Protein Interactions

Exchange factors for Rho proteins are known to activate their downstream targets through direct binding to Rho GTPases. Hence, the molecular basis for the observed genetic interaction between pebble and Rho1 may be a physical interaction between Pbl and Rho1 proteins. To test this hypothesis, a yeast two-hybrid assay was carried out. Fusions of Pbl (full-length and amino-terminally truncated) as well as Drosophila Rho proteins, Rho1, Rac1, and Cdc42, were constructed with both the GAL4 DNA-binding domain (DBD) and GAL4 activation domain (AD). Plasmids were transformed in various combinations, and the resulting colonies were tested for the ability to activate HIS3 and lacZ reporters. Only colonies that carry plasmids expressing both Pbl (full-length or DeltaPbl325-853, amino-terminally truncated Pbl) and Rho1 are able to grow on a medium that lacks His, Leu, and Trp, suggesting that the HIS3 gene is induced. Therefore, it is suggested that Pbl and Rho1 proteins interact in vivo and that this interaction is specific for Rho1, but not for Rac1 or Cdc42. Furthermore, the DH domain, but not the amino terminus of Pbl (containing BRCT domains and NLS), is essential for this interaction, because it is abolished by a small deletion within the DH domain, but not by the amino-terminal truncation of Pbl. These results indicate that Pbl and Rho1 form a protein complex in vivo, and that the basis for the genetic interaction between pbl and Rho1 may be a direct interaction between the two proteins (Prokopenko, 1999).

Cytokinesis, the final step in cell division, involves the formation and constriction of an actomyosin-based contractile ring. The mechanism that positions the contractile ring is unknown, but derives from the spindle midzone. An interaction between Pebble [a Rho GTP exchange factor (GEF)], and the Rho family GTPase-activating protein, RacGAP50C, has been shown to connect the contractile ring to cortical microtubules at the site of furrowing in D. melanogaster cells. Pebble regulates actomyosin organization, while RacGAP50C and its binding partner, the Pavarotti kinesin-like protein, regulate microtubule bundling. All three factors are required for cytokinesis. As furrowing begins, these proteins colocalize to a cortical equatorial ring. It is proposed that RacGAP50C-Pavarotti complexes travel on cortical microtubules to the cell equator, where they associate with the Pebble RhoGEF to position contractile ring formation and coordinate F-actin and microtubule remodeling during cytokinesis (Somers, 2003).

In Drosophila melanogaster embryonic epithelial cells, constriction occurs during anaphase B and telophase to generate two daughter cells, each containing one set of the recently separated sister chromatids. Constriction of the cleavage furrow proceeds through the activity of the myosin II motor protein acting on an F-actin network. Members of the Rho subfamily of small G proteins are potent regulators of the actin cytoskeleton in a variety of contexts. Like all small G proteins, Rho1 is active when GTP is bound and inactive when GDP is bound. Activation is mediated by guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP and the uptake of GTP, whereas inactivation is regulated by GTPase-activating proteins (GAPs) that stimulate the intrinsic GTPase activity of the G protein (Somers, 2003).

Molecular and genetic studies have shown that the D. melanogaster RhoGEF, Pebble (PBL), and its mammalian ortholog, the protooncogene ECT2, are required for cytokinesis. pbl mutant embryos proceed normally through the first 13 syncytial mitotic cycles following fertilization and cellularize normally during G2 phase of cycle 14, but they fail to undergo cytokinesis during the fourteenth and subsequent division cycles. Pbl binds to Rho1, but not Rac1 or Cdc42, and sensitized pbl mutant alleles show strong genetic interactions with Rho1 but not Rac1 or Cdc42 alleles. During cytokinesis in epithelial cells of the embryo, Pbl accumulates in the contractile ring during furrowing, where it appears to stimulate Rho1-mediated organization and activity of the actomyosin contractile ring (Somers, 2003).

Reorganization of the actomyosin contractile apparatus occurs coincident with reorganization of the microtubule network. During anaphase, the mitotic spindle is remodeled to form a midzone bundled microtubule structure referred to as the central spindle, which is further compacted into a late cytokinetic structure termed the midbody. Curiously, another regulator of Rho family G protein activity, the Caenorhabditis elegans CYK-4 GAP, is required for microtubule bundling, because microtubule reorganization fails in cyk-4 mutant embryos. It also fails in embryos mutant for the zen-4/CeMKLP1 gene, which encodes a kinesin-like protein that forms a complex with CYK-4. This complex has been shown to bundle microtubules in vitro. The CYK-4 and ZEN-4 proteins and their respective mammalian orthologs localize to the central spindle and are all essential for cytokinesis, as is Pavarotti (Pav), the D. melanogaster ortholog of ZEN-4 (Somers, 2003).

It is not known how remodeling of the microtubule and F-actin networks is coordinated during cytokinesis. Although initial studies focused on their role in F-actin remodeling, recent studies have now linked Rho family members to microtubule organization. For example, depolymerization of microtubules results in an increase in the amount of active RhoA and the formation of contractile actin bundles, while microtubule polymerization results in an increase in the amount of active Rac1 and the formation of lamellipodia. RhoA can also mediate selective microtubule stabilization, while the Rac1/Cdc42 effector PAK is capable of activating the microtubule destabilizer Stathmin. It is possible, therefore, that Rho family members play roles in both F-actin and microtubule organization during dynamic processes such as cytokinesis. An important but poorly understood aspect of the relationship between the microtubule and F-actin networks is the nature of the signal that positions the contractile ring and initiates furrowing. It is now generally accepted that the signal originates from the midzone of the anaphase microtubule network, although the nature of the stimulus is unknown (Somers, 2003).

A complex has been identified between two Rho family regulators, the RhoGEF Pbl and RacGAP50C, the D. melanogaster ortholog of the CYK-4 Rho family GAP. A ring of RacGAP50C and Pav, associated with cortical microtubules, colocalizes with Pbl in dividing embryonic epithelial cells, forming a link between the actomyosin and microtubule networks. These observations suggest a molecular model for contractile ring positioning and function whereby RacGAP50C-Pav complexes, positioned at the equatorial cortex of the cell by their association with microtubules, interact with cortical Pbl to activate Rho1, initiate formation of the contractile ring, and coordinate F-actin and microtubule dynamics during furrowing (Somers, 2003).

Thus, in Drosophila embryonic epithelial cells at the onset of cytokinesis, the two Rho family regulators are part of a cortical double-ring structure at the site of cleavage furrowing. The RacGAP50C ring is associated with cortical microtubules, presumably through its interaction with the Pavarotti kinesin-like protein. Pav colocalizes with RacGAP50C, and coimmunoprecipitation experiments have shown that they form a complex in vivo. The RacGAP50C-Pav ring appears to abut or overlap the Pbl-containing contractile ring. The Pav kinesin-like protein, RacGAP50C and Pbl RhoGEF trimolecular complex simultaneously associates with, and has the capacity to control, both the actin and microtubule cytoskeletons as they are remodeled during cytokinesis. Furthermore, this complex appears to be a conserved feature of animal cytokinesis, since the mammalian Pbl and RacGAP50C orthologs, the protooncogene ECT2 and MgcRacGAP, bind to each other in a yeast two-hybrid assay and colocalize during mitosis (Somers, 2003).

The interaction between Pbl and RacGAP50C occurs through an extended BRCT domain of Pbl and an N-terminal coiled-coil domain of RacGAP50C. RacGAP50C binds Pav through sequences adjacent to the Pbl-interacting domain, indicating the presence, in Drosophila, of the so-called centralspindlin complex (Mishima, 2002) first identified from analysis of CYK-4 and ZEN-4, the C. elegans RacGAP50C and Pav orthologs (Somers, 2003).

RacGAP50C-Pav complexes were found to be cytoplasmic at prophase, associated with mitotic spindles during metaphase, concentrated in the spindle midzone during anaphase, and localized to the midbody at cytokinesis and to the nucleus during interphase. During late anaphase and early telophase in Drosophila epithelial cells, RacGAP50C-Pav complexes not only localize to the overlapping microtubules of the centrally located anaphase spindle, but also to distinct cortical microtubules. Cortical microtubules have been reported in dividing Drosophila neuroblasts (Savoian, 2002), and they can be seen in all D. melanogaster anaphase cells examined. Localization of the RacGAP50C-Pav complexes to the microtubule midzone is independent of its interaction with Pbl, since RacGAP50C is found to localize appropriately in Pbl mutant cells. However, localization of RacGAP50C is dependent on the Pav kinesin-like protein. The affinity of the RacGAP-KLP complex for microtubules, the cortical localization of the microtubules, and the plus end-directed nature of the Pav kinesin-like motor protein appear sufficient to account for localization of the complex to an equatorial cortical ring (Somers, 2003).

The molecular signal that positions the contractile ring and initiates furrowing remains to be elucidated. A number of studies have shown that the signal derives from the overlapping midzone microtubules that form during anaphase. One of the most striking aspects of the formation of the Pbl-RacGAP50C ring is that it is present in the earliest examples of furrowing that were observed. The existence of this ring at the onset of cytokinesis suggests a molecular model for the positioning and regulation of the contractile ring. Specifically, it is proposed that the initiation signal corresponds to the microtubule-mediated arrival of the RacGAP50C-Pav kinesin-like protein complex at its equatorial ring and establishment of the interaction with the Pbl RhoGEF. It is proposed that this interaction results in activation of RhoGEF activity. Rho1 would then be activated to initiate contractile ring formation and furrowing through activation of factors such as Diaphanous and myosin. This model accounts for the role of microtubules in positioning the contractile ring, because microtubules deliver the RacGAP50C-Pav complexes to their interaction site with Pbl. It also accounts for the conclusion, made by Gatti and colleagues from their studies of cytokinesis, that there is a requirement for both the central spindle and a cortical Pbl-containing apparatus for the onset of cytokinesis (see Somma, 2002; Somers, 2003).

There is strong support for this model beyond the evidence described here. Importantly, consistent with the observation that DRacGAP50C RNAi-treated S2 cells show no furrowing, pav and pbl mutant cells fail to form a contractile ring and do not undergo furrow ingression. However, contradictory evidence has come from C. elegans, where cyk-4 and zen-4 mutant cells initiate but fail to complete furrowing. Two possible explanations are suggested for these contradictory observations. The first is that D. melanogaster epithelial cells may use a different cytokinesis mechanism than that used in the early C. elegans embryo. In support of this, the cell types are very different in size and exhibit differences in microtubule organization during anaphase and telophase. Alternatively, it is possible that the C. elegans phenotypes do not represent the true null phenotype. The cyk-4 allele used to determine the phenotype is a temperature-sensitive allele, which may not abolish all activity at the restrictive temperature. The zen-4 allele used to generate germline mutant clones is a premature truncation that would eliminate all function. However, to observe the phenotype, germline clones were generated, perhaps requiring zen-4 activity to undergo the previous division. Some of the product may therefore have persisted to produce the partial furrowing observed in the mutant embryos. It therefore remains to be seen whether the model proposed here is applicable to cytokinesis in all animal cells (Somers, 2003 and references therein).

Midzone microtubule bundles have been shown to be required continuously for cytokinesis in cultured cells. The cortical Pbl-RacGAP50C-Pav ring, which persists and narrows as cytokinesis proceeds, is ideally positioned to coordinate actomyosin contraction and the bundling of microtubules. Actin filament activity is regulated by Pbl, which is required for establishment and/or maintenance of the contractile ring through activation of the Rho1 GTPase. Microtubule bundling activity has been demonstrated for CYK-4 and ZEN-4. It is likely, therefore, that the complex between the Pbl RhoGEF, RacGAP50C and the Pav kinesin-like protein functions to coordinate F-actin and microtubule remodeling during contractile ring constriction (Somers, 2003).

While actomyosin regulation and microtubule bundling may be the primary regulatory roles of these factors, there are additional ways that the Pbl RhoGEF and RacGAP50C could influence both the actin and microtubule-based cytoskeleton. Rho downstream effectors have been shown to regulate both cytoskeletal systems. For example, the Rho1 target, Diaphanous, mediates actin reorganization but also affects the stability of microtubules (Somers, 2003).

The CYK-4 and ZEN-4 microtubule bundling activity does not require the presence of any of the small G proteins, but the site-directed mutant analysis described in this study suggests a requirement for the GTPase-activating domain of RacGAP50C. Consistent with this, a GAP domain-defective form of MgcRacGAP appears to act as a dominant-negative protein, inducing cytokinetic defects. If such a target of RacGAP50C GAP activity exists, it has still not been identified. However, the evidence is inconsistent with RacGAP50C acting as the Rho1 GAP that opposes Pbl, based on the synergistic nature of pbl and RacGAP50C genetic interactions and on the absence of genetic interactions between RacGAP50C and Rho1. Consistent with this, in vitro assays show that the CYK-4 and MgcRacGAP homologs target Rac and Cdc42 with far greater efficiency than Rho1 (Somers, 2003 and references therein).

Ths study has identified complexes between the RhoGEF Pbl and the Rho family GAP, RacGAP50C, and between RacGAP50C and the kinesin-like protein, Pav, that connect the contractile ring to cortical microtubules during cytokinesis. During late stages in anaphase and during telophase, these proteins localize to a cortical ring where furrowing is initiated, constricting as furrowing proceeds. These observations suggest a model for the molecular control of cytokinesis in animal cells, whereby microtubule-dependent cortical equatorial localization of RacGAP50C-Pav kinesin-like protein complexes is the positioning signal generated by the central spindle microtubules, and formation of complexes with the Pbl RhoGEF allows coordination of F-actin and microtubule remodeling (Somers, 2003).

Citron Kinase is an essential effector of the Pbl-activated Rho signalling pathway in Drosophila

Pebble (Pbl)-activated RhoA signalling is essential for cytokinesis in Drosophila melanogaster. The Drosophila citron gene, [a. k. a. sticky (sti)], encodes an essential effector kinase of Pbl-RhoA signalling in vivo. Drosophila citron is expressed in proliferating tissues but is downregulated in differentiating tissues. Citron can bind RhoA and localisation of Citron to the contractile ring is dependent on the cytokinesis-specific Pbl-RhoA signalling. Phenotypic analysis of mutants showed that citron is required for cytokinesis in every tissue examined, with mutant cells exhibiting multinucleate and hyperploid phenotypes. Strong genetic interactions were observed between citron and pbl alleles and constructs. Vertebrate studies implicate at least two Rho effector kinases, Citron and Rok, in cytokinesis. By contrast, no evidence was found of a role for the Drosophila ortholog of Rok in cell division. It is concluded that Citron plays an essential, non-redundant role in the Rho signalling pathway during Drosophila cytokinesis (Shandala, 2004).

RNA interference-mediated silencing of sticky/citron in cultured cells causes them to become multinucleate. Components of the contractile ring and central spindle are recruited normally in such Sticky-depleted cells that nevertheless display asymmetric furrowing and aberrant blebbing. Together with an unusual distribution of F-actin and Anillin, these phenotypes are consistent with defective organization of the contractile ring. sti shows opposite genetic interactions with Rho and Rac genes, suggesting that these GTPases antagonistically regulate Sticky functions. Similar genetic evidence indicates that RacGAP50C inhibits Rac during cytokinesis. Antagonism between Rho and Rac pathways may control contractile ring dynamics during cytokinesis (D'Avino, 2004).

Citron has been proposed to act downstream of Rho in the regulation of cytokinesis. However, little in vivo evidence has been found to support this proposition. To test whether Citron participates in Rho signalling, genetic interactions were examined between citron and a known regulator of the Rho pathway, the Rho-GEF-encoding gene, pebble. The first assay chosen was the ability to modify the moderate citron embryonic PNS phenotype. pbl mutants were chosen rather than Rho mutants because Pbl appears to be a specific Rho activator for cytokinesis, whereas loss of Rho also affects many other processes. Removing one copy of pbl in cit mutants results in a significant reduction in the overall number of cells in the PNS, while most of the remaining cells (52%) appear to be multinucleate. Therefore, a mild reduction in Pbl-mediated Rho activation during cytokinesis results in a significant enhancement of the cit mutant embryonic PNS defects (Shandala, 2004).

A complementary approach monitored whether under- or over-expression of citron could modify a loss-of-Pbl phenotype. Since strong pbl phenotypes arise too early and are too drastic to be of use, an RNAi construct was generated to inhibit Pbl synthesis later in development. Expression of this pblRNAi construct in the posterior half of the wing resulted in a decrease in the size of the corresponding region. Analysis of the affected area revealed that more than 67% of cells produce multiple hairs in contrast to the invariably single-haired cells in wild-type, a phenotype observed when cytokinesis is blocked, for example by inhibition of RacGAP50C. As expected, co-staining of pupal wings with phalloidin and the DNA stain Hoechst 33258 revealed that the pblRNAi-expressing cells were abnormally large and typically multinucleate, resembling the embryonic phenotype of pbl mutants. The intermediate nature of the en-GAL4>UAS-pblRNAi wing size and multiple hair phenotypes allowed detection of enhancement and suppression by prospective interactors. To test the specificity of this assay system, the pblRNAi phenotype was examined in a RhoA/+ background. Significant diminution of the pbl-depleted region of the wing shows that the pblRNAi phenotype is enhanced by removal of one copy of wild-type RhoA, as seen in other genetic assays for pbl function. The multiple hair phenotype was quantified in a defined wing region posterior to vein L5. A significant increase in the proportion of multihaired cells from 67% to 84% upon loss of one copy of RhoA shows that this assay could detect reductions in the dose of cytokinesis effector genes. Removal of one copy of wild-type citron also reduces the size of the posterior half of the wing in en-GAL4>UAS-pblRNAi flies and enhances the multiple hair phenotype. Identical effects were observed in Df(3)iro-2 heterozygous mutants . The genetic interactions between loss of function citron and pbl phenotypes support the role of Citron as a Rho effector in cytokinesis. Ectopic expression of citron in various Drosophila tissues generates no dramatic phenotype in wild-type or pblRNAi backgrounds, suggesting that the activity of Rho is rate limiting for Citron function (Shandala, 2004).

Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis

The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. A collection of genes was examined that causes defects in cytokinesis and revealed (with live cell imaging) two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. These findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex (Dean, 2005).

This study has discovered three steps in the myosin II localization/activation process that involve distinct groups of genes: (1) an initial recruitment of myosin II to the equatorial cortex that is independent of F-actin and centralspindlin but requires rho1 signaling; (2) a secondary stabilization of myosin II at the midzone that requires F-actin and a second set of genes that are likely involved in building a specific type of actin network, and (3) the activation of furrowing once myosin II is localized that depends on centralspindlin (Dean, 2005).

Rho1, its activating guanidine nucleotide exchange factor pebble, and rho kinase are each required for the initial recruitment of myosin II to the equatorial cortex. Rho1 has been implicated in two pathways that are important for cytokinesis. In the first pathway, rho1 signals to F-actin through the formin diaphanous. However, proteins on this F-actin pathway, including F-actin itself, are not essential for the initial myosin II recruitment to the equatorial cortex. However, rho kinase, another downstream target of rho1, is essential. Because rho kinase phosphorylates the myosin II RLC, it is possible that phosphorylation of the RLC is essential for myosin II recruitment to the furrow. This hypothesis could not be directly tested, because the myosin II heavy chain forms large aggregates when the RLC is depleted by RNAi (Dean, 2005).

Phosphorylation of the RLC both activates the motor domain and, in some myosins, increases bipolar thick filament formation. Because F-actin is not required for myosin II recruitment, activation of the motor is unlikely to be the mechanism by which phosphorylation of the RLC would cause recruitment of myosin II to the equatorial cortex. It is quite possible, however, that the rho kinase-mediated myosin II phosphorylation leads to thick filament assembly and that this assembly is important for localization of myosin to the equatorial cortex. Indeed, in Dictyostelium, it is clear that bipolar thick filament formation is sufficient for myosin II localization to the midzone of a mitotic cell. The nonactin-based mechanism of recruitment of myosin II filaments remains unknown (Dean, 2005).

In contrast to the lack of F-actin involvement in the early recruitment of myosin II to the equatorial cortex at anaphase, F-actin disruption by Latrunculin A results in a failure to maintain myosin II in the equatorial region. Interestingly, the downstream rho1 effectors diaphanous/formin and chickadee/profilin are also necessary for myosin II maintenance at the equatorial midzone. Although the loss of these genes could deplete F-actin, phalloidin staining has shown that F-actin is still present in all of the RNAi-treated cells. In addition, these RNAi-treated cells still contract, unlike when F-actin is completely disrupted with LatA. Thus, myosin II appears to be interacting with F-actin in the cortex as it disperses in dynamic patches throughout the cortex of these diaphanous- or chickadee-depleted cells (Dean, 2005).

It is suggested that the role of diaphanous/formin and chickadee/profilin in maintaining the myosin II contractile ring is through the creation of specific F-actin structures. In particular, formin- and profilin-mediated nucleation results in unbranched actin filaments because profilin promotes the barbed-end growth of formin-capped actin filaments. Indeed, electron microscopy has shown that F-actin in the cleavage furrow mainly consists of unbranched, bundled filaments. These parallel filaments contrast with Arp2/3-mediated nucleation, which creates a highly branched actin filament network. Indeed, Arp2/3, although essential for lamellipodia formation, is not required for cytokinesis in Drosophila cells. The hypothesis here is that once myosin II is recruited to the equatorial cortex of the cell by a rho kinase-dependent mechanism, possibly localized activation of RLC phosphorylation, it is retained there because of its higher affinity for parallel, unbranched actin filaments than to branched actin networks. Consistent with this hypothesis, myosin II is depleted from the lamellipodia in migrating cells where Arp2/3 is localized and branched F-actin networks are formed but is enriched in the lamella where F-actin filaments are more likely to be aligned in parallel bundles. Thus, it is proposed that high rho1 signaling to Diaphanous at the cleavage furrow maintains a higher concentration of parallel actin filaments in this region compared with the rest of the cortex, and these parallel filaments serve to selectively retain myosin II at the equator to form a stable contractile ring. In the absence of these parallel actin filaments, myosin II can bind branched F-actin throughout the cortex, perhaps occasionally organizing them into parallel bundles that cause increased myosin recruitment corresponding to the flashes of cortical myosin accumulation, but these interactions are unstable (Dean, 2005).

Live-cell imaging shows that when pavarotti or racGAP50c are depleted, the cells do not display significant contractions despite recruiting myosin II to the equatorial cortex. Although there is some modest membrane contractile activity in these cells, it is clear that significant contraction or furrowing requires both components of the centralspindlin complex. It is surprising that only these proteins were found to be necessary for cortical contraction at sites of myosin II localization. Data from fixed cells, as well as earlier studies, indicated that Drosophila cells do not undergo equatorial contractions during mitosis when Diaphanous or Chickadee is depleted. However, live-cell imaging shows that when either of these two genes is depleted in S2 cells, not only is myosin II transiently localized to the equatorial cortex before dispersing, but cells do indeed display transient equatorial contraction. It is difficult to recognize these events in fixed cells because of their transient nature and the somewhat irregular shapes of cells depleted of these proteins. This work highlights the importance of live-cell imaging in the study of dynamic processes such as cytokinesis (Dean, 2005).

In addition to the suppression of furrowing, depletion of centralspindlin also leads to an inability to retain F-actin exclusively at the equatorial cortex during cytokinesis. This similar phenotype of the centralspindlin complex and the F-actin affecting proteins suggests that centralspindlin may be an upstream regulator of F-actin filament formation. Indeed kinase-dead mutants of Pavarotti have been shown to accumulate at the spindle poles and are associated with an abnormal accumulation of F-actin near the centrosomes. Centralspindlin may be acting indirectly by helping to localize an important actin-affecting protein at the central spindle, or it may act more directly on the cortex. Because RacGAP50c has been shown to bind Pebble in vitro, it has been hypothesized that centralspindlin affects the F-actin cortex through rho1 signaling by the localization and/or activation of Pebble. However, RacGAP50c depletion does not lead to a lack of myosin II recruitment as does Pebble or Rho1 depletion, and, thus, centralspindlin must act in a rho1-independent manner. For instance, the racGAP activity of centralspindlin may itself be important for signaling to the F-actin cortex. Finally, centralspindlin cannot be the major actomyosin ring positioning signal because myosin II is properly recruited in its absence (Dean, 2005).

Tum/RacGAP50C provides a critical link between anaphase microtubules and the assembly of the contractile ring in Drosophila melanogaster

A central question in understanding cytokinesis is how the cleavage plane is positioned. Although the positioning signal is likely to be transmitted via the anaphase microtubule array to the cell cortex, exactly how the microtubule array determines the site of contractile ring formation remains unresolved. By analysing tum/RacGAP50C mutant Drosophila embryos it has been shown that cells lacking Tumbleweed (Tum) do not form furrows and fail to localise the key cytokinetic components Pebble (a RhoGEF), Aurora B kinase, Diaphanous, Pav-KLP and Anillin. The GAP activity of Tum is required for cytokinesis: in its absence cytokinesis fails early even though Tum is present on microtubules at the cell equator where the furrow should form. Disruption of the Pebble-interacting domain leaves Tum localised to the cell equator on cortically associated microtubules, again with no evidence of furrowing. These data support a model in which Tum/RacGAP, via its interaction with Pbl, provides a critical link between the anaphase microtubule spindle and cytokinetic furrow formation in Drosophila cells (Zavortink, 2005).

Cells lacking detectable Tum progress through the mitotic cycle, successfully assembling a metaphase spindle and undergoing anaphase A and B. However, they fail to form or maintain a distinct central spindle or establish a cytokinetic furrow. Telophase cells possessed variable numbers of microtubule bundles, but these were rarely organised into a central spindle. This phenotype is similar to that seen in cells depleted of the Tum binding partner, Pav-KLP (Zavortink, 2005).

Significantly, and consistent with the absence of furrowing, none of the other cytokinetic components that have been checked to date (AurB, Dia, Anillin, Pbl and Pav-KLP) localise correctly in the absence of Tum. These observations show that Tum is required at the very earliest stages of furrow formation, consistent with suggestions that the central spindle and/or bundled midzone microtubules direct the earliest events of cytokinesis (Zavortink, 2005).

It was initially proposed that Tum interacts with Pbl at the cell equator and initiates changes in Rho activity, culminating in formation of the furrow. When the Pbl-interaction-domain deletion protein, TumDeltaPbl, is the only Tum protein present, cells proceed to a point in cytokinesis where assembled centralspindlin complexes are found at the cortex, bridging bundles of microtubules from opposite poles, but they do not proceed beyond this stage, suggesting that the Pbl-Tum interaction becomes critical at this time. This observation satisfies one important prediction of the model, that disruption of the Tum-Pebble interaction prevents cytokinesis but does not affect earlier centralspindlin-based microtubule localisation, supporting the proposal that the interaction between Tum and Pbl is the bridge between the anaphase mitotic spindle and contractile ring assembly (Zavortink, 2005).

Microtubule function in tum mutant cells is perturbed at anaphase, leading to the loss or disruption of the central spindle. It has been suggested that central spindle microtubules are unstable in the absence of bundling so the concomitant loss of Pav-KLP with Tum could explain this loss of microtubule organisation. However cases in cells overexpressing mutant forms of Tum were observed in which central spindle organisation is disrupted even in the presence of apparently stable centralspindlin complexes. If Tum does not interact with Pbl, or if GAP activity is compromised, central spindles fail to form or are unstable, even though the modified Tum is still delivered to the ends of microtubules associated with the cell cortex. Equatorial-cortex-associated microtubule bundles and misdirected non-cortical bundles form, but normal central spindles are rarely seen in these cells even though TumDeltaPbl apparently can bind Pav-KLP. Loss of the central spindle has also been observed in S2 cells depleted of Pbl by RNAi and in spermatocyte-specific pbl alleles (Zavortink, 2005).

Pbl and Tum may exert these effects on central spindle microtubule organisation by directly affecting the bundling activity and higher order structure of centralspindlin. Alternatively, they may act indirectly, influencing centralspindlin function by regulating the local concentration of active GTPases, a possibility that is supported by the failure of GAP-specific mutations to form or maintain a central spindle. However, the possibility cannot be ruled out that the TumDeltaPbl, TumDeltaEIE and TumDeltaYRL (the latter two with a deletions in the gap domain) constructs delete critical amino acid residues required for centralspindlin functions that are unrelated to GTPase regulation (Zavortink, 2005).

Although the central spindle is perturbed in the absence of a Tum-Pbl interaction or if Tum GAP activity is altered, a subpopulation of microtubules still contacts the cell cortex at the equator and both TumDeltaPbl and TumDeltaYRL can accumulate there, highlighting one difference between this microtubule population and the central spindle. Specific subpopulations of microtubules are beginning to be identified during anaphase and there is some evidence that a subpopulation of microtubules is stabilised at the equatorial cortex. Although a number of studies have provided links between Rho family GTPases and the stabilisation of microtubule cortex and microtubule-kinetochore interactions in mammalian cells, the presence of TumDeltaYRL at the ends of microtubules at the cell equator indicates that if Tum plays a role in the stabilisation of these microtubules at the furrow site it can do so in the absence of GAP activity (Zavortink, 2005).

Experiments with two different GAP deletions demonstrate that GAP activity is required for cytokinesis in the ectoderm. The DeltaEIE deletion removes three amino acids at the end of the 'A helix', which positions the catalytic, or finger loop containing the essential arginine (Arg417 in Tum). In vitro analysis of a similar deletion in n-chimaerin eliminates all GAP activity but results in a protein with higher affinity for GTPase than a wild-type protein. An equivalent deletion in Cdc42GAP also lacks GAP activity but, by several biochemical measures, has similar stability and structure to the wild-type protein. The YRL deletion removes the essential catalytic arginine and the two amino acids around it; in n-chimaerin a similar deletion eliminates all GAP activity. The localisation of these two proteins during mitosis in tum mutant cells differs. The EIE deletion seems to affect the behaviour of Tum more severely, resulting in diffuse localisation of the protein during anaphase, whereas TumDeltaYRL protein reaches the cell equator at anaphase but no cytokinetic constriction occurs. Irrespective of these differences, neither GAP-deficient form of Tum is capable of rescuing the tum cytokinetic defect (Zavortink, 2005).

These results differ from a recent analysis of Tum function in the larval nervous system, where Arg417 substituted Tum was able to rescue cytokinesis in larval neuroblasts. A different balance of cytokinetic mechanisms that contributes to cytokinesis may have evolved to meet the special requirements for asymmetric cell divisions in the CNS or cell divisions in an epithelial sheet. It is interesting to note, however, that in vitro studies in which the GAP DeltaEIE or DeltaYRL triplet mutations were compared to single arginine substitution mutants, the single arginine mutations always have significant GTPase-activating ability remaining. Indeed structural and biochemical studies of several GAP domains have emphasised that stabilisation of the switch 1 and switch 2 loops of bound GTPase by the GAP protein contributes significantly to GAP activity, suggesting the possibility that Arg417-substituted Tum may still have sufficient GAP activity to support cytokinesis, at least in some cell types (Zavortink, 2005).

Understanding the role of Tum and its orthologs, Cyk-4 and MgcRacGAP, in cytokinesis is complicated by conflicting evidence from different experimental systems. In contrast to the current observations, C. elegans eggs and mammalian tissue culture cells in which centralspindlin members are depleted, initiate cytokinesis, but the furrow regresses. The predominant aberrant phenotype produced by expression of GAP deletion mutants in the current experiments was a cell arrested before a furrow was evident, demonstrating that Tum is needed early in furrow formation. Some apparent late-stage cytokinesis failures were observed in embryos expressing TumDeltaYRL like those seen in mammalian cells, indicating that there is a second critical phase for Tum GAP activity in cytokinesis. It is suggested that late-stage defects may reveal an ancestral function of Tum and its homologs, with an earlier cytokinetic function adopted in Drosophila. There is a growing consensus that some of the differing observations between cell types and species reflect real differences in the way cells perform cytokinesis, rather than differences in the efficacy of RNAi, the perdurance of proteins, redundancy of protein functions or other experimental variables, but this issue remains unresolved (Zavortink, 2005).

Mammalian Tum, MgcRacGAP, exhibits several functions not seen in the current study. Expression of a MgcRacGAP GAP-defective mutant protein in mammalian cells affects chromosome attachment to the spindle at prometaphase via regulation of Cdc42, generating cells arrested in prometaphase or cells with micronuclei. Micronucleation was not seen in the current in this study, but multipolar anaphase cells were seen in tum mutants, indicating that Tum-deficient cells that have failed cytokinesis can transit the cell cycle and successfully enter anaphase again, suggesting that Tum does not have a critical role in prometaphase in these cells (Zavortink, 2005).

Expression of a MgcRacGAP GAP-defective protein also affects the cell cortex, causing blebbing during anaphase in mammalian cells. Blebbing in both Dictylostelium and mammalian cells is suppressed by substrate attachment and it is suggested that this phenotype, if it exists in Drosophila embryos, could be suppressed in cells in an epithelial sheet. All previous studies of Tum function in flies have noted non-cytokinetic functions revealed as aberrations of wingless signalling, EGFR signalling and axon migration, some of which might be indirect results of perturbation of cortical organisation (Zavortink, 2005).

Tum protein is required for the localisation of all cytokinetic components tested so far, including Pbl and Dia, two components that mark the earliest events in contractile ring formation. Disruption of the Pbl-interacting domain of Tum leaves centralspindlin at the cell equator, on cortically associated, bundled microtubules that are unable to induce furrowing, supporting the model for Tum-directed positioning of Pbl and, consequently, the cytokinetic furrow in Drosophila cells. A Tum protein with a defective GAP domain also arrives at the equatorial cortex at the ends of microtubules and remains there, but furrows do not form, demonstrating that Tum GAP activity is required at this early stage of cytokinesis. These results demonstrate the critical role Tum plays in initiation of cytokinetic furrowing in Drosophila cells and provides further evidence for the importance of the Tum-Pbl interaction in this process (Zavortink, 2005).

Expression of the Rho-GEF Pebble is regulated by the UBE3A E3 ubiquitin ligase

Genetic tools available in Drosophila have been applied to identify candidate substrates of the UBE3A ubiquitin ligase (See Drosophila Ube3a), the gene responsible for Angelman syndrome (AS). Human UBE3A was expressed in Drosophila heads to identify proteins differentially regulated in UBE3A-expressing versus wild-type extracts. Using two-dimensional gel and MALDI-TOF analysis, 20 proteins were detected that were differentially regulated by over-expression of human UBE3A in Drosophila heads. One protein responsive to UBE3A was the Rho-GEF Pebble (Pbl). Three lines of evidence are presented suggesting that UBE3A regulates Pbl. First, genetic evidence is shown that UBE3A and the Drosophila de-ubiquitinase Fat facets (Faf) exert opposing effects on Pbl function. Secondly, it was found that both Pbl and ECT2, the mammalian orthologue of Pbl called epithelial cell transforming sequence 2 oncogene, physically interact with their respective ubiquitin E3 ligases. Finally, it was shown that Ect2 expression is regulated by Ube3a in mouse neurons, since the pattern of Ect2 expression is dramatically altered in the hippocampus and cerebellum of Ube3a null mice. These results suggest that an orthologous UBE3A post-translational regulatory pathway regulates neuronal outgrowth in the mammalian brain and that dysregulation of this pathway may result in neurological phenotypes including AS and possibly other autism spectrum disorders (Reiter, 2006).

Identifying neurologically relevant substrates of the ubiquitin E3 ligase UBE3A is essential to understand the phenotypes associated with AS. However, the results from this study may also have implications for other learning and behavior disorders. For example, maternally derived interstitial duplication of the 15q11-q13 region, which includes the UBE3A gene, consistently results in an ASD phenotype. Although there have been rare cases of paternally inherited 15q11-q13 that result in developmental impairments there is an overwhelming preference for maternal inheritance of this duplication. This implies that the UBE3A gene, which is maternally imprinted in both the hippocampus and cerebellum, is likely to be the key gene in this region responsible for the ASD phenotype. It is proposed, therefore, that at least a subset of idiopathic autism cases may be the result of dysregulation of UBE3A substrates. It may be possible to address this hypothesis by performing association studies on UBE3A substrates like ECT2 in autism families (Reiter, 2006).

ECT2 is the first candidate substrate of UBE3A with an obvious relevance to the neurological phenotypes observed in AS and ASD patients. While the dysregulation of UBE3A substrates like ECT2 in the hippocampus may explain the general learning and behaviour defects observed in both AS and ASD patients, the findings of a Purkinje cell phenotype may provide yet another link between AS and ASD. For example, AS patients exhibit ataxia and motor control problems, which could be explained by the dysregulation of ECT2 and/or other UBE3A substrates in cerebellum. Similarly, a strong correlation has been found between ASD and non-progressive congenital ataxia, whereas a link has been found between sensory-motor deficits and ASD. Perhaps, more telling in terms of understanding ASD pathology is the possibility that cerebellar defects may explain some of the emotion recognition and expressive language problems observed in ASD individuals (Reiter, 2006).

It is hypothesized that UBE3A may play a role in regulating growth of neuronal processes or synapse formation through the degradation or cellular localization of various proteins, such as Pbl/ECT2. The observation that the intracellular distribution of Ect2 is controlled by Ube3a parallels previous studies in which it was observed that Ect2 undergoes a cell cycle-dependent redistribution from the nucleus to cytoplasm, which is controlled by N-terminal sequences distinct from the Rho-GEF domain. Furthermore, mutations in pbl have also been shown to adversely affect neuronal outgrowth in post-mitotic cells. Thus, gross dysregulation of Pbl may lead to defects in neuronal pathfinding and/or synaptogenesis. Perhaps, UBE3A regulates the sub-cellular localization of ECT2 in post-mitotic neurons to ensure that ECT2 is delivered to the tips of growing axons or dendrites only under appropriate conditions. Given the critical role that Pbl and the Rho/Rac/Cdc42 system plays in axonal navigation and synapse formation in Drosophila, it seems highly likely that the gross dysregulation of this exquisitely dosage sensitive regulator in the hippocampus and cerebellum of Ube3a null mice would result in aberrant neuronal development, connectivity, or function. Such primary phenotypes in turn may underlie part of the observed learning defects and central nervous system features of this murine model for AS. These data are also consistent with growing evidence that the ubiquitin pathway is a key regulator of synaptic growth/stabilization and function (Reiter, 2006).

It has been reported that activity of ECT2 during G2/M phase of the cell cycle is regulated by phosphorylation, however, the data provides the first evidence that ECT2 may also be regulated through the ubiquitin proteasome system via its interaction with UBE3A. One unresolved question is whether ubiquitination of Ect2 would act primarily by marking this protein for degradation, modulating its function or cellular distribution, or whether it acts in both of these capacities. Interestingly, ubiquitination has been implicated in the regulation of both cellular trafficking and intercellular signalling in addition to protein stability. These recent observations are intriguing in light of the results that cellular distribution of Ect2 was altered in response to deletion of Ube3a in the murine brain. However, it is still not clear whether the levels of Ect2 protein per cell increase substantially overall in Ube3a–/– brains since in specific regions, such as the cerebellum or hippocampus, there appears to be substantial redistribution of Ect2 protein into regions in which Ect2 is not detectable in wild-type littermates. Therefore, it is possible that the primary defect in Ect2 regulation in these mice is the cellular relocalization of the UBE3A candidate substrate from the perinuclear region of the cell body to axonal or dendritic processes, rather than control of total protein levels. Further investigation of the role of ubiquitination in regulating Ect2 stability, activity and subcellular localization will be necessary in order to discriminate between these possible mechanisms (Reiter, 2006).

In summary, the combined approach that was taken, which exploits the strengths of both Drosophila and mouse models, strongly suggests that Pbl/ECT2 is a direct substrate of the ubiquitin ligase UBE3A and that ECT2 is the most compelling putative substrate identified to date that could be relevant to neurological disorders. Given that increased levels of UBE3A have also been implicated in the pathogenesis of ASD, continued identification and characterization of the multiple substrates regulated by UBE3A in the brain could have far reaching clinical impact for the most common forms of learning defects in humans (Reiter, 2006).

Rho-dependent control of anillin behavior during cytokinesis

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

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

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

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

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

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

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

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

The effects of anillin RNAi on MRLCSqh-GFP localization were tested. MRLCSqh-GFP recruitment and furrow initiation appeared normal, but within a few minutes of initiation, furrows became laterally unstable and oscillated back and forth across the cell cortex, parallel to the spindle axis, in repeated cycles, each lasting ~1-2 min and eventually subsiding to yield binucleate cells after ~20 min. The phenotype was very similar to that reported for anillin RNAi in HeLa cells and represents a requirement for anillin at an earlier stage than previously noted in Drosophila. Thus, a conserved function of anillin is to maintain furrow positioning during ingression (Hickson, 2008).

In LatA, anillin RNAi did not prevent equatorial MRLCSqh-GFP recruitment, but instead of appearing as persistent linear structures distorting the cell surface, a more reticular and dynamic structure lacking cell surface protrusions was observed. Thus myosin II can localize independently of both anillin and F-actin but the filamentous appearance of myosin II in the presence of LatA requires anillin, indicating that anillin can influence myosin II behavior in the absence of F-actin, whereas myosin II appeared capable of influencing anillin behavior only in the presence of F-actin (Hickson, 2008).

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

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

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

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

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

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

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

Regulation of the Rac GTPase pathway by the multifunctional Rho GEF Pebble is essential for mesoderm migration in the Drosophila gastrula

The Drosophila guanine nucleotide exchange factor Pebble (Pbl) is essential for cytokinesis and cell migration during gastrulation. In dividing cells, Pbl promotes Rho1 activation at the cell cortex, leading to formation of the contractile actin-myosin ring. The role of Pbl in fibroblast growth factor-triggered mesoderm spreading during gastrulation is less well understood and its targets and subcellular localization are unknown. To address these issues, a domain-function study in the embryo was performed. Pbl is shown to be localized to the nucleus and the cell cortex in migrating mesoderm cells and it was found that, in addition to the PH domain, the conserved C-terminal tail of the protein is crucial for cortical localization. Moreover, the Rac pathway plays an essential role during mesoderm migration. Genetic and biochemical interactions indicate that during mesoderm migration, Pbl functions by activating a Rac-dependent pathway. Furthermore, gain-of-function and rescue experiments suggest an important regulatory role of the C-terminal tail of Pbl for the selective activation of Rho1-versus Rac-dependent pathways (van Impel, 2009).

The Rho GEF Pbl provides one of the few molecular links between the proximal FGF receptor signalling events and the regulation of cell shape changes. Loss-of-function phenotype of pbl mutants have shown that Pbl acts in a pathway downstream or in parallel to Htl-dependent MAP kinase activation. This study used genetics and biochemistry to determine the regulation of Pbl and its downstream Rho GTPase pathways in migrating cells. The data demonstrate that Pbl partially localizes to the cell cortex of mesoderm cells and functionally interacts with Rac GTPases in this process (van Impel, 2009).

The tandem DH-PH domain of Pbl are shown to be essential for cell migration and employs not only Rho1, but also the Rac pathway. Several lines of evidence strongly suggest that Pbl acts through Rac GTPases during mesoderm migration. The dominant rough eye phenotype induced by PblDH-PH is sensitive to gene doses of Rac GTPases. Expression of constitutively active or dominant-negative Rac1 but not Rho1 enhances the mesoderm phenotype in the hypomorphic pbl11D allele. Moreover, co-expression of Rac1, but not of Rho1, promotes the suppression of mesoderm migration defects by PblδBRCT in pbl-null mutants. In addition, biochemical data is provided that strongly suggest the Rac pathway as a direct target of Pbl (van Impel, 2009).

Pbl has previously been reported to localize to the nucleus in interphase cells. Nuclear localization was interpreted as a means of storing the protein until rapid release at mitosis. In cultured cells and C. elegans zygotes, homologues of Pbl localize at the cell cortex, e.g. cell junctions or the anterior cortex in the nematode zygote. This study detected functional Pbl-HA in the nucleus and the cytoplasm, including membrane protrusions. These data are consistent with the model that Pbl activates Rac GTPases at the cell cortex during cell migration (van Impel, 2009).

This study identified two domains, the conserved C-terminal tail and the PH domain, as candidates to mediate the association of Pbl with the cell cortex in interphase cells. The use of N-terminally deleted constructs facilitated these studies, because the respective proteins were excluded from the nucleus as they lack the NLS. Either domain alone is sufficient to localize to the cell cortex, and deletion studies suggest that both domains are crucial for cortical localization. It is proposed that the PH domain and the C-terminal tail might cooperate in localizing Pbl to the cell cortex. DH domain associated PH domains are essential for GEF function and are known to promote binding to specific membrane subdomains enriched in phosphoinositides. An attractive model therefore is that the PH domain provides specificity by targeting Pbl to membrane domains enriched for particular phospholipids, whereas the C-terminal tail functions in anchoring Pbl to the cell cortex. In addition, binding to phospholipids might promote the specific exchange activity of the tandem DH-PH domain, as described for other Dbl family GEFs (van Impel, 2009).

It is difficult to address the issue of whether cortical localization is important for the function of Pbl in mesoderm migration. The reduced rescuing capability of PblδC-term is consistent with a correlation of cortical localization through the C-terminal domain and the function of Pbl in cell migration. A more stringent experiment would involve the generation of a construct that lacks the PH and C-terminal domains for membrane association. However, as PH domains are essential for DH domain function in vivo, deletion of the PH domain will abolish activity in any case, as has been shown for the constitutively active DH-PH construct. Such an analysis would require a way to uncouple the activities of the PH domain that promote the exchange activity and membrane-phospholipid binding. It will therefore remain important to determine whether the function of the PH domain involves its interaction with lipid substrates or directly promotes the activity of the DH domain in migrating cells (van Impel, 2009).

The inhibition of invagination and cytokinesis by PblδNterm is probably caused by disruption of the local activation of Rho1 at the cell cortex. During invagination and cytokinesis, the Rho1 pathway is activated locally: either 1) in the apical domain of the mesoderm cells to trigger apical constriction or 2) at the cell equator of the dividing cell to promote assembly of the contractile ring. Since PblδNterm strongly accumulates at the cortex in a non-polarized fashion, it might activate Rho1 ectopically throughout the cell cortex and thereby overriding any polarizing cues for local activation (van Impel, 2009).

The dramatic differences in the overexpression phenotypes of PblDH-PH or PblδNterm suggest an important function of the C-terminal tail in controlling the biochemical activities of the tandem DH-PH domain. Strikingly, PblδNterm genetically interacts with Rho1, but not with Rac GTPases, supporting the idea that the C-terminus promotes the exchange activity towards Rho1. It is proposed that in the mesoderm cells this activity of the C-terminal domain is antagonized to activate the Rac rather than to the Rho1 pathway. In the presence of the NLS and PEST motifs, the cytoplasmic levels of Pbl are low and allow for this regulation to occur, whereas the oncogenic forms lacking these motifs are present in the cytoplasm at high levels and might escape regulation. Thus, constructs that lack the C-terminal tail promote interaction with Rac and rescue Rac-dependent mesoderm migration. This model is also supported by the observation that the C-terminal domain is essential for Rho1 activation, but not for Pbl localization in dividing cells. The same construct, PblδC-term, is still able to rescue Rac-dependent migration defects. Thus, deletion of the C-terminal tail uncouples activation of Rho1-dependent from Rac-dependent processes and suggests that in the absence of the negative interaction with the C-terminal tail, the tandem DH-PH domain promotes activation of Rac (van Impel, 2009).

Although many receptor tyrosine kinases signal through Rho GTPases, only few FGF receptors have been reported to regulate Rho GEFs. One attractive model is that FGF signalling mediates post-translational modification of the C-terminal tail to trigger the switch in the differential interaction with Rho1 and Rac GTPases. The sequence of the C-terminal tail contains several conserved putative phosphorylation sites that might represent targets for FGF signalling. Interestingly, the exchange factor specificity of oncogenic ect2 for GTPase substrates depends on the C-terminal tail of the protein. Identification of proteins that interact with the C-terminal domain might shed light on its role in controlling selectivity for distinct GTPase pathways. Such studies will be important to advance our understanding of the mechanism of the transforming potential of Pbl, as well as its mechanism of action in cell polarity and cell migration (van Impel, 2009).

A small GTPase molecular switch regulates epigenetic centromere maintenance by stabilizing newly incorporated CENP-A

Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. This study shows that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, it was found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A (Lagana, 2010).

Epigenetic regulation of genome activity is critical during development and stem cell maintenance, and increasing amounts of evidence highlight its importance in cancers. However, mechanisms controlling epigenetic regulation during a single cell cycle are generally less well understood, compared with those involved in transcriptional programmes. Centromere specification is an epigenetic regulatory event that controls genome activity at singular chromosomal loci and occurs each cell cycle. Nucleosomes that contain CENP-A are thought to epigenetically define centromeres. During DNA replication, centromere identity is maintained by segregating CENP-A equally to the two daughter chromosomes. Before the subsequent S-phase, additional CENP-A must be incorporated at centromeres, thus propagating the centromere epigenetic mark. Critical to this cycle is maintenance of the proper amount of CENP-A; too little or too much CENP-A incorporation could result in either loss of centromere identity or errors in chromosome segregation. This study describes a mechanism to ensure maintenance of the proper CENP-A levels during the cell cycle regulated by a Rho family small GTPase molecular switch (Lagana, 2010).

Proteomics and quantitative imaging assays were used to identify a previously unknown step in centromere maintenance. MgcRacGAP, together with the GEF ECT2, and their cognate small GTPase Cdc42 (or possibly Rac) specifically maintain CENP-A at centromeres. MgcRacGAP localization to centromeres at the end of G1 is incongruous with a role in CENP-A loading and strongly suggests that MgcRacGAP acts in maintenance and not licensing or loading of CENP-A. Pulse-chase analysis revealed that MgcRacGAP is required specifically for maintenance of newly incorporated CENP-A as old CENP-A from the previous cell cycle was present at normal levels at centromeres. Reciprocal immunoprecipitation of MgcRacGAP did not isolate HsKNL2, probably because of a large excess of MgcRacGAP bound to other known interacting proteins in the cytoplasm (data not shown). These results support the conclusion that a minor subset of MgcRacGAP is bound to HsKNL2 for a brief period each cell cycle and imply that non-overlapping MgcRacGAP-containing protein complexes function in cells. Overall, this work defines a new event in epigenetic centromere regulation and reveals its control by a small GTPase molecular switch (Lagana, 2010).

A model is proposed wherein the HsKNL2–Mis18 complex licenses centromeres for loading of new CENP-A by the combined activities of HJURP and CAF1. After loading (approximately 8–12 h after anaphase onset), HsKNL2–Mis18 recruits Cdc42. The activity of Cdc42 is required for preservation of newly incorporated CENP-A and thus finalizes centromere repopulation. Cdc42 activity requires GTPase cycling facilitated by MgcRacGAP and the GEF ECT2. The results predict that newly incorporated CENP-A is distinct from CENP-A remaining from the previous cell cycle and can be recognized and removed. It is proposed that Cdc42 activity modifies (by either adding or removing a mark on) newly incorporated CENP-A, rendering it identical to old CENP-A. The manifestation of this mark could be any distinguishing modification, including but not limited to, recruitment of an additional protein, conformational change of the CENP-A nucleosome, or any of a range of post-translational modifications. New CENP-A that is not modified would be recognized as erroneously incorporated and removed from chromatin during a late-G1 surveillance step, or during DNA replication (Lagana, 2010).

In budding yeast, excess CENP-A (CSE-4) mislocalized to the chromosome arms is removed and selectively degraded through a proteasome-based mechanism. If this mechanism is conserved in human cells, it is expected to be less stringent, as overexpressed CENP-A localizes diffusely to chromosome arms without causing obvious defects in cell division. Alternatively or additionally, centromere maintenance could involve the chromatin remodelling protein RSF-1, which is required for CENP-A nucleosome stability. However, because RSF-1 is proposed to function in mid-G1 before MgcRacGAP and Cdc42 localize to centromeres, it is unlikely to be the downstream target of small GTPase activity at centromeres (Perpelescu, 2009). Regardless of the removal mechanism, it is proposed that a GTPase switch is spatially and temporally restricted through regulated localization to centromeres precisely after CENP-A doubling to promote the removal of spurious CENP-A (either excess at centromeres, or outside true centromere loci). By restricting centromere size, this 'quality control' mechanism helps to ensure proper centromere function and kinetochore assembly, thus preventing aneuploidy. Furthermore, it is possible that this mechanistic theme will apply to other epigenetic events that contribute to genomic regulation (Lagana, 2010).

Regulation of Drosophila mesoderm migration by phosphoinositides and the PH domain of the Rho GTP exchange factor Pebble

The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants, the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B was identified. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, PtdInsP-binding of Pbl was examined, along with the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(DeltaPH) mutants. Taken together, these results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration (Murray, 2012).

pebble: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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