InteractiveFly: GeneBrief

Striatin interacting protein: Biological Overview | References

BIOLOGICAL OVERVIEW

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. This study demonstrates that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. This study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization (Sakuma, 2016).

Since the Hippo (Hpo) pathway was discovered as the key regulator to ensure the appropriate final tissue size by coordinating cell proliferation and cell death, large-scale genetics studies have identified numerous regulators of the Hpo pathway. While most pathway components identified thus far are positive regulators of Hpo, some negative regulators were recently reported. One such negative regulator is the STRIPAK (STRiatin-Interacting Phosphatase And Kinase) complex, which is evolutionarily conserved and regulates various cellular processes including cell cycle control and cell polarity (Hwang, 2014). The core component of the STRIPAK complex is the striatin family of proteins: striatins serve as B‴ subunits (one of the subfamily of regulatory B subunits) of the protein phosphatase 2A (PP2A) complex. Beyond this, the A and C subunits of PP2A, Mob3, Mst3, Mst4, Ysk1, Ccm3, Strip1, and Strip2 form the core mammalian STRIPAK complex. It has been previously reported that Strip, the Drosophila homolog of mammalian Strip1 and 2, is involved in early endosome formation, which is essential for axon elongation (Sakuma, 2014). Building on these findings, it was hypothesized that the Strip-Hpo pathway may also be involved in neuronal synaptic development (Sakuma, 2016).

The Drosophila larval neuromuscular junction (NMJ) is an ideal model for studying synaptic development because of its identifiable, stereotyped morphology, accessibility, broad complement of available reagents, and suitability for a wide range of experimental approaches. Furthermore, the Drosophila NMJ, like vertebrate central synapses, is glutamatergic, suggesting that the molecular mechanisms that regulate synaptic development in Drosophila NMJ might be applicable to vertebrates. Motor neuron axons are genetically hardwired to target specific muscles by the end of the embryonic stage. There, axonal growth cones subsequently differentiate into presynaptic termini, called boutons, each of which contains multiple active zones). During the larval stage, muscle size increases nearly 100-fold and boutons are continuously and proportionately added to maintain constant innervation strength. Various molecules can negatively or positively regulate the growth of synaptic termini. Amongst the many factors, elements of the actin cytoskeleton are key effectors of morphological change, functioning downstream of several cell surface receptors and signaling pathways. Of the two types of actin filaments (branched and linear), the activity of Arp2/3 complex, responsible for nucleation of branched F-actin, the first step of actin polymerization, should be strictly regulated. Arp2/3 hyperactivation results in synaptic terminal overgrowth characterized by excess small boutons emanating from the main branch that are termed satellite boutons (Sakuma, 2016).

This study shows that Strip negatively regulates the synapse terminal development through tuning the activity of the core Hpo kinase cassette. Loss or reduction of strip function in motor neurons increased the number of satellite boutons, which could be suppressed by reducing the genetic dosage of hpo. Similarly, activation of the core Hpo kinase cassette also increased satellite boutons. In this context, the presumptive downstream target of the core Hpo kinase cassette is Enabled (Ena), a regulator of F-actin assembly and elongation that was reported to antagonize the activity of Arp2/3. The canonical downstream transcriptional co-activator, Yorkie (Yki), appears dispensable for Hpo-mediated synaptic terminal development. It is proposed that the evolutionarily conserved Strip-Hpo pathway regulates local actin organization by modulating Ena activity during synaptic development (Sakuma, 2016).

This study has identified Strip and components of the Hpo pathway as regulators of synaptic morphology. In addition to the intensely investigated function of Hpo in growth control in mitotic cells, a few postmitotic roles of the Hpo pathway have recently been uncovered, such as dendrite tiling in Drosophila sensory neurons (Emoto, 2006) and cell fate specification of photoreceptor cells in Drosophila retina (Jukam, 2013). This study now finds an additional postmitotic role for Hpo in synaptic terminal development. The results indicate that Strip and the core Hpo kinase cassette regulate satellite bouton formation by regulating the activity of Ena, an actin regulator that is involved in the initiation, extension, and maintenance of linear actin filaments at the barbed end (Winkelman, 2014). Although it cannot be excluded that there might be other targets of Yki in motor neurons than diap1 or bantam whose transcriptional activations were not observed in this study, Yki, a well-known downstream target of the core Hpo kinase cassette, was dispensable for proper synaptic morphology. Ena phosphorylation causes its inactivation; therefore, it was reasoned that Strip can act as a positive regulator of Ena by inactivating the Hpo pathway. A model is proposed for the regulation of satellite bouton formation by Strip and Hpo pathway components (see The Strip-Hpo pathway regulates satellite bouton formation with Ena, a regulator of F-actin organization). As the presynaptic localization of endogenous Strip was punctate and non-uniform, it is expected that Strip localization could be critical for regulating the phosphorylation status of Hpo, Wts, and Ena, which locally alters actin organization and eventually specifies the position of satellite bouton formation that could be a marker for new bouton outgrowth. When Strip is present, the core Hpo kinase cassette is inactivated, which in turn locally increases the expression of the active (unphosphorylated) form of Ena. However, the core Hpo kinase cassette can be activated in the absence of Strip, which subsequently phosphorylates and inactivates Ena. Ena prevents Arp2/3-induced branching, suggesting that Ena inactivation activates Arp2/3 and results in satellite bouton formation, similar to Rac activation. It is reported that Arp2/3 is involved in bouton formation and axon terminal branching downstream of WAVE/SCAR complex in NMJ (Koch, 2014). Indeed, the cureent findings support this hypothesis. F-actin organization was altered by strip knockdown in motor neurons. When the GFP-moe reporter was expressed in motor neuron, the GFP fused to the C-terminal actin-binding domain of Moesin, which is widely used as an F-actin reporter. The intensity of actin puncta became higher and puncta were unevenly distributed when strip was knocked down. This data suggests that Strip function is important for the proper organization of F-actin (Sakuma, 2016).

There are many indications that Strip and other STRIPAK components (Mst3, Mst4, and Ccm3) regulate the actin network. For example, Strip1, Strip2, Mst3, and Mst4 regulate the actomyosin contractions which regulate cell migration in cancer cells. In addition to regulating the actin network, STRIPAK has been suggested to function in microtubule organization. Mutants of Drosophila Mob4, a member of the core STRIPAK complex and homolog of mammalian Mob3, show abnormal microtubule morphology at NMJs and muscles (Schulte, 2010). Furthermore, it has been reported that Strip forms a complex with Glued, the homolog of mammalian p150^Glued (Sakuma, 2014), a component of the dynactin complex required for dynein motor-mediated retrograde transport along microtubules. Strip also affects microtubule stability (Sakuma, 2015). As previously mentioned, microtubules are also key effectors of synaptic development downstream of several receptors and signaling pathways. Taken together, the STRIPAK complex can act as a regulatory hub for multiple cellular signals including Hpo pathway-mediated actin organization, endocytic pathway-dependent BMP signals, and microtubule stability for proper synaptic development (Sakuma, 2016).

The Hpo pathway has been reported to act as a sensor of the local cellular microenvironment, such as mechanical cues, apico-basal polarity and actin architecture to balance cell proliferation and cell death. Although synaptic morphogenesis is a postmitotic process, it is very plastic and depends on a dynamically changing extracellular environment, as exemplified by the nearly 100-fold expansion of muscle size during larval development. Thus, this study demonstrates an intriguing function for the Strip-Hippo pathway in the homeostatic control of neuronal synaptic morphology and function (Sakuma, 2016).

A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability

During neural development, regulation of microtubule stability is essential for proper morphogenesis of neurons. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was revealed to be involved in diverse cellular processes. However, there is little evidence that STRIPAK components regulate microtubule dynamics, especially in vivo. This study shows that one of the core STRIPAK components, Strip, is required for microtubule organization during neuronal morphogenesis. Knockdown of Strip causes a decrease in the level of acetylated alpha-tubulin in Drosophila S2 cells, suggesting that Strip influences the stability of microtubules. It was also found that Strip physically and genetically interacts with tubulin folding cofactor D (TBCD), an essential regulator of alpha- and beta-tubulin heterodimers. Furthermore, a genetic interaction was demonstrated between strip and Down syndrome cell adhesion molecule (Dscam), a cell surface molecule that is known to work with TBCD. Thus, it is proposed that Strip regulates neuronal morphogenesis by affecting microtubule stability (Sakuma, 2015).

To form a functional neural circuit, the morphology of each neuron should be strictly regulated to wire with the correct synaptic partners. Microtubules are fundamental structural components of axons and dendrites and their dynamics directly affect neuronal morphology. This report shows that Strip, one component of the STRIPAK complex, affects microtubule stability in Drosophila S2 cells. Furthermore, Strip regulates dendrite branching and axon elongation by interacting with TBCD in the olfactory projection neurons. Moreover, it was observed genetic interactions between strip and Dscam in the mushroom body neurons, suggesting that Strip and TBCD regulate microtubule stability in the downstream of Dscam (Sakuma, 2015).

We recently found that TBCD can bind to the intracellular domain of Dscam and it is likely that TBCD mediates Dscam functions by affecting microtubule dynamics. Although Dscam has been extensively studied, the downstream pathways are not well known. The Dock/Pak signaling pathway, one of the known pathways acting downstream of Dscam, does not seem to be required for dendrite targeting and axon guidance of olfactory projection neurons. Strip seems to act with TBCD in the downstream of Dscam during neuronal morphogenesis, and link outer cellular environment with microtubules (Sakuma, 2015).

strip and TBCD mutants show similar phenotypes in axon elongation and dendrite branching, and Strip and TBCD genetically and physically interact with each other. TBCD assists in the formation of tubulin heterodimers, and affects microtubule stability by controlling the availability of tubulin subunits because concentration of free tubulin dimers can affect microtubule dynamics. Although mammalian TBCD does not bind to microtubules, Alp1, a homolog of TBCD in Schizosaccharomyces pombe, is colocalized with microtubules. Furthermore, overproduction of Cin1p, the homolog of TBCD in Saccharomyces cerevisiae, resulted in an increased sensitivity to benomyl, an antifungal compound that weakly inhibits microtubule assembly. Thus, TBCD and its homologs have been shown to regulate microtubule dynamics (Sakuma, 2015).

Strip seems to affect microtubule stability by interacting with several molecules in addition to TBCD. Strip also forms a complex with Glued, the homolog of mammalian p150^Glued,. Glued is one of the components of the dynactin complex required for dynein motor-mediated retrograde transport along microtubules. Glued has the CAP-Gly domain that is common in several +TIPs and regulates initiation of retrograde transport in neuronal cells. Interestingly, mammalian TBCB, another member of tubulin-folding cofactor family, also has the CAP-Gly domain and interacts with p150^Glued. +TIPs are a structurally and functionally diverse group of proteins that are distinguished by their specific accumulation at microtubule plus ends, or growing ends. +TIPs control different aspects of microtubule dynamics and form links between microtubule ends and other cellular structures. Some of +TIPs also participate in microtubule-actin crosstalk, such as CLIP-170–formin interaction and EB1-RhoGEF2 interaction. There is accumulating evidence that Strip and also other STRIPAK components (Mst3, Mst4, and Ccm3) regulate the actin network. For example, Strip1 and 2, homologs of Drosophila Strip, were recently identified as regulators of the actomyosin contraction that regulates cell migration in cancer cells. Taken together, Strip and STRIPAK seem to form a giant complex with TBCD and Glued at growing ends of microtubule to stabilize them and serve as a linker between microtubules and actin networks to regulate proper neurite branching and elongation (Sakuma, 2015).

strip knockdown resulted in a decrease in the level of acetylated α-tubulin. Therefore, this study examined the possibility that Strip directly affects the acetylated tubulin level by regulating acetylation or deacetylation enzymes. The genetic interaction between strip and HDAC6, one of the enzymes responsible for the deacetylation of α-tubulin was investigated, however, the shorter axon phenotype of strip knockdown was not suppressed when combined with a HDAC6 mutation. Thus, Strip does not seem to directly affect the acetylation of α-tubulin, but affect the stabilization of microtubule by interacting with TBCD, Glued, and other molecules. Although controversial, some reports indicate that acetylation is the result, and not the cause, of stabilization (Sakuma, 2015).

Many diseases are linked to microtubules. For example, defects in retrograde transports along microtubules cause neurodegenerative diseases, such as motor neuropathy 7B and Perry syndrome. Furthermore, Dscam is implicated in the cognitive disabilities in Down syndrome. Moreover, STRIPAK complexes have been linked to a number of clinical conditions and diseases. Cancer genome sequencing also identified frequent mutations in human STRIP2, and based on the mutation frequency and types, STRIP2 was classified as an oncogene. Thus, further investigation of Strip, STRIPAK, and the microtubule complex would yield new insights into the mechanisms of various diseases (Sakuma, 2015).

Drosophila Strip serves as a platform for early endosome organization during axon elongation

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. This study shows that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis (Sakuma, 2014).

A forward genetic mosaic screen was performed to identify genes for their cell autonomous functions in dendrite and axonal development, and an evolutionarily conserved protein, Striatin-interacting protein (Strip), was identified that works as a molecular linker between retrograde transport and Rab5 activity in early endosome organization. Strip and its orthologues were reported to be a component of Striatin-interacting phosphatase and kinase (STRIPAK) complex (Huang, 2014; Ribeiro, 2010; Ashton-Beaucage, 2014); however, the function of Strip has not yet been reported. Interestingly, this study found that Strip forms the protein complex with both Glued, the orthologue of mammalian p150Glued, and Sprint, the orthologue of RIN-1. Glued is an essential component of the dynactin complex and regulates the initiation of retrograde transport on microtubule, and Sprint is supposed to be a guanine nucleotide exchange factor (GEF) for Rab5. This study found that Strip affects the transport of early endosomes by forming complex with Glued in developing neurons. Furthermore, clustering of early endosomes is defective in strip-knockdown Drosophila S2 cells and HeLa cells; early endosomes become smaller and scattered as reported in dynein-inhibited HeLa cells. Moreover, similar to other GEFs, Sprint and Strip have a higher affinity for guanosine diphosphate (GDP)-bound forms of Rab5 than for guanosine triphosphate (GTP)-bound form. In addition, Strip seems to stabilize protein level of Sprint. Finally, the expression of constitutively active form of Rab5 in strip-mutant neurons suppresses the axon elongation defect. These data demonstrate that Strip coordinates dynein-dependent transport and Rab5 activation at the clustering and fusion of early endosomes, which are required for axon elongation (Sakuma, 2014).

During neural development, axon termini are exposed to a constantly changing surrounding environment. Thus, the spatio-temporal regulation of the amount of adhesion molecules and receptors at the axon growth cone is essential. This study identified an uncharacterized protein Strip that forms complexes with Glued and Sprint, and positively regulates clustering (retrograde transport along microtubule) of early endosomes. To form mature early endosomes from internalized small early endosomes, the coordination between clustering and fusion of early endosomes is crucial. For this, Strip serves as a molecular linker by interacting with Glued in clustering and with Sprint, Rab5 and probably with Vps45 in fusion. Vps45 is a Sec-1/Munc18 (SM) protein that targets Avl (Syntaxin 7) to promote fusion of early endosomes, which seems to function with Strip in the wing development. As this study also observed genetic interaction between sprint and Glued in axon elongation, Strip seems to be a platform for early endosome organization by forming a dynein-Glued-Strip-Sprint-Rab5 complex. The following model to explain the physiological role of Strip in the organization of early endosomes. During axon elongation, the strength of the elongation signal could be properly regulated by the dynein-Glued-Strip-Sprint-Rab5 complex by affecting the organization of mature early endosomes. Mature early endosomes are essential vesicular structures for cell signalling, serving both as signalling centres and as sorting centres to degrade or recycle cargoes. In vertebrate neurons, target-derived nerve growth factor (NGF) mediates axon elongation via retrograde axon transport of TrkA-containing signalling endosomes locally or to the cell body to stimulate the expression of downstream target genes essential for long-term axon growth and target innervation. Furthermore, the amount of cell adhesion molecules might also be regulated by Strip-mediated early endosome organization. It has been reported that the regulation of the amount and localization of molecules such as L1 and integrin in the axon growth cone is essential for growth cone motility (Sakuma, 2014).

Further investigations are still needed to identify which Strip domains are important for the formation of the dynein-Glued-Strip-Sprint-Rab5 complex. As Glued and Sprint were identified using a yeast two-hybrid screen, it seems that the binding between Glued and Strip, and that between Sprint and Strip, are likely to be direct. Furthermore, it is thought that Glued and Sprint bind to Strip simultaneously and form a tertiary complex for two reasons; (1) Sprint and Glued showed genetic interaction in PNs and exhibited a phenotype similar to that of stip^dogi PNs and (2) Vps45 and Glued exhibited genetic interaction at the wing vein formation. Thus, Strip seems to serve as a molecular linker between early endosome clustering and fusion by simultaneously forming a complex with Glued and Sprint. In addition, Strip seems to stabilize Sprint protein level. Unfortunately, it was not possible to produce a usable anti-Sprint antibody; therefore, Sprint localization could not be studied. However, it is speculated that Sprint stabilized by Strip could determine the spatio-temporal localization and the activity of Rab5. Regarding the binding between Strip and Glued, it is hypothesized that Strip may act as an adaptor between early endosomes and dynein. Unlike the highly diverse kinesin superfamily proteins (KIFs), which encompass 45 genes, cytoplasmic dyneins have only two heavy-chain family members in mammals. Thus, adaptor proteins for the dynein motor are thought to be important to determine the cargo selectivity, and some adaptor proteins have already been reported. Although Kif16b links Rab5 and microtubules, no specific molecule linking the dynein heavy chain with Rab5-positive early endosomes has been reported yet. Therefore, it is speculated that Strip could serve as an adaptor protein between Rab5-positive early endosomes and the dynein/dynactin complex by binding to Glued, similar to RILP and ORP1L that serve as adaptors between late endosomes and the dynein/dynactin complex (Sakuma, 2014).

This study has provided genetic evidences that Strip, Glued, Sprint and Rab5 are required for both axon elongation and dendrite branching in PNs. However, the expression of a constitutively active form of Rab5 partially suppressed axon elongation and did not suppress dendrite branching defects of strip^dogi PNs. It is considered that the level and the spatio-temporal pattern of Rab5 activity are crucial. Axon elongation and dendrite branching may require different levels or patterns of Rab5 activity. Furthermore, the failure to suppress the dendrite phenotype could also be explained by the fact that not only formation but also localization of mature early endosomes is required for dendrite branching. In Drosophila dendritic arborization (da) neurons, the proximal branching phenotype has been observed in the dendrite of Rab5², dynein light intermediate chain (dlic) or the kinesin heavy chain (khc) mutant. Consistent with this, dendrite proximal branching phenotype was also observed in Rab5² or strip^dogi PNs. The additional dendrite branched out from the dendrite stalk at a more proximal side in Rab5² or strip^dogi PNs than in wild-type PNs. Therefore, it is hypothesized that the constitutively active form of Rab5 could not suppress the dendrite phenotype in strip^dogi PNs, as the expression of the constitutively active form of Rab5 in strip^dogi PNs could promote early endosome maturation in axon and dendrites, but could not adjust early endosome subcellular localization, which is crucial for dendrite branching (Sakuma, 2014).

Strip is structurally and functionally conserved. Branching and elongation defects in strip^dogi PNs were rescued by the expression of the mouse homologue, Strip1. In addition, Strip1 was found to be involved in neuronal migration in the mouse developing cerebral cortex. In the mouse developing cerebral cortex, the Rab5-dependent endosomal trafficking pathway is required for neuronal migration, rather than for neurite extension, probably because it occurs before dendrite formation and axon elongation in vivo. Electroporation of Strip1 shRNA into the cerebral cortex at E14 caused migration defects, similar to those observed when Rab5 shRNAs were used. Thus, strip/Strip1 is involved in Rab5-dependent neural development including neuronal morphogenesis and migration events in both Drosophila and the mouse (Sakuma, 2014).

During this study of Strip function, Strip was reported as a member of the STRIPAK complex (Ribeiro, 2010; Ashton-Beaucage, 2014; Goudreault, 2009; Hwang, 2014). The core component of the PP2A phosphatase complex shows phosphatase activity, whereas the regulatory subunit determines the substrate specificity. Thus, PP2A phosphatase targets specific molecules by changing the regulatory subunit. When Strip is included in the PP2A complex, Cka serves as a regulatory subunit and targets Hippo and probably RAF that belongs to the RAS-MAPK pathway. As early endosome organization is important for many signal-transduction pathways, it might be possible that axon elongation and dendrite targeting require both the dynein-Glued-Strip-Sprint-Rab5 complex and the Strip-STRIPAK (Cka)-Hippo or the Strip-STRIPAK (Cka)-RAS-MAPK complex, but this does not seem possible, at least, in neuronal morphogenesis of PNs. When PNs homozygous for Cka were generated, no defects were found in axon elongation or dendrite branching. Thus, it is thought that the Strip-STRIPAK complex might not have a major role in neuronal morphogenesis (Sakuma, 2014).

The disruption of dynein or dynactin function causes neurodegeneration in many species, and this has been suggested to reflect impaired transport of signalling endosomes. In humans, G59S and G59A missense mutations of p150^Glued have been discovered in motor neuropathy 7B and Perry syndrome, respectively. Both mutations are located within the p150^Glued cytoskeleton-associated protein glycine-rich (CAP-Gly) domain, which is an MT-binding domain required for efficient retrograde transport. Progressive motor neuron degeneration is observed in p150^Glued transgenic mice, with pathological similarities to amyotrophic lateral sclerosis (ALS), including muscle atrophy, axonal swelling, disrupted intracellular trafficking and proliferation of degenerative organelles such as lysosomes and autophagosomes. Furthermore, motor neurons in Als2 (juvenile ALS-associated gene, Rab5GEF)-deficient mice show axonal degeneration. Moreover, the primary neurons in this mouse model show disturbed endosomal trafficking of the insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF) receptors. These observations suggest that retrograde transport and Rab5GEF may play a particularly important role in axonal health by transmitting information about the local environment from the axon to the soma. As Strip affects the early endosome organization by forming a complex with Gl and Spri, studying the functions of Strip may provide new insights into both developmental and pathological processes (Sakuma, 2014).

STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. This study identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice.These factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate (Neal, 2020).

Broad expression of the Pax6-type retinal determination factors Eyeless and Twin-of-eyeless initially prime the disc to give rise to the eye primordium. This event is closely linked to the acquisition of distinct morphological characteristics between the facing portions of the disc epithelium. Cells on one side of the epithelium adopt a columnar morphology and begin to express the retinal determinant Eyes absent (Eya), thus initiating eye formation; cells on the apposed side adopt the squamous and cuboidal morphology typical of the peripodial epithelium (PE). This binary choice between PE and retina developmental potential is a first step in a complex regional specification program that progresses in larvae over a 4-day period while the disc epithelium grows from fewer than 100 cells to more than 50,000 cells (Neal, 2020).

While the development of the eye, from primordium establishment to differentiation of the various retinal cell types, has been extensively studied, much less is known about PE formation. During metamorphosis, the PE is necessary for the morphogenetic events that transform a pair of apparently simple epithelia into the complex fly head. However, there is evidence that PE is more broadly required for the development of eye-antennal disc-derived organs, in particular the retina. In fact, the PE is essential for cellular growth in the eye primordium and for retinal neurogenesis; for example, PE ablation during photoreceptor neuron formation results in an arrest of retinogenesis. Notwithstanding these important functions of the PE, very little is known about the genes and molecular mechanisms that specify PE fate. This study has focused on the initial step, when retina potential is fostered within a portion of the eye disc epithelium but suppressed elsewhere, in favor of PE formation (Neal, 2020).

Heterotrimeric protein phosphatase 2A (PP2A) holoenzymes are an abundant group of cellular phosphatases that are highly conserved across Metazoa. They consist of A, B and C subunits; the catalytic (C) subunit and adaptor subunit (A) are each encoded by unique genes in Drosophila (mts and PP2A-29B, respectively). In contrast, the interchangeable substrate-specifying and activity-gating B subunits are divided into four families (B, B', B'' and B''') and are encoded by numerous genes in flies and vertebrates. Cka encodes the sole atypical B''' subunit that is orthologous to the mammalian striatins. PP2A holoenzymes containing Cka are found in the striatin-interacting phosphatase and kinase (STRIPAK) complex, which also includes other scaffolding and substrate-associated factors such as Strip. The contribution of this major cellular phosphatase to developmental fate decisions is yet to be determined (Neal, 2020).

This study demonstrates that STRIPAK-PP2A is essential for PE cell fate specification in the Drosophila eye disc. Loss of function of either Cka or Strip results in the emergence of an ectopic retinal field within the presumptive PE. STRIPAK-PP2A acts by negatively regulating the activity of the Hippo kinase (Hpo) and its effector Warts (Wts), thereby releasing the PE-promoting activity of the transcriptional co-activator Yorkie (Yki). Thus, this study elucidates the genetic pathway through which STRIPAK-PP2A promotes PE fate and provide a first example of a STRIPAK-PP2A-controlled cell fate decision in metazoan development (Neal, 2020).

The results demonstrate a clear role for STRIPAK-PP2A in regulating the adoption of distinct cell fates in the eye disc. PE was absent when Cka function was reduced, and instead the development of a mirror-image ectopic retina was detected within the presumptive PE cell layer. Similar phenotypes were observed when the loss of function of the STRIPAK-PP2A subunits Strip and SLMAP were induced. Furthermore, a dominant enhancement was observed of the Cka RNAi phenotype by two different loss-of-function alleles of the PP2A catalytic subunit, Mts. In all tested cases, including temperature modulation and the co-expression of pro-proliferation and anti-apoptotic factors, changes in disc size did not correlate with the PE-to-retina transformation and development of the ectopic retinal field proceeded in an apparently normal pattern (Neal, 2020).

Interestingly, PE fate was restored by the removal of one wild-type copy of hpo in Cka- or SLMAP-deficient discs, and by hpo RNAi in Strip-deficient discs, strongly suggesting that the latter factors act genetically upstream of Hpo. Given the direct roles of Strip and SLMAP in the recruitment of Hpo as a PP2A substrate in cell culture, the findings strongly point to the Hpo kinase as the target of negative regulation by STRIPAK-PP2A in the control of PE cell fate (Neal, 2020).

Furthermore, the loss-of-Cka phenotype was also suppressed by genetic reduction of Wts and by overexpression of Yki. As the Wts kinase is a direct intermediate between Hpo and Yki, this evidence links STRIPAK-PP2A to the eye disc PE fate-promoting factor Yki. Therefore, it is postulated that during the induction of PE fate, high levels of Yki activity are maintained in the presumptive PE via the negative regulation of the Hpo-Wts kinase axis by a STRIPAK-PP2A complex containing Cka, Strip and SLMAP. Notably, the ubiquitous role of the Hpo-Yki signaling axis in regulating tissue size appears to be in parallel to, and genetically separable from, its role in determining PE fate. Hence, STRIPAK-PP2A represents one effector of the developmental regulation of Yki activity in fate specification (Neal, 2020).

PE fate specification was first characterized in the wing imaginal disc, where the factor Bowl is a driver of PE fate. In addition, ectopic activation of Wg signaling or expression of Lines within the presumptive PE domain suppress PE fate by repressing the accumulation of Bowl (Neal, 2020).

Previous work demonstrated that the transcription co-activator Yki, with its DNA-binding partners Sd and Hth, appeared to be necessary for the establishment of PE in the eye disc, based on phenotypes induced by RNAi-mediated gene silencing (Zhang, 2011). This was confirmed by showing that ykiB5/ykiB5 mutant eye discs generated by MARGE are nearly identical to yki-RNAi discs, and also demonstrate that Yki's role in the PE-retina cell fate choice is independent of its nearly universal role in proliferation and cell survival (Neal, 2020).

Interestingly, these mechanisms of PE versus non-PE fate specification are not conserved between the eye and wing discs. Whereas a similar role for Wg has been proposed for the leg and haltere PE, based on the induction of columnar cell morphology by ectopic Wg in the PE, the same effect is not observed in the eye antennal disc. Lines overexpression within the eye disc PE also has no effect on cell fate, whereas in the retinal progenitor region of the disc, Lines repression of Bowl initiates precocious eye morphogenesis. Thus, Wg, Lines and Bowl do not appear to be required for PE specification in the eye disc. Conversely, extensive analyses of Yki and Sd function in the wing imaginal disc have not revealed roles for Yki in the PE versus wing-proper fate choice, consistent with the specific role for Yki in suppressing ectopic retina formation that is documented in this study (Neal, 2020).

Thus, the acquisition of PE fate results from different genetic determinants in the eye and wing discs, and is mechanistically distinct (Neal, 2020).

Given the plethora of cellular kinases that regulate myriad biological processes, it has been appreciated that cellular phosphatases might play equally important and diverse biological roles. However, there are far fewer cellular phosphatases, and each is known to target a broad range of cellular substrates. PP2A is no exception to this; it is essential for cell proliferation and viability. Hence, it is difficult to investigate potential roles of this phosphatase in more specific biological processes, such as cell fate specification. This paper reports the involvement of STRIPAK-PP2A in regulating Hippo-Yki signaling in the PE fate choice within the developing eye disc. This role of STRIPAK-PP2A was revealed by targeted silencing of complex-specific PP2A co-factors (Cka-B'', Strip, SLMAP), and the link to PP2A was confirmed by genetic interaction testing using alleles of the vital catalytic subunit (mts) (Neal, 2020).

Few studies in metazoans have identified contexts in which PP2A complexes may play a role in fate specification during development. Screens in Drosophila and Caenorhabditis elegans have identified mutant alleles of fly mts (PP2A-C) and worm sur-6 (a PP2A-B-proper subunit) as modifiers of the MAPK pathway in the R7 photoreceptor and vulval fate choices, respectively. In the fly, mts mutant alleles dominantly enhanced the effects of activated Ras signaling and dominantly suppressed the effects of activated Raf signaling, whereas in the worm, sur-6 mutant alleles dominantly suppressed the effects of activated Ras signaling. Yet, homozygous loss-of-function conditions for these PP2A components could not be shown to affect either fate in an otherwise wild-type background (Neal, 2020).

Two additional phenotypes suggestive of potential changes in cell fate have also been linked to loss-of-function alleles of tws, which encodes the sole Drosophila PP2A-B-proper subunit. First, loss of tws led to the overgrowth and duplication of the pouch in the developing wing imaginal disc, and second, hypomorphic alleles of tws were shown to yield flies with atypical sensory bristles that display supernumerary shaft and socket cells with coincident loss of their single neuronal and sheath cells. However, neither study substantiated a true switch in cell fate vis-à-vis aberrant proliferation or reorganization of patterning cues, nor did they establish a connection between the PP2A catalytic subunit and the putative fate-switch phenotypes. The molecular substrates of PP2A-Tws also remain to be determined in these contexts. Nonetheless, these data hint at the possibility that specific PP2A holoenzymes, STRIPAK-PP2A and PP2A-Tws/Sur-6, may direct different cell fate choices and do so via the modulation of distinct signaling pathways (Neal, 2020).

Whereas there is a lack of evidence in the literature for PP2A- and/or STRIPAK-regulated fate decisions, Hippo (MST1/2) signaling and Yki (YAP/TAZ) have been implicated in fate decisions in different developmental contexts. Arguably the best studied example also involves the Drosophila eye, where Hpo-Yki-Sd signaling has been shown to instruct the choice between Rh5- versus Rh6-expressing R8 photoreceptor subtypes (Neal, 2020).

Perhaps more intriguing is that, during the development of the vertebrate eye, a fate paradigm strikingly similar to the PE/retina fate choice has been described to involve the Yki orthologs YAP and TAZ. In vertebrates, the primordia of the retina and the retinal pigment epithelium (RPE) are specified at the optic vesicle stage, when the vesicle epithelium is partitioned into two fields, one that generates neural retina and one that develops into non-neural RPE in the optic cup. Miesfeld and colleagues demonstrated that YAP and its paralog TAZ play important roles in promoting RPE fate in zebrafish, as loss of these factors leads to transdifferentiation of RPE into retina-like cells (Miesfeld, 2015). A similar role for YAP in RPE fate specification was subsequently demonstrated in mice (Kim, 2016), where loss of YAP produces optic cups with mirror-image retinae, consisting of a pseudostratified epithelium that expresses markers of retinal progenitor cells in the presumptive RPE (Neal, 2020).

The contribution of STRIPAK-PP2A to cell fate decisions in these models has not been investigated. However, given the similar fate-determining roles for Yki and YAP/TAZ, particularly in the latter example, it will be of great interest to determine whether STRIPAK-PP2A-mediated regulation of YAP and/or TAZ activity through the MST2 (Hpo)-LATS1/2 (Wts) kinase cascade is equally important in the patterning of the vertebrate optic vesicle, and possibly in other Yki/YAP/TAZ-linked cell fate decisions (Neal, 2020).

Daytime CLOCK dephosphorylation is controlled by STRIPAK complexes in Drosophila

In the Drosophila circadian oscillator, the CLOCK/CYCLE complex activates transcription of period (per) and timeless (tim) in the evening. PER and TIM proteins then repress CLOCK (CLK) activity during the night. The pace of the oscillator depends upon post-translational regulation that affects both positive and negative components of the transcriptional loop. CLK protein is highly phosphorylated and inactive in the morning, whereas hypophosphorylated active forms are present in the evening. How this critical dephosphorylation step is mediated is unclear. This study shows that two components of the STRIPAK complex, the CKA regulatory subunit of the PP2A phosphatase and its interacting protein STRIP, promote CLK dephosphorylation during the daytime. In contrast, the WDB regulatory PP2A subunit stabilizes CLK without affecting its phosphorylation state. Inhibition of the PP2A catalytic subunit and CKA downregulation affect daytime CLK similarly, suggesting that STRIPAK complexes are the main PP2A players in producing transcriptionally active hypophosphorylated CLK (Andreazza, 2015).

Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling

The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the pro-growth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, this study identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development (Ribeiro, 2010).

Search PubMed for articles about Drosophila Strip

Andreazza, S., Bouleau, S., Martin, B., Lamouroux, A., Ponien, P., Papin, C., Chelot, E., Jacquet, E. and Rouyer, F. (2015). Daytime CLOCK dephosphorylation is controlled by STRIPAK complexes in Drosophila. Cell Rep 11: 1266-1279. PubMed ID: 25981041

Ashton-Beaucage, D., Udell, C. M., Gendron, P., Sahmi, M., Lefrancois, M., Baril, C., Guenier, A. S., Duchaine, J., Lamarre, D., Lemieux, S. and Therrien, M. (2014). A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila. PLoS Biol 12: e1001809. PubMed ID: 24643257

Emoto, K., Parrish, J. Z., Jan, L. Y. and Jan, Y. N. (2006). The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance. Nature 443: 210-213. PubMed ID: 16906135

Goudreault, M., D'Ambrosio, L. M., Kean, M. J., Mullin, M. J., Larsen, B. G., Sanchez, A., Chaudhry, S., Chen, G. I., Sicheri, F., Nesvizhskii, A. I., Aebersold, R., Raught, B. and Gingras, A. C. (2009). A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics 8: 157-171. PubMed ID: 18782753

Hwang, J. and Pallas, D. C. (2014). STRIPAK complexes: structure, biological function, and involvement in human diseases. Int J Biochem Cell Biol 47: 118-148. PubMed ID: 24333164

Jukam, D., Xie, B., Rister, J., Terrell, D., Charlton-Perkins, M., Pistillo, D., Gebelein, B., Desplan, C. and Cook, T. (2013). Opposite feedbacks in the Hippo pathway for growth control and neural fate. Science 342: 1238016. PubMed ID: 23989952

Kim, J. Y., Park, R., Lee, J. H., Shin, J., Nickas, J., Kim, S. and Cho, S. H. (2016). Yap is essential for retinal progenitor cell cycle progression and RPE cell fate acquisition in the developing mouse eye. Dev Biol 419(2): 336-347. PubMed ID: 27616714

Koch, N., Kobler, O., Thomas, U., Qualmann, B. and Kessels, M. M. (2014). Terminal axonal arborization and synaptic bouton formation critically rely on abp1 and the arp2/3 complex. PLoS One 9: e97692. PubMed ID: 24841972

Miesfeld, J. B., Gestri, G., Clark, B. S., Flinn, M. A., Poole, R. J., Bader, J. R., Besharse, J. C., Wilson, S. W. and Link, B. A. (2015). Yap and Taz regulate retinal pigment epithelial cell fate. Development 142(17): 3021-3032. PubMed ID: 26209646

Neal, S. J., Zhou, Q. and Pignoni, F. (2020). STRIPAK-PP2A regulates Hippo-Yorkie signaling to suppress retinal fate in the Drosophila eye disc peripodial epithelium. J Cell Sci. PubMed ID: 32184260

Ribeiro, P. S., Josue, F., Wepf, A., Wehr, M. C., Rinner, O., Kelly, G., Tapon, N. and Gstaiger, M. (2010). Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling. Mol Cell 39: 521-534. PubMed ID: 20797625

Sakuma, C., Kawauchi, T., Haraguchi, S., Shikanai, M., Yamaguchi, Y., Gelfand, V. I., Luo, L., Miura, M. and Chihara, T. (2014). Drosophila Strip serves as a platform for early endosome organization during axon elongation. Nat Commun 5: 5180. PubMed ID: 25312435

Sakuma, C., Okumura, M., Umehara, T., Miura, M. and Chihara, T. (2015). A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability. Sci Rep 5: 17769. PubMed ID: 26644129

Sakuma, C., Saito, Y., Umehara, T., Kamimura, K., Maeda, N., Mosca, T. J., Miura, M. and Chihara, T. (2016). The Strip-Hippo pathway regulates synaptic terminal formation by modulating actin organization at the Drosophila neuromuscular synapses. Cell Rep 16: 2289-2297. PubMed ID: 27545887

Schulte, J., Sepp, K. J., Jorquera, R. A., Wu, C., Song, Y., Hong, P. and Littleton, J. T. (2010). DMob4/Phocein regulates synapse formation, axonal transport, and microtubule organization. J Neurosci 30: 5189-5203. PubMed ID: 20392941

Winkelman, J. D., Bilancia, C. G., Peifer, M. and Kovar, D. R. (2014). Ena/VASP Enabled is a highly processive actin polymerase tailored to self-assemble parallel-bundled F-actin networks with Fascin. Proc Natl Acad Sci U S A 111: 4121-4126. PubMed ID: 24591594

Zhang, T., Zhou, Q. and Pignoni, F. (2011). Yki/YAP, Sd/TEAD and Hth/MEIS control tissue specification in the Drosophila eye disc epithelium. PLoS One 6(7): e22278. PubMed ID: 21811580

date revised: 18 February 2024

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.