BIOLOGICAL OVERVIEW

The Cactus-Dorsal interaction demonstrates how the cytoplasmic protein (Cactus) can regulate the localization of a transcription factor (Dorsal) to either the cytoplasm or the nucleus. Adequate vertebrate homology exists for purposes of comparison. In vertebrates, I kappa B protein is homologous to Cactus. It regulates the nuclear entry of NF kappa B, which is the vertebrate homolog to Dorsal in Drosophila.

Cactus and Dorsal are part of an activation cascade responsible for dorsal-ventral polarity in the fly. Preceding Cactus activity in this cascade of events is Toll. The Toll receptor is first acted upon by its ligand, Spätzle. This occurs in the ventral portion of the young embryo. The Toll signal then regulates destruction of Cactus, thus allowing nuclear transport of Dorsal.

Signal dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to signaling, and that disruption of the Dorsal/Cactus complex is a secondary result of Cactus degradation. Neither is the PEST sequence required for the signal-dependent degradation of Cactus. However, Cactus degradation does require tube and pelle. These two genes are also necessary for the nuclear localization of Dorsal.

Signal independent degradation of Cactus occurs when Cactus is not complexed with Dorsal. This type of degradation is PEST dependent. In the absence of Dorsal, Cactus is synthesized and degrades much more rapidly. The presence of Dorsal serves to stabilize Cactus in the cytoplasm. The normal Cactus-Dorsal interaction produces a trimer consisting of two molecules of Dorsal for each molecule of Cactus. It is not known how phosphorylation regulates the Cactus-Dorsal interaction. In the mammalian system, rapid phosphorylation of I kappa B, the Cactus homolog, has been observed in response to signaling by IL-1 and TNF-alpha, but phosphorylation of Cactus in response to Toll signaling has not been observed (Belvin, 1995).

Thus the generation of dorso-ventral polarity in Drosophila relies on the formation of a nuclear gradient of the rel/nuclear factor kappa B transcription factor Dorsal in the pre-cellular syncytial embryo by a process of differential nuclear localization. It is thought that the gradient is formed by activation of Toll's membrane receptor at ventral positions. This in turn causes the local dissociation of Dorsal from the cytoplasmic anchor protein Cactus.

Cactus and Dorsal play a critical role in a hemocyte-dependent function in Drosophila. In invertebrates such as Drosophila, hematopoietic stem cells are located in the lymph glands. They give rise to progenitors of at least two lineages, plasmatocytes and crystal cells. Plasmatocytes are the predominant form of hemocytes in the wild-type larval hemolymph and, like mammalian macrophages or neutrophils, they perform phagocytic functions. Plasmatocytes are small, spherical and non-adhesive, and engulf bacteria and cell debris. Plasmatocytes also secrete extracellular matrix components. When a larva experiences an immune challenge, plasmatocytes become stimulated, increase in number and, depending on the nature of infection, engage in phagocytosis or differentiate into discoidal and adhesive lamellocytes. Lamellocytes do not show any capacity for phagocytosis. Instead, they form multilayered capsules around foreign invaders or objects that are too large for phagocytosis. These capsules get melanized by the activities of crystal cells. Crystal cells house the substrates and enzymes for melanization reactions. In the absence of an immune challenge, plasmatocytes of a normal larva differentiate into lamellocytes at the onset of pupariation. However, in certain Drosophila mutants, lamellocytes form melanotic capsules around self tissue, even in the absence of an immune challenge. The mechanisms that control the production, differentiation and functions of these cells in wild-type and mutant Drosophila are poorly understood but they appear to represent constitutive activation of the immune system (Qiu, 1998).

Cactus and Dorsal play a critical role in a hemocyte-dependent function in Drosophila. The zygotic lethal phenotype of cact was examined. The absence of Cactus results in a highly penetrant overproliferation of hemocytes. To identify the lethal phase of cact mutants, the null (cact E8/cact D13 ) and hypomorphic (cact E8/cact IIIG ) larvae were examined through different larval and pupal stages. These larvae are viable at 24 and 48 hours after fertilization. By 72 hours, there is some larval lethality and delay in the rate of development of the null animals when compared to their heterozygous siblings. A clear difference between null and hypomorphic animals in their viability and melanotic capsule phenotype becomes evident by 120 hours. Whereas only 40% of the expected cact null animals survive to 120 hours, more than 80% of the expected hypomorphic animals are alive. With time this difference is even more pronounced: only 4 out of 100 cact null larvae make white prepupae; the remaining animals persist as larvae and eventually die. In contrast, more than 80% of the hypomorphs progress into pupal stages. However, less than 1% of these animals eclose, while the majority die as pupae (Qiu, 1998).

To determine if the incidence of encapsulation in cact animals correlates with zygotic lethality and if capsules are present in null larvae, larvae of different allele classes were examined for the presence of dark spots. Neither larvae nor adults of the V4, V3 or gain-of-function cact classes have melanotic capsules. In contrast, about 40% of the hypomorphic larvae and almost all (over 90%) of the null larvae bear melanotic capsules. The high penetrance of capsule formation in cact null animals and the strong correlation of encapsulation with lethality suggest that encapsulation of self tissue is at least one of the primary zygotic phenotypes of cact. To identify the tissues or organs where cact function may be required, capsules and accompanying tissues were examined from mutant larvae. Aggregates of hemocytes are often found in association with the larval fat body. Melanization in the cact capsules appears to initiate at discrete spots. As larvae grow, the size of capsules and the area of the melanized foci become larger. Cells of the cact fat body show variable loss of intercellular adhesion. In addition to these defects, salivary glands appear atrophied and sometimes show melanization that is not accompanied by hemocytic encapsulation. These phenotypic defects are strikingly similar to those observed when Dorsal is overexpressed, suggesting that they represent a hyperactive immune system. To determine if the high incidence of melanotic capsules in cact mutants is caused by an increase in hemocyte concentration, by increased hemocyte differentiation, or by both these processes, the hemocyte concentration and differentiation were compared in mutants and heterozygotes. The number of hemocytes/ml in cact - hemolymph is more than ten-fold higher than in the hemolymph of heterozygous siblings or wild-type larvae (Qiu, 1998).

Dominant mutations in Toll or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals can be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. These results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva (Qui, 1996).

Since effects of the Toll/Cactus pathway mutants appear to be confined to the regulation of hemocyte density in the larva, other basal and regulatory signals must contribute more directly to lineage specification, hemocyte turnover and differentiation. Phenotypes of other hematopoietic mutants in Drosophila range from the absence of lymph glands to the presence of massively overgrown lymph glands. Analysis of some of these mutants suggests that these genes play specific roles in hemocyte specification, differentiation or turnover. For example, recent studies on the constitutive JAK mutant hop Tum-l (which results in lymph gland overgrowth and hematopoietic neoplasm) suggest that, like the Toll/Cactus pathway, the JAK/STAT signaling pathway also regulates hematopoiesis and hemocyte density in the Drosophila larva. It is possible that multiple regulatory inputs, such as the Toll/Cactus and JAK/STAT signals, are integrated with, or superimposed on one another, to ensure that hematopoietic precursors survive and divide, and progress normally through their developmental program to give rise to mature hemocytes in a consistently controlled manner (Qiu, 1998 and references).

NF-kappaB, IkappaB, and IRAK control glutamate receptor density at the Drosophila NMJ

NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. This study shows that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. Mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, evidence is provided that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon these data it is speculated that Dorsal, Cactus, and Pelle function together, locally at the postsynaptic density, to specify GluR levels (Heckscher, 2007).

NF-κB signaling has been implicated in the mechanisms of neural plasticity, learning, epilepsy, neurodegeneration, and the adaptive response to neuronal injury. The data presented in this study advance the understanding of neuronal NF-κB signaling in two ways. First, multiple lines of evidence are presented that NF-κB/Dorsal signaling is required for the control of GluR density at the NMJ. These data provide a synaptic function for NF-κB signaling that may be directly relevant to the diverse activities ascribed to NF-κB in the nervous system. Second, molecular and genetic evidence is provided that Dorsal, Cactus, and Pelle may function posttranscriptionally, at the postsynaptic side of the NMJ, to specify GluR density during postembryonic development (Heckscher, 2007).

Several independent lines of experimentation suggest that Cactus, Dorsal, and Pelle function together at the PSD to specify GluR density. Evidence is provided that Cactus and Dorsal localize to a similar postsynaptic domain. In addition, overexpression of a GFP-tagged Pelle protein that is sufficient to rescue a pelle mutation, can traffic to the PSD where Cactus and Dorsal reside. Next, genetic evidence is presented that cactus, dorsal, and pelle function together, in the same genetic pathway, to control GluR density. It is particularly surprising that mutations in cactus behave similarly to dorsal and pelle. In other systems (embryonic patterning and immunity), Cactus inhibits Dorsal-mediated transcription by binding and sequestering cytoplasmic Dorsal protein. As a result, in these other systems, cactus mutations cause phenotypes that are opposite to those observed in dorsal mutations. This study used the same cactus and dorsal mutations that previously have been observed to generate the predicted opposing phenotypes during embryonic patterning, and yet it was observed that cactus phenocopies the dorsal mutations. In addition, genetic epistasis experiments indicate that these genes function together to facilitate GluR density. Thus, at the NMJ, Cactus functions in concert with, rather than in opposition to, Dorsal (Heckscher, 2007).

One explanation for this observation could be that Dorsal does not function as a nuclear transcription factor during the control of GluR levels. In support of this idea it has been demonstrated that (1) Dorsal protein is not detected in the nucleus, (2) reporters of Dorsal-dependent transcription fail to show activity in muscle nuclei, and (3) mutation of the Dorsal transactivation domain, dl^U5 does not impair GluR abundance even though this same mutation has been shown to impair transcription-dependent patterning during embryogenesis. An alternative explanation for the observation that dorsal and cactus have similar phenotypes at the NMJ could be that Cactus and Dorsal act synergistically to control the transcription of GluRs at the NMJ. Indeed, there is evidence in other systems that IκB can shuttle with NF-κB to the nucleus. A previous study shows Cactus accumulation in Drosophila larval muscle nuclei in a dorsal mutant background (Cantera, 1999). However, this result could not be repeated despite examination of Cactus localization in five allelic combinations of dorsal. Furthermore, the data from vertebrate systems suggest that IκB should shuttle into the nucleus with NF-κB, not in its absence. Thus, a model is favored in which Dorsal and Cactus function together at the postsynaptic membrane to facilitate GluR abundance during development (Heckscher, 2007).

If this model is correct, then it is predicted that NF-κB does not control GluR density through transcriptional regulation. This prediction is supported by two experimental observations: (1) GluR transcript levels (assessed by QT PCR) are not statistically different from wild-type in dorsal and cactus mutations that cause an ~50% decrease in GluR abundance; (2) it was demonstrated that overexpression of a myc-tagged GluRIIA cDNA using a heterologous, muscle-specific promoter is not able to restore synaptic GluRIIA levels in either the cactus or dorsal mutant backgrounds. These data are consistent with Dorsal and Cactus acting posttranscriptionally to control GluR density at the NMJ. There are two general mechanisms by which GluR levels could be controlled posttranscriptionally: (1) altered receptor delivery to the NMJ or (2) altered receptor internalization/degradation. If receptor internalization/degradation were enhanced in the cactus, dorsal, or pelle mutant backgrounds, one might expect GluRIIA-myc overexpression to overcome this change and restore normal receptor levels. In addition, less myc-tagged protein might be seen in the mutants in comparison to wild-type. This is not what was observed. Therefore, the hypothesis is favored that Cactus, Dorsal, and Pelle function together to promote the delivery of glutamate receptors to the NMJ during development (Heckscher, 2007).

The possibility that Cactus, Dorsal, and Pelle act posttranscriptionally to control GluR density raises many questions. For example, do Dorsal and Cactus exist as a protein complex at the PSD? If so, is this complex regulated and how might such a complex influence GluR density? Since pelle kinase-dead mutants impair GluR density, it is possible that Dorsal and Cactus recruit Pelle to the PSD. If so, what are the targets of Pelle kinase that are relevant to establishing or maintaining the proper density of glutamate receptors at the PSD? Finally, the demonstration that cytoplasmic NF-κB/Dorsal can influence GluR density does not rule out the possibility that NF-κB/Dorsal may also translocate to the muscle nucleus at the Drosophila NMJ under certain stimulus conditions. Indeed, in both the vertebrate central and peripheral nervous systems NF-κB is found within neuronal and muscle nuclei, and nuclear translocation can be stimulated by neuronal activity, glutamate, injury, and disease. For nuclear entry of Dorsal, two events must occur: (1) Cactus must be degraded and (2) Dorsal must be phosphorylated. It remains possible that one or both of these criteria are not met during the normal development of the Drosophila NMJ but could be met under as-yet-to-be-identified stimulus conditions. The possibility that NF-κB acts both locally at the synapse and globally via the nucleus is not unique to this signaling pathway. A similar organization has been documented for wingless/wnt signaling where noncanonical cytoplasmic signaling can impact cytoskeletal organization while canonical signaling involves the nuclear translocation of downstream beta-catenin and TCF-dependent gene transcription (Heckscher, 2007).

It remains unknown how NF-κB signaling is activated at the Drosophila NMJ. In Drosophila embryonic patterning and innate immunity, NF-κB signaling is initiated through activation of Toll or Toll-like receptors. There are nine Toll and Toll-like receptors encoded in the Drosophila genome. However, none of these receptors appear to be present in Drosophila larval muscle. The Toll receptor is expressed in a subset of embryonic muscle fibers, but is absent from larval muscle. None of the Toll-like receptors are expressed in Drosophila embryonic muscle and none appear to be expressed in larval muscle. An alternative possibility is that TNF-α receptors activate NF-κB in Drosophila muscle as has been observed in vertebrate skeletal muscle. Indeed, a TNF-α receptor homolog (Wengen) has been identified, and it is expressed in Drosophila skeletal muscle. The possibility that TNF-α signaling is mediated via NF-κB is intriguing given the recent demonstration that TNF-α regulates GluR abundance in the vertebrate central nervous system. In both cultured neurons and hippocampal slices glial-derived TNF-α signaling is required for the increase in postsynaptic AMPA receptor abundance observed following chronic activity blockade. Thus, the current data in combination with work in the vertebrate CNS raise the possibility that a conserved TNFα/NF-κB signaling system controls GluR abundance at both neuromuscular and central synapses during development and in response to chronic activity blockade (Heckscher, 2007).

Yorkie drives supercompetition by non-autonomous induction of autophagy via bantam microRNA in Drosophila

Mutations in the tumor-suppressor Hippo pathway lead to activation of the transcriptional coactivator Yorkie (Yki), which enhances cell proliferation autonomously and causes cell death non-autonomously. The mechanism by which Yki causes cell death in nearby wild-type cells, a phenomenon called supercompetition, and its role in tumorigenesis remained unknown. This study shows that Yki-induced supercompetition is essential for tumorigenesis and is driven by non-autonomous induction of autophagy. Clones of cells mutant for a Hippo pathway component fat activate Yki and cause autonomous tumorigenesis and non-autonomous cell death in Drosophila eye-antennal discs. This study found that mutations in autophagy-related genes or NF-κB genes in surrounding wild-type cells block both fat^-induced tumorigenesis and supercompetition. Mechanistically, fat mutant cells upregulate Yki-target microRNA bantam, which elevates protein synthesis levels via activation of TOR signaling. This induces elevation of autophagy in neighboring wild-type cells, which leads to downregulation of IκB Cactus and thus causes NF-κB-mediated induction of the cell death gene hid. Crucially, upregulation of bantam is sufficient to make cells to be supercompetitors and downregulation of endogenous bantam is sufficient for cells to become losers of cell competition. These data indicate that cells with elevated Yki-bantam signaling cause tumorigenesis by non-autonomous induction of autophagy that kills neighboring wild-type cells (Nagata, 2022).

The data reveal that the Hippo pathway mutant fat clones cause supercompetition by inducing autophagy-mediated cell death in surrounding wild-type cells via NF-κB-mediated induction of hid. The autophagy induction in wild-type cells depends on Yki-bantam-mediated activation of TOR signaling in neighboring fat mutant cells. This mechanism is similar to what was observed in the elimination of ribosomal protein or Hel25E mutant loser clones when surrounded by wild-type cells. This is particularly interesting in two ways: first, it suggests that different types of cell competition, namely elimination of unfit cells by wild-type cells and elimination of wild-type cells by supercompetitors, are driven by the common mechanism, and second, it indicates that induction of autophagy in loser cells is non-autonomous, as even wild-type cells elevate autophagy when juxtaposed to supercompetitors. Although the mechanism by which autophagy is induced in loser cells nearby winner cells remains unknown, observations in this study in conjunction with the previous data on the elimination of ribosomal protein or Hel25E mutant clones suggest the possibility that relative difference in protein synthesis levels between cells plays a critical role in autophagy induction (Nagata, 2022).

The mechanism by which elevated autophagy induces hid expression via NF-κB still remains to be elucidated. Elevated autophagy results in downregulation of IκB protein Cactus. IκB is known to be degraded by the ubiquitin-proteasome system. On the other hand, elevated autophagy by starvation or rapamycin treatment was shown to cause degradation of IκB and thus activate NF-κB in mouse fibroblast. Together, the data suggest the possibility that IκB is degraded by selective autophagy in losers of cell competition (Nagata, 2022).

The observations of this studsy intriguingly show that non-autonomous cell death in wild-type cells promotes fat-induced tumorigenesis. This supports the idea that cancer cells expand their territories within the tissue by cell competition during malignant progression of tumors. While the mechanism by which wild-type cell death fuels neighboring tumorigenesis is an important open question, it may involve compensatory proliferation triggered by mitogenic factors secreted from dying cells. Intriguingly, it has been reported in Drosophila eye-antennal discs that clones of malignant tumors caused by Ras activation and cell polarity defects induce autophagy in surrounding wild-type cells, which in this case do not cause cell death but provide nutrient such as amino acids to neighboring tumors to promote their growth. Clones of cells overexpressing activated form of Yki were also shown to induce autophagy in neighboring cells, but in this case non-autonomous autophagy does not have a role in promoting tumorigenesis. Thus, non-autonomous autophagy may have multiple roles and mechanisms in regulating tissue homeostasis and tumorigenesis (Nagata, 2022).

Given that the Hippo pathway is conserved throughout evolution and that YAP-mediated cell competition occurs in mammalian systems as well, autophagy-mediated cell death may play an important role in mammalian cell competition. Notably, in a mouse liver cancer model, hyperactivation of YAP in peritumoral hepatocytes triggers regression of primary liver tumors and melanoma-derived liver metastases. Thus, further studies on the mechanism of Hippo-signaling-mediated supercompetition in Drosophila may provide a novel therapeutic strategy against human cancers (Nagata, 2022).

GENE STRUCTURE

Two Cactus transcripts have been detected. The 2.2 kb transcript (maternal/zygotic) is more abundant and is present throughout development. A 2.6 kb transcript (zygotic) is present from 4 hours of development onward. Both these transcripts initiate from the same start site but differ at their 3' ends (Kidd, 1992).

PROTEIN STRUCTURE

Amino Acids - 482 (zygotic); 500 (maternal/zygotic)

Structural Domains

Among the maternally active genes of Drosophila, cactus is the only one whose loss of function mutations specifically produce ventralized embryos. Its product inhibits nuclear translocation of the Dorsal morphogen in the dorsal region of the embryo. cactus encodes an acidic, cytoplasmic protein with six ankyrin repeats. The sequence has similarity to the I kappa B proteins that inhibit the vertebrate transcription factor NF-kappa B. By analogy to results obtained with I kappa B and NF-kappa B, bacterially expressed Cactus protein can inhibit DNA binding of Dorsal protein in vitro (Geisler, 1992). There is an N-terminal acidic domain, six ankyrin repeats in the C-terminal (involved in the interaction with Dorsal), followed by a PEST sequence conferring rapid degradation (Kidd, 1992).

date revised: 20 June 2023 

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