peanut
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).
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 (www.sbg.bio.ic.ac.uk/~3dpssm). 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).
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).
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).
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).
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).
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).
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).
Adams, R. R., et al. (1998). pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 12(10): 1483-1494.
Adam, J. C., Pringle, J. R. and Peifer, M. (2000). Evidence for functional differentiation among Drosophila septins in
cytokinesis and cellularization. Mol. Biol. Cell 11: 3123-3135.
Afshar, K., Stuart, B. and Wasserman, S. A. (2000). Functional analysis of the Drosophila Diaphanous FH protein in early embryonic
development. Development 127(9): 1887-1897
Amberg, D. C., et al. (1997). Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites. Mol. Biol. Cell 8(4): 729-753.
Barral, Y., et al. (1999). Nim1-related kinases coordinate cell cycle progression with the
organization of the peripheral cytoskeleton in yeast. Genes Dev. 13(2): 176-187.
Beites, C. L., et al. (1999). The septin CDCrel-1 binds syntaxin and inhibits exocytosis. Nat. Neurosci. 2(5): 434-9. 10321247
Carroll, C. W., et al. (1998). The septins are required for the mitosis-specific activation of the gin4 kinase. J. Cell Biol. 143(3): 709-17.
Castillon, G. A., et al. (2003). Septins have a dual role in controlling mitotic exit in budding yeast. Curr. Biol. 13: 654-658. 12699621
Chant, J. (1996). Septin scaffolds and cleavage planes in Saccharomyces. Cell 84(2): 187-190
Chesnokov, I. N., Chesnokova, O. N. and Botchan, M. (2003). A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci. 16: 9150-9155. 12878722
Cvrckova, F., et al. (1995). Ste20-like protein kinases are required for normal localization of cell
growth and for cytokinesis in budding yeast. Genes Dev. 9(15): 1817-1830.
D'Avino, P. P., et al. (2008). Interaction between Anillin and RacGAP50C connects the actomyosin contractile ring with spindle microtubules at the cell division site.
J. Cell Sci. 121(Pt 8): 1151-8. PubMed citation: 18349071
De Virgilio, C., DeMarini, D. J. and Pringle, J. R. (1996). SPR28, a sixth member of the septin gene family in Saccharomyces cerevisiae that is expressed specifically in sporulating cells. Microbiology 142( Pt 10): 2897-2905.
Dobbelaere, J., et al. (2003). Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Developmental Cell 4: 345-357. 12636916
Echard, A., Hickson, G. R., Foley, E. and O'Farrell, P. H. (2004).
Terminal cytokinesis events uncovered after an RNAi screen.
Curr. Biol. 14(18): 1685-93. PubMed citation: 15380073
Fares, H., Peifer, M. and Pringle, J. R. (1995). Localization and possible functions of Drosophila septins. Mol. Biol. Cell 6(12): 1843-1859.
Fares, H., Goetsch, L. and Pringle, J. R. (1996). Identification of a developmentally regulated septin and involvement of the septins in spore formation in Saccharomyces cerevisiae. J. Cell Biol. 132(3): 399-411.
Field, C. M. and Alberts, B. M. (1995). Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J. Cell Biol. 131: 165-178. PubMed citation: 7559773
Field, C. M., et al. (1996). A purified Drosophila septin complex forms filaments and exhibits GTPase activity. J. Cell Biol. 133(3): 605-616.
Finger, F. P., Kopish, K. R. and White, J. G. (2003). A role for septins in cellular and axonal migration in C. elegans. Dev. Biol. 261: 220-234. 12941631
Giot, L. and Konopka, J. B. (1997). Functional analysis of the interaction between Afr1p and the Cdc12p septin, two proteins involved in pheromone-induced morphogenesis. Mol. Biol. Cell 8(6): 987-998.
Halme, A., et al. (1996). Bud10p directs axial cell polarization in budding yeast and resembles a transmembrane receptor. Curr. Biol. 6(5): 570-579.
Hickson, G. R. and O'Farrell, P. H. (2008). Rho-dependent control of anillin behavior during cytokinesis. J. Cell Biol. 180(2): 285-94. PubMed citation: 18209105
Hime, G. R., Brill, J. A. and Fuller, M. T. (1996). Assembly of ring canals in the male germ line from structural components of the
contractile ring. J. Cell Sci. 109( Pt 12): 2779-2788.
Hsu, S. C., et al. (1998). Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 20(6): 1111-22. 9655500
Kinoshita, M., et al. (1997). Nedd5, a mammalian septin, is a novel cytoskeletal component interacting with actin-based structures. Genes Dev. 11(12): 1535-1547.
Kinoshita, M., Field, C. M., Coughlin, M. L., Straight, A. F. and Mitchison, T. J. (2002). Self- and actin-templated assembly of mammalian septins. Dev. Cell 3: 791-802. PubMed citation: 12479805
Kremer, B. E., Adang, L. A. and Macara, I. G. (2007). Septins regulate actin organization and cell-cycle arrest through nuclear accumulation of NCK mediated by SOCS7. Cell 130(5): 837-50. Medline abstract: 17803907
Lippincott, J. and Li, R. (1998). Sequential assembly of Myosin II, an IQGAP-like protein, and
filamentous actin to a ring structure involved in budding yeast
cytokinesis. J. Cell Biol. 140(2): 355-366.
McKie, J. M., et al. (1997). A human gene similar to Drosophila melanogaster peanut maps to the DiGeorge syndrome region of 22q11. Hum. Genet. 101(1): 6-12.
Mendoza, M., Hyman, A. A. and Glotzer, M. (2002). GTP binding induces filament assembly of a recombinant septin. Curr. Biol. 12: 1858-1863. 12419187
Momany, M. and Hamer, J. E. (1997). The Aspergillus nidulans Septin Encoding Gene, aspB, Is Essential for Growth. Fungal Genet. Biol. 21(1): 92-100.
Neufeld, T. P. and Rubin, G. M. (1994). The Drosophila peanut gene is required for cytokinesis and encodes
a protein similar to yeast putative bud neck filament proteins. Cell 77(3): 371-379.
Ozsarac, N., et al. (1995). The SPR3 gene encodes a sporulation-specific homologue of the
yeast CDC3/10/11/12 family of bud neck microfilaments and is
regulated by ABFI. Gene 164(1): 157-162.
Park, H.-O., et al. (1997). Two active states of the Ras-related Bud/Rsr1 protein bind to different effectors to determine yeast cell polarity. Proc. Natl. Acad. Sci 94: 4463-4468.
Schnorr, J. D., et al. (2001). Ras1 interacts with multiple new signaling and cytoskeletal loci in Drosophila eggshell patterning and morphogenesis. Genetics 159: 609-622. 11606538
Somma, M. P., Fasulo, B., Cenci, G., Cundari, E. and Gatti, M. (2002). Molecular dissection of cytokinesis by RNA interference in Drosophila cultured cells. Mol. Biol. Cell 13: 2448-2460. PubMed citation: 12134082
Straight, A. F., Cheung, A., Limouze, J., Chen, I., Westwood, N. J., Sellers, J. R. and Mitchison, T. J. (2003). Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299: 1743-1747. PubMed citation: 12637748
Straight, A. F., Field, C. M. and Mitchison, T. J. (2005). Anillin binds nonmuscle Myosin II and regulates the contractile ring. Mol. Biol. Cell 1: 193-201. PubMed citation; Online text
Tada, T., et al. (2007). Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons. Curr. Biol. 17(20): 1752-8. PubMed citation: 17935993
Takahashi, Y., Iwase, M., Konishi, M., Tanaka, M., Toh-e, A. and Kikuchi,
Y. (1999). Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component
of septin rings at the mother-bud neck in budding yeast. Biochem. Biophys.
Res. Commun. 259: 582-587. 10364461
Takahashi, Y., Toh-e, A. and Kikuchi, Y. (2001). A novel factor required for
the SUMO1/Smt3 conjugation of yeast septins. Gene 275: 223-231. 11587849
Xie, Y., et al. (2007). The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology. Curr. Biol. 17(20): 1746-51. PubMed citation: 17935997
Yang, S., Ayscough, K. R. and Drubin, D. G. (1997). A role for the actin cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection. J. Cell Biol. 136(1): 111-123.
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
peanut:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 15 April 2008
Society for Developmental Biology's Web server.