cells that connect neighboring egg chambers. In border cells, a subset of follicle cells, Sep1, like actin, appears to be concentrated at the plasma membranes and may also be present in the cytoplasm (Fares, 1995). Note: Copy below to spectrin

Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells. Ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. In males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids Hime, 1996).

Effects of Mutation or Deletion

peanut was discovered in a search for factors that interact with seven in absentia, a gene required for the induction of neural fate in the presumptive R7 cells. peanut behaves as a dominant enhancer of sina. Homozygous peanut mutants do not survive to adulthood but instead die shortly after pupation. peanut mutants have severely reduced or no imaginal discs. Such disc-less, pupal-lethal phenotypes often reflect an underlying defect in mitosis. In mitotic mutants, early divisions during embryogenesis are presumably supported by maternally supplied gene products, but later proliferation of imaginal tissues is dependent on the mutant zygotic genome. peanut mutant brains contain a large number of polyploid and multinucleate cells. Such cells have multipolar mitotic spindles and extra centrosomes (Neufeld, 1994).

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, attempts were made to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. Two methods were used, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut+ transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method complete early syncytial development and begin cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows becomes progressively more abnormal, and the embryos display widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins has shown that Sep1 is not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 is still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function; that there is functional differentiation among the Drosophila septins, or both (Adam, 2000).

Maternally contributed Pnut appears to be essential for embryogenesis, and a zygotic copy of the gene cannot rescue the lethality due to depletion of the maternal supply. As a first step in evaluating embryonic morphogenesis in the Pnut-depleted embryos, the cuticles produced by mutant embryos were examined prior to their death. The cuticle is an exoskeleton deposited by epidermal cells of embryos shortly prior to hatching; its structure reflects the differentiated identities of the underlying epidermal cells. Wild-type cuticles typically fill the eggshell, possess distinguishable head and tail structures, and have discrete bands of denticle-containing and denticle-free cuticle (corresponding to the larval segments) along the ventral surface. pnut germline-clone embryos display a characteristic but variable cuticle phenotype. The cuticles are always much smaller than those from wild type and do not usually fill the eggshell. This suggests that the Pnut-deficient embryos develop or maintain fewer epidermal cells than do normal embryos. The head and tail structures also are severely defective. In addition, although some denticles could be identified and were usually grouped into clusters that might be called bands, these bands vary considerably in number (from 0 to 5, with 3 about average), shape, size, and position. Thus, the pnut germline-clone embryos experience major (albeit somewhat variable) defects in morphogenesis (Adam, 2000).

Because Pnut and other septins are concentrated at the leading edge of the furrow during cellularization, the effect of Pnut depletion on this process was examined. Similar results were obtained with germline-clone embryos and with embryos derived from HS-pnut+ females. To look for possible global defects, the development of living Pnut-depleted embryos was examined. Events prior to gastrulation usually appear to occur normally: a layer of cleared cytoplasm like that in wild-type embryos forms at the cortex of mutant embryos, indicating that nuclei arrive and are maintained at the cortex, and cellularization begins and proceeds evenly over the entire embryo at a normal speed and to a normal depth, although the cellularization front typically appears somewhat less well defined than in wild-type embryos. Global defects are generally not evident until gastrulation onset, when characteristic morphogenetic features associated with gastrulation are observed to be abnormal; for example, an ectopic fold is often seen anterior to the cephalic furrow. In addition, the posterior midgut plate often migrates to the embryo's left or right instead of dorsally. The apparently successful completion of cellularization suggests either that Pnut is dispensable for this process or that it is required for some aspect(s) of furrow structure or function that is not visible in living embryos, where only the overall progress of the leading edge can be observed. In the latter case, the defects in gastrulation might be (at least in part) consequences of the cellularization defect(s) (Adam, 2000).

To investigate these possibilities, Pnut-depleted embryos were stained at different stages for F-actin. Actin organization during events prior to cellularization, such as the syncytial blastoderm divisions and formation of the pole cells (the future germ cells), is approximately normal. In addition, the embryos are able to organize a normal-looking actin cytoskeleton at the beginning of cellularization and retain normal-looking actin organization through the slow phase of this process. However, during the subsequent fast phase, although most embryos appear grossly normal, close examination of the leading edge of the furrow reveals defects in the organization of F-actin at the bases of the forming cells. At this stage in wild-type embryos, actin is concentrated in a ring surrounding the base of each cell, with smaller amounts present elsewhere in the plane of the leading edge. In contrast, in Pnut-deficient embryos, the actin does not resolve into discrete rings and is more uniformly distributed in the plane of the leading edge, except for the presence of concentrated "bars" of actin between some pairs of cell bases. About 10% of the germline-clone embryos exhibited regions where the bases of cells were unevenly closed. In the abnormal regions of such embryos, there is an associated disruption of the actin cytoskeleton along the lateral surfaces of the forming cells and occasional abnormal displacement of nuclei from the cortex. Later, in the fast phase, at the stage when a wild-type embryo has actin organized in a discrete ring around the yolk plug at each cell base, the abnormality of actin organization around the bases of Pnut-deficient cells is more consistent and more conspicuous, with the actin displaying a punctate distribution throughout the plane of the leading edge and actin bars are no longer visible between pairs of cell bases. In summary, it appears that the cellularization machinery is initially assembled normally but does not maintain its organization as cellularization proceeds (Adams, 2000).

The synthesis of dorsal eggshell structures in Drosophila requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. Advantage of this process was taken to identify genes that link patterning and morphogenesis; lethal mutations on the second chromosome were screened for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. These genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61ß, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway: Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a 'neurospecific' receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype (Schnorr, 2001).

The current evidence concerning the function of Peanut and Smt3 links these proteins to signaling and the cytoskeleton. peanut (pnut) is one of five Drosophila septin genes and was originally identified as a genetic enhancer of a weak allele of seven in absentia (sina). pnut is required for cytokinesis and is localized to the cleavage furrow of dividing cells. How does heterozygous Pnut enhance a weak Ras1 phenotype? Both Pnut and Ras1 have been implicated in cell division and a reduction in these proteins may inhibit cell division early in oogenesis resulting in later effects on egg morphogenesis. This possibility seems remote, however, since no obvious defects are observed in the follicular layer (Schnorr, 2001).

Alternatively, septins may be critical for the organization of the cytoskeleton and/or the localization of signal transduction components in the follicle cells. Septins are found in the cytoplasmic bridges during spermatogenesis, but their distribution in oogenesis differs. In nurse cells, septins are not part of the ring canals or intracellular bridges but are found in the cytoplasm, while in follicle cells, septins are specifically localized to baso-lateral surfaces. Septins also localize to areas of cortical reorganization and thus may be important for follicle cell shape changes and migrations. In yeast, septins function to localize signaling molecules to the future bud site. Thus, peanut may be critical for the correct localization, anchoring, and stability of the Ras1 signal transduction machinery in the follicle cells (Schnorr, 2001).

Together, these results suggest that effective Ras1 signaling during eggshell morphogenesis depends on molecules that control the dynamic cytoskeleton. As described above, molecules such as Chic, Tec29, Pnut, Smt3, and Dock are involved in cytoskeletal reorganization. The Ras1 pathway may require a properly assembled cytoskeletal scaffold to achieve adequate signaling levels to correctly pattern the egg. Alternatively, the Ras1 signal may induce reorganization of the follicle cell cytoskeleton during the later cell migrations and subsequent secretion of the eggshell. Such large-scale reorganization may depend on a number of these Ras1 Enhancers (Schnorr, 2001).


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peanut: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 October 2013

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