InteractiveFly: GeneBrief

Ras association domain family member 8: Biological Overview | References

Collective cell migration is a key driver of tissue morphogenesis and cancer invasion. This study identified the tumor suppressor Ras association domain-containing protein 8 (RASSF8) as a WAVE interactor required for border cell migration. RASSF8 colocalizes with F-actin and cell adhesion molecules at border cell- border cell contacts. Loss of RASSF8 function results in border cell cohesion defects, a phenotype associated with changes in the localization of the Echinoid (Ed) and Coracle (Cora). Cell-type-specific RNA interference (RNAi) experiments suggest that cohesion defects are caused by changes in localization of Ed rather than E-cadherin. Gain-of-function experiments further revealed reciprocal functional interactions between RASSF8 and WAVE controlling collective border cell movement. Thus, a dual function is proposed of RASSF8 in coordinating border cell cluster behavior. RASSF8 is thought to regulate the collective movement of border cells by restricting WAVE function, while it controls the epithelial cluster integrity by regulating cell-cell adhesion and septate junction molecules such as Ed and Cora (Hohne, 2025).

Important cell-cell adhesion molecules (CAMs) controlling collective movements include cadherins, immunoglobulin superfamily members, integrins, but also intracellular scaffolding and signaling proteins. Drosophila border cells are an interesting example of invasive collective cell migration that highly depends on cell-cell adhesion. SiHohner to cells that undergo epithelial to mesenchymal transitions (EMT), border cells delaminate from a monolayer of polarized follicle epithelial cells, dissolve contacts with neighboring cells, and start to migrate. However, border cells never lose contact with their neighbors and retain their epithelial identity. As a small group of epithelial cells, border cells also retain apicobasal polarity, while exhibiting a clear front-rear polarity like mesenchymal cells. Migration and cohesion of border cell clusters require core apicobasal polarity components as well as E-cadherin (Hohne, 2025).

This study identified the tumor suppressor protein RASSF8 as an important regulator of border cell migration. A dual function of RASSF8 is suggested in regulating the collective behavior of border cell clusters. Gain-of-function experiments revealed reciprocal functional interactions between RASSF8 and WAVE controlling collective migration of border cells. Previous studies support the importance of Rac-WAVE-Arp2/3 pathway in controlling protrusion formation driving border cell migration. The data suggest that RASSF8 is not a simple activator of WAVE regulating border cell movement but rather indicate that the protein level of RASSF8 is critical for proper border cell migration. Forced overexpression of RASSF8 seems to restrict WAVE function. Too low or too high ratios of branched F-actin correlate with loose or tight border cell clusters, respectively. As a consequence, stalled migration was observed similar to overexpression of a membrane-targeted, activated WAVE protein. Previous studies also indicate that the Rac-WAVE-Arp2/3 pathway drives not only lead cell protrusions but also promotes follower-cell motility which is important for cluster compactness and movement. Thus, border cell movement and cluster cohesion are intimately connected. ras^sf8 mutants show indeed prominent defects in cluster cohesion, a phenotype, however, that depends not primarily on WAVE function. Loss of RASSF8 function rather impairs the localization of the CAM Ed, a binding partner of many PDZ proteins and E-cadherin, along border cell contacts. Consistently, loss of RASSF8 function induces cohesion defects more similar to depletion of Ed than E-cadherin. Depletion of both RASSF8 and Ed were found to lead to increased cohesion defects, suggesting that the lagging border phenotype might be caused by changes in Ed localization. Given that WAVE-dependent branched actin filaments also stabilize cell-cell junctions and support the polarized transport of adhesion molecules such as E-cadherin, it is proposed that RASSF8-WAVE interaction might further contribute to Ed-mediated junctional cell adhesion or the recycling of Ed to AJs as previously reported in wing epithelium (Hohne, 2025).

RASSF8 physically interacts with two other conserved junctional scaffold proteins, ASPP and membrane-associated guanylate kinase inverted protein (MAGI), in Drosophila eye epithelial cells. This trimeric protein complex is supposed to promote E-cadherin stability at AJs by recruiting the Par3 ortholog Bazooka. In ras^sf8 mutant border cell cluster, significant changes could not be found neither in E-cadherin nor in Bazooka localization. However, depletion of both the ASPP and the MAGI in border cell cluster using the c306-Gal4 driver phenocopy ras^sf8 mutants, suggesting that ASPP and MAGI function are required in border cell cohesion. As an archetype of scaffolding proteins with tumor suppressor property, MAGI forms a multiple protein-protein interaction module (WW and PDZ domains) involved in the development and maintenance of cell polarity, cell-cell adhesion, cell-cell communication, as well as signal transduction across species. In the Drosophila eye epithelium, the junctional localization of RASSF8 strongly depends on MAGI. In Caenorhabditis elegans, however, the localization of MAGI at the apical junctions again depends on SAX-7/L1CAM, the ortholog of Neuroglian (Nrg) in flies, an immunoglobulin domain-containing CAM that mediates cell-cell adhesion by forming homo- or heterophilic interactions, for example with Ed. More recent studies have shown that a few core SJ components such as Nrg and the conserved FERM domain protein Cora have nonoccluding functions required for egg elongation, border cell migration, and cohesion. This study found loss of RASSF8 affects the localization of Cora and Nrv2. This suggests that defective distribution of Ed in ras^sf8 mutants might also cause the mislocalization of SJ components which contributes to border cell cohesion defects observed in ras^sf8 mutants (Hohne, 2025).

The vertebrate ortholog of Nrg, the L1 CAM was originally discovered as a major player in the development of the nervous system but it also plays an important role in the malignancy of human tumors, especially in metastasis of many human cancers. The human neural cell adhesion molecule L1 (L1CAM) identified as a prognostic marker for a wide spectrum of malignancies including melanoma, neuroblastoma, pancreatic, prostate, ovarian, colorectal, breast, head and neck, non-small cell lung cancer, consistent with its role in cell-cell adhesion and EMT. Thus, this suggests that RASSF8 expression may control diverse heterophilic interactions between different CAMs in diverse epithelial tissue suppressing metastasis (Hohne, 2025).

Previous studies identified RASSF proteins, Ed, and their interacting partners such as ASPP not only as tumor suppressors with functions in cell-cell adhesion but also as signaling molecules controlling cell growth. Both RASSF proteins and Ed can act as upstream regulators of the Hippo signaling pathway driving tissue overgrowth. As shown for many other junctional scaffolding and cytoskeletal proteins such as WAVE, RASSF8 undergoes liquid-liquid phase separation and resides in YAP-associated membraneless condensates, which also engage several Ras and Rho GTPases. However, how spontaneous phase separation into biomolecular condensates contributes to apical junction assembly remains to be determined. Recent studies suggest that phase separation may contribute to the regulation of distinct cellular mechanosensitive signaling pathways that require a connection to the actin cytoskeleton. Mechanical forces are transmitted across AJs, and it will be interesting to determine whether an interaction between RASSF8 and WAVE might also play an important role in biomolecular condensates that guide cell adhesion and growth (Hohne, 2025).

RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. This study shows that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. The exocyst component Sec15 was identfied as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, these results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis (Chan, 2021).

Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division

Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. This study shows that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery (Banerjee, 2017).

Polarity is a fundamental feature of most cells and tissues. It is evident both at the level of individual cells and groups of cells (e.g. planar cell polarity (PCP) in epithelia. However, despite the fact that different cell types use a common set of molecules to establish and maintain polarity (Par complexes, Fz-PCP pathway), the organization of polarized cells and cell assemblies varies dramatically across different species and tissues. This implies the existence of factors that act in a cell or tissue-specific manner to modulate/rewire the core polarity machinery into the appropriate organization. Despite many advances in understanding of polarity in unicellular and multicellular contexts, little is known about the identity or function of such factors (Banerjee, 2017).

An example of polarity remodeling is the process of asymmetric cell division (ACD), where cells need to rearrange their polarity determinants into a machinery capable of asymmetrically segregating cell fate determinants, vesicles and organelles, as well as controlling the orientation of the mitotic spindle. ACDs result in two daughter cells of different fates and occur in numerous cell types and across species. Well-studied examples include budding in Saccharomyces cerevisiae, ACD in the early embryo of Caenorhabditis elegans, or ACD of progenitor cells in the mammalian stratified epidermis and neural stem cells in the mammalian neocortex. In Drosophila melanogaster, the study of germline stem cells, neuroblasts (neural stem cells) and sensory organ precursors (SOPs) has greatly contributed to understanding of the cell biology and molecular mechanisms of ACD (Banerjee, 2017).

SOPs (or pI cells) divide asymmetrically within the plane of the epithelium into pIIa and pIIb daughter cells. pIIa and pIIb themselves divide asymmetrically to give rise to the different cell types of the external sensory organs (bristles), which are part of the peripheral nervous system and allow the adult fly to sense mechanical or chemical stimuli. Individual SOPs are selected by Notch-dependent lateral inhibition from multicellular clusters of epithelial cells expressing proneural genes (proneural clusters) (Banerjee, 2017).

The unequal segregation of cell fate determinants (the Notch pathway modulators Numb and Neuralized), which specifies the different fates of the daughter cells, requires their asymmetric localization on one side of the cell cortex prior to mitosis. This is achieved by remodeling the PCP and apical-basal polarity systems in the SOP, and by orienting the spindle relative to the tissue axis. The epithelial sheet that forms the pupal notum (dorsal thorax), where the best-studied SOPs are located, is planar polarized along the anterior-posterior tissue axis, with the transmembrane receptor Frizzled (Fz) and its effector Dishevelled (Dsh) localizing to the posterior side of the cell cortex, while the transmembrane protein Van Gogh (Vang, also known as Strabismus) and its interactor Prickle (Pk) are found anteriorly. The apical-basal polarity determinants central to SOP polarity are the PDZ domain-containing scaffold protein Bazooka (Baz, or Par3), atypical Protein Kinase C (aPKC) and Partitioning defective 6 (Par6), which localize apically in epithelial cells and the basolaterally localized membrane-associated guanylate kinase homologues (MAGUK) protein Discs-large (Dlg). In most epithelial cells, these proteins localize uniformly around the cell cortex, whereas in SOPs they show a striking asymmetric localization during mitosis: the Baz-aPKC-Par6 complex is found at the posterior cell cortex, opposite an anterior complex consisting of Dlg, Partner of Inscuteable (Pins) and the G-protein subunit Gαi. The Fz-Dsh complex provides the spatial information for the Baz-aPKC-Par6 complex, while Vang-Pk positions the Dlg-Pins-Gαi complex (likely through direct interaction between Vang and Dlg). The asymmetric distribution of the polarity determinants then directs the positioning of cell fate determinants at the anterior cell cortex. Additionally, Fz-Dsh and Pins orient the spindle along the anterior-posterior axis by anchoring it on both sides of the cell via Mushroom body defective (Mud, mammalian NuMA) and Dynein (Banerjee, 2017).

The planar symmetry of the Baz-aPKC-Par6 complex in SOPs is initially broken in interphase via Fz-Dsh, and is independent of the Dlg-Pins-Gαi complex. Once this initial asymmetry is established, the core PCP components become dispensable for Par complex polarization at metaphase due to the mutual antagonism between the opposing polarity complexes, which then maintains asymmetry during cell division. Indeed, Baz is still polarized in fz mutants during mitosis, but losing both pins and fz results in Baz spreading uniformly around the cortex. Crucially, it is unclear how Fz-Dsh can transmit planar information to the Baz-aPKC-Par6 complex in SOPs but not in neighboring epithelial cells. The cell-type dependent coupling between PCP and apical-basal polarity suggests the involvement of unknown SOP-specific factors in this process (Banerjee, 2017).

The four N-terminal RASSFs (Ras association domain family) in humans (RASSF7-10) have been associated with various forms of cancer, but the exact processes in which these scaffolding proteins act remain mostly elusive. Drosophila RASSF8, the homologue of human RASSF7 and RASSF8, is required for junctional integrity via Baz. Interestingly, human RASSF9 and RASSF10 were found in an interaction network with Par3 (the mammalian Baz homologue) and with several PCP proteins. The Drosophila genes CG13875 and CG32150 are believed to be homologues of human RASSF9 and RASSF10, respectively and remarkably, CG32150 mRNA is highly enriched in SOPs (Banerjee, 2017).

This study shows that Meru, encoded by CG32150, is an SOP-specific factor, capable of linking PCP and apical-basal polarity. Meru localizes asymmetrically in SOPs based on the polarity information provided by Fz/Dsh, and is able to recruit Baz to the posterior cortex (Banerjee, 2017).

PCP provides the spatial information for the initial polarization of SOPs at interphase, resulting in the planar polarization of Baz, which is uniformly localized prior to SOP differentiation. How Fz/Dsh communicate with Baz and enable its asymmetric enrichment was unknown. Based on the current results and previous findings, the following model is proposed for the role of Meru in SOP polarization. Upon selection and specification of SOPs, Meru expression is transcriptionally activated by the AS-C transcription factors. At interphase, planar-polarized Fz/Dsh recruit Meru to the membrane and hence direct its polarization. Meru in turn positions and asymmetrically enriches Baz, promoting the asymmetry of aPKC-Par6. Upon entry into mitosis, Meru is also required to retain laterally localized Baz, thus supporting the antagonism between the opposing Dlg-Pins-Gα_i and Baz-aPKC-Par6 complexes, ultimately enabling the correct positioning of cell fate determinants (Banerjee, 2017).

The meru mutant cell fate phenotype (bristle duplication or loss) is weaker than the baz loss-of-function phenotype, which results in loss of entire SOPs. This is likely due to two factors: (1) unlike meru mutants, the full baz mutant phenotype is the result of a complete loss of Baz in all cells of the SOP lineage, which is known to cause multiple defects including apoptosis of many sensory organ cells as well as cell fate transformations; (2) since a small amount of Baz is retained at the cortex of some meru mutant cells, it is likely that this residual Baz can still be polarized through the antagonistic activity of Pins at metaphase and thus partially rescues SOP polarization. Indeed, it was observed that reduction of pins or baz levels by RNAi strongly enhanced the meru cell specification phenotype. Conversely, supplying excess levels of Baz in a meru mutant background presumably restores sufficient Baz at the cortex to rescue the meru specification defect, as long as Pins is present to drive asymmetry at mitosis. (Banerjee, 2017).

While a decrease in cortical Baz can account for the cell specification defects in meru mutants, it does not explain the spindle orientation phenotypee. This abnormal spindle alignment could either be due to a decrease in Fz/Dsh levels/activity, or a decrease in the ability of Dsh to recruit the spindle-tethering factor Mud. No gross abnormalities were detected in Fz levels in meru mutants, though the presence of Fz in all neighboring cells would make it difficult to detect subtle decreases in SOPs. Further work will be required to understand Meru's role in spindle orientation (Banerjee, 2017).

Analysis of Meru in Drosophila is in agreement with the association of human RASSF9 and RASSF10 with both Par3 and PCP proteins previously reported. However, while the interaction with Dsh is conserved between the fly and human proteins, the transmembrane protein Vangl1 (the mammalian homologue of Vang), rather than its antagonist Fz was recovered in the mammalian proteomic analysis. This could reflect species-specific differences or altered polarity in the transformed human embryonic kidney 293 cells used for the mammalian work. Although Meru (CG32150) was classified as a potential homologue of RASSF10, alignment of the protein sequences showed similar sequence identities for both human RASSF9 (31%) and RASSF10 (26%). Thus, further functional work on Meru, its Drosophila paralogue CG13875, as well as mammalian RASSF9 and RASSF10 is required to understand the evolutionary and functional relationships between these proteins (Banerjee, 2017).

Little is known about the in vivo functions of either RASSF9 or RASSF10 in other species. Xenopus RASSF10 is prominently expressed in the brain and other neural tissues of tadpoles, potentially indicating a function in neurogenesis, a process where ACDs are known to take place. Interestingly, mouse RASSF9 shows a cell-specific expression in keratinocytes of the skin and loss of RASSF9 results in differentiation defects of the stratified epidermis. Considering that Par3 is required for ACD of basal layer progenitors of the stratified epidermis this raises the exciting prospect that RASSF9 might regulate ACD in the mammalian skin (Banerjee, 2017).

The polarization of cells and tissues is essential for their architecture and ultimately allows them to fulfill their function. The polarity machinery can be considered as a series of modules that are combined in a cell or tissue-specific manner, and hence requires specific factors that can create a polarity network appropriate to each tissue and cell type. This study has identified Meru as an SOP-specific factor, which is able to link PCP (Fz-Dsh) with apical-basal polarity (Baz). The PCP proteins Vang and Pk promote the positioning of the opposing Dlg-Pins-Gα_i complex. Although Vang can directly bind to Dlg, the SOP and neuroblast-specific factor, Banderuola (aka Wide Awake) was recently shown to be required for Dlg localization and could thus constitute a link between the two polarity systems on the opposite side of the cortex (Banerjee, 2017).

There is increasing evidence that cell-type specific rewiring of the polarity modules may be a widespread phenomenon. For instance, in different parts of the embryonic epidermis, Baz is planar polarized by Rho-kinase or by the Fat-PCP pathway, while in the retina, Vang is responsible for Baz polarization. Apical-basal polarity can also operate upstream of PCP in some systems, as in Drosophila photoreceptor specification, where aPKC restricts Fz activity by inhibitory phosphorylation in a subset of photoreceptor precursors. Thus, tissue-specific factors are likely to operate in a number of different contexts (Banerjee, 2017).

The interplay between PCP and apical-basal polarity is also evident in other species, as Dishevelled has been reported to promote axon differentiation in rat hippocampal neurons by stabilizing aPKC, while Xenopus Dishevelled is required for Lethal giant larvae (Lgl) basal localization in the ectoderm. Interestingly, both mammalian Par3 and the Vang homologue Vangl2 are required for progenitor cell ACD in the developing mouse neocortex, raising the question as to whether PCP and apical-basal polarity are also connected in mammalian ACDs. It is therefore proposed that tissue-specific factors such as Meru might enable the diversity and plasticity observed across different polarized cells and tissues by rewiring the core polarity systems (Banerjee, 2017).

Magi is associated with the Par complex and functions antagonistically with Bazooka to regulate the apical polarity complex

The mammalian MAGI proteins play important roles in the maintenance of adherens and tight junctions. The MAGI family of proteins contains modular domains such as WW and PDZ domains necessary for scaffolding of membrane receptors and intracellular signaling components. Loss of MAGI leads to reduced junction stability while overexpression of MAGI can lead to increased adhesion and stabilization of epithelial morphology. However, how Magi regulates junction assembly in epithelia is largely unknown. This study investigated the single Drosophila homologue of Magi to study the in vivo role of Magi in epithelial development. Magi is localized at the adherens junction and forms a complex with the polarity proteins, Par3/Bazooka and aPKC. A Magi null mutant was generated and found to be viable with no detectable morphological defects even though the Magi protein is highly conserved with vertebrate Magi homologues. However, overexpression of Magi results in the displacement of Baz/Par3 and aPKC and leads to an increase in the level of PIP3. Interestingly, it was found that Magi and Baz function in an antagonistic manner to regulate the localization of the apical polarity complex. Maintaining the balance between the level of Magi and Baz is an important determinant of the levels and localization of apical polarity complex (Padash Barmchi, 2016).

A common component of junctional and polarity complexes is modular scaffolding proteins that are capable of binding to each other as well as recruiting other proteins to the complex. Magi proteins are evolutionarily conserved scaffolding proteins and contain multiple domains including a N-terminal catalytically inactive GUK domain, two WW domains and five to six PDZ (PSD95/Dlg/ZO-1) domains. There are three MAGI proteins in vertebrates (MAGI-1,2,3) all with multiple splice isoforms. MAGI-1 and MAGI-3 are relatively ubiquitously expressed and localize to a range of junctions including epithelial tight junctions. MAGI-2 (also known as AIP1/S-SCAM/ARIP1) is expressed in the nervous system as a synaptic protein and within glomerular podocytes in the kidney and plays important role in scaffolding synaptic proteins such as NMDA receptors and Neuroligin, the tip-link protocadherin Cadherin, the Kir4.1 K(+) channel, as well as kidney proteins such as nephrin and JAM4 (Padash Barmchi, 2016).

Within epithelia and endothelia, MAGI-1 and -3 are localized at tight junctions and form a structural scaffold for the assembly of junctional complexes. MAGI-1 also localizes and plays a role in modulating adherens junction adhesion through scaffolding beta-catenin and PTEN. MAGI-1 overexpression stabilizes adherens junctions and epithelial cell morphology through increased E-cadherin and β-catenin recruitment. Silencing of MAGI-1 has the opposite effect with decreased adherens junction adhesion and reduced focal adhesion formation leading to anchorage-independent growth and migration in vitro. MAGI-1 overexpression suppresses the invasiveness of MDCK cells, as well as suppresses tumor growth and spontaneous lung metastasis through the increased recruitment of PTEN or β-catenin and E-cadherin (Padash Barmchi, 2016).

Overall, MAGI proteins play important roles in the stabilization of cell-cell interactions and as such Magi is a key target in polarized epithelia during cell death and viral infection. For instance, MAGI-1 is cleaved by activated caspases during apoptosis, a process thought to mediate the disassembly of cell-cell contacts. MAGI proteins are also targeted by a number of oncogenic viruses: it is aberrantly sequestered in the cytoplasm by Adenovirus E4orf1, and is targeted for degradation by the E6 oncoprotein of high-risk human papillomavirus. E6-mediated degradation of MAGI-1 in cultured epithelial cells leads to loss of tight-junction integrity (Padash Barmchi, 2016 and references therein).

There is a high degree of conservation of protein structure and function in the invertebrate homologues of Magi in particular with regards to epithelial junction formation and maintenance. In C. elegans, Magi-1 plays a role in the segregation of different cell adhesion complexes into distinct membrane domains along the lateral plasma membrane. In Drosophila, Magi binds Ras association domain protein 8 (RASSF8) and modulates adherens junctions remodeling in late eye development during interommatidial cell (IOC) rearrangements. In this context Magi function is necessary to recruit the polarity protein Par-3 (Drosophila Bazooka, Baz) to the remodeling adherens junction. However, the association of Drosophila Magi or any Magi homologue with any components of the Par polarity complex in stable epithelia has not been determined (Padash Barmchi, 2016).

The Par complex consisting of Par-3/Par-6/aPKC localizes to tight junctions where MAGI is present in vertebrate epithelial cells and is necessary for assembly of this junctional complex as well as for separation of the apical region of the plasma membrane from the basolateral domain. In Drosophila epithelial cells, the Par complex localizes to the apicolateral membrane and demarcates the boundary between the apical and basolateral membrane regions. Mutant embryos for any member of this complex show loss of apicobasal polarity and disruption in the integrity of epithelia. Although the members of the Par complex are important for the establishment of cell polarity, some of the core components of this complex such as Baz are dispensable for the maintenance of cell polarity during later stages of development. Baz localizes to adherens junction and mutant clones of baz in wing imaginal discs are fully viable with no polarity or adherens junction defects. Similarly, Magi function in AJ stability has been determined in many systems, but surprisingly loss of Drosophila Magi has no effect on established, stable AJs. Little is known about the convergence of Magi and Par complex function at the adherens junctions and it is possible that Baz and Magi function in established epithelia are redundant. Therefore this study investigated the role of Magi in the established and stable epithelia of the Drosophila wing imaginal disc to test the potential interactions between Magi and members of the Par complex (Padash Barmchi, 2016).

Drosophila Magi was found associated with the PAR polarity complex and is localized at the adherens junction with Baz, Par-6, and aPKC. Overexpression of Magi resulted in the reduction of apical polarity proteins from the membrane and these interactions required the second half of the Magi protein containing the four PDZ domains. Overexpression of Baz resulted in a reduction of Magi from the membrane but an increase in aPKC and Par-6. While Magi mutants were viable with no polarity defects, Magi levels were found to be antagonistic with Baz, and a balance between the two was found to be necessary to regulate the level and localization of Par complex (Padash Barmchi, 2016).

PDZ domain-containing proteins form scaffolding protein complexes with a wide range of roles including cell polarity and signaling. As a MAGUK protein, Magi is part of a scaffold that interacts with members of the polarity complex at the adherens junctions in the epithelia of the imaginal disc. The scaffolding function of Magi has been well established in other systems. In vertebrates epithelial cells MAGI-1 has been shown to act as structural scaffold at tight junctions and adherens junctions. In C. elegans, Magi-1 localizes apical to adherens junction and functions as an organizer to ensure that different cell adhesion complexes are segregated into distinct membrane domains along the lateral plasma membrane. In neuronal cells MAGI-2/S-SCAM was also shown to cluster the cell adhesion molecule Sidekick, and the AMPA and NMDA glutamate receptors at the synapse (Padash Barmchi, 2016).

Given the strong conservation of the Magi protein it is surprising that null mutants of Drosophila Magi exhibit no lasting cellular defects (other than transient defects in the interommatidial cells of the pupal eye and null animals are fully viable. Similarly in C. elegans, magi-1 null worms are healthy with only a few embryos (1.3%) with defects during the ventral enclosure stage. As Magi is highly conserved, it is plausible that Magi may only act in response to cell stress, DNA damage or some other trigger. For example, loss of p53 does not disrupt cellular function under normal conditions and p53 null flies or mice are viable with no cellular defects. However, the role of p53 in response to DNA damage is well established and when these animals are exposed to irradiation apoptosis is not induced. Alternatively, Magi function might be redundant with other components of the apical polarity complex or another protein and that loss of both is necessary for the disruption of cellular function. Core scaffolding components of the apicobasal polarity complex are dispensable for maintaining polarity in the wing imaginal disc epithelia supporting the idea of redundancy in this system. For instance, somatic clones of loss of function mutations in crb, sdt and baz have no effect on the polarity in the wing disc epithelia of the 3rd instar larvae. Baz is a strong candidate for redundancy with Magi given the localization to the adherens junction and function as a PDZ scaffolding protein. As loss of baz in the wing imaginal disc does not disrupt the polarity of wing disc epithelia this leads to the hypothesis that Baz and Magi are redundant. However, somatic clones of a baz null mutant in a Magi mutant background did not lead to a loss of cell polarity or apoptosis. While the two scaffolding proteins do not appear to functionally interact, it was observed that Magi and Baz are in a protein complex and their close proximity within the wing columnar epithelia also suggests a common complex. Overexpression of Magi displaces Baz and aPKC from the apical membrane and, likewise overexpression of Baz displaces Magi from the membrane. The simultaneous over-expression of Magi and Baz suppresses the changes caused by their individual expression, suggesting a balance or competition between the two proteins. The maintenance of a balance between Magi and Baz might be due to a direct physical competition between these two proteins or opposite effects on a common mediator or interactor (Padash Barmchi, 2016).

Baz and vertebrate MAGI proteins bind the lipid phosphatase PTEN and thus the Magi-Baz interaction and balance could be influenced by changes in the level of phosphoinositides such as PtdIns(4,5)P2 (PIP2) or PtdIns(3,4,5)P3 (PIP3). In polarized epithelia, PIP2 is found within the apical domain and PIP3 restricted to the basal-lateral domain. Baz localization in polarized epithelia depends on PIP2 and on the PI4P5 kinase Skittles. Baz in turn can be a positive regulator of PIP2 levels at the plasma membrane by local recruitment of the lipid phosphatase PTEN. This study observed an increase in PIP3 levels with increased expression of Magi, which may reflect the loss of Baz and a loss of PTEN recruitment to the membrane. This study was not able to assess changes in PTEN levels at the membrane with available antibodies. However it was observe that the recruitment of Magi or Baz was not affected in Pten mutant cells. Similarly the changes in PIP3 levels are unlikely to be the cause of Baz loss in the presence of increased Magi as co-expression of PTEN and Magi still resulted in the loss of Baz from the membrane. Prior studies on Magi in Drosophila in the pupal eye did not detect any physical interaction between Drosophila Magi and Pten, and the phenotypes generated by overexpression of Magi in the Drosophila eye were not affected by Pten mutants. Therefore it is likely that loss of Baz in the presence of increased Magi in the wing imaginal disc and vice versa is through competition for a protein component (Padash Barmchi, 2016).

In the developing eye Magi forms a protein complex with RASSF8 (the N-terminal Ras association domain-containing protein) and ASPP (Ankyrin-repeat, SH3-domain, and proline-rich-region containing protein), and this complex plays a role during remodeling of the adherens junctions in the interommatidial cells (IOCs) (Zaessinger, 2015). When IOCs rearrange to create the pupal lattice, this process requires regulation of the E-Cadherin complex where RASSF8 and ASPP regulate adherens junction remodeling and integrity through regulation of Src kinase activity. Magi recruits the RASSF8-ASPP complex in the process of adherens junction remodeling and there are defects in IOC rearrangement in Magi mutants where AJs are frequently interrupted. In the eye the Magi-RASSF8-ASPP complex is necessary for the cortical recruitment of Baz and of the adherens junction proteins α- and β-catenin. A model has been proposed where Magi-RASSF8-ASPP complex functions to localize Baz to remodeling junctions to promote the recruitment or stabilization of E-Cad complexes (Zaessinger, 2015). However, it is not thought that the RASSF8-ASPP complex is the point of competition between Magi and Baz within the wing imaginal disc. In the wing imaginal disc Magi and the RASSF8-ASPP complex are localized to the adherens junction domain independently (Zaessinger, 2015) and while RASSF8 mutants have a wing rounding phenotype, Magi mutants do not. Furthermore no differences were observed in Baz, Ecad or Arm distribution in Magi somatic loss of function clones in the wing imaginal disc. Finally the Magi WW domains are required for the interaction with RASSF8 (Zaessinger, 2015), while the overexpression of the Magi transgene that contains the PDZ domains led to a reduction in Baz suggesting that second half of the Magi protein containing the PDZ domains contains the important sites for this competition (Padash Barmchi, 2016).

Therefore, a strong possibility to explain the reciprocal effects of overexpression is that Baz and Magi compete for a common binding site. Magi was found to interacte with both Baz and aPKC; the latter two are known to interact directly. However, it is unlikely that the shared site is through physical scaffolding of aPKC, as high levels of wild type aPKC had no effect on either Magi or Baz and was not able rescue the changes in Baz levels and localization caused by Magi overexpression. In addition the overexpression of Magi also led to a reduction in aPKC. It is unlikely that the loss of Baz is responsible for this displacement as aPKC is not mislocalized in Baz clones and Baz is not mislocalized in Par-6, aPKC or Cdc42 null clones. Further investigation is required to explore the mechanisms that underlie Magi interactions with components of the apical polarity complex and the adherens junction complex (Padash Barmchi, 2016).

Drosophila MAGI interacts with RASSF8 to regulate E-Cadherin-based adherens junctions in the developing eye

Morphogenesis is crucial during development to generate organs and tissues of the correct size and shape. During Drosophila late eye development, interommatidial cells (IOCs) rearrange to generate the highly organized pupal lattice, in which hexagonal ommatidial units pack tightly. This process involves the fine regulation of adherens junctions (AJs) and of adhesive E-Cadherin (E-Cad) complexes. Localized accumulation of Bazooka (Baz), the Drosophila PAR3 homolog, has emerged as a critical step to specify where new E-Cad complexes should be deposited during junction remodeling. However, the mechanisms controlling the correct localization of Baz are still only partly understood. This study shows that Drosophila Magi, the sole fly homolog of the mammalian MAGI scaffolds, is an upstream regulator of E-Cad-based AJs during cell rearrangements, and that Magi mutant IOCs fail to reach their correct position. They uncovered a direct physical interaction between Magi and the Ras association domain protein RASSF8 through a WW domain-PPxY motif binding, and showed that apical Magi recruited the RASSF8-ASPP complex during AJ remodeling in IOCs. Further, this Magi complex was required for the cortical recruitment of Baz and of the E-Cad-associated proteins α- and β-catenin. They propose that, by controlling the proper localization of Baz to remodeling junctions, Magi and the RASSF8-ASPP complex promote the recruitment or stabilization of E-Cad complexes at junction sites (Zaessinger, 2015).

As Magi is the sole Drosophila homolog of the three vertebrate MAGI scaffolds, it offers a powerful system with which to investigate the functions of these important proteins. Using newly generated null alleles, this study has shown that Magi coordinates the number and packing of IOCs in the developing Drosophila pupal eye by regulating AJ dynamics. Magi is necessary in the IOCs to localize the RASSF8-ASPP complex correctly during their junctional remodeling. This ensures the integrity of E-Cad-based junctions and the correct localization of Baz, α- and β-catenin. Based on these observations and on the growing evidence of a role for Baz in AJ remodeling, a model is proposed whereby, during AJ remodeling in IOCs, Magi recruits the RASSF8-ASPP complex, which helps to localize Baz at the membrane and regulates the sites of E-Cad accumulation (Zaessinger, 2015).

Junction remodeling is a key step during morphogenesis, in which cells in a tissue change position and neighbors. For instance, in the developing pupal eye, IOCs found between ommatidia organize as a single row of cells. During this process existing contacts are eliminated and new ones are established by remodeling E-Cad-based junctions. In Magi mutants, rearrangement defects and some incorrect localization of IOCs were observed. At the same time, E-Cad-based AJs were interrupted in Magi mutant cells. It is proposed that this defect in AJ remodeling leads to IOCs remaining at the wrong place in the lattice. The most parsimonious model is that the defects in AJ remodeling trigger the defects in cell numbers seen in Magi mutants by preventing apoptosis, although it was not possible to fully substantiate this as the effect of Magi on apoptosis was not statistically significant. If the model is correct, it still remains unclear how disrupted junctions would lead to a failure in apoptosis. One possibility is that IOCs only receive the correct 'death signal' when they have rearranged to contact the correct cells. Thus, in Magi mutants, the defective AJs would lead to apoptosis failure because the IOCs did not attain their position in the 'death zone' to receive the killing signal (Zaessinger, 2015).

These junctional defects are reminiscent of those seen for magi-1 mutants in the nematode C. elegans, in which magi-1 loss of function enhanced the defects caused by cadherin and catenin mutations and disrupted cell migration during enclosure. MAGI scaffolds are thus implicated in the fine regulation of AJs in both flies and nematodes. A similar role has been suggested for MAGI proteins in mammalian epithelial cells. In overexpression studies, human MAGI1 reduced the Src-induced invasiveness of MDCK cells and stabilized E-Cad-mediated intercellular aggregation. By analogy, the overexpression phenotype of Drosophila Magi could thus be due to stronger AJs, although this remains to be experimentally tested. The overexpression effects of MAGI-1b were sensitive to PTEN and AKT activities and mammalian MAGI scaffolds have also been implicated in PTEN activation through their direct binding to PTEN. However, this study did not detect any physical interaction between Drosophila Magi and Pten, and the overexpression phenotype of Magi, at least in the Drosophila eye, appeared insensitive to Pten. Although these are negative observations, they suggest that in Drosophila Magi and Pten do not form a complex to regulate AJs (Zaessinger, 2015).

Despite its effects on eye development, Magi mutants exhibit slightly enlarged wings. Whether this is dependent on E-Cad belt integrity and AJ dynamics remains to be established. The fact that ASPP shows a very similar wing phenotype supports this model (Zaessinger, 2015).

Rather than binding and modulating the activity of Pten, this analysis supports a model whereby Magi, by binding to the RASSF8-ASPP complex, recruits and stabilizes Baz at the membrane. Accumulation of Baz has been shown to specify and initiate the formation of new AJs both in cellularizing embryos and in photoreceptors. It is proposed that Baz recruited at the membrane of IOCs will in turn promote the stabilization or the proper distribution around the cell cortex of AJ material. Since biochemical and genetic experiments suggest that RASSF8 and Magi act together in a complex, it is proposed that the effects of Magi on AJs and on Baz membrane recruitment are mediated by RASSF8, and are thus likely to involve ASPP. Indeed, mammalian ASPP2 binds PAR3 and is required for PAR3 localization at junctions both in cell culture and in the mouse neuroepithelium. This suggests that Magi might control Baz localization through ASPP. However, Baz membrane recruitment is unlikely to be the only step to form correct AJs downstream of Magi/RASSF8/ASPP. Previous studies have implicated C-terminal Src kinase (Csk) and its action on Src kinase, and the relationships between Magi, Baz and Csk should be investigated in the future (Zaessinger, 2015).

During IOC remodeling, Magi therefore appears to be a crucial upstream regulator of AJs. However, the mechanisms governing Magi membrane localization are still unknown. One hypothesis is that the membrane recruitments of different AJ components and regulators are dependent on each other in stabilization loops. However, this is unlikely to be the case for Magi as it is still perfectly localized at the membrane in ASPP, RASSF8 and baz mutants, and in ASPP; RASSF8 double mutants (Zaessinger, 2015).

Another possibility is that Magi would require mature AJs with E-Cad to be at the membrane. No direct correlation was found between E-Cad accumulation around the apical membrane and Magi membrane localization. For instance, in ASPP; RASSF8 double-mutant cells, E-Cad belt interruptions were detected either without or with Magi, indicating that Magi localization does not require E-Cad directly. Furthermore, an extensive domain mapping of Magi failed to identify a single domain (WW or PDZ) that would be required for Magi recruitment, suggesting that it might be independent of these domains or that several redundant mechanisms may be at play. The nature of the signal required for Magi membrane localization thus remains to be uncovered (Zaessinger, 2015).

Even though Magi binds to RASSF8 directly and both proteins function together during Drosophila eye morphogenesis, their mutant phenotypes are not identical. First, RASSF8 mutants have a wing rounding phenotype, which is absent in Magi mutants. Second, whereas RASSF8 has a significant role in the global developmental apoptosis rate in the pupal eye, no significant effect could be detected for Magi and ASPP. Taken together, this suggests that the assembly of a Magi-RASSF8-ASPP complex might be context dependent or that RASSF8 has Magi-independent functions (Zaessinger, 2015).

Although the human N-terminal RASSF (RASSF7-10) proteins lack any PPxY motifs, one is present in ASPP2 and has been shown to bind to MAGI1. It is therefore possible that MAGI-ASPP complexes are formed in all organisms but the precise mode of interaction differs: mediated by RASSF8 in the fly, but direct in humans (Zaessinger, 2015).

MAGI scaffolds have been suggested to play a role in tumorigenesis. First, they are bound and inactivated by several viral oncoproteins. Second, MAGI1 has been shown to exhibit tumor suppressor activity in colorectal cancer cell lines in xenograft model. Finally, mutations in MAGI2 and MAGI3 are reported in colon, prostate and breast cancers. Documented alterations include deletion of the second WW motif of MAGI2 and a MAGI3:AKT3 fusion leading to a disruption of MAGI3. Based on the current work, it is proposed that these are loss-of-function mutations. It would be interesting to investigate whether changes in AJ dynamics are associated with these MAGI mutations in human cancers and whether they contribute to tumorigenesis (Zaessinger, 2015).

The dASPP-dRASSF8 complex regulates cell-cell adhesion during Drosophila retinal morphogenesis

Adherens junctions (AJs) provide structure to epithelial tissues by connecting adjacent cells through homophilic E-cadherin interactions and are linked to the actin cytoskeleton via the intermediate binding proteins beta-catenin and alpha-catenin. Rather than being static structures, AJs are extensively remodeled during development, allowing the cell rearrangements required for morphogenesis. Several 'noncore' AJ components have been identified that modulate AJs to promote this plasticity but are not absolutely required for cell-cell adhesion. dASPP has been identified as a positive regulator of dCsk (Drosophila C-terminal Src kinase). This study shows that dRASSF8, the Drosophila RASSF8 homolog, binds to dASPP and that this interaction is required for normal dASPP levels. genetic and biochemical data suggest that dRASSF8 acts in concert with dASPP to promote dCsk activity. Both proteins specifically localize to AJs and are mutually required for each other's localization. Furthermore, abnormal E-cadherin localization is observed in mutant pupal retinas, correlating with aberrant cellular arrangements. Loss of dCsk or overexpression of Src elicited similar AJ defects. Because Src is known to regulate AJs in both Drosophila and mammals, it is proposed that dASPP and dRASSF8 fine tune cell-cell adhesion during development by directing dCsk and Src activity. The dASPP-dRASSF8 interaction is conserved in humans, suggesting that mammalian ASPP1/2 and RASSF8, which are candidate tumor-suppressor genes, restrict the activity of the Src proto-oncogene (Langton, 2009).

Cell-cell contacts are essential for development and adult life of multicellular organisms. The best-characterized form of cell-cell contact is the adherens junction (AJ), which links neighboring cells via homotypic E-cadherin (E-Cad) interactions. The highly conserved intracellular domain of E-Cad binds to β-catenin, which itself binds to α-catenin. Transient interactions between α-catenin and actin filaments link AJs to the cytoskeleton, though the exact nature of this connection remains controversial. AJs are particularly important for the integrity of epithelial tissues. In addition to establishing and maintaining cell-cell adhesion, AJs regulate several aspects of cellular behavior, including cytoskeletal rearrangement and transcription. Inappropriate disruption of cell-cell contacts can lead to excess proliferation and is a hallmark of the metastatic process (Langton, 2009).

Dynamic remodeling of AJs occurs during all major morphogenetic events involving movement and rearrangement of epithelial cells, including convergent extension and gastrulation. AJ remodeling is necessary for the generation of epithelial structures with extremely precise patterns, such as the hexagonal array of ommatidia in the Drosophila compound eye (Langton, 2009).

SRC signaling is a major cellular pathway known to promote AJ remodeling in development and metastasis. Cellular SRC (c-SRC) is a member of the SRC family kinases (SFKs), which include c-SRC, FYN, and YES. Activated c-SRC is known to regulate AJs by several mechanisms. For example, c-SRC can induce the ubiquitylation of E-Cad by an E3 ubiquitin ligase called Hakai, promoting E-Cad internalization or degradation. In Drosophila, Src42A (one of two c-Src homologs) genetically interacts with E-Cad (encoded by shotgun [shg] in Drosophila), localizes to AJs, and forms a ternary complex with E-Cad and Armadillo (Drosophila β-catenin). Furthermore, Src42A activation leads to decreased E-Cad protein levels and concurrent stimulation of E-Cad transcription by Armadillo and TCF, which is thought to be important for AJ turnover during morphogenesis (Langton, 2009).

The C-terminal region of c-SRC and other SFKs is targeted by C-terminal SRC kinase (CSK), which negatively regulates c-SRC by phosphorylating a conserved tyrosine residue (Tyr527 in avian c-SRC). Drosophila CSK (dCsk) appears to function analogously to mammalian CSK as a negative regulator of SFKs. dCsk is a negative regulator of tissue growth; mutants die as giant pupae and imaginal discs are enlarged as a result of increased proliferation. These observations are seemingly at odds with studies showing that Src activation in Drosophila tissues stimulates proliferation but also leads to considerable apoptosis. A recent report attempted to reconcile this discrepancy, suggesting that lower levels of Src activation induce proliferation and protection from apoptosis, whereas high levels lead to apoptosis and invasive migration (Vidal, 2007; Langton, 2009 and references therein).

It has been shown that dCsk activity is modulated by dASPP, the Drosophila homolog of mammalian ASPP1 and ASPP2, which physically interacts with dCsk and enhances its capacity to phosphorylate Src42A. Accordingly, dASPP phenotypes are enhanced by reducing dCsk gene dosage and are rescued by complete removal of Src64B, which functions redundantly with Src42A. This study identifies dRASSF8 as a new dASPP regulator. dRASSF8 is the homolog of mammalian RASSF7/8 (Ras association domain family 7/8). Ras association (RA) domain-containing proteins are putative Ras effectors; they specifically bind the activated (GTP-bound) form of Ras family GTPases, which function in numerous signal transduction pathways regulating proliferation, apoptosis, and differentiation. Mammalian RASSF family members 1–6 are characterized by their domain structure, with a C-terminal RA domain, a C1-like zinc finger, and a SARAH (Salvador-RASSF-Hippo) domain (van der Weyden, 2007). Mammalian RASSF7–10 are atypical RASSF proteins because they contain an N-terminal RA domain and lack a C1-like or SARAH domain. Recently, Xenopus RASSF7 was shown to be required for completing mitosis. Human RASSF8 is a putative tumor-suppressor gene; when expressed in lung cancer cells, RASSF8 inhibits anchorage-independent growth (Falvella, 2006). Importantly, the molecular function of RASSF8 has not been elucidated (Langton, 2009).

Two RASSF family proteins are encoded by the Drosophila genome. dRASSF is similar to human RASSF1–6 and has been linked to the Hippo pathway. dRASSF8 is similar to human RASSF7 and RASSF8, having an N-terminal RA domain. Published genome-wide yeast two-hybrid data suggested that dRASSF8 interacts with dASPP, prompting an investigation of the relationship between these proteins. Based on genetic and biochemical data, it is suggested that the dASPP-dRASSF8 complex regulates AJs by directing the activity of dCsk and Src (Langton, 2009).

dRASSF8 is the sole Drosophila homolog of mammalian RASSF7 and RASSF8, which are so-called N-terminal RASSF proteins and the least-studied members of the RASSF family. This study demonstrates that dRASSF8 binds to dASPP in Drosophila cells and that RASSF8 binds to ASPP1 and ASPP2 in human cells, indicating that an evolutionarily conserved relationship between these proteins has been uncovered. The function of RASSF8 is currently unknown, and this study thus provides new insights into the function of N-terminal RASSF proteins (Langton, 2009).

Future experiments will determine whether RASSF7 also binds ASPP family proteins or whether this function is specific to RASSF8. RASSF7 has been studied in Xenopus and was found to associate with centrosomes and to be required for completing mitosis. In contrast, the current data suggest that dRASSF8 is not required for cell-cycle progression because null mutants for dRASSF8 are viable. These findings are suggestive of divergent functions for RASSF7 and RASSF8 in vertebrates, with dRASSF8 being functionally analogous to RASSF8 rather than RASSF7. Indeed, GFP-tagged RASSF7 localizes to the nucleus and centrosomes in Xenopus embryos, whereas this study never observed nuclear localization of dRASSF8. Further studies of N-terminal RASSF proteins in vertebrates should clarify whether RASSF7 and RASSF8 have overlapping or independent functions (Langton, 2009).

In vivo data point at a close relationship between dRASSF8 and dASPP, which colocalize and are required for each other's presence at AJs in epithelial cells. dRASSF8 posttranscriptionally regulates the levels of dASPP protein in epithelia. Thus, it seems likely that binding to dRASSF8 stabilizes dASPP and prevents its degradation, which can be observed for many protein complexes. Overall, these data provide compelling evidence for a functional link between dRASSF8 and dASPP, which is likely to be conserved through to their closest mammalian counterparts, RASSF8 and ASPP1/2 (Langton, 2009).

The data suggest that dRASSF8 has some dASPP-independent roles. For example, dRASSF8 mutant wings are large and broadened, whereas dASPP mutant wings are large but of normal shape. In addition, the dRASSF8 adult eye phenotype is more marked than that of dASPP mutants. Accordingly, it was found that dRASSF8, but not dASPP, is required for apoptosis of excess IOCs in the developing pupal retina. It therefore appears that the dRASSF8 eye phenotype results from both reduced apoptosis of IOCs and cell-cell adhesion defects. The subtle differences between the dASPP and dRASSF8 phenotypes indicate unknown functions for dRASSF8, which are not due to its effects on dASPP. Future efforts will be aimed at elucidating these functions (Langton, 2009).

These data are consistent with a model in which dRASSF8 binds to and positively regulates dASPP and, in this way, promotes dCsk activity indirectly. Coimmunoprecipitation experiments support this idea, showing that dRASSF8 and dASPP associate and that dASPP and dCsk associate. However, no detect interaction was detected between dRASSF8 and dCsk, indicating that dRASSF8 does not directly associate with dCsk. The proposed model is also supported by genetic data; the dRASSF8-dCsk genetic interaction is weaker than the dASPP-dCsk interaction, suggesting that dASPP is the primary regulator of dCsk. The weaker genetic relationship between dRASSF8 and dCsk can be explained by the observation that some dASPP protein persists in dRASSF8 mutant tissue. These observations suggest that dRASSF8 regulates dCsk via dASPP (Langton, 2009).

Retinal morphogenesis involves dynamic changes in cell-cell contacts to create the final ordered array of photoreceptors and accessory cells. dASPP and dRASSF8 are required for normal E-Cad localization in 26-27 hr APF retinas, providing an explanation for the patterning defects in mutant eyes. It is proposed that the abnormal E-Cad localization in dASPP mutant eyes results from increased Src activity based on several lines of evidence. dASPP binds to and positively regulates dCsk, leading to Src inhibition; therefore, loss of dASPP increases Src activity, which is known to reduce cell-cell adhesion by promoting the internalization and degradation of E-Cad. In agreement with this, it was shown that loss of dCsk or overexpression of either Drosophila Src leads to loss of AJ material in 26-27 hr APF retinas. This claim is further supported by the fact that the dASPP eye phenotype is suppressed by loss of Src64B. Thus, the presence of the dASPP-dRASSF8 complex at AJs may be required to locally prevent inappropriate Src activation and dissolution of AJs (Langton, 2009).

The fact that dASPP and dRASSF8 mutants are homozygous viable implies that these genes are dispensable for the majority of morphogenetic processes occurring during development. Therefore, the regulation of AJs by dASPP and dRASSF8 may be restricted to the eye. However, as they are expressed in other epithelial tissues, a closer examination of dASPP and dRASSF8 mutants may reveal subtle defects in other morphogenetic processes (Langton, 2009).

It is suggested that dASPP and dRASSF8 are new noncore AJ components and part of the machinery that ensures the fine regulation of AJs by Src during development. This regulation is crucial to provide precisely the right amount of junctional plasticity to allow cell-cell rearrangements and patterning to take place while limiting this plasticity to maintain epithelial coherence and prevent cell delamination. Because the interaction between these proteins is conserved in mammals, this finding is likely to be relevant to mammalian development and to the metastatic process, which is associated with downregulation of E-Cad and loss of cell-cell adhesions. Indeed, ASPP1 knockout mice present defects in the assembly of lymphatic vessels consistent with a potential adhesion defect. This suggests that regulation of cell-cell adhesion may underlie the function of ASPP1/2 and RASSF8 as mammalian tumor suppressors (Langton, 2009).

Functions of RASSF8 orthologs in other species

A novel serum biomarker Enhancer RNA RASSF8-AS1 promotes the progression of gastric cancer

Gastric cancer (GC) is globally recognized as one of the most widespread malignant tumors. As the symptoms of patients with early GC are ambiguous, the majority of patients are given a diagnosis of advanced GC. Therefore, this necessitates the search for new biomarkers to be utilized in the early diagnosis and screening of GC. Enhancer RNA (eRNA) is a non-coding RNA in transcription by enhancers that is tumor-specific and has a critical function in cancer progression. This research investigates new eRNAs as bio-diagnostic markers for GC. Four eRNAs with good differential expression in GC were screened by TCGA and University of California, Santa Cruz databases. Quantitative real-time PCR was utilized for testing the level of RASSF8-AS1 (antisense RASSF8). The diagnostic effect of RASSF8-AS1 was evaluated using the receiver operating characteristic (ROC). Functional experiments were used to detect the ability of RASSF8-AS1 to affect the metastasis and proliferation in GC cells. The expression of RASSF8-AS1 was obviously elevated in both GC tissues and serum, whereas it was decreased in the serum levels of postoperative GC patients. ROC showed that RASSF8-AS1 was more diagnostically efficient than common diagnostic biomarkers for GC and that diagnostic effectiveness could be better than combining them. The findings of in vitro experiments showed that knocking down the level of RASSF8-AS1 clearly suppressed the ability of growth and metastasis in GC cells. Studies have shown that serum RASSF8-AS1 has the potential to contribute to the progression of GC as a biomarker for diagnosis and prognostic monitoring of GC (Li, 2025).

Complex interplay between RAS GTPases and RASSF effectors regulates subcellular localization of YAP

RAS GTPases bind effectors to convert upstream cues to changes in cellular function. Effectors of classical H/K/NRAS are defined by RBD/RA domains which recognize the GTP-bound conformation of these GTPases, yet the specificity of RBD/RAs for over 160 RAS superfamily proteins remains poorly explored. This study has systematically mapped interactions between BRAF and four RASSF effectors, the largest family of RA-containing proteins, with all RAS, RHO and ARF small GTPases. 39 validated complexes reveal plasticity in RASSF binding, while BRAF demonstrates tight specificity for classical H/K/NRAS. Complex between RASSF5 and diverse RAS GTPases at the plasma membrane can activate Hippo signalling and sequester YAP in the cytosol. RASSF8 undergoes liquid-liquid phase separation and resides in YAP-associated membraneless condensates, which also engage several RAS and RHO GTPases. The poorly studied RASSF3 has been identified as a first potential effector of mitochondrial MIRO proteins, and its co-expression with these GTPases impacts mitochondria and peroxisome distribution. These data reveal the complex nature of GTPase-effector interactions and show their systematic elucidation can reveal completely novel and biologically relevant cellular processes (Singh, 2024).

CircCERS6 Suppresses the Development of Epithelial Ovarian Cancer Through Mediating miR-630/RASSF8

Accumulating evidence have demonstrated that circular RNAs (circRNAs) exert important roles in tumor initiation and progression. Nevertheless, the role and mechanism of circRNA ceramide synthase 6 (circCERS6) in epithelial ovarian cancer (EOC) remain to be clarified. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, RNA and protein expression were measured. The effects of circCERS6/microRNA-630 (miR-630)/Ras-association domain family member 8 (RASSF8) axis was analyzed in cell proliferation, migration, and invasion by 5-Ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, scratch test, and transwell assay. Using RNA-pull down assay and dual-luciferase reporter assay, the interaction between miR-630 and circCERS6 or RASSF8 was verified. The in vivo role of circCERS6 in tumor growth was analyzed using xenograft mice model. CircCERS6 expression was markedly reduced in EOC tissues and cell lines. CircCERS6 overexpression hampered the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of EOC cells. CircCERS6 directly interacted with miR-630. miR-630 expression was up-regulated in EOC tissues and cell lines. CircCERS6 overexpression-induced anti-tumor effects in EOC were largely reversed by the overexpression of miR-630. RASSF8 was a direct target of miR-630. RASSF8 level was decreased in EOC tissues and cell lines. CircCERS6 up-regulated RASSF8 expression by acting as a molecular sponge for miR-630 in EOC cells. CircCERS6 overexpression-mediated suppressive effects in EOC cells were largely overturned by the silence of RASSF8. CircCERS6 overexpression blocked tumor growth in vivo. CircCERS6 overexpression hampered the proliferation, migration, invasion, and EMT of EOC cells by up-regulating RASSF8 via sponging miR-630 (Li, 2022).

MicroRNA-320a Promotes Epithelial Ovarian Cancer Cell Proliferation and Invasion by Targeting RASSF8

MicroRNAs (miRNAs) play important roles in tumorigenesis by controlling target gene expression. With opposing roles as a tumor suppressor or oncogene, microRNA-320a (miR-320a) was found to participate in tumor genesis and progression and also identified as a potentially useful marker in cancer diagnosis, treatment, and prognosis. To better understand the role of miR-320a in ovarian cancer, this study investigated miR-320a expression in epithelial ovarian cancer (EOC) specimens as well as EOC cell lines, and correlations between miR-320a expression and processes associated with EOC progression were analyzed. The miR-320a level in EOC specimens was found to be associated with ovarian cancer progression and infiltration. Through in vitro and in vivo studies, this study found that miR-320a significantly promoted the proliferation, migration, and invasion of EOC cells, and RASSF8 was identified as a target gene of miR-320a that was downregulated in EOC tissues and cell lines. In vitro downregulation of RASSF8 promoted the growth, migration, and invasion of EOC cells. Together these findings indicate that RASSF8 is a direct target of miR-320a, through which miR-320a promotes the progression of EOC (Zang, 2021).

MicroRNA-322 Regulates Self-renewal of Mouse Spermatogonial Stem Cells through Rassf8

Spermatogonial stem cells (SSCs) are essential for spermatogenesis and male fertility. MicroRNAs (miRs) are key regulators of gene expression involved in self-renewal, differentiation, and apoptosis. However, the function and mechanisms of individual miR in regulating self-renewal and differentiation of SSCs remain unclear. This study reports for the first time that miR-322 regulates self-renewal of SSCs. Functional assays revealed that miR-322 was essential for SSC self-renewal. Mechanistically, miR-322 promoted SSC self-renewal by targeting RASSF8 (ras association domain family 8). Moreover, the WNT/beta-catenin signaling pathway was involved in the miR-322-mediated regulation. Furthermore, miR-322 overexpression increased GFRalpha1, ETV5 and PLZF expression but decreased STRA8, C-KIT and BCL6 expression. This study provides not only a novel insight into molecular mechanisms regulating SSC self-renewal but also a basis for the diagnosis, treatment, and prevention of male infertility (Wang, 2019).

REFERENCES

Search PubMed for articles about Drosophila RASSF8

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Banerjee, J. J., Aerne, B. L., Holder, M. V., Hauri, S., Gstaiger, M. and Tapon, N. (2017). Meru couples planar cell polarity with apical-basal polarity during asymmetric cell division. Elife 6. PubMed ID: 28665270

Bo, X., Chen, Y., Sheng, W., Gong, Y., Wang, H., Gao, W., Zhang, B. (2018). The regulation and function of microRNA-377/RASSF8 signaling axis in gastric cancer. Oncol Lett, 15(3):3630-3638 PubMed ID: 29456730

Chan, E. H. Y., Zhou, Y., Aerne, B. L., Holder, M. V., Weston, A., Barry, D. J., Collinson, L. and Tapon, N. (2021). RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering. Development 148(20). PubMed ID: 34532737

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Huang, Y., Li, Y., Wang, F. F., Lv, W., Xie, X., Cheng, X. (2016). Over-Expressed miR-224 Promotes the Progression of Cervical Cancer via Targeting RASSF8. PLoS One, 11(9):e0162378 PubMed ID: 27626930

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Zhang, L., Chen, H., He, F., Zhang, S., Li, A., Zhang, A., Zhang, A. (2021). MicroRNA-320a Promotes Epithelial Ovarian Cancer Cell Proliferation and Invasion by Targeting RASSF8. Front Oncol, 11:581932 PubMed ID: 33718138 Biological Overview

date revised: 30 March 2026

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