DRacGAP gives rise to a unique transcript of 2.3 kb present throughout development. The expression pattern of DRacGAP is highly dynamic. It is ubiquitous during the initial stages of embryogenesis, suggesting maternal expression, and becomes restricted, after germ band retraction, to the central and peripheral nervous system. In the early second instar wing imaginal disc, DRacGAP expression occurs mostly in the presumptive wing region . Afterwards, DRacGAP mRNA accumulation is widespread in all imaginal discs. At late third instar DRacGAP is expressed in the eye-antenna disc in two stripes of cells located at, and ahead of, the morphogenetic furrow and in several rings of cells in the presumptive antenna. In the wing disc, DRacGAP mRNA accumulates in the presumptive interveins, where it persists in pupae, being stronger at the vein/intervein boundaries (Sotillos, 2000).

Effects of Mutation or Deletion

Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac; Genetic interactions of Citron kinase with RacGAP50C

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).

Genetic evidence that Rac genes dominantly suppress the sti RNAi phenotype suggests the possibility that Rac GTPases may play an inhibitory role during cytokinesis. Loss of function studies in Drosophila and other animals have not previously implicated Rac in cytokinesis. These studies, however, could only indicate that Rac GTPases are not essential for this process, but do not rule out the possibility that the activity of these GTPases needs to be down-regulated during cytokinesis. Identification of a Rac repressor that is essential for cytokinesis would provide further evidence for such an inhibitory role. The Drosophila RacGAP50C has been implicated in cytokinesis, but its target GTPase has not been clearly identified yet. Thus, whether RacGAP50C could inhibit Rac activity was investigated in vivo. Expression of RacGAP50C dsRNA in developing imaginal tissues led to the formation of multinucleate cells and its RNAi-mediated silencing during eye development resulted in a significant reduction of the adult eye. These eyes contained large and disorganized ommatidia, very similar to sti RNAi mutants. A chromosome carrying mutations in all three Rac genes, (triRac), dominantly suppressed this phenotype, whereas Rho mutations acted as strong enhancers. These observations are consistent with RacGAP50C inhibiting Rac and promoting Rho activity. Finally, the strongest sti mutation available, sti3, failed to significantly enhance or suppress the RacGAP50C eye phenotype. This result suggests that sti does not function in a pathway between Rho and RacGAP50C (D'Avino, 2004).

Genetic interaction experiments indicate that genes function in the same biological process, but not necessarily in the same pathway. However, because in the current experiments the suppression of sti and RacGAP50C RNAi phenotypes by Rac mutations is dominant, it is conceivable that they act in the same, rather than in a parallel, pathway. In this scenario, RacGAP50C might be expected to inhibit Rac through a direct protein-protein interaction mechanism. In contrast, two opposite explanations exist for the relationship between sti and Rac: either Rac represses Sti or Sti inhibits Rac. Under the second hypothesis, however, Rac inhibition by Sti should be indirect, since GTPase activity is generally regulated by cofactors (i.e., GEFs and GAPs) and not by phosphorylation. Because the experiments indicate that RacGAP50C represses Rac activity, the second hypothesis implicates a linear pathway in which Rho activates Sti, which in turn activates RacGAP50C, which ultimately inhibits Rac GTPases. This is in contrast with findings that sti does not enhance the RacGAP50C RNAi phenotype whereas Rho1 does, suggesting that these factors do not function in a simple linear pathway. For these reasons the alternative model is favored. In this model, RacGAP50C plays a key role by inhibiting Rac and promoting Rho activity, probably through its interaction with the PBL/ECT2 GEF. The two GTPases then antagonistically regulate Sti activity. This regulation could be direct, since both GTPases bind CIT-K in vitro, but the current genetic data do not exclude the possibility that Rac regulates Sti through one or more intermediates. The hypothesis that Rac inhibits the function of proteins that are activated by Sti cannot be excluded. Further functional and structural analysis of Sti will be required to understand the molecular mechanisms that control the activity of this kinase. One implication of the current model is that even slight variations in the equilibrium of the factors could easily alter the dynamics of contractile ring components during cytokinesis. For example, RacGAP50C activation could promote RhoA activity and consequently actomyosin filament assembly and contraction. Conversely, RacGAP50C inhibition would both down-regulate Rho and activate Rac to repress Sti thereby promoting filament disassembly (D'Avino, 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).

DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc

A novel Drosophila gene, DRacGAP/RacGAP50C has been identified that behaves as a negative regulator of Rho-family GTPases Rac1 and Cdc42. Reduced function of DRacGAP or increased expression of Rac1 in the wing imaginal disc cause similar effects on vein and sensory organ development and cell proliferation. These effects result from enhanced activity of the EGFR/Ras signalling pathway. In the wing disc, Rac1 enhances EGFR/Ras-dependent activation of MAP Kinase in the prospective veins. Interestingly, DRacGAP expression is negatively regulated by the EGFR/Ras pathway in these regions. During vein formation, local DRacGAP repression would ensure maximal activity of Rac and, in turn, of Ras pathways in vein territories. Additionally, maximal expression of DRacGAP at the vein/intervein boundaries would help to refine the width of the veins. Hence, control of DRacGAP expression by the EGFR/Ras pathway is a previously undescribed feedback mechanism modulating the intensity and/or duration of its signalling during Drosophila development (Sotillos, 2000).

Evidence is presented for the cooperation of Rac and Ras signaling pathways in the context of a whole organism. A Drosophila gene, DRacGAP, is described that encodes a putative GAP for Rac and Cdc42 GTPases. Both DRacGAP and DRac1 are involved in the control of cell proliferation. Moreover, reduced activity of DRacGAP or overexpression of DRac1 in the wing imaginal disc cause similar defects: widening of veins, development of extra sensory organs (SOs), apoptosis and the appearance of enlarged cells that differentiate multiple hairs with abnormal polarity. These phenotypes result from DRac1 enhancement of epidermal growth factor receptor (Egfr)/Ras signaling. The Egfr/Ras pathway pathway, which operates through activation of the Ras/Raf/MEK/MAP kinase cascade controls multiple developmental processes and is accurately regulated. Indeed, this pathway controls the expression of its own negative and positive regulators. Interestingly, expression of DRacGAP is repressed by Egfr/Ras signaling in the prospective veins and accumulates at the vein/intervein boundaries. These results suggest that control of DRacGAP expression by the Egfr/Ras pathway provides a new mechanism to modulate the intensity of this pathway during Drosophila development (Sotillos, 2000).

Overexpression of DN DRacGAP or Rac1 causes vein enlargement and the appearance of extra SOs, two structures requiring Egfr/Ras/Raf/MAPK activity. Since Rac and Ras pathways cooperate in mammalian cells in the control of cell proliferation, an investigation was carried out to see whether the phenotypes associated with increased Rac signaling could be due to overactivity of the Ras pathway. This appears to be the case, since a reduction in Egfr signaling (by expression of DN Raf) reduces vein, wing notching and large cell phenotypes. Similarly, when levels of the Egfr activators rhomboid and vein (vn) are decreased, the wing notching associated with DN DRacGAP expresson is substantially corrected. In contrast, activation of Egfr signaling enhances the mutant phenotype of DN DRacGAP flies. Thus, although flies heterozygous for mutant argos, a repressor ligand of Egfr, are phenotypically wild type, its combination with DN DRacGAP significatively increases the number of campaniform sensilla of DN DRacGAP flies. These results further suggest that the efficacy of the Egfr pathway is enhanced by increased Rac activity. Rac signaling ultimately activates MAPK since expression of DN DRacGAP widens the domains of accumulation of dp-ERK in the presumptive veins and spreads them into the interveins. This effect is enhanced by coexpression of Rac1 (Sotillos, 2000).

Expression of DRacGAP appears to be decreased in the domains of Egfr activation. This is most apparent in the notum region of second instar wing disc, where the Egfr activator vein is expressed, and in the presumptive veins of third instar wing disc, territories of maximal Egfr signaling. This observation suggests that the Egfr pathway may repress the expression of DRacGAP. In agreement with this notion, expression of DRacGAP is either decreased or enhanced in cells ectopically expressing Ras V12 or argos in which the EFGR/Ras pathway is activated or repressed, respectively (Sotillos, 2000).

The enlarged cell size of wing cells overexpressing Rac and the small size of wings with reduced Rac function indicate a role for Rac in cell proliferation control. In mammalian cells, Ras and Rac pathways cooperate in stimulating cell proliferation. Similarly, Drosophila Rac1 and Ras also appear to cooperate in this process since the reduced size of the wings of DN Raf-expressing flies is largely normalized by reduction of DRacGAP function or by overactivity of Rac1. The induction of cell death by DN DRacGAP, where Rac activity is upregulated, is in apparent contradiction with these results. However, note that in mammalian cells, quantitative variations in the level of Ras signaling can cause very different effects. Thus, instead of cell proliferation, high levels of Ras signaling may induce cell cycle arrest at G1 and apoptosis as part of a cell self protective mechanism. In that situation, G1 arrest is caused by Raf-dependent induction of p21 WAF1/CIP1 expression, which inhibits Cdk activity and indirectly represses CycE transcription. The situation appears to be very similar in Drosophila. Cell death of DN DRacGAP flies could be attributed to their arrest at the G1 stage, since the phenotype is rescued by expression of CycE. Moreover, the accumulation of p21 causes apoptosis, enlargement of cell size and polarity defects, phenotypes that are corrected by expression of CycE. Accordingly, it is hypothesised that induction of cell cycle arrest and apoptosis by overactivity of the Rac pathway could be a consequence of increased Ras signaling. Rac would potentiate the reduced Raf signaling occurring in DN Raf flies, allowing wing disc cells to proliferate, but in the presence of wild-type Raf, overactivity of Rac would enhance Raf signal to such a high level as to induce p21 expression and ultimately, apoptosis. This interpretation is supported by the partial rescue of the wing notching phenotype of DN DRacGAP flies in a ve;vn heterozygous background and in DN Raf flies where Egfr/Ras signaling is reduced (Sotillos, 2000).

Interestingly, in the Drosophila wing disc, DRacGAP transcription is repressed by the Egfr/Ras pathway. Activity of this pathway is finely tuned by its control of the expression of its own inhibitors and activators. The results indicate that Ras signaling can self-stimulate through activation of the Rac pathway by repression of DRacGAP. During vein formation, the Egfr/Ras pathway, once it has attained a certain threshold, should repress expression of DRacGAP in the prospective vein regions, thus ensuring maximal activity of Rac and, in turn, of Ras pathways in these territories of the imaginal wing disc, which should trigger vein differentiation. In contrast, maximal expression of DRacGAP at the vein/intervein boundaries should locally decrease Rac and Ras signaling, and in collaboration with Notch and Dpp pathways help to refine the final width of the veins. Hence, this regulatory loop is another feedback mechanism modulating the activity of the Egfr/Ras pathway during Drosophila development (Sotillos, 2000).

RacGap50C negatively regulates wingless pathway activity during Drosophila embryonic development

In a genetic screen to identify mutations that suppress or enhance the mutant phenotype of the wg temperature-sensitive allele wgIL114, two EMS-induced mutations were isolated that subtly modify the wgIL114 mutant cuticle pattern. These modifier mutations, AR2 and DH15, fail to complement each other and thus identify a single complementation group, linked to wg on the second chromosome. These nonsense mutations are shown to disrupting the RacGap50C locus. The two alleles produce identical phenotypes. Both mutations show no increase in severity when placed in trans to a deficiency for the region and so are likely to represent loss-of-function alleles (Jones, 2005).

wgIL114 embryos at the restrictive temperature are much smaller than wild-type embryos and secrete the 'lawn of denticles' cuticle pattern typical of low pathway activity. The modifier lines AR2 and DH15 were found to alter the wgIL114 phenotype in a similar way, increasing the spacing between denticles and the overall body size of the doubly mutant embryos. This phenotype indicates that the modifier mutations considerably reduce the severity of the wg loss-of-function phenotype, resulting in a larger, healthier embryo even though the cuticle pattern is only subtly altered. Both AR2 and DH15 also modify other wg alleles, including wgCX4, an RNA null allele of wg. The cuticle pattern defects of weak wg alleles are more dramatically suppressed by the AR2 and DH15 mutations. The wgPE2 hypomorphic allele produces a protein with a lower affinity for the receptor complex. wgPE2 homozygous mutants show segmental denticle diversity, but little or no naked cuticle separating the belts. The wgPE2 AR2 doubly homozygous mutants show an increase in the amount of naked cuticle, indicating that some Wg signaling is restored (Jones, 2005).

In an otherwise wild-type background, the AR2 and DH15 mutations are recessive lethal, and homozygous embryos show an excess specification of naked cuticle at the expense of denticles. Wild-type embryos secrete an average of 5.95 ± 0.15 rows of denticles/belt in abdominal segments 3-6. In contrast, AR2 mutant embryos secrete an average of only 3.95 ± 0.64 rows of denticles/belt. The mutant denticles also differ morphologically from the wild type. Wild-type denticles show a wide range in size, with those in row 5 being largest. The AR2 and DH15 mutant denticle belts contain fewer of these large denticles: most denticles observed are similar in size to the smaller denticles normally found in rows 1-4 of the wild-type belt pattern. The slight suppression of wg mutant phenotypes and the ectopic specification of naked cuticle are consistent with an increase in Wg pathway activity, suggesting that the AR2 and DH15 mutations might disrupt a negative regulator of the pathway (Jones, 2005). RacGap50C interacts genetically with nkd and appears to act at the same level or downstream of Axin in the control of Arm stabilization. The data indicate that RacGap50C probably does not act through Rac1 to negatively regulate Wg activity, nor are other GTPases likely to be involved in this aspect of epidermal patterning since the cuticle defects of mutant embryos can be rescued by a form of RacGap50C that lacks catalytic residues in the GTPase-activating domain. Moreover, previous work shows that other Rho family members are unlikely to be involved in Wg-mediated patterning. Overexpressing either constitutively active or dominant-negative Rho, Rac, or cdc42 transgenes disrupts dorsal closure but does not appear to affect ventral patterning. Loss of maternal Rho activity has been found to alter embryonic segmental pattern, but this is due to an early effect on establishing segmentation gene expression patterns. Ras activation through the EGF signaling cascade has been found to affect epidermal patterning, but in a way that counteracts Wg signaling. Thus a GTPase-activating protein would be expected to positively influence Wg-mediated patterning if it acted through Ras, rather than the negative influence observed for RacGap50C (Jones, 2005).

For these reasons, it is believed that the role of RacGap50C in Wg signaling may instead parallel its role in cytokinesis, where it seems to function primarily as an adaptor molecule. RacGap50C was identified by the Saint laboratory through a yeast two-hybrid screen for molecules that interact with pebble, a RhoGEF that is essential for cytokinesis (Somers, 2003). Subsequently, it was shown that RacGap50C protein also binds to Pavarotti, a kinesin-like molecule, thus forming a bridge between the microtubule and actin cytoskeletons. This adaptor function appears critical for the proper positioning of the acto-myosin contractile ring at the end of mitosis (Jones, 2005).

One could imagine that a structural role for RacGap50C in linking the microtubule and actin cytoskeletons might be relevant to its regulation of Wg pathway activity. Apc2, a scaffolding molecule that is an essential component of the destruction complex, is known to interact with both microtubules and the cortical actin cytoskeleton. Mutations that disrupt the cortical localization of Apc2 compromise function of the destruction complex, suggesting that subcellular localization of the complex may be critical. Furthermore, recent work demonstrates that Axin, another scaffolding component in the complex, changes its subcellular localization in response to Wg signaling and is consequently degraded. Thus the positioning of the destruction complex and/or some of its subunits may play a critical role in regulating its Arm-degrading activity. RacGap50C may be involved directly in linking the destruction complex to the cell cortex to promote its proper activity or may restrict movement of Axin to the cortex for its Wg-mediated destruction. In either case, loss of RacGap50C function would reduce the normal degradation of Arm and thereby cause ectopic Wg pathway activity (Jones, 2005).

Another possible explanation for RacGap50C's effects on Wg pathway activity would not require direct interaction with any pathway component. Rather, some coordination between the microtubule and actin cytoskeletons may be generally required for many different cellular processes. The loss of this adaptor molecule would 'loosen' the connection between these two filamentous networks and compromise many cellular events indirectly. It may be that cytokinesis and Wg signal transduction are particularly sensitive to such perturbations or that they simply produce the earliest or most easily detected phenotypes in response to them. A general requirement for microtubule and actin network coordination in destruction complex function could be of great importance in understanding oncogenic Wnt pathway activity. Current work on this problem focuses on distinguishing whether RacGap50C interacts specifically with the destruction complex or acts more generally by controlling cellular architecture (Jones, 2005).

Decreased RacGAP50C levels lead to defects in Nb proliferation and axon guidance

A genome-wide transgenic RNAi-based screen in Drosophila to identify RhoGAPs essential for neuronal morphogenesis identified two RhoGAPs that function in the development or maintenance of normal morphology of mushroom body (MB) neurons in the Drosophila brain (Billuart, 2001). The first, Drosophila p190 RhoGAP, is critical for inhibiting RhoA activity to maintain the stability of axon branches (Billuart, 2001). The second RhoGAP identified in the RNAi screen was RacGAP50C (Goldstein, 2005).

RacGAP50C is known for its function in cytokinesis. RacGAP50C and its homologues bind directly to a mitotic kinesin-like protein, named Pavarotti (Pav) in Drosophila, to form a complex known as centralspindlin (Mishima, 2002, Somers, 2003). Based on their in vitro behavior, the RhoGAP and kinesin form a heterotetrameric complex that bundles microtubules, and both are necessary for the formation of midzone antiparallel microtubules critical for cytokinesis (Mishima, 2002; Somers, 2003; Jantsch-Plunger, 2000). In vitro evidence indicates that the vertebrate and worm homologues of RacGAP50C, mgcRacGAP and CYK-4, have preferential GAP activity toward Rac1 and Cdc42 (Jantsch-Plunger, 2000; Toure, 1998; Kawashima, 2000), but, upon phosphorylation of the GAP domain, mgcRacGAP's activity is increased for RhoA (Minoshima, 2003). Recent work has begun to shed light on the function of the centralspindlin complex during cytokinesis (Mishima, 2004), as well as its potential function in regulating cellular polarity (Portereiko, 2004); however, whether this RhoGAP–kinesin complex also functions in postmitotic neurons is unclear. The expression of the mammalian homologue of RacGAP50C (mgcRacGAP) in postmitotic neurons (Van de Putte, 2001), the uncoordinated phenotype seen with temperature-sensitive alleles of the Caenorhabditis elegans homologue of RacGAP50C (Jantsch-Plunger, 2000), and the disruption of dendritic/axonal polarity with inhibition of the mammalian homologue of Pav in vitro (Yu, 2000) all hint at a postmitotic function for the RhoGAP-kinesin complex in neurons. Through the use of both RNAi and loss-of-function analysis, the function of RacGAP50C and Pav in neuronal development has been directly examined (Goldstein, 2005).

Intrinsic neurons of the Drosophila MB (referred to as MB neurons) were used as model neurons to study RacGAP50C. The MB is a center for olfactory learning and memory and represents an ideal structure to examine neuronal morphogenesis because its axonal projection pattern and development have been well characterized. MB neurons each have a single ventrally projecting process that extends dendrites close to the cell body in a spherical mass called the calyx. The main process continues through an axonal peduncle to reach its target region, where the axonal processes form distinct lobular regions segregated by neuronal type. The ~2,500 neurons of each MB hemisphere are generated from four MB Nbs that sequentially generate three subclasses of MB neurons. The earliest born gamma neurons have one major medially projecting axon branch that elaborates in the gamma lobe. The second-born alpha'/beta' neuron innervates two distinct dorsal and medial projecting lobes. Alpha/beta neurons are born last and, similarly, have a major dorsal and medial projecting axon branch that can be distinguished from alpha'/beta' neurons by its strong expression of the cell adhesion protein FasII. The GAL4 enhancer trap, GAL4-OK107, is strongly expressed in all three MB neuron subclasses and effectively drives RNAi expression, along with a cellular marker, mCD8::GFP, to simultaneously knock down gene expression and visualize the gross morphology of the MB complex. In addition, with MB neurons many different aspects of neural development can be examined, including Nb proliferation, dendrite morphogenesis, and axon growth, guidance, branching, and stability, all of which are regulated by Rho GTPases (Goldstein, 2005).

Knockdown of RacGAP50C in MB neurons results in reduction of cell number and axon overextension. The enlarged cells and reduction of neurons derived from the Nbs are reminiscent of cytokinesis defects seen in MB neurons that are homozygous mutant for small GTPase RhoA and is consistent with work identifying RacGAP50C and its homologues as a critical regulator of cytokinesis (Somers, 2003; Jantsch-Plunger, 2000; Toure, 1998; Kawashima, 2000; Minoshima, 2003). In addition to the cytokinetic phenotype, the normal axon trajectory is disrupted with RacGAP50C reduction. Dorsal projecting axon branches are normally confined within a morphologically distinct dorsal lobe that extends toward the dorsal surface of the brain. However, with reduced RacGAP50C, axons extend beyond the dorsal lobe and project toward the midline of the brain. Occasionally, processes also target incorrectly and extend ventromedially from the dendritic region of the MB. This phenotype suggests a previously unrecognized role for RacGAP50C activity in the postmitotic neuron: regulating neuronal projections (Goldstein, 2005).

To confirm the RNAi phenotype of RacGAP50C and expand phenotypic analysis, genetic loss-of-function mutants were sought. Because phenotypes caused by RNAi-based knockdown of gene activity are predicted to be enhanced by losing one copy of the corresponding endogenous gene, this strategy was used to look for candidate RacGAP50C alleles in the cytological position 50C region where RacGAP50C is mapped. Several candidates were tested from the MB axon guidance screen, as well as candidates from a dendritic development screen. It was found that heterozygosity of the tum mutant chromosome significantly enhances the rough eye RNAi phenotype caused by knockdown of RacGAP50C. The ability of candidate chromosomes to enhance the RacGAP50C RNAi phenotype in the MB was tested, and it was similarly found that only tum enhances the neuronal knockdown phenotype. These data suggests that tum is a strong candidate for the RacGAP50C gene (Goldstein, 2005).

To directly test this hypothesis, the RacGAP50C region of two alleles of tum (tum1 and tum347) was sequenced and it was found both alleles contain nonsense mutations. This key piece of evidence, combined with the genetic enhancement of RacGAP50C RNAi phenotype by tum mutation, the similarity of tum loss-of-function and RacGAP50C RNAi phenotypes, and the transgenic rescue of tum phenotypes by RacGAP50C genomic DNA, establishes unequivocally that RacGAP50C is encoded by tum. According to conventions of fly genetics, RacGAP50C is referred to as Tumbleweed (Tum). Because both tum alleles are nonsense mutations before the catalytic RhoGAP domain and the predicted diacyl glycerol-binding domain, both alleles are likely null (Goldstein, 2005).

To examine the role of Tum and its binding partner, Pav, in the dividing Nbs and in postmitotic neurons, MARCM clones were generated in newly hatched larvae. MB Nb clones generated at this stage produce all three neuronal types. Loss of Tum or Pav causes arrest of Nb proliferation and prevents the continued generation of later-born alpha '/beta' and alpha/beta neurons. The presence of enlarged cells is indicative of cytokinesis defects consistent with the Tum RNAi phenotype and the function of Tum and Pav in regulating cytokinesis (Goldstein, 2005).

Single-cell MB neuron clones that are homozygous mutant for tum or pav are grossly normal in their axonal and dendritic projections. Because of the severe proliferation phenotypes, it was not possible to generate Nb clones large enough to encompass later-born alpha '/beta' or alpha/beta MB neurons that project to the dorsal lobes. Therefore, the axon misrouting phenotypes due to tum knockdown by RNAi and loss-of-function phenotypes cannot be directly compared. However, axon overextension defects were observed in both Tum and Pav Nb clones expressing UAS-cDNA transgenes that partially rescue the cytokinetic phenotype, supporting the notion that optimal Tum activity is required for correct axon projection (Goldstein, 2005).

Because of the limited ability of the GAL4-dependent transgenes to rescue the Nb proliferation phenotype in MARCM clones, transgenic flies were generated by using genomic DNA encompassing the tum locus (gTum). One copy of WT gTum rescues the lethality of tum-/- and fully restores Nb proliferation as judged by increased cell number, the loss of enlarged cell bodies, and the generation of late-born neurons from homozygous mutant clones. By contrast, gTum transgenes with a deletion of the predicted Pav-binding region of Tum fail to rescue the proliferation phenotype associated with tum Nb clones, supporting the necessity of Pav binding for Tum function in cytokinesis. Introduction of a point mutation (gTumR417L) of the conserved arginine residue critical for GAP activity abolishes the ability of gTum to rescue the lethality of tum but, surprisingly, is able to largely rescue the Nb proliferation phenotype. More than 80% of Nb clones are able to generate the latest born neuron class, and 50% of Nb fully rescue cell-proliferation defects (Goldstein, 2005).

With the rescue of Nb proliferation, it is possible to examine a larger population of neurons for their axon targeting. Restoration of later born neuron types by gTumWT or gTumR417L in tum Nb clones allows for normal axon guidance of later born neuron types. Most medial projecting axons of the MB extend toward the midline and stop. A small fraction of WT Nb clones extend their axons, mostly the alpha/beta class, beyond the midline. With loss of Tum's RhoGAP activity, the later born neurons are twice as likely to extend beyond their normal target region in the medial lobe compared with control Nb clones, and the degree of overextension is more severe. Dorsal lobe overextension similar to the RNAi phenotypes is also occasionally seen. Five of the 12 Nb clones with axon overextensions exhibit full rescue of Nb proliferation phenotypes as judged by the lack of enlarged cells and the full innervation of the lobes targeted by the latest born neurons, alpha/beta lobes. This finding suggests that the axon phenotype of tum is not a secondary consequence of cytokinesis defects; rather, it reflects a role of tum in regulating axon extension in postmitotic neurons. In addition, regulation of axon extension appears to depend more on the GAP activity of Tum compared with regulation of cell proliferation (Goldstein, 2005).

In conclusion, this study shows that Tumblweed/RacGAP50C and its binding partner, Pavarotti, are both required for MB Nb proliferation, consistent with previously characterized functions of this protein complex in regulating cytokinesis in C. elegans, Drosophila, and mammals. Structure-function analysis suggests that Tum's binding to Pav, but not its GAP activity, is essential for its function in cytokinesis. Recent evidence has found that the Rho-specific guanine nucleotide exchange factor, Pebble, binds directly to Tum's coiled-coil domain (Somers, 2003). Pebble itself is critical in cytokinesis to activate RhoA and generate the contractile ring. It has been proposed that Pebble's association with Tum allows for its localization to the midzone to promote the initiation of cytokinesis (Somers, 2003). The function of the GAP domain of Tum in promoting cytokinesis has remained enigmatic. The GAP activity of the mammalian and C. elegans homologues of Tum have little or no RhoA-specific GAP activity but preferentially regulate Rac1 and Cdc42 activity. The finding that Tum with a mutation that disrupts the conserved arginine finger necessary for RhoGAP activity allows dividing Nb to generate >500 MB neurons demonstrates that the GAP activity of Tum is not essential for its function during cytokinesis. This observation supports the model that in cytokinesis, Tum acts simply as a scaffolding protein that brings Pebble to the midzone formed by the bundling of antiparallel microtubules through association of Tum with Pav (Goldstein, 2005).

Several lines of evidence are provided that in addition to the function of the Tum-Pav complex in cytokinesis, Tum and Pav also have a function in regulating morphogenesis of postmitotic neurons and that this function depends on the GAP activity of Tum. (1) RNAi knockdown of Tum results not only in defects of Nb proliferation, but also in axon extension beyond their normal boundary. (2) Introducing a tum transgene with a GAP domain mutation into tum-/- Nb clones largely restores normal proliferation but results in the overextension of its axons beyond their normal boundary. The inability to limit axon extension in the MB neuropil suggests a GAP-dependent role of Tum that is reminiscent of the neuronal function attributed to Tum through RNAi knockdown. (3) Tum and Pav regulate each other's subcellular localization, consistent with the notion that they form a complex in postmitotic neurons. (4) The same point mutation in the GAP domain of Tum abolishes its axon disruption activity in Tum/Pav coexpression experiments, consistent with the GAP activity being essential in regulating axon development (Goldstein, 2005).

Given the potential role of Tum as a GAP in regulating axon development, it is somewhat surprising that the protein should have a predominant nuclear localization when expressed in postmitotic neurons. The Pav coexpression experiments shed some light: It was found that accumulation of Tum in the cytoplasm leads to severe disruption of axon development. The phenotypes are highly reminiscent of loss-of-function mutant phenotypes for the Rac GTPases, where axons collect in a ball at the base of or misroute from the cell body region, suggesting that cytoplasmic accumulation of Tum down-regulates Rac activity. This observation is consistent with a previous report that RacGAP50C regulates wing disc development by regulating Drosophila Rac1 (Sotillos, 2000). In contrast, loss of RhoA or Cdc42 in MB neurons does not affect axon guidance. It is speculated that in postmitotic neurons, the distribution of Tum contributes to the spatial regulation of the Rac GTPase activity essential for proper morphogenesis. Because Rac GTPases are also essential for axonal growth, limited and localized cytoplasmic Tum could reduce the activity of Rac GTPases and hence restrict axon growth, consistent with the finding that complete loss of Tum results in axon overgrowth. As a kinesin shown to bind to and bundle microtubules, Pav could act to transport Tum along microtubules to modulate Rac activity in axon growth and guidance (Goldstein, 2005).

Both Tum and Pav function have been implicated in dendritic morphogenesis. tum was originally identified as a mutation that leads to tangled dendritic branches in the sensory neurons of the Drosophila embryo. Disruption of the mammalian form of Pav, CHO1/MKLP1, in postmitotic and differentiated neurons in culture led to the rearrangement of microtubule polarity and the subsequent loss of dendrites. No defects were observed in dendritic arborization or targeting in single-cell MARCM clones that are homozygous mutant for tum or pav in either the MB neurons or the second-order olfactory projection neurons. There are several possible explanations: (1) the dendritic defects seen in tum homozygous embryos may be a secondary consequence of cytokinesis defects or due to nonautonomous effects of tum in neighboring cells; (2) the Tum/Pav pathway may be redundant with other pathways in MB or projection neurons in regulating dendritic morphogenesis; (3) perdurance of Tum/Pav proteins in single-cell clones may be sufficient to allow proper dendritic morphogenesis. Future studies using neuronal types that have more elaborate and stereotyped dendritic trees may help distinguish these possibilities (Goldstein, 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).

Cell division requires a direct link between microtubule-bound RacGAP and Anillin in the contractile ring

The mitotic microtubule array plays two primary roles in cell division. It acts as a scaffold for the congression and separation of chromosomes, and it specifies and maintains the contractile-ring position. The current model for initiation of Drosophila and mammalian cytokinesis postulates that equatorial localization of a RhoGEF (Pbl/Ect2) by a microtubule-associated motor protein complex creates a band of activated RhoA, which subsequently recruits contractile-ring components such as actin, myosin, and Anillin. Equatorial microtubules are essential for continued constriction, but how they interact with the contractile apparatus is unknown. This study reports the first direct molecular link between the microtubule spindle and the actomyosin contractile ring. The spindle-associated component, RacGAP50C, which specifies the site of cleavage, interacts directly with Anillin, an actin and myosin binding protein found in the contractile ring. Both proteins depend on this interaction for their localization. In the absence of Anillin, the spindle-associated RacGAP loses its association with the equatorial cortex, and cytokinesis fails. These results account for the long-observed dependence of cytokinesis on the continual presence of microtubules at the cortex (Gregory, 2008).

Live imaging of tagged Anillin and RacGAP revealed that they accumulate rapidly at the equatorial cortex and, in the case of RacGAP, on the central spindle in early anaphase. At the onset of constriction, RacGAP was found in discrete short longitudinal stripes across the equator, which are the site of bundles of astral microtubules, as well as on interpolar microtubules. Anillin was not seen internally at any stage, being restricted to a cortical ring. The equatorial RacGAP was seen to abut with Anillin, although Anillin was also found between the stripes of RacGAP (Gregory, 2008).

To test whether the juxtaposition of RacGAP with Anillin reflected a molecular interaction, fluorescence resonance energy transfer (FRET) by acceptor photobleaching was used, which detects very tightly apposed proteins (<10 nm) and correlates well with high-affinity direct interaction. To test the assay system, it was shown that FRET could be detected between actin and Anillin, which are known to interact. A significant increase in Anillin signal is observed when the actin marker is photobleached, indicating that this known biochemical interaction can be detected in situ. Antibodies directed against Anillin and RacGAP were used to determine whether they would produce a similar FRET signal. Anillin also gave a significant FRET signal with RacGAP, suggesting that the two proteins are in extremely close proximity during cytokinesis. No significant difference in the RacGAP-Anillin FRET signal was observed between early-constricting anaphase cells, midconstriction telophase cells, and compact midbodies present at the terminal stage of cytokinesis, indicating that the association persists throughout cytokinesis (Gregory, 2008).

A direct interaction between Anillin and RacGAP was confirmed by yeast two-hybrid assay. RacGAP was found to interact with full-length Anillin via amino acids 83 to 309, whereas deletion of amino acids 245 to 311 of RacGAP abolished interaction with Anillin. The N-terminal half of Anillin did not interact with any RacGAP constructs. Specific RacGAP deletions known to abolish Pebble or MKLP binding did not affect the interaction with Anillin (Gregory, 2008).

To test whether Anillin is required for RacGAP localization, tagged RacGAP was followed in proliferating larval cells expressing double-stranded RNA (dsRNA) to remove Anillin. In fixed brain cells lacking detectable Anillin, RacGAP was found transiently at the equator and in the central spindle in early anaphase but in late anaphase was only seen in the interpolar region. Some dsRNA interference (dsRNAi)-treated cells that retained detectable amounts of Anillin showed Anillin localization to small stretches near the equator, and in these cells RacGAP was also found in these locations. Of the dividing cells that lacked any detectable Anillin, 16 of 18 had already clearly failed cytokinesis; the remaining two were still in early anaphase. All telophase cells that lacked detectable Anillin also lacked equatorial RacGAP; conversely, all cells with detectable equatorial Anillin also retained equatorial RacGAP (Gregory, 2008).

These results suggested that the equatorial RacGAP required Anillin to maintain contact with the cortex. To test this, tagged RacGAP was followed in live cells depleted for Anillin. As expected from the fixed tissue, RacGAP was seen at the equatorial cortex and central spindle at the onset of cytokinesis, suggesting that the initial localization mechanism was unaffected. As constriction of the ring began, however, loss was observed of equatorial RacGAP from the cortex, concomitant with the collapse of furrowing (Gregory, 2008).

Previous work has shown that initiation of contractile-ring formation is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed microtubule protein Pav-KLP, which accumulates at the equatorial cortex during anaphase. This study reports another crucial interaction involving RacGAP, this time relating to the stability of the contractile ring once its position has been set by equatorial microtubules. It was expected that there must be some link between the microtubule-bound positioning complex and the cortical ring, made of cytoskeletal polymers like actin, myosin, and septins. Previous models proposed that this link is through activated Rho: equatorial RacGAP bringing Pebble to activate Rho, which then recruits the actomyosin contractile apparatus via Diaphanous and Rho-dependent kinases. However, this model does not explain why a newly formed ring remains where it began unless there is something structurally connecting the nascent ring to the spindle. In fact, the relationship between the position of microtubules and the ring is extremely robust, as demonstrated by classic manipulation experiments . Furthermore, inhibitor studies have shown that an ongoing interaction between microtubules and the cortex is critical for cytokinesis to proceed. This study presents the first structural connection between the actin-based ring and the microtubule based positioning mechanism that can explain their relationship. RacGAP, localized to the equatorial microtubules by Pav-KLP, induces contractile-ring formation via Pebble activation of Rho and then anchors the forming contractile ring by binding Anillin, which binds both myosin and actin. It is anticipated that this link ensures not only the stable localization of the ring but also the continued delivery of Rho-activating signals by the RacGAP-linked RhoGEF Pebble (Gregory, 2008).

In the absence of Anillin, phenotypes such as ring slippage have been observed that are strikingly similar to those seen when microtubules are removed, consistent with the model for its role in attaching the ring to cortical microtubules so that the cleavage position is dictated by the spindle. The loss of Anillin phenotype described, in which RacGAP is found only on interpolar microtubules, can also be seen in human cells. RacGAP and Pav-KLP are normally found on equatorial microtubules, and this localization is critical for cytokinesis, at least in Drosophila. Evidence from vertebrate cells indicates that contractile-ring formation is similarly dependant on cortical RacGAP. Recent evidence for the role for cytokinesis failure in the generation of aneuploid cells and the promotion of tumorigenesis and the strong conservation of the Anillin sequence across phyla suggests that this newly discovered link between centralspindlin and the contractile ring is likely to be broadly significant (Gregory, 2008).

RacGAP50C directs perinuclear gamma-tubulin localization to organize the uniform microtubule array required for Drosophila myotube extension

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. A novel postmitotic function has been identified for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. The localization of the MT nucleator gamma-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and this change in localization depends on RacGAP50C. RacGAP50C and gamma-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants gamma-tubulin remains dispersed throughout the cytoplasm. Furthermore, the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute gamma-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process (Guerin, 2009).

The reorganization of the actin and MT cytoskeletons during myogenesis has long been observed in cell culture as individual myoblasts fuse into multinucleated myotubes. Although the actin cytoskeleton has been shown to be indispensable for mediating myoblast fusion, little is known about how the MT network is remodeled during muscle development or the developmental significance of this event. This study provides evidence that RacGAP, a known regulator of the MT cytoskeleton during cytokinesis, and Pav, a kinesin-like RacGAP-binding protein, play a novel and important role in MT organization in vivo by localizing γ-tubulin to perinuclear sites in myotubes. Furthermore, the organization of the MT network in multinucleated myotubes is important for muscle attachment site (MAS) selection. Muscles that are mutant for RacGAP or pav have defects in MT polarity and fail to properly extend towards their attachment sites, resulting in defects in somatic muscle patterning (Guerin, 2009).

The current model for MT organization in differentiated myotubes has come primarily from cell culture studies, which describe MTs that run parallel to the long axis of the cell and do not appear to be directly associated with any one organizing center. Further studies have demonstrated that proteins involved in MT organization, such as γ-tubulin, are redistributed from the centrosome of individual myoblasts to discrete cytoplasmic puncta as well as along the nuclear membrane in multinucleated myotubes and that these sites are associated with MT growth (Bugnard, 2005; Musa, 2003). The diffuse cytoplasmic distribution of γ-tubulin that is observed in Drosophila myoblasts differs from that in cultured vertebrate myoblasts, in which γ-tubulin is associated with centrosomes. Nonetheless, in both cases, the MT cytoskeleton must be reorganized from either a centrosomal or broadly distributed array in individual myoblasts, to a parallel array in multinucleated myotubes with the plus ends directed outwards. This study shows that RacGAP plays a crucial role in this reorganization. In the absence of RacGAP, MTs are not uniform in their polarity and γ-tubulin remains dispersed throughout the cytoplasm rather than accumulating at the nuclear periphery of multinucleated myotubes. Furthermore, in pav mutants, mislocalization of RacGAP is sufficient to redistribute γ-tubulin to the ends of myotubes (Guerin, 2009).

To date, the perinuclear localization of γ-tubulin in myotubes has only been weakly detected in vitro (Bugnard, 2005). This study shows that the association of γ-tubulin with the nucleus also occurs in vivo and is dependent at least in part on RacGAP. What is the function of γ-tubulin localization to the nuclear periphery in myotubes? One likely possibility is to anchor MT minus ends. Because the nuclei in multinucleated myotubes cluster in the interior of the myotube, this would allow for the polarization of the MT network, which is aligned along the axis of cell migration, with the plus ends at the leading edge. What is the purpose of MT polymerization at the ends of myotubes? Although, conventionally, the driving force for cell motility has been thought to be provided mainly by the reorganization of the actin cytoskeleton, there is increasing evidence that MTs are indispensable for cell migration. It has been hypothesized that MTs form longitudinal arrays in bipolar myotubes in order to facilitate elongation by 'active crawling' of the two ends of the myotube during MAS selection (Musa, 2003). The data point to a MT-based mechanism for myotube extension and MAS selection. In the absence of RacGAP or Pav, the MT network shows non-uniform polarity and many muscle fibers are abnormally shaped and display guidance errors. The effect of RacGAP and pav mutations on muscle morphology is consistent with previous findings in which both RacGAP and Pav have been implicated in regulating axonal outgrowth and maintaining dendritic morphology. RacGAP was identified in a genetic screen by the increased dendritic branching phenotype observed in tum mutants. RacGAP and Pav have also been shown to play a role in regulating the morphogenesis of postmitotic mushroom body neurons in the Drosophila brain. In addition, disruption of the mammalian form of Pav, KIF23 (CHO1; MKLP1), in postmitotic cultured neurons resulted in the rearrangement of MT polarity and in the disruption of dendrite morphology (Guerin, 2009).

There is increasing evidence that morphological processes require regulated coordination of the cytoskeleton by linking actin and MTs. For example, in Drosophila the Rho activator RhoGEF2 is implicated both in Myosin II localization and MT organization via the localization of the plus-end protein Eb1. Likewise, RacGAP provides a connection between the actomyosin ring and the peripheral central-spindle MTs during cytokinesis via its interaction with the actin-binding protein Anillin. In addition, proper formation of the cleavage furrow is dependent on a complex between RacGAP, the Rho activator Pebble, and the plus-end-directed MT protein Pav. The current data show that similar to its function during cytokinesis, the function of RacGAP in postmitotic myotubes depends on its association with the MT-binding protein Pav. However, the role of RacGAP in regulating γ-tubulin distribution appears to be independent of its interaction with Anillin and the actin cytoskeleton. scraps mutants do not show defects in muscle patterning. Furthermore, the organization of the actin cytoskeleton and two known actin-dependent processes, myoblast fusion and muscle attachment, are not significantly affected in RacGAP mutants. These findings demonstrate a newly described function for RacGAP that is restricted to the modulation of MTs, but not the actin cytoskeleton, in postmitotic cells (Guerin, 2009).

What is the developmental significance of the actin-independent function of RacGAP in myotube extension? The answer might lie in the complex process of myogenesis itself. Building a mature muscle fiber requires the coordination of many morphological processes, including myoblast fusion, myotube extension and muscle attachment. The uncoupling of actin- and MT-based cytoskeletal processes might allow for actin-based myoblast fusion and MT-based myotube elongation to occur simultaneously. This idea is supported by previous findings showing that myoblasts continue to fuse as the myotube elongates to find its attachment sites. In addition, fusion-defective mutant FCs have been observed to extend and attempt to migrate to their targets, demonstrating that the migration machinery is not perturbed in mutants in which fusion is disrupted (Guerin, 2009).

It is not yet clear what serves as the trigger for MT reorganization upon myoblast fusion or how RacGAP is recruited for this process. It also remains to be determined whether RacGAP promotes the nucleation of new MTs at the nuclear periphery, or reorganizes existing MTs from fusing myoblasts. Changes in MT architecture could be regulated through a direct physical interaction between RacGAP and γ-tubulin, or indirectly through a complex with downstream targets of the GAP domain of RacGAP (Guerin, 2009).

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. Two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. This nuclear localization is essential for their role in Wnt regulation. Thus, two modulators of the Wnt pathway have been identified that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis (Jones, 2010).

This study presents evidence that Tum and its binding partner, Pav, are required for negative regulation of Tcf-mediated transcription. In retrospect, it seems likely that their pleiotropic effects on cytokinesis have obscured their role in regulation of the Wnt pathway until now. For example, neither of these genes was identified in genomics-based screens for Wg pathway components in cultured cells. Reduction of Tum or Pav function cannot be easily assessed in cell culture because tum- or pav-deficient cells cease to divide. The fly embryo, which uses maternally loaded Tum and Pav until midway through development, provided a fortuitous and genetically tractable model that allowed the detection of their involvement in Wnt modulation. This raises the question of how many pathway components have been overlooked because they have other essential cellular functions (Jones, 2010).

The roles of Tum and Pav in cytokinesis are genetically separable from their roles in Wnt signaling. The GAP activity of Tum is required for cytokinesis: TumδEIE, mutated in the gap domain, was unable to support normal cell division. However, TumδEIE had a significant capacity to repress TOPflash activity in cultured cells and to rescue pattern defects in tum mutant fly embryos. Similarly, the Pav-binding domain of Tum is essential for cytokinesis, but TumδPav retained a modest ability to repress Wg/Wnt signaling in cultured cells, indicating that some of the Tum regulatory function is independent of Pav. Conversely, nuclear localization of Pav is irrelevant to its role in cytokinesis, whereas it was found that nuclear localization of both Tum and Pav was required for rescuing pattern defects in embryos and for repressing TOPflash activity in cultured cells. Thus the Tum-Pav interaction is important for both cytokinesis and full Wnt regulation, but the cellular activity of the complex differs both spatially and enzymatically in the two processes (Jones, 2010).

Most molecules that negatively regulate the Wnt pathway act through the ubiquitylation-based pathway that controls βcat stability. However, Tum and Pav were able to repress TOPflash induced by the constitutively active δGSK-βcat and to diminish pattern disruption caused by the analogous mutant form of Arm in fly embryos. This observation places Tum and Pav activity downstream of the well-characterized control exerted by the destruction complex. Tum and Pav did not prevent βcat from entering the nucleus, but did prevent a βcat-Lef fusion protein from successfully activating gene expression. Therefore a model is favored where Tum and Pav have a constitutive role that impedes binding of the βcat-Tcf complex to DNA or that dampens transcriptional activation by the DNA-bound complex. Curiously, the Tum homolog in humans MgcRacGAP has been connected with transcriptional regulation via HIF-1α, with which it interacts physically (Lyberopoulou, 2007). MgcRacGAP has also been associated with nuclear translocation and activation of STAT transcription factors (Kawashima, 2009). Tum itself was shown to regulate activity of the EGF pathway in the Drosophila wing. These observations suggest that Tum/MgcRacGAP has a broader role within the nucleus, interacting with transcription factors in other cellular pathways (Jones, 2010).

Since Drosophila Tum and Pav are able to repress the Wnt response in human cells, this raises the possibility that MgcRacGAP and MKLP-1, their vertebrate counterparts, might also regulate Wnt signaling. It is tempting to speculate that a direct connection between Wnt regulation and cell division is relevant to oncogenesis, in much the same way that Wnt signaling and cell adhesion are integrally connected through the shared component βcat. Recent work has demonstrated that cell-division defects leading to tetraploidy might ultimately lead to cancer. Multinucleate cells are more likely to experience genetic instability, increasing the risk of transformation to malignancy. Thus, any genetic or epigenetic change that reduces the function of Tum/MgcRacGAP or Pav/MLKP-1 could have a double impact on the cell, compromising the cytokinesis machinery and elevating activity of the Wnt pathway (Jones, 2010).


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RacGAP50C: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 18 June 2017

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