Immunofluorescent analysis with this antibody reveals that, during the late syncytial divisions, Nuf is highly concentrated at the centrosomes during prophase and is cytoplasmic during the other stages of the nuclear cycle. Nuf is also concentrated at the centrosomes during interphase of nuclear cycle 14 and throughout cellularization. Double stains of wild-type embryos with Nuf and tubulin verify the centrosomal localization of Nuf. The Nuf antibody does not stain centrosomes in equivalently staged nuf-derived embryos. To show that this lack of staining was not due to an inability of the antibodies to access the centrosomes in the nuf-derived embryos, normal and nuf-derived embryos were stained with anti-centrosomin, a Drosophila centrosomal antibody. Both normal and nuf-derived embryos reacted strongly with this antibody. This demonstrates that nuf embryos contain centrosomes. Wild-type embryos were double stained with phalloidin and anti-Nuf antibodies to correlate actin dynamics with Nuf centrosomal localization. This analysis revealed that the localization of Nuf to the centrosomes during prophase of the syncytial divisions occurs during the most dramatic reorganization of actin from caps to furrows. Nuf centrosome staining during cellularization occurs as the actin enters cellularization furrows and as the furrows extend (Rothwell, 1998).
Nuf concentrates at the centrosomes during prophase and diffusely localizes throughout the cytoplasm during the remainder of the cell cycle (Rothwell, 1998). To visualize the cell cycle dynamics of Nuf in real time, a GFP-Nuf transgenic line was constructed. The GFP-Nuf construct completely rescues nuf-induced maternal lethality. Live analysis of a GFP-Nuf–expressing embryo indicates that during interphase, Nuf accumulates at each of the separating centrosomes. During prophase, immediately before nuclear envelope breakdown, Nuf accumulation peaks, concentrating around the base of the astral microtubules radiating away from the centrosomes. Nuf is absent on the side of the centrosome adjacent to the nuclear envelope. Significantly, maximal Nuf localization at prophase corresponds to the time of metaphase furrow invagination. Nuf localization is correlated with areas of high astral microtubule density. This is in accord with the finding that Nuf pericentriolar localization requires intact microtubules. Although the pericentriolar concentration of Nuf significantly diminishes during metaphase and anaphase, a small fraction of Nuf remains tightly associated with the centrosomes. During telophase, immediately after nuclear envelope reformation, Nuf begins accumulating at the newly duplicated centrosome pair. Low magnification images dramatically highlight the cell cycle regulation of Nuf subcellular localization. It is not known if Nuf is maintained in constant levels throughout the nuclear cycle and is simply cycling from the cytoplasm to the centrosomes, or if Nuf levels change throughout the cell cycle. The localization of Nuf during cellularization at nuclear cycle 14 differs significantly from its localization during the syncytial divisions. During the syncytial divisions, Nuf is present in lower levels at the centrosome during interphase, reaches its maximal concentration, and is highly dynamic during prophase. In contrast, during cellularization, Nuf reaches its maximal concentration during interphase and is relatively motionless, forming few flares and puncta. During the syncytial divisions, Nuf concentrates only in the region of the centrosome facing away from the nuclear envelope. However, during cellularization, Nuf is more evenly distributed around the centrosome, forming an intact ring. The differences between Nuf behavior during the syncytial divisions and cellularization may be a consequence of the more stable microtubule arrays that form during the prolonged interphase of nuclear cycle 14 (Riggs, 2003).
Nuf is extremely dynamic at the centrosome during prophase. Detailed imaging reveals that Nuf forms dynamic puncta and flares that rapidly migrate from the centrosomes. The puncta form, travel a short distance from the centrosome, then disappear. As described below, Nuf is associated with the recycling endosomal compartment. Therefore, this movement may reflect endosomal dynamics (Riggs, 2003).
The mammalian homolog of Nuf, Arfo2, physically associates and colocalizes with Rab11, a key component of the recycling endosome (Hickson, 2003). Rab11 is required for the integrity of the recycling endosome, and is believed to mediate transport of vesicles from the RE to the TGN, early endosome, and plasma membrane via a 'slow' recycling route (Ullrich, 1996; Ren, 1998). The pattern of Rab-protein 11 localization in the developing Drosophila oocyte has been characterized (Dollar 2002). Rab11 localizes at the posterior pole and is necessary for proper microtubule organization and Oskar mRNA localization. The pattern of Rab11 localization have been examined during the cortical divisions in the early Drosophila embryo. Rab11 exhibits a diffuse punctate localization that concentrates around the nuclei. As the embryos progress into prophase, Rab11 maintains its punctate morphology, but exhibits significantly increased concentration at the centrosomes. During metaphase, the centrosomal concentration of Rab11 decreases and there is a concomitant dispersal of Rab11 throughout the cytoplasm encompassing each chromosome-spindle complex. This trend continues as the nuclei enter anaphase. Even though the nuclear envelope is substantially broken down during metaphase and anaphase, Rab11 does not enter the interior nuclear space. During telophase, Rab11 puncta concentrate around the newly formed nuclear envelope. There is a slight increase in the concentration of Rab11 puncta at the centrosomes. Cellularization occurs during the prolonged interphase of nuclear cycle 14. At this time, Rab11 is highly concentrated around the pair of apically located sister centrosomes (Riggs, 2003).
The pericentriolar concentration of Rab11 in Drosophila embryos is equivalent to Rab11 localization observed in mammalian cells. In CHO cells, Rab11 is primarily localized to a discrete pericentriolar region with a lower concentration of puncta distributed throughout the cell (Ullrich, 1996). Colocalization experiments with internalized transferrin have indicated that Rab11 localizes to the pericentriolar RE (Ullrich, 1996; Sheff, 2002). GFP-Rab11 also exhibits a pericentriolar localization and colocalizes with the transferrin receptor (Sonnichsen, 2000). Given the equivalent staining patterns in Drosophila, it is concluded that Rab11 also localizes to the RE in syncytial and cellularized Drosophila embryos (Riggs, 2003).
Immunofluorescent analyses using anti-Nuf and anti-Rab11 antibodies reveal that during prophase, when both antigens are highly concentrated in the pericentriolar region, areas of maximal Nuf localization correspond to areas of maximal Rab11 localization. Almost without exception, Nuf colocalizes with Rab11. However, the converse is not true, and in regions more distal from the centrosome, Rab11, but not Nuf, is present. During cellularization at interphase of nuclear cycle 14, Nuf and Rab11 exhibit high pericentriolar concentrations and extensive colocalization. As observed for prophase of the cortical divisions, Nuf always colocalizes with Rab11, but there are regions of Rab11 localization in which Nuf is not present. Given that Rab11 is an excellent marker of the RE, these results support the notion that Nuf localizes to the RE during cortical syncytial divisions and during cellularization at interphase of nuclear cycle 14 (Riggs, 2003).
The nuf maternal-effect mutation specifically disrupts syncytial nuclear divisions only after the nuclei migrate to the cortex (Sullivan, 1993). These nuclear defects are a consequence of incomplete metaphase furrow formation, which allows inappropriate fusions between nonsister nuclei (Rothwell, 1998). Although the interphase actin caps form normally, large gaps are present in the metaphase and cellularization furrows. The gaps are observed in the earliest stages of furrow formation, suggesting that Nuf disrupts recruitment of actin to the furrows rather than in stabilization of actin once at the furrows. To determine if reduced maternal supplies of Rab11 produce cortical phenotypes similar to those observed in nuf mutations, rab11 transheterozygotes were used. The nuclear phenotype is equivalent to nuf. In rab11-derived embryos, nuclear distribution and morphology is normal in premigration and early cortical blastoderm embryos. However, during the late cortical divisions when the nuclei are more densely packed, the nuclear distribution and morphology is disrupted. In premigration and early cortical embryos, 8% (2/23) exhibit disrupted nuclear morphology. During the late cortical divisions, 65% (31/48) exhibit severely disrupted nuclear morphology. This is indicative of defects in the metaphase furrows that serve to separate neighboring nonsister nuclei (Riggs, 2003).
To examine the role of Rab11 in organizing the cortical cytoskeleton and metaphase furrows, wild-type, nuf-derived, and rab11-derived cortical nuclear cycle 12 embryos were double stained for DNA and actin. During interphase, actin organizes into caps apically positioned above each nucleus. In nuf- and rab11-derived embryos, actin cap formation occurs normally. As the embryos progress into prophase, the actin caps are dismantled and actin reorganizes into furrows encompassing each prophase nucleus and its developing spindle. As the nuclei progress into metaphase, these furrows become more pronounced and tightly focused. The actin-based furrow defects in rab11-derived embryos are strikingly similar to those observed in nuf-derived embryos. In both, the hexagonal furrow network is riddled with gaps. The gaps are present at prophase during the initial stages of furrow formation, suggesting defects in the initial actin recruitment. nuf and rab11 mutations also produce similar defects during cellularization at nuclear cycle 14, although defects in nuf-derived embryos are much more extensive than observed in rab11-derived embryos. This difference may be a result of partial zygotic rescue by the paternally supplied rab+ allele (Riggs, 2003).
nuf-derived embryos disrupt recruitment of membrane components during furrow invagination. The Drosophila protein Discontinuous actin hexagon (Dah) tightly associates with the plasma membrane as well as actin, and is thought to link cortical microfilaments to the plasma membrane (Zhang, 1996). In cortical Drosophila embryos, Dah localizes to the plasma membrane as well as to vesicles that concentrate at the leading edge of the invaginating furrows. Analysis of Dah mutations indicates that incorporation of these vesicles into the plasma membrane contributes to furrow invagination (Rothwell, 1999). To determine the role of Rab11 and Nuf in Dah-associated vesicle delivery, wild-type, nuf-derived, and rab11-derived embryos were double stained for actin and Dah. In nuf-derived embryos, incorporation of Dah into the metaphase furrows is dramatically reduced. Although Dah vesicles are observed, they are more randomly distributed throughout the cytoplasm. A similar defect is observed in rab11-derived embryos; incorporation of Dah into the invaginating metaphase furrows is disrupted. However, in contrast to nuf, Dah staining is not observed in the furrow regions and few Dah-staining vesicles are visible (Riggs, 2003).
nuclear fallout (nuf) is a maternal effect mutation that specifically disrupts the cortical syncytial divisions during Drosophila embryogenesis. The nuf gene encodes a highly phosphorylated novel protein of 502 amino acids with C-terminal regions predicted to form coiled-coils. During prophase of the late syncytial divisions, Nuf concentrates at the centrosomes and is generally cytoplasmic throughout the rest of the nuclear cycle. In nuf-derived embryos, the recruitment of actin from caps to furrows during prophase is disrupted. This results in incomplete metaphase furrows specifically in regions distant from the centrosomes. The nuf mutation does not disrupt Anillin or Peanut recruitment to the metaphase furrows, indicating that Nuf is not involved in the signaling of metaphase furrow formation. These results also suggest that Anillin and Peanut localization are independent of actin localization to the metaphase furrows. nuf also disrupts the initial stages of cellularization and produces disruptions in cellularization furrows similar to those observed in the metaphase furrows. The localization of Nuf to centrosomal regions throughout cellularization suggests that it plays a similar role in the initial formation of both metaphase and cellularization furrows. A model is presented in which Nuf provides a functional link between centrosomes and microfilaments (Rothwell, 1998).
Nuf is a member of a growing class of proteins that concentrate at the centrosome in a cell-cycle-specific manner. Immunofluorescent analysis demonstrates that Nuf is diffusely localized throughout the cortical cytoplasm from early metaphase through interphase. During prophase, Nuf concentrates at the centrosomes. Other Drosophila centrosomal proteins that exhibit a cell-cycle specific centrosomal localization include CP60 and CP190. These proteins localize to the nuclei during interphase and the centrosome at mitosis and are members of a larger protein complex. Separable domains within these proteins have been identified that are responsible for their nuclear and cytoplasmic localization (Rothwell, 1998 and references therein).
Given the difference in the cell cycle timing of the centrosomal localization between Nuf and the CP60/190 complex, it is likely that they are involved in different cellular functions. The recently characterized Drosophila Abnormal spindle protein localizes to regions near the centrosome during prophase through anaphase and concentrates at the midbody during telophase. Mutations in Asp disrupt microtubule organization. As with many proteins that localize to the centrosome, Asp and Nuf both include C-terminal regions with a high probability of forming coiled-coils (Rothwell, 1998).
In mutations that lack Centrosomin (Cnn), a protein that localizes to the centrosome throughout the Drosophila syncytial nuclear cycles, Nuf no longer localizes to the centrosome. Conversely, Cnn localization is not disrupted in nuf-derived embryos. These results suggest that Nuf is not a core component of the centrosome. In nuf-derived embryos, prior to migration, the nuclear divisions are normal. This phenotype is also in accord with the idea that Nuf is not a core component of the centrosome (Rothwell, 1998).
In nuf-derived embryos, there are no obvious defects in the interphase actin caps. During metaphase, however, the furrows are incomplete in regions near the metaphase plate. Through both live and fixed analysis, this defect is observed during the earliest stages of furrow formation at prophase. This suggests a defect in the recruitment of actin to these regions of the furrow rather than the stabilization of actin already present in these furrow regions. It is not known whether or not Nuf interacts directly with actin. Insight into the mechanism of Nuf action will require identifying interacting proteins and other components involved in the process. A potential interactor is Dah, a Drosophila protein with some homology to dystrophin. dah-derived embryos have syncytial and cellularization phenotypes similar to nuf, indicating that they may be involved in a common pathway (Rothwell, 1998).
Nuf is concentrated at the centrosomes during prophase and is cytoplasmic throughout the rest of the cell cycle. It is not known when, in the division cycle, Nuf is required for actin recruitment to the furrows. For example, cytoplasmic Nuf may regulate actin dynamics and the localization of Nuf to the centrosome may serve to sequester Nuf in an inactive state. Alternatively, Nuf may influence actin dynamics while it is localized to the centrosomes. Nuf localizes to the centrosomes during prophase, specifically when the most extensive reorganization of actin from caps to furrows is occurring. In addition, Nuf is concentrated at the centrosomes from early interphase of nuclear cycle 14 through to the completion of cellularization. nuf-derived embryos produce a dramatic cellularization phenotype with extensive gaps in the furrows and it is likely that, as found for the metaphase furrows, this is a result of failed actin recruitment. These results suggest that, at least during cellularization, the centrosomal localization of Nuf is important for its effect on actin localization (Rothwell, 1998).
The prophase centrosomal localization of Nuf coincides with the timing of actin reorganization and supports a model in which Nuf is acting at the centrosomes to organize cortical actin. Nuf may influence microfilament organization by organizing microtubules. Although previous analysis failed to reveal disruption in microtubule organization in the nuf mutant, it remains possible that subtle microtubule defects have been overlooked. Alternatively, Nuf may act more directly to influence microfilament organization. For example, centrosomal Nuf may facilitate the cap-to-furrow transition by stimulating severing or depolymerization of the microfilaments. Another possibility is that the cap-to-furrow transition involves loading and transport of components onto and along the microtubules and Nuf may be involved in this process (Rothwell, 1998).
Although nuf1 only partially disrupts the formation of metaphase furrows, molecular analysis indicates that it is a null allele. One explanation for the fact that the nuf1 phenotype is not more severe is that Nuf may be a member of a protein complex that is involved in the recruitment of actin to the metaphase furrows. The absence of Nuf compromises, but does not eliminate, function in this complex. During cellularization, the furrow defect is much more severe in nuf1-derived embryos and extensive regions of the embryo lack cellularization furrows. It may be that, during cellularization, greater demands are placed on the functioning of this complex. Alternatively, the partially disrupted metaphase furrows of nuf1-derived embryos may reflect the fact that this process involves redundant mechanisms. Null alleles of another maternal effect mutation, dah, also produce slight defects in the metaphase furrows and severe cellularization defects. In addition to actin, the effect was examined of the nuf mutation on Anillin (Drosophila name: Scrapes) and Peanut localization. Anillin, Peanut and many other proteins are common to both metaphase and more conventional cleavage furrows. Therefore, many of the lessons learned in one system will likely apply to the other. During the initial stages of furrow formation in nuf-derived embryos, Anillin and Peanut localize to furrow regions in which actin fails to localize. These results demonstrate that, during the initial stages of furrow formation, proper localization of Anillin and Peanut to the furrow is independent of the proper localization of actin to the furrow. These results also suggest that Nuf functions in a pathway that is downstream or independent of anillin and peanut localization. Because these initial events of furrow formation occur normally in nuf-derived embryos, the signals for positioning and timing of furrow formation are probably intact. Although during prophase the localization of Anillin and Peanut is independent of actin localization, as the embryos progress into metaphase, Anillin and Peanut fail to maintain their localization in regions of the furrows in which actin fails to localize (Rothwell, 1998). It is likely that one of the furrow components that fail to be recruited in nuf-derived embryos, possibly actin, is required for stabilization of the furrow during the prophase to metaphase transition (Rothwell, 1998).
nuf-derived embryos produce a dramatic cellularization phenotype in which the gaps in the furrows are so extensive that the furrows usually encompass multiple nuclei. It is likely that the origin of this phenotype is equivalent to that of the metaphase furrows. This phenotype is strikingly similar to that observed for the zygotic mutations nullo and serendipity alpha. Analysis of these genes demonstrates that they encode novel proteins that localize to the invaginating cellularization furrow. The nullo mutation disrupts the localization of Serendipity, but the serendipity mutation does not disrupt the localization of Nullo. This indicates that Serendipity functions downstream of Nullo. Since the cellularization furrows initiate normally in the nullo mutation, the Nullo protein apparently is not required for the initial stages of cellularization but is required for the stabilization of the growing cellularization furrow. Serendipity is also probably not required for the initial stages of cellularization, since it functions downstream of Nullo. In contrast, nuf disrupts the initial formation of the cellularization furrows. This indicates that the maternally supplied Nuf acts upstream to the zygotic genes nullo and serendipity in initiating and establishing the cellularization furrow (Rothwell, 1998).
During mitosis of the Drosophila cortical syncytial divisions, actin-based membrane furrows separate adjacent spindles. Genetic analysis indicates that the centrosomal protein Nuf is specifically required for recruitment of components to the furrows and the membrane-associated protein Dah is primarily required for the inward invagination of the furrow membrane. Recruitment of actin, Anillin and Peanut to the furrows occurs normally in dah-derived embryos. However, subsequent invagination of the furrows fails in dah-derived embryos and the septins become dispersed throughout the cytoplasm. This indicates that stable septin localization requires Dah-mediated furrow invagination. Close examination of actin and Dah localization in wild-type embryos reveals that they associate in adjacent particles during interphase and co-localize in the invaginating furrows during prophase and metaphase. The Nuf centrosomal protein is required for recruiting the membrane-associated protein Dah to the furrows. In nuf-mutant embryos, much of the Dah does not reach the furrows and remains in a punctate distribution. This suggests that Dah is recruited to the furrows in vesicles and that the recruiting step is disrupted in nuf mutants. These studies lead to a model in which the centrosomes play an important role in the transport of membrane-associated proteins and other components to the developing furrows (Rothwell, 1999).
nuf and dah, identify distinct steps in the process of metaphase furrow formation in syncytial blastoderm embryos. During the initial stage, actin and other components are recruited to the furrow regions. Following this, furrow invagination occurs. Nuf is specifically involved in recruiting components to the furrow while Dah is required for furrow invagination. In nuf-mutants, actin recruitment to regions distant from the centrosomes often fails. This results in the inability of furrows to form in these regions (Rothwell, 1998). Alternatively, the partial formation of furrows in the nuf null mutation could be due to incomplete activity of a Nuf-containing multi-protein complex. Furrow regions closest to the centrosomes, in which actin and the other furrow components are properly recruited, undergo normal invagination. This indicates that Nuf is specifically required for recruitment of furrow components and is not involved in the subsequent stages of furrow formation. In contrast, recruitment of furrow components occurs normally in dah-derived embryos but furrow extension fails. Thus, Dah functions primarily in the invagination process. It is likely that other cortical components will fall into one of these classes or play roles in other distinct steps in furrow formation. For example, the unconventional myosin, 95F-myosin, appears to play a role in the invagination process; injection of antibodies directed against 95F myosin results in metaphase furrows that do not invaginate as deeply as those in normal embryos (Rothwell, 1999).
During cytokinesis, the cleavage furrow contracts through an actomyosin based mechanism. Actomyosin based contraction may also play a role in formation of the metaphase and cellularization furrows of the early Drosophila embryo. Myosin II is present at the tips of both the invaginating metaphase and cellularization furrows indicating that invagination may involve contractile processes. Myosin II localizes to metaphase furrows specifically during membrane invagination, leaving the furrow tips once they are fully formed. Injection of antimyosin II antibodies into syncytial embryos disrupts cortical nuclear organization and inhibits subsequent cellularization (Rothwell, 1999).
Several lines of evidence indicate that formation of cellularization furrows also requires membrane addition. The plasma membrane above each nucleus contains microvilli-like projections that increase in number during the initial, slow phase of cellularization and disappear in the later fast phase. Therefore, the early phase of cellularization may involve membrane recruitment at the cell surface for microvilli formation while the fast phase utilizes the excess membrane for invagination. However, calculations indicate that the membrane supplied by these microvilli-like projections is not sufficient to complete cellularization. Therefore, other mechanisms of membrane addition may be involved in the cellularization process. In support of this, coated pits and multilamellar bodies decorate the furrows. Some EM studies describe cellularization as a process involving vesicle alignment at the future furrow site followed by their fusion to form double membranes. It is suggested that that the slow and fast phases utilize different vesicle populations. Zygotic mutations that specifically effect the slow or fast phases support the idea that they occur through different mechanisms (Rothwell, 1999).
Genetic studies also support the idea that cellularization requires significant membrane addition. Syntaxins are a family of membrane proteins that are thought to provide specificity for targeting of vesicles to specific membrane compartments. Germline clones of Drosophila syntaxin produce extensive defects in cellularization. The Drosophila temperature-sensitive mutation shibire disrupts the gene encoding Dynamin, a protein required for endocytosis. In addition to neuronal defects, shibire also disrupts cellularization. At the restrictive temperature, cellularization furrows do not form and vesicles accumulate in the cytoplasm. These data suggest that processes related to endocytosis are required for cellularization (Rothwell, 1999).
Evidence is provided that metaphase furrows, a process very similar to cellularization, may also form through significant addition of membrane. Dah is a furrow component required for metaphase furrow formation and cellularization (Zhang, 1996). Diochemical analysis demonstrates that Dah is membrane-associated protein (Zhang, 2000). In the absence of Dah, although recruitment of furrow components occurs normally, furrow invagination fails. In addition, Dah localizes to membrane containing particles that often concentrate at the leading edge of the furrows. These results suggest that invagination of the metaphase furrows occurs through membrane addition and that Dah is a membrane-associated protein required for this process (Rothwell, 1999).
95F-myosin (Jaguar) has also been hypothesized to play a role in recruiting particles necessary for furrow formation. The phenotype of embryos injected with antibodies against 95F-myosin is extremely similar to that of dah-mutant embryos and the protein products of these genes share similar localization patterns. Therefore, 95F-myosin and Dah may act together in the process of furrow extension. An attractive hypothesis is that 95F-myosin delivers vesicles containing Dah and other necessary furrow components to the furrow region (Rothwell, 1999).
nuf-derived embryos form incomplete furrows (Sullivan, 1993; Rothwell, 1998). To determine the role of contraction in metaphase furrow formation, invagination of the free ends of these incomplete furrows was examined; the free ends of metaphase furrows are stable and invaginate to the same extent as intact normal furrows. This result would not be expected if long range actomyosin-based contraction played a major role in furrow formation. This result is consistent with alternative mechanisms based on membrane addition. One potential mechanism is vesicle fusion (Rothwell, 1999).
The centrosome plays a key role in the formation of the actin caps and the metaphase furrows in the early Drosophila embryo. The centrosomal protein, Nuf, has been shown to be required for proper actin recruitment to the furrows (Rothwell, 1998). This study demonstrates that Nuf is also involved in recruiting membrane to the furrows. In wild-type embryos, membrane-bearing particles containing the Dah protein are recruited to the site of furrow formation as the furrows initiate formation during prophase. In similarly staged nuf-mutant embryos, many of these membrane containing particles are not properly recruited and remain in a perinuclear punctate distribution. Close examination of Dah and actin localization in wild-type embryos reveals that they localize to adjacent particles that lie between the nuclei and concentrate ahead of the furrow tips. At metaphase, the majority of Dah and actin co-localize in the furrows. Since Dah is not required for proper actin recruitment to the furrows, it is likely that Dah itself does not play a role in the transport mechanism. A model consistent with these observations is that Nuf acts at the centrosome to initiate the transport of vesicle-associated components along the microtubules to the furrow regions. This model accounts for the observation that furrow formation fails primarily in those regions most distant from the centrosomes in which the greatest demand would be placed on the transport process (Rothwell, 1998). Similar models have been proposed for membrane transport along the cytoskeleton in other systems. Long range transport of vesicles is thought to utilize kinesins and occur along microtubules; once at the cell periphery, the vesicles are transferred to actin filaments and actin-based motors carry them to their final destinations at the plasma membrane. The recent finding that a microtubule-based motor (conventional kinesin) and an actin motor (myosin V) directly interact is providing insight into the mechanism whereby the same vesicle can move along different cytoskeletal tracks. Transport of vesicles necessary for furrow formation in the early Drosophila embryo may also occur through coordinated microtubule and actin-based transport systems. In support of this model, drug studies show that disruption of microtubules by injection of colchicine into Drosophila embryos halts transport of particles to the cellularization furrows resulting in inhibition of membrane invagination. In addition, injection of antibodies against 95F-myosin disrupts invagination of the metaphase furrows that form during syncytial development (Rothwell, 1999).
Anillin and Peanut (a Drosophila septin) are conserved components of the metaphase and cytokinesis furrows and show a similar pattern of localization, both localizing early during the process of furrow formation. Mutational analysis of nuf derived embryos indicates that recruitment of these proteins to the developing furrows occurs independently of actin. Once the furrows are formed at metaphase, however, peanut and anillin are not maintained in regions where actin is not localized and furrow formation has failed (Rothwell, 1998). These results indicate that stable furrow localization of these proteins either requires actin or normally invaginated membranes (Rothwell, 1999).
Analysis of dah-derived embryos in which actin localization occurs normally but membrane invagination fails helps distinguish between these alternatives. In dah-derived embryos, prophase occurs relatively normally but membrane invagination during metaphase fails. Actin, Peanut and anillin localize normally during prophase in dah-derived embryos. However, during metaphase, Peanut localization specifically fails while actin and Anillin remain localized in the un-invaginated furrows. One interpretation of these results is that the septins require intact plasma membrane for stable localization. This interpretation is supported by co-localization and biochemical studies indicating that septins are intimately associated with membrane. Anillin, on the other hand, has a more diverse localization pattern in that it cycles between the nucleus and the cortex (Rothwell, 1999).
Reference names in red indicate recommended papers.
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date revised: 25 November 2008
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