nullo: Biological Overview | References
Gene name - nullo
Cytological map position- 6F1-6F1
Function - cytoskeleton
Keywords - cellularization, junctions
Symbol - nullo
FlyBase ID: FBgn0004143
Genetic map position - X: 6,943,454..6,944,417 [+]
Classification - novel; myristoylprotein with a cluster of basic amino acids
Cellular location - membrane
During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila ß-catenin homolog Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. The basal accumulation of Arm colocalizes with E-cadherin and alpha-catenin, and corresponds to a region of tight membrane association, which is referred to as the basal junction. Although the two junctions (apicolateral and basal) are similar in components and function, they differ in their response to the novel cellularization protein Nullo. The nullo gene encodes a predicted protein of 213 amino acids, a large proportion of which is basic (Rose, 1992). nullo transcripts are first detectable at nuclear cell cycle 11, peak in accumulation at the end of cycle 13, and disappear rapidly as cellularization begins. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation (Hunter, 2000).
During cellularization, the syncytial blastoderm is converted to a monolayer of cells that display many of the features of a polarized epithelium, including distinct apical and basolateral surfaces that are separated by a belt-like ZA. The formation of the ZA has been well documented: it begins with an accumulation of Arm and E-cadherin to spot-like adhesive contacts in the newly formed apicolateral membrane. During late cellularization, these accumulations cluster apically to form the apical spot-junctions, which give rise to the ZA during gastrulation. In addition to its apical accumulation at the ZA, Arm protein is also localized to the leading edge of the cleavage furrows. This localization is observed at the onset of cellularization and is maintained as the membrane invaginates into the interior. When cellularization is completed, this early Arm accumulation is found at the basal-most region of the lateral membrane. It was of interest to enquire whether the localization of Arm to the basal tip of the cleavage furrow might indicate a novel requirement for adherens junctions during cellularization. As in a traditional adherens junction, the Arm protein at the cellularization front colocalizes with E-cadherin and alpha-catenin, and electron micrographs show that the membranes just above the furrow canal are more closely apposed than other regions of the lateral membranes. Although this region lacks the electron dense plaques of a mature ZA, it is similar in nature to the cell-cell contacts seen at the apical spot-junctions; this adhesive zone is therefore referred to as the basal junction (Hunter, 2000).
To position the basal junction relative to the furrow canal, the localization of Arm with respect to myosin in the cleavage furrow was examined. In cross section, embryos initiating cellularization show spot-like accumulations of Arm at the sites of somatic bud contact. This is similar to the accumulation of Arm seen in pseudo-cleavage furrows. The spots of Arm staining in the nascent cleavage furrows are spatially distinct from the early accumulation of myosin, suggesting the basal junction and furrow canal form as separate domains. Many embryos that have a clear accumulation of Arm, but lack detectable levels of myosin: this suggests that the formation of the basal junction might precede the completion of myosin localization to the cellularization front. As cleavage furrows extend, it is clear that myosin and Arm are present in nonoverlapping regions of the cellularization front. During late cellularization, the furrow canals widen and it becomes apparent that the basal Arm population marks the boundary between the existing lateral membrane and the expanding basal membrane. At this stage, embryos also begin to accumulate Arm at the apical junction. As the embryo initiates gastrulation, the basal accumulation of Arm, and other junctional proteins, is gradually lost, while the apical population continues to coalesce into the mature ZA (Hunter, 2000).
In summary, cleavage furrows of the Drosophila blastoderm form two distinct adherens junctions: a transient junction at the boundary of the basal and lateral membrane domains, and a permanent junction at the boundary of the presumptive apical and basolateral domains. Like the apical-spot junction, the basal junction contains coincident accumulations of Arm, E-cadherin, and alpha-catenin, and corresponds to a region of tight membrane apposition (Hunter, 2000).
The rapid rate of cellularization requires that the apical and basal junctions be formed in close spatial and temporal proximity, without coalescing to form a single junctional complex. The first indication of how this might be achieved came from observations of Arm protein distribution in nullo mutant embryos. In nullo mutant embryos, a subset of somatic bud contacts fails to accumulate actin and myosin, and no cleavage furrows form at these positions (Simpson, 1990). The distribution of myosin and Arm in the remaining furrows was examined, and it was found that myosin shows its typical localization to the furrow canal, but the early Arm protein is not restricted to the basal junction. Instead, Arm extends apically along the lateral membrane, suggesting that in the absence of Nullo protein, the basal Arm population moves toward the apical junction. Interestingly, the apical junction is not affected by the nullo mutation: during late cellularization the existing cleavage furrows form apical spot-junctions that coalesce to form ZAs. The observation that Nullo is required for basal, but not apical, junction formation is supported by the fact that Nullo protein is normally found at the basal tip of the cleavage furrow, and is degraded before apical junction formation (Hunter, 2000).
To pinpoint the onset of the basal junction defects, the distribution of Arm protein was examined in embryos initiating cellularization. Since wild-type and nullo mutant embryos are phenotypically identical at this stage, anti-HA-Nullo immunostaining was used to identify the embryos. During the first phase of cellularization, cleavage furrows form at the slight infoldings of membrane where adjacent somatic buds abut each other. Surface views of wild-type embryos during this stage show a diffuse hexagonal pattern of Arm that corresponds to the infoldings of plasma membrane. The Arm staining gradually resolves into sharp lines as basal junctions form, but this does not occur synchronously across the embryo. At early stages a given region contains both diffuse and sharp lines of Arm staining. This process is completed by the onset of cleavage furrow invagination, at which point all of the cleavage furrows have sharp lines of Arm accumulation at the level of the basal junction. As in apical junctions, this staining is strongest at the lateral cell-cell contacts, and Arm is depleted from the vertices of the hexagonal array (Hunter, 2000).
nullo mutant embryos show a normal formation of somatic buds above each nucleus and the same diffuse pattern of Arm protein as wild-type embryos. Arm also begins the same gradual transition to form sharp lines of staining at the level of the basal junction. However, some interfaces fail to establish a focused concentration of Arm protein, so that the partially resolved Arm network characteristic of early stages is still present when the cleavage furrows begin to invaginate. Those regions that fail to form a basal junction do not give rise to cleavage furrows. Areas with a basal junction invaginate, but Arm protein does not remain restricted to the basal junction, as described above. It is interesting to note that the defects in Arm distribution can be observed before nullo mutant embryos develop visible morphological defects, and often can be seen before the visible accumulation of myosin to the furrow canals. This may suggest that the failure to establish a basal junction is the primary defect in nullo mutant embryos (Hunter, 2000).
Based on these observations, it is proposed that the Nullo protein is required to maintain the early accumulation of Arm at the basal junction. In the absence of Nullo, a subset of somatic bud contacts contains only a diffuse accumulation of Arm protein, and fails to initiate cleavage furrows. In those furrows that do invaginate, Arm protein is not restricted to the basal junction but spreads apically along the nascent lateral membrane (Hunter, 2000).
The nullo mutation was originally thought to primarily affect the actin-myosin network that forms at the cellularization front. Given the apparent involvement of nullo in the formation of the basal junction, the localization of Nullo with respect to actin, myosin, and Arm was examined. The Nullo protein had been shown to be concentrated at the leading edge of the cleavage furrow during cellularization (Postner, 1994), but the fixation conditions required to detect the protein precluded most colocalization studies. Therefore a nullo transgene was constructed containing a triple-HA tag at the COOH terminus. The protein produced by this transgene rescues the nullo mutant phenotype and shows the same temporal and spatial localization as the wild-type protein, allowing Nullo to be detected under a wider range of conditions (Hunter, 2000).
A comparison of Nullo and actin shows a strong colocalization during early cellularization. Both Nullo and actin are initially distributed apically, beneath the surface of the somatic buds, and then localize to the nascent cleavage furrow as it begins to invaginate. Nullo and actin maintain their colocalization at the cellularization front until Nullo is degraded during late cellularization. To determine if the concentration of Nullo at the cellularization front corresponds to the basal junction, the furrow canal, or both, the distribution of Nullo was examined with respect to myosin and Arm. It was found that Nullo and Arm protein distributions overlap at the basal junction, whereas the region just below this contains only Nullo protein. Counterstaining with myosin confirms that this region corresponds to a population of Nullo protein in the furrow canal. Thus, the Nullo protein colocalizes not only with actin and myosin in the furrow canal, but also with actin and Arm at the basal junction (Hunter, 2000).
The colocalization of Nullo with actin, Arm, and myosin is maintained until late cellularization, at which point Nullo protein is rapidly degraded. At the onset of gastrulation, the basal junction is also lost, and myosin relocalizes from the furrow canal to the apical region of the cell. This first occurs in the cells that form the ventral furrow and therefore it was of interest to see whether Nullo protein is also degraded more rapidly in these cells. By examining embryos in cross-section, the loss of Nullo protein from the cellularization front was observed to occur more rapidly on the ventral surface of the embryo (Hunter, 2000).
Ectopic Nullo expression gives rise to a striking defect in the later localization of Arm to the apical junctions. The first differences are observed at the point when wild-type embryos lose Nullo protein and accumulate Arm along the apicolateral surface. At this stage, embryos expressing UASnullo maintain a normal localization of Arm at the basal junction, but fail to establish a concentrated localization of Arm in the apicolateral region. Instead, low levels of Arm protein are distributed along a broad region of the lateral membrane, and fail to coalesce into a junctional structure during gastrulation. Similar defects are also observed in the distribution of alpha-catenin and E-cadherin (Hunter, 2000).
The junctional defects lead to irregularities in cell morphology and a failure to form the ventral furrow. Although the ventral cells of embryos expressing ectopic Nullo undergo a normal basal to apical shift in myosin localization and rapidly lose the basal accumulation of Arm, they are unable to invaginate. The cells do appear to initiate cell-shape changes and occasionally produce a wide, shallow furrow on the ventral surface, but they fail to complete ventral furrow formation. In contrast, the cephalic furrow, which forms at the same time, appears normal. Since the ventral surface is normally the first region to degrade Nullo protein, it may be especially sensitive to continued Nullo expression. This may indicate that the rapid, coordinated cell constriction that forms the ventral furrow has a more stringent requirement for apical spot-junctions than other movements of early gastrulation (Hunter, 2000).
These findings suggest that the rapid degradation of Nullo protein in late cellularization is critical for the establishment of the apical junction and the formation of the ventral furrow. Although Nullo protein is required to stabilize the accumulation of Arm in the basal junction, it appears to block the coalescence of Arm that gives rise to the apical spot-junctions. It is also shown that ectopic Nullo blocks only the de novo formation of apical adherens junctions that occurs as epithelial polarity is first established during cellularization (Hunter, 2000).
The formation of an adherens junction between the basal and lateral membrane compartments is unusual, and may reflect a unique need to separate these membrane domains during cellularization. The cytokinesis that takes place during cellularization is a two-step process: the lateral membrane is generated during the initial invagination of the cleavage furrow and the basal membrane is produced by the later expansion of the furrow canal. Therefore, the basal membrane of the furrow canal must be isolated as the lateral cell surface elongates. The furrow canal is known to constitute a separate membrane domain (Lecuit, 2000) that accumulates a specific set of proteins. It also maintains a larger intercellular space, which may prevent lateral contacts that could block furrow canal expansion. In this respect, the basal junction separates the noncontacting basal membrane from the adherent lateral membrane in a manner similar to the separation of the apical (noncontacting) and lateral (contacting) membrane by the ZA. The tight adhesion at the basal junction may also insulate the nascent lateral junctions from the outward pull that generates the basal cell surface. The basal junction therefore acts to define membrane domains and reinforce cell-cell contact in a manner similar to the traditional apical adherens junction (Hunter, 2000 and references therein).
During embryonic development, spot-junctions are often created by the delivery of cadherin-catenin complexes to regions of cell-cell contact. This appears to be the case for the basal junction: unlike Nullo and actin, which are initially present along the apical surface, Arm first appears as dots of staining at sites where somatic buds abut. The lack of overlap between Arm and myosin suggests that Arm is restricted to the small region of membrane contact between the embryo surface and the noncontacting domain of the furrow canal. This small area of localization may allow junctional components to bypass the clustering step that typically follows the delivery of the cadherin-catenin complexes. Examination of the basal Arm domain reveals that its size does not change appreciably between the onset of cleavage furrow invagination and late cellularization (Hunter, 2000).
The absence of the clustering step may, in fact, be critical for the formation of the basal junctions. Unlike a typical adherens junction, which is formed at sites of cell-cell contact, the basal junction forms at sites where a single membrane folds inward and contacts itself. Junctional complexes form on opposite sides of the shallow fold and establish extracellular contacts, but they are also separated by an extremely small intracellular space. In this situation, clustering is problematic: it might allow the two sides of a junction to collapse into a single complex, or allow the recruitment of cadherins and catenins into neighboring furrow canals. A similar problem is faced in mid-cellularization, when the coalescence of the apical junction takes place in close proximity to the existing basal junction (Hunter, 2000).
The Drosophila embryo must have a mechanism to preserve the local accumulations of junctional components when multiple adherens junctions are formed within a common membrane. It is proposed that the presence of Nullo stabilizes the accumulation of Arm in the basal junctions and prevents its recruitment into neighboring complexes or the coalescing apical junctions. In the absence of Nullo a subset of furrows fails to focus Arm protein into a stable basal junction, perhaps due to the recruitment of junctional components into neighboring complexes. The remaining basal junctions elongate during cellularization, suggesting that Arm is being recruited into the coalescing apical junctions. Nullo does not appear to be required for maintenance of the basal junction once it has moved below the region of apical junction synthesis. Although Nullo is degraded during mid-cellularization, the basal junction persists into early gastrulation, and its life is not extended in the presence of prolonged Nullo expression (Hunter, 2000).
A striking feature of nullo is its stringent developmental regulation: by mid-cellularization nullo gene expression has ceased, and the Nullo protein is rapidly degraded (Simpson, 1990). Extending the period of nullo expression into late cellularization prevents the formation of the apical adherens junctions. Instead, Arm, alpha-catenin, and E-cadherin accumulate along a broad apicolateral region, which appears to correspond to the zone where new membrane is inserted into the cleavage furrow. This suggests that the junctional components are delivered to the lateral membrane, but fail to cluster towards the apicolateral boundary. It is proposed that, as in basal junction formation, the ectopic Nullo protein stabilizes the accumulation of cadherins and catenins as they are delivered to regions of lateral membrane contact. Although the depth of the contacting membrane is <1 µm when the basal junction is formed, it has expanded to >20 µm by the onset of apical junction formation. Therefore, cadherins and catenins targeted to this large area must undergo conventional clustering movements to form a concentrated accumulation at the apicolateral boundary. The continued presence of Nullo protein blocks this clustering, and instead preserves the transitional state in which junctional components are broadly distributed along the lateral membrane. Ectopic expression of Nullo during late embryogenesis does not disrupt development, suggesting that once epithelial polarity is established, the presence of Nullo does not affect adherens junctions. The existence of mature apical and basolateral domains may provide cues for the targeting of cadherins and catenins, making the formation of subsequent junctions less reliant on large clustering movements, and therefore less susceptible to the effects of Nullo protein (Hunter, 2000).
During cellularization, the Drosophila embryo undergoes a transition from syncytial to cellular blastoderm with the de novo generation of a polarized epithelial sheet in the cortex of the embryo. This process couples cytokinesis with the establishment of apical, basal, and lateral membrane domains that are separated by two spatially distinct adherens-type junctions. In nullo mutant embryos, basal junctions fail to form at the onset of cellularization, leading to the failure of cleavage furrow invagination and the generation of multinucleate cells. Nullo is a novel protein that appears to stabilize the initial accumulation of cadherins and catenins as they form a mature basal junction. This study characterized a nullo homologue from D. virilis, and conserved domains of Nullo were identified that are required for basal junction formation. Nullo is a myristoylprotein and the myristate group acts in conjunction with a cluster of basic amino acids to target Nullo to the plasma membrane. The membrane association of Nullo is required in vivo for its role in basal junction formation and for its ability to block apical junction formation when ectopically expressed during late cellularization (Hunter, 2002; full test of article).
The wild-type Nullo protein is rapidly degraded before the completion of cellularization and the formation of the apical spot junctions (Postner, 1994). When expression is artificially prolonged, Nullo prevents the clustering of cadherins and catenins into apical spot junctions, leading to defects in cell morphology and a failure of ventral furrow formation (Hunter, 2000). To determine the effect of the N-terminal mutations on the localization and activity of the ectopic Nullo protein, DeltaM, DeltaP, and DeltaMP (respectively lacking the myristoylation site, the positively charged cluster and a combination of the sites) were placed under the control of a GAL4 responsive UAS and expression was driven using GAL4-VP16 under the control of the maternal alpha-tubulin promoter. This results in Nullo expression, which begins during cellularization, peaks during gastrulation, and declines gradually during late embryogenesis (Hunter, 2002).
Prolonging the expression of full-length Nullo at levels characteristic of early cycle 14 is sufficient to produce the ectopic phenotype. When expressed during late cellularization, Nullo protein accumulates along the entire plasma membrane and in punctate structures in the basal region of the newly formed epithelial cells. Although DeltaM under the endogenous promoter localizes to the membrane before cellularization and later becomes nuclear, UAS-DeltaM expressed during late cellularization shows no membrane association and is enriched in the nucleus. Ectopic UAS-DeltaP shows weak membrane localization, along with a diffuse cytoplasmic staining but no visible punctate staining, as seen with the endogenous DeltaP protein. UAS-DeltaMP shows no membrane staining and no observed enrichment in the nucleus versus the cytoplasm (Hunter, 2002).
The activity of a given mutant transgene was assayed by measuring the degree of lethality caused by its ectopic expression. Using the full-length transgene to elevate Nullo protein levels during late cellularization to 69% of that observed at the beginning of cycle 14 caused lethality in 87% of embryos and blocked the formation of apical junctions. UAS-DeltaP at similar levels caused lethality in only 43% of embryos and resulted only in a partial blockage of apical junction formation. UAS-DeltaP therefore shows a reduction in activity but can still interfere with apical junction formation. The ectopic expression of UAS-DeltaM and UAS-DeltaMP, in contrast, has no effect on viability and does not block apical junction formation. The failure of these mutant proteins to localize to the cell surface suggests that membrane localization is absolutely required for Nullo's interference with apical junction formation (Hunter, 2002).
Before the cloning of DV Nullo, little was known about the aspects of the Nullo protein involved in subcellular localization and basal junction formation. No Nullo point mutations had been isolated in screens for embryonic-lethal mutations on the X-chromosome, and no homologues had been identified in any other species. Given the unique nature of cellularization and the fact that nullo expression is tightly restricted to this process, it was likely that nullo homologues would be found only in insects that undergo synchronous cellularization. For this reason, D. virilis, which undergoes cellularization but provides sufficient evolutionary divergence (~60 million years), was used to examine nullo conservation (Hunter, 2002).
Like DM nullo, DV nullo appears to require a relatively small region of upstream DNA for proper expression. The DV nullo gene also lacks introns, a characteristic of DM nullo and the other zygotic cellularization genes bottleneck and serendipity-alpha (Vincent, 1985; Schejter and Wieschaus, 1993). This may reflect the need to produce large amounts of transcript in the short time between the onset of zygotic transcription and cellularization. The DV nullo gene is expressed in the same temporal pattern as DM nullo, with zygotic transcription beginning just before cellularization and degradation of the transcript taking place during late cellularization. The spatial pattern is also similar, with uniform expression until midcellularization and the presence of several stripes of expression during late cellularization. As with sry-alpha, the positions of the stripes differ between D. melanogaster and D. virilis. However, previous studies suggest that exact pattern of nullo expression along the anterior-poterior axis is not functionally relevant (Hunter, 2002).
Comparison of the DM Nullo and DV Nullo predicted proteins revealed several interesting features. The first of these is the conservation of the N-terminal myristoylation site and the presence of an adjacent unconserved, but highly basic, region. This suggested that the N-terminal myristoylation site was important for Nullo localization or function and that it likely acted as part of a 'myristate plus basic' motif used in membrane localization. The second feature was the presence of five distinct conserved domains, separated by short nonconserved stretches. This pattern of conservation is evident even among closely related Drosophila species. In the nullo genes of D. oreana, D. lutescens, and D. yakuba the N-terminus and C-terminal conserved blocks have a high degree of sequence identity, whereas the C-terminal 'linker' regions have already begun to diverge (Hunter, 2002).
Nullo is one of ~35 sequences in the D. melanogaster genome that contains the MGXXXS/T consensus site for N-terminal myristoylation. Although the majority of these genes have been characterized genetically and developmentally, only a handful have actually been shown to be myristoylated, and the in vivo role of the myristoylation site has been studied only in the neural protein Numb. Using a yeast system, this study demonstrated that Nullo is modified by the addition of a myristoyl group, and this is prevented by the substitution of alanine for glycine in position 2. It is believed that this in vitro demonstration of myristoylation is relevant to the in vivo modification of Nullo for two reasons. (1) The consensus target for N-terminal myristoyltransferase (NMT) activity appears to be well conserved between Drosophila and other organisms. Both Drosophila embryonic extracts and Schnieder cells have been shown to have an NMT activity that modifies myristoyl proteins from other organisms in a specific manner, and the corresponding NMT gene has been shown to be expressed before cellularization. (2) The glycine to alanine mutation which blocks myristoylation in vitro has a striking effect on the subcellular localization of Nullo in vivo, suggesting that the N-myristoylation site is important for the targeting of Nullo to the plasma membrane (Hunter, 2002).
During metaphase of nuclear cycle 13, Nullo lacking a myristoylation site (DeltaM) still shows normal localization to the plasma membrane of the metaphase furrows. However, at the onset of cycle 14, DeltaM is lost from the cellularization front and begins to accumulate in the nucleus where it remains until midcellularization. UAS-DeltaM expressed during late cellularization showed no membrane association, but was observed in the nucleus. The difference in DeltaM localization before and after the onset of cellularization suggests that some aspect of Nullo targeting or membrane association changes at this point. The fact that DeltaMP, which lacks both N-terminal domains, shows no early membrane association suggests that the initial association of Nullo with the plasma membrane requires the presence of a positive charge and that the positive charge is no longer sufficient for membrane localization after the onset of cellularization. The electrostatic interaction may be disrupted by changes in the phospholipid content of the apical membrane as new membrane is inserted during cellularization. In contrast, endogenous and ectopic expression of DeltaP demonstrates that the myristoylation site alone is sufficient for partial membrane localization throughout cellularization (Hunter, 2002).
After cycle 13, DeltaM accumulates in the nucleus, which is surprising, because Nullo does not contain a predicted nuclear localization signal, and wild-type Nullo protein has not been observed in the nucleus. It is possible, however that the basic cluster confers a nuclear localization that is normally overridden by the membrane targeting of the N-terminal myristoyl group. In the absence of the myristoyl group, Nullo would associate with the nucleus via the polybasic region, and as was observed, the association with the nucleus would be diminished by the removal of the positively charged cluster (DeltaMP). A similar observation has been made with the Moloney MuLV Gag protein, where mutation of the N-terminal myristoylation site roughly doubles the amount of protein found in the nucleus (Hunter, 2002).
Although the DeltaM, DeltaP, and DeltaMP proteins all retained some ability to rescue the nullo mutant phenotype, only DeltaP was able to partially block apical junction formation when expressed ectopically during late cellularization. One explanation for the difference in activity may lie in the levels of functional protein required in each of these assays. As seen with the LII27.32R5 allele, the rescue of the nullo phenotype requires levels of Nullo protein much lower than those normally seen at the start of cycle 14. In contrast, the ectopic effects on apical junction formation require at least half the protein level seen at the onset of cellularization. In the case of DeltaM the difference in activity may also result from the fact that this protein retains membrane localization during the cycle 13 to cycle 14 transition when basal junctions are formed but lacks any detectable membrane localization when ectopically expressed during late cellularization. The fact that both assays showed a decline in Nullo activity that correlated with the loss of Nullo from the plasma membrane suggests that the 'myristate plus basic' motif controls membrane localization and that the membrane association of Nullo is essential for its role in junction formation (Hunter, 2002).
The fact that both DeltaM and DeltaP retained some ability to associate with the plasma membrane is unusual, as removal of either the myristoyl group or the basic region is usually enough to prevent plasma membrane association in 'myristate plus basic' proteins. Interestingly, mutation of the myristoylation site of Numb also failed to affect membrane localization, although truncation of the first 40 amino acids of the N-terminus caused the protein to accumulate in the cytoplasm. It remains to be seen if Numb contains a bipartite localization motif similar to that seen in Nullo and whether the independent membrane targeting activity of myristoyl groups and basic clusters is a common occurrence in Drosophila (Hunter, 2002).
Loss of Nullo has its primary effect on the formation of basal junctions from the small points of membrane contact that arise at the start of cellularization. The results presented in this article argue that Nullo is not an essential component of the junction itself but is required to stabilize nascent junctions, especially at higher temperatures where the membrane contacts may be more fluid. Ectopic expression of Nullo during late cellularization may also stabilize junctional components as they reach the plasma membrane, but in this case it would prevent the clustering required to form the apical adherens junctions (Hunter, 2000). The effect of Nullo on adherens junctions is compatible with numerous direct and indirect modes of action. However, the structural studies presented in this study suggest that Nullo's activity requires its cell-surface localization and therefore may involve a physical interaction between Nullo and some component of the basal junction, or the accompanying cytoskeleton (Hunter, 2002).
In early Drosophila embryos, several mitotic cycles proceed with aborted cytokinesis before a modified cytokinesis, called cellularization, finally divides the syncytium into individual cells. This study finds that scission of endocytic vesicles from the plasma membrane (PM) provides a control point to regulate the furrowing events that accompany this development. At early mitotic cycles, local furrow-associated endocytosis is controlled by cell cycle progression, whereas at cellularization, which occurs in a prolonged interphase, it is controlled by expression of the zygotic gene nullo. nullo mutations impair cortical F-actin accumulation and scission of endocytic vesicles, such that membrane tubules remain tethered to the PM and deplete structural components from the furrows, precipitating furrow regression. Thus, Nullo regulates scission to restrain endocytosis of proteins essential for furrow stabilization at the onset of cellularization. It is proposed that developmentally regulated endocytosis can coordinate actin/PM remodeling to directly drive furrow dynamics during morphogenesis (Sokac, 2008).
Early morphogenetic events are accomplished by maternal cellular machinery that is developmentally controlled by expression of the zygotic genome. The zygotic gene product Nullo acts as a developmental switch at cycle 14 and targets the endocytic machinery to cellularize the embryo. This study assayed endocytic dynamics by following fluorophore-conjugated wheat-germ agglutinin (Alexa 488-WGA) internalization in living embryos and Amphiphysin (Amph) tubulation in fixed embryos. WGA is a general marker for endocytosis, whereas Amph tubules are more specifically associated with the initial ingression of PM furrows. Several findings support the hypothesis that these Amph tubules are endocytic intermediates. First, their structure is tubular rather than sheet like, consistent with a role in endocytosis. Second, perturbation of Dynamin, a catalyst of endocytic scission, increases the number of Amph tubules at cellularization furrows. Third, in living nulloX and Cyto-D-treated embryos, WGA is internalized in long, PM-tethered tubules that resemble Amph tubules. Fourth, DPATJ enters Amph tubules in nulloX embryos and accumulates at early endosomes. Thus, Amph tubules were used as quantifiable reporters of endocytosis at furrows (Sokac, 2008).
The membrane furrows that form during the early mitotic cycles regress, whereas those that form at cellularization stably ingress. It is proposed that endocytosis is differentially controlled to achieve these distinct morphogenetic events. During the mitotic cycles, metaphase furrows are transient, ingressing only ~5 μm before completely regressing. Restrained endocytosis, detected by both WGA internalization and Amph tubules, accompanies the initial furrow ingression that occurs at prophase/metaphase. This is followed by a fast wave of vigorous WGA endocytosis that traverses the embryo surface when metaphase furrows regress at anaphase/telophase. The endocytosis accompanying furrow regression is not associated with high levels of Amph tubules. Although this endocytosis may not recruit Amph, a model is favored whereby endocytic scission is more efficient at this time, precluding the capture of tubule intermediates by fixation. Thus, endocytosis at furrow regression may be mechanistically distinct from endocytosis at furrow ingression. It may also be functionally distinct and could even drive furrow regression, as endocytosis adjusts the surface area of both motile and dividing cells. Thus, throughout the early mitotic cycles, alternating and distinct endocytic dynamics are regulated by cell cycle progression and correlate with specific furrow events (Sokac, 2008).
At the onset of cellularization, furrows form in a way that resembles metaphase furrows, but then assemble furrow canals that stabilize the furrow and sustain ingression over ~40 μm. Endocytosis, marked by both WGA internalization and Amph tubules, also accompanies the initial ingression of cellularization furrows, but ceases by the time furrows reach 5 μm in length and furrow canals fully assemble. Since cellularization occurs during interphase, cell cycle progression cannot regulate this endocytosis. Instead, zygotic expression of Nullo aids endocytic scission, and this has the effect of limiting membrane dynamics at the tip of the incipient cellularization furrow, so that proteins, including Myosin-2, Septin, and DPATJ, that concentrate there are retained there. As a result, furrow canals assemble and stabilized furrows ingress to cellularize the embryo. Thus, Nullo regulation of endocytic dynamics could promote the developmental transition from transient furrowing that maintains the syncytium to stable furrowing that generates the primary epithelial cell sheet (Sokac, 2008).
Nullo activity facilitates endocytic scission, such that budding vesicles are rapidly released from the PM. When scission is impaired, some budding vesicles are distended into long Amph tubules that remain persistently tethered to the PM. This phenotype is mimicked when F-actin levels are reduced with Cyto-D, and Nullo regulates cortical F-actin. Thus, it is suggested that Nullo aids scission via its regulation of F-actin. How Nullo regulates actin remains elusive, since it is a small (213 amino acids), highly basic (pI 11.4), myristoylated protein with no readily identifiable globular domains to suggest interaction partners. Instead, sequence composition and hydropathy character suggest that Nullo is “natively unfolded,” containing 63% disorder-promoting amino acids (T, R, G, Q, S, N, P, D, E, and K) over its entire length and a disordered run of 50 consecutive amino acids. Other disordered proteins have been identified that control F-actin organization, such as MARCKs, MARCKs-related proteins, and GAP43, either by direct interaction with actin or by locally sequestering the actin-regulator PIP2 within the PM. Although sequence comparison and lack of characteristic acidic regions do not suggest that Nullo is MARCKs related, it was found that Nullo interacts with PIP2 in in vitro binding assays. Nullo may then concentrate PIP2 locally to regulate actin and/or to couple actin to components of the endocytic machinery that also interact with PIP2 at the PM (Sokac, 2008).
Nullo may aid endocytic scission via F-actin by either active or passive mechanisms. In yeast, F-actin actively drives endocytic scission by exerting polymerization and myosin-based forces to lengthen, and eventually break, the budding vesicle neck. In Drosophila hemocytes and mammalian cells, F-actin also contributes to a late step in endocytosis that just precedes vesicle release and that may be either bud invagination or scission. Additionally, cortical F-actin passively regulates endocytic dynamics by reinforcing the plasma membrane (PM) and thus antagonizing PM deformation. In the case of Bin-Amph-Rvs (BAR) domain activity, drug-mediated reduction of F-actin levels enhances PM tubulation. Reduced levels of cortical F-actin in nulloX mutants result in the appearance of more Amph tubules. However, fewer vesicles are released in living nulloX embryos, and Amph tubulation does not expand to regions beyond the furrow, arguing that there is not more endocytosis in mutants. Thus, impaired scission generates the appearance of more tubules, and it takes almost three times longer for budding vesicles to release from the PM in nulloX versus wild-type embryos. Consistently, dynamin defects enhance PM tubulation in cultured cells, and BAR-induced membrane tubulation is antagonized by coexpression of dynamin. Thus, Nullo may regulate a population of F-actin that either actively aids scission or stiffens the cortex and somehow contributes to endocytosis (i.e., if breaking the bud neck is aided by the PM being under cortically maintained tension) (Sokac, 2008).
This analysis strongly supports that membrane trafficking is differentially controlled at specific sites and times within embryos to achieve distinct morphogenetic events. This was previously suggested for fly embryos by the membrane-labeling analysis that demonstrates that exocytosis occurs at specific sites along cellularization furrows and thus helps establish apical/basal polarity in these cells. Two additional observations are relevant to the results described here. First, membrane labeling of the furrow canal is only possible at very early cellularization. After that time, the furrow canal persists as a stable membrane compartment in which no new membrane is either added or taken away. The current data also support the finding that membrane turnover at the furrow canal region is restricted to the very beginning of cellularization. In fact, endocytic dynamics are tightly controlled there to establish and/or maintain the concentration of proteins at the furrow canal. Second, it has been observed that the membrane label is cleared from the apical PM, and it has been suggested that clearing is mediated by endocytosis. At furrow lengths > 5 μm, WGA vesicles are seen moving away from the apical PM. But when perivitelline injections are done with higher lectin concentrations and at furrow lengths < 5 μm, they reveal WGA endocytosis from the tips of incipient furrows. In fixed embryos in which spatial resolution is better, Amph tubules clearly extend only from furrow tips. Thus, this analysis shows that, in addition to apical endocytosis, local endocytosis also occurs where furrows first ingress (Sokac, 2008).
During cytokinesis in some mammalian cells, membrane endocytosed at sites remote from the furrow is later delivered to the division plane via the endocytic pathway. At cellularization furrows in the fly embryo, the exocytosis of membrane derived from recycling endosomes suggests a similar pathway. In these cases, endocytosis from one site can provide a store of membrane to feed growth somewhere else. However, the observation that endocytosis occurs at the furrow itself is counterintuitive, since endocytosis would be expected to reduce surface area, whereas furrow ingression requires surface expansion. Nonetheless, there are now several reports that endocytic proteins including Clathrin, Clathrin adaptor-2, and Dynamin concentrate in cytokinesis furrows. In addition, endocytosis has been directly visualized at furrows in dividing zebrafish embryos, cultured cells, and fission yeast, although the function of this endocytosis remains unclear (Sokac, 2008).
Endocytosis occurs at the tips of both metaphase and cellularization furrows when the furrows are first ingressing, suggesting that it confers some temporally and spatially specific function. Both the PM and actin are significantly remodeled at these sites as furrows form. At the onset of cellularization in particular, the F-actin/Myosin-2 furrow canals are assembling at this place and time. Actin remodeling is intimately coupled to endocytosis in other cell types. Endocytic proteins control actin dynamics, and actin-binding proteins are required for endocytosis. Also, endocytic and actin-binding proteins are regulated by the same phosphoinositide pools at the PM. It follows that local endocytosis could influence local actin organization during furrow formation in fly embryos. This study shows that actin conversely provides developmental regulation of endocytic dynamics. This analysis leads to a speculation that the coupled regulation of actin and endocytosis can effectively coordinate actin/PM remodeling to drive furrow dynamics, and thus shape cells during morphogenesis (Sokac, 2008).
Search PubMed for articles about Drosophila Nullo
Hunter, C. and Wieschaus, E. (2000). Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm. J. Cell Bio. 150: 391-402. PubMed ID: 10908580
Hunter, C., Sung, P., Schejter, E. D. and Wieschaus, E. (2002). Conserved domains of the Nullo protein required for cell-surface localization and formation of adherens junctions. Mol. Biol. Cell 13(1): 146-57. PubMed ID: 11809829
Postner, M. A., and Wieschaus, E. F. (1994). The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos. J. Cell. Sci. 107: 1863-1873. PubMed ID: 7983153
Rose, L. S. and Wieschaus, E. (1992). The Drosophila cellularization gene nullo produces a blastoderm-specific transcript whose levels respond to the nucleocytoplasmic ratio. Genes Dev. 6(7): 1255-68. PubMed ID: 1378418
Simpson, L. and Wieschaus, E. (1990). Zygotic activity of the nullo locus is required to stabilize the actin-myosin network during cellularization in Drosophila embryos. Development 110: 851-863. PubMed ID: 2088725
Sokac, A. M. and Wieschaus, E. (2008). Local actin-dependent endocytosis is zygotically controlled to initiate Drosophila cellularization. Dev. Cell 14: 775-786. PubMed ID: 18477459
date revised: 15 August 2008
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