InteractiveFly: GeneBrief

cheerio: Biological Overview | References

BIOLOGICAL OVERVIEW

Filamin is a scaffolding protein that functions in many cells as an actin-crosslinker. FLN90, an isoform of the Drosophila ortholog Filamin/cheerio that lacks the actin-binding domain, is shown in this study to govern the growth of postsynaptic membrane folds and the composition of glutamate receptor clusters at the larval neuromuscular junction. Genetic and biochemical analyses reveal that FLN90 is present surrounding synaptic boutons. FLN90 is required in the muscle for localization of the kinase dPak and, downstream of dPak, for localization of the GTPase Ral and the exocyst complex to this region. Consequently, Filamin is needed for growth of the subsynaptic reticulum. In addition, in the absence of filamin, type-A glutamate receptor subunits are lacking at the postsynapse, while type-B subunits cluster correctly. Receptor composition is dependent on dPak, but independent of the Ral pathway. Thus two major aspects of synapse formation, morphological plasticity and subtype-specific receptor clustering, require postsynaptic Filamin (Lee, 2016).

Proper postsynaptic function depends on appropriate localization of receptors and signaling molecules. Scaffolds such as PSD-95/SAP90 and members of the Shank family are critical to achieving this organization. While usually without intrinsic enzymatic activity, scaffolds recruit, assemble, and stabilize receptors and protein networks through multiple protein-protein interactions: they can bind to receptors, postsynaptic signaling complexes, and the cytoskeleton at the postsynaptic density. Mutations in these proteins are associated with neuropsychiatric disorders. While understanding synapse assembly has begun, much remains to be investigated (Lee, 2016).

The Drosophila larval neuromuscular junction (NMJ) is a well-studied and genetically accessible glutamatergic synapse. Transmission is mediated by AMPA-type receptors, and several postsynaptic proteins important for its development and function have related proteins at mammalian synapses, including the PDZ-containing protein Discs-Large (DLG) and the kinase Pak. In addition, the postsynaptic membrane forms deep invaginations and folds called the subsynaptic reticulum (SSR), which are hypothesized to create subsynaptic compartments comparable to dendritic spines. Recently, it was found that the SSR is a plastic structure whose growth is regulated by synaptic activity (Teodoro, 2013). This phenomenon may be akin to the use-dependent morphological changes, such as growth of dendritic spines, that occur postsynaptically in mammalian brain. The addition of membrane and growth of the SSR requires the exocyst complex to be recruited to the synapse by the small GTPase Ral; the SSR fails to form in ral mutant larvae. Moreover, the localization of Ral to a region surrounding synaptic boutons is likely to direct the selective addition of membrane to this domain. Ral thus provided a tractable entry point for better understanding postsynaptic assembly. The mechanism for localizing the Ral pathway, however, was unknown. The present study determined that Ral localization is dependent on cheerio, a gene encoding filamin, which is critical for proper development of the postsynapse (Lee, 2016).

Filamin is a family of highly conserved protein scaffolds with a long rod-like structure of Ig-like repeats. With over 90 identified binding partners, some of which are present also at the synapse, mammalian filamin A (FLNA) is the most abundant and commonly studied filamin. Filamin can bind actin and has received the most attention in the context of actin cytoskeletal organization (Nakamura, 2011). Drosophila filamin, encoded by the gene cheerio (cher), shares its domain organization and 46% identity in amino acid sequence with human FLNA. Drosophila filamin has a well-studied role in ring canal formation during oocyte development, where it recruits and organizes actin filaments (Li, 1999; Robinson, 1997; Sokol 1999). This study now shows that filamin has an essential postsynaptic role at the fly NMJ. An isoform of this scaffold protein that lacks the actin-binding domain acts via dPak to localize GluRIIA receptors and Ral; filamin thereby orchestrates both receptor composition and membrane growth at the synapse (Lee, 2016).

Filamin is a highly conserved protein whose loss of function is associated with neurodevelopmental disorders. In humans, mutations in the X-linked FLNA cause periventricular heterotopia, a disorder of cortical malformation with a wide range of clinical manifestations such as epilepsy and neuropsychiatric disturbances. Studies in rodent models have shown that abnormal filamin expression causes dendritic arborization defects in a TSC mouse (Zhang, 2014) and that filamin influences neuronal proliferation (Lian, 2012). Filamin is present in acetylcholine receptor clusters at the mammalian NMJ, but its function there is unknown. In lysates of the mammalian brain, filamin associates with known synaptic proteins such as Shank3, Neuroligin 3, and Kv4.2. A recent report indicated that filamin degradation promotes a transition from immature filopodia to mature dendritic spines (Segura, 2016), a phenomenon that is likely to be related to the actin-bundling properties of the long isoform of filamin. Data in the present study have uncovered a novel pathway that does not require the actin-binding domain of filamin. In this pathway, postsynaptically localized filamin, via Pak, directs two distinct effector modules governing synapse development and plasticity: (1) the Ral-exocyst pathway for activity-dependent membrane addition and (2) the composition of glutamate receptor clusters. These pathways determine key structural and physiological properties of the postsynapse (Lee, 2016).

Although loss of filamin had diverse effects on synapse assembly, they were selective. Muscle-specific knockdown or the cher^Q1415sd allele disrupted type-A but not type-B GluR localization at the postsynaptic density. Likewise, the phenotypes for muscle filamin were confined to the postsynaptic side: the presynaptic active zone protein Brp and overall architecture of the nerve endings were not altered by muscle-specific knockdown. The specificity of its effect on particular synaptic proteins, and the absence of the actin-binding region in FLN90, suggests that filamin's major mode of action here is not overall cytoskeletal organization, but rather to serve as a scaffold for particular protein-protein interactions (Lee, 2016).

Analysis of the distribution of the SSR marker Syndapin and direct examination of the subsynaptic membrane by electron microscopy revealed that formation of the SSR required filamin. Genetic analysis uncovered a sequential pathway for SSR formation from filamin to the Pak/Pix/Rac signaling complex, to Ral, to the exocyst complex and consequent membrane addition. The SSR is formed during the second half of larval life and may be an adaptation for the low input resistance of third-instar muscles. Like dendritic spines, the infoldings of the SSR create biochemically isolated compartments in the vicinity of postsynaptic receptors and may shape physiological responses, although first-order properties of the synapse, such as mini- or EPSP amplitude are little altered in mutants that lack an SSR. The formation of the SSR requires transcriptional changes driven by Wnt signaling and nuclear import, proteins that induce membrane curvature (such as Syndapin, Amphiphysin, and Past1), and Ral-driven, exocyst-dependent membrane addition (Teodoro, 2013). The activation of Ral by Ca2+ influx during synaptic transmission allows the SSR to grow in an activity-dependent fashion. The localization of Ral to the region surrounding the bouton appears crucial to determining the site of membrane addition because Ral localization precedes SSR formation and exocyst recruitment and because exocyst recruitment occurred selectively surrounding boutons even when Ca2+-influx occurred globally in response to calcimycin (Teodoro, 2013). This study has now shown that Ral localization, and consequently exocyst recruitment, membrane growth, and the presence of the SSR marker Syndapin, are all dependent on a local action of filamin at the synapse. FLN90, the filamin short isoform, localized to sites of synaptic contact and indeed surrounded the boutons just as does Ral. When this postsynaptic filamin was removed by muscle-specific filamin knockdown or the cher^Q1415sd allele, the downstream elements of the pathway, Pak, Rac, Ral, the exocyst, and Syndapin, were no longer synaptically targeted. The mislocalization is not a secondary effect of loss of the SSR but likely a direct consequence of filamin loss, as Pak and Ral can localize subsynaptically even in the absence of the SSR (Teodoro, 2013). Unlike the likely mode of action of nuclear signaling by Wnt, the delocalization of Ral was not a consequence of altered protein production; its expression levels did not change. Thus filamin may be viewed as orchestrating the formation of the SSR and directing it to the region surrounding synaptic boutons (Lee, 2016).

The second major feature of the filamin phenotype was the large reduction in the levels of the GluRIIA receptor subunit from the postsynaptic membranes. GluRIIA and GluRIIB differ in their electrophysiological properties and subsynaptic distribution (DiAntonio, 1999; Marrus, 2004). Because type B GluRs, which contain the IIB subunit, desensitize more rapidly than type A, the relative abundance of type A and type B GluRs is a key determinant of postsynaptic responses and changes with synapse maturation. The selective decrease in GluRIIA at filamin-null NMJs is likely a consequence of dPak mislocalization: filamin-null NMJs lack synaptic dPak, and dPak null NMJs lack synaptic GluRIIA (Albin, 2004; Parnas, 2001). Moreover, the first-order electrophysiological properties at NMJs lacking filamin resembled those reported at NMJs missing dPak (Parnas, 2001). In the current study, though, only the change in mEPSP frequency was statistically significant. At filamin-null NMJs, the decrease in GluRIIA is accompanied by an increase in GluRIIB, suggestive of a partial compensation that could account for the relatively normal synaptic transmission. Because the IIA and IIB subunits differ in desensitization kinetics and regulation by second messengers (DiAntonio, 2006), functional consequences of filamin loss may become more apparent with more extensive physiological characterizations at longer time scales (Lee, 2016).

While both SSR growth and receptor composition required the kinase activity of dPak, receptor composition was independent of Ral and thus represents a distinct branch of the pathway downstream of dPak. As with Ral, the loss of GluRIIA from the synapse was due to delocalization and not a change in expression of the protein, consistent with unaltered GluRIIA transcripts in dPak null animals (Albin, 2004). Thus filamin, via dPak, alters proteins with functional significance for the synapse as well as its structural maturation (Lee, 2016).

Mammalian filamin, via its many Ig-like repeats, has known scaffold functions in submembrane cellular compartments (Popowicz, 2006; Stossel, 2001) and filamin is therefore likely also to serve as a scaffold at the fly NMJ. These epistasis data indicate that filamin recruits Ral through recruitment of a signaling complex already known to function at the fly NMJ: dPak and its partners dPix and Rac (Albin, 2004; Parnas, 2001). Mammalian filamin is reported to directly interact with Ral during filopodia formation (Ohta, 1999), however the details of their interaction at the fly NMJ are less clear. Because Ral localization requires filamin to recruit dPak and dPix and specifically requires the kinase activity of dPak, it is possible that either Ral or filamin need to be phosphorylated by dPak to bind one another. Mammalian filamin interacts with some components of the Pak signaling complex (Bellanger, 2000; Ohta, 1999) and is a substrate of Pak (Vadlamudi, 2002). This study has now shown that Drosophila filamin and PAK interact when coexpressed in HEK cells, and thus a direct scaffolding role for FLN90 in the recruitment of Pak and the organization of the postsynapse is likely (Lee, 2016).

The overlapping but different distributions of filamin and its downstream targets indicate that its scaffolding functions must undergo regulation by additional factors. The proteins discussed in this study take on either of two patterns at the synapse. Some, like Ral, Syndapin, and filamin itself, are what can be termed subsynaptic and, like the SSR, envelope the entire synaptic bouton. Others, like dPak and its partners and the GluRIIA proteins, are concentrated in much smaller regions, immediately opposite presynaptic active zones, where the postsynaptic density (PSD) is located. It is hypothesized that filamin interacts with additional proteins, including potentially transsynaptic adhesion proteins, that localize filamin to the subsynaptic region and also govern to which of the downstream effectors it will bind. Indeed, it appears paradoxical that dPak, though predominantly at the postsynaptic density is nonetheless required for Ral localization throughout the subsynaptic region. If dPak is needed to phosphorylate either filamin or Ral to permit Ral localization, the phosphorylations outside the PSD may be due to low levels of the dPak complex in that region; synaptic dPak was previously shown to be a relatively mobile component of the PSD (Lee, 2016).

Filamin was the first nonmuscle actin-crosslinking protein to be discovered. With an actin-binding domain at the N terminus, the long isoform of filamin and its capacity to integrate cellular signals with cytoskeletal dynamics have subsequently been the focus of the majority of the filamin literature (Nakamura, 2011; Popowicz, 2006; Stossel, 2001). At the NMJ, however, this was not the case. Several lines of evidence indicated that the short FLN90 isoform of filamin, which lacks the actin-binding domain, plays an essential role in postsynaptic assembly. First, the short FLN90 isoform was the predominant and perhaps the only isoform of filamin found expressed in the muscles. Second, both endogenous and overexpressed FLN90 localized subsynaptically. Third, loss of the short isoform disrupted localization of postsynaptic components while lack of just the long isoform had little or no effect. Lastly, exogenous expression of just the short isoform in filamin null background sufficiently rescued the defect in SSR growth. The modest postsynaptic phenotypes of the cher1allele, which predominantly disrupts the long isoform, may be due to small effects of the allele on expression of the short isoform or may be an indirect consequence of the presence of the long isoform in the nerve terminals (Lee, 2016).

The existence of the short isoform has been reported in both flies and mammals and may be produced either by transcriptional regulation or calpain-mediated cleavage. The short isoform can be a transcriptional co-activator, but its functional significance and mechanisms of action have been largely elusive. The short isoform has little or no affinity for actin, but most of the known sites for other protein-protein interactions are shared by both isoforms. Thus the structure of FLN90, with nine predicted Ig repeats and likely protein-protein interactions, is consistent with a scaffolding function to localize key synaptic molecules independent of interactions with the actin cytoskeleton (Lee, 2016).

This study has introduced filamin as a major contributor to synapse development and organization. The severity of the phenotypes indicates filamin has a crucial role that is not redundant with other scaffolding proteins. The effects of filamin encompass several much-studied aspects of the Drosophila NMJ: the clustering and subunit subtype of glutamate receptors and the plastic assembly of specialized postsynaptic membrane structures. The pathways that govern these two phenomena diverge downstream of Pak kinase activity and are dependent on filamin for the proper localization of key signaling modules in the pathways. By likely acting as a scaffold protein, the short isoform of filamin may function as a link between cell surface proteins, as yet unidentified, and postsynaptic proteins with essential localizations to and functions at the synapse. Because many of the components of these pathways at the fly NMJ are also present at mammalian synapses and can interact with mammalian filamin, a parallel set of functions in CNS dendrites merits investigation (Lee, 2016).

Evidence for the mechanosensor function of filamin in tissue development

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin (Cheerio in Drosophila) function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. This study tested how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, these results show that an appropriate level of mechanical sensitivity is required for filamins' function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components (Huelsmann, 2016).

The concept of mechanical regulation of tissue development, mechanotransduction, has been proven in several experimental settings. In adult mesenchymal stem cells, the elastic modulus of the culture substrate regulates cell differentiation along different lineages. In muscles, mechanical stretching generates various responses that affect cell proliferation and differentiation. Different sensor molecules, including many cytoskeleton-linked proteins, elicit these cellular responses according to the mechanical cues they perceive. For instance, the actin filament - plasma membrane linkers talin and vinculin regulate the mechanical tuning of cell adhesion strength by sensing the ECM rigidity and the sarcomeric ruler titin modulate the mechanical signaling in muscles. Filamins cross-link actin filaments and anchor them to membranes, and pulling forces regulate protein-interaction sites within their C-terminal immunoglobulin-like domains. The C-terminal mechanosensor region (MSR) of filamin has two protein interaction sites that are masked by neighbouring sequences (closed conformation, and masking is released by small forces of 2-5 pN. In cell culture models, the cytoplasmic tails of integrin adhesion receptors preferentially bind to open filamins as indicated by reduction of the interaction decay time when myosin is active. Rare mutations in humans and animal models demonstrate that filamins are involved in three-dimensional tissue morphogenesis and the maintenance of muscle integrity. This study tests whether the mechanosensor function of filamins has a role within tissues (Huelsmann, 2016).

This paper shows that filamin mechanosensing-altering mutations disrupt ring canal development in the Drosophila ovary and lead to small, partially sterile eggs. Of note, changing the spring properties of the mechanosensor site in either direction: releasing (open MSR mutation) or tightening (closed MSR), caused similar ring canal phenotypes, yet distinct dynamics of filamin in the ring canal. These data fit with a model in which filamins have a mechanosensory function during animal tissue development (Huelsmann, 2016).

Definite mutations that alter spring properties of a cytoskeletal proteins have not been tested rigorously in animal models. For example, deletions in titin M-band region cause muscle development and regeneration defects in mouse, but it is unclear to what extent altered mechanosensitivity contributes to these phenotypes. The unique structure of filamin MSR allowed mutations to be made that do not change the interaction sites themselves, but change how they are exposed. Previous structural and functional information allowed engineering of mutations that either require less or more force for opening than the normal structure. The same mutations that were made in this study in the Drosophila filamin Cheerio have been earlier used in human Filamin A for biochemical studies and single molecule studies. SAXS analysis suggested that the Cheerio MSR has similar shape parameters as the human Filamin A and that the open MSR mutation had changed the structure as expected. In the absence of external force the overall structure of the closed MSR mutant fragment was similar as WT (Huelsmann, 2016).

For the in vivo experiments, the open and closed MSR substitution mutations were made n the genomic locus of cheerio. This allowed study of the effects of mutations at normal gene copy number without interference from the WT gene. Although it has been previously shown that some mutations disrupting the structure filamins cause aggregation of the mutant protein in skeletal or cardiac muscle and that filamin unfolding may trigger chaperone-assisted autophagocytosis in muscle, the MSR mutations used here did not cause filamin aggregates or decreased the protein amount. In contrast, immunoblotting experiments showed that all mutants were expressed at similar protein levels as the WT (Huelsmann, 2016).

In addition to the finding that mechanosensing modifying filamin mutants disrupted the maturation of ring canals, the second main finding in the current study was that the mutants showed altered dynamics in the ring canal: open MSR mutant had markedly reduced recovery rate, whereas the closed MSR or ΔMSR mutant recovered faster than the WT filamin. This is consistent with the hypothesis that the open MSR interacts more strongly with ring canal components, whereas the interaction of closed MSR and ΔMSR mutants is mainly mediated by other regions of filamins, presumably the actin binding domain. Unfortunately, it was not possible to analyse the dynamics of the MSR mutants in the ring canals at late developmental stages, as the ring canals disintegrate in the MSR mutants (Huelsmann, 2016).

How can the apparently conflicting results that the open and closed MSR mutants cause similar ring canal phenotypes, but yet have dramatically different levels of exchange at their site of function? These observations do not fit with a strictly structural role of the filamin MSR in the ring canal. If that were the case, it would be expected that the MSR interaction- stabilizing mutant would also abnormally stabilize the ring canal structure. This was not observed. Instead, a model is favored in which different domains of filamin coordinate structural and mechanosensory roles during oogenesis. The C-terminal MSR region regulates the dynamics of filamins and is essential for maintaining the protein at ring canals. In contrast, the N-terminal actin-binding domain is not essential for localisation, but for ring canals growth: filamins with a non-functional actin binding domain localise to ring canals, but ring canals stay small. Furthermore, the results suggest that to be fully functional the MSR must oscillate between open and closed conformations during ring canal maturation and growth: both, open and closed MSR mutants, destabilised the structure at the stage when it was supposed to grow. Thus, the data fit with a regulatory, mechanosensory function of filamin in ring canals. It would be predicted that growth of the ring canal would therefore involve membrane tension driven oscillations in the amount force within the structure. When under high tension filamin would bind tightly and perhaps also recruit new components to expand the structure. As the ring canal enlarges the tension would reduce, allowing filamin to redistribute within the expanded structure (Huelsmann, 2016).

In mammalian model systems and in human patients, mutations in FLNA, FLNB and FLNC genes have been associated with developmental or regeneration abnormalities in vasculature, cartilage, bone and muscle. The current study suggests that some of these filaminopathies with either truncations of the C-terminal parts of filamins or point mutations at or near the MSR, may be caused by defects in mechanosensor function. For instance, missense mutations in the MSR of FLNC have been recently linked hypertrophic cardiomyopathy (Huelsmann, 2016).

In conclusion, the current results suggest that normal filamin function requires mechanically regulated conformational changes within developing intact tissues. This fits with the model that filamin has a mechanosensing function during tissue growth that is be conserved from flies to mammals (Huelsmann, 2016).

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. This study shows that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) (Cheerio) and CryAB. NUAK biochemically and genetically interacts with the cochaperone Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. It was further demonstrated that Fil/Cheerio is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, these results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases (Brooks, 2020).

Proteins must fold into an intrinsic three dimensional structure to perform distinct cellular functions. Denatured or misfolded proteins can be refolded by chaperones or are subject to degradation by the ubiquitin-proteasome system (UPS) and/or the autophagosome-lysosome pathway (ALP). The accumulation of misfolded proteins upon genetic mutation or decreased chaperone function causes protein aggregates that are not effectively cleared by the UPS or the ALP. Environmental insults or aging may exacerbate this accumulation of misfolded proteins, resulting in disease and eventual cell death (Brooks, 2020).

A specialized autophagy pathway, termed chaperone-assisted selective autophagy (CASA), has been verified in both Drosophila and mammalian systems. The CASA complex includes BAG3 in concert with the chaperones HSC70/HSPA8 (HSP70 family), HSPB8 (small HSP family), and the ubiquitin (Ub) ligase CHIP/STUB1. CASA regulates the removal and degradation of Fil from the Z-disc in striated muscle or actin stress fibers in non-muscle cells. The N-terminal actin-binding domain (ABD) in Fil is followed by multiple immunoglobulin (Ig)-like repeats which bind numerous proteins to link the internal cytoskeleton to the sarcolemma. Tension exerted by contractile muscle tissue requires continuous folding and refolding of individual Ig-like domains in Fil, eventually damaging the ability of the protein to sense and transmit mechanical strain. The BAG3-HSC70 protein complex binds to the mechanosensor region (MSR) of Fil and upon detection of protein damage, CHIP ensures the addition of polyubiquitin (polyUb) moieties. Rather than promoting delivery to the proteasome, these Ub chains instead recruit the autophagic Ub adapter protein p62/SQSTM1. p62 interacts with Atg8a/LC3 to induce autophagophore formation and the subsequent clearance of Fil through lysosomal degradation. Fil aggregates and a block in autophagosome-lysosome fusion are present in lysosomal associated membrane protein 2 (LAMP2)-deficient muscles, thus linking impaired autophagy to abnormal protein deposits (Brooks, 2020).

Drosophila NUAK encodes for a conserved serine/threonine kinase that is homologous to the mammalian kinases NUAK1/ARK5 and NUAK2/SNARK. These proteins comprise a family of twelve AMP-activated protein kinase (AMPK)-related kinases (NUAK1 and 2, BRSK 1 and 2, QIK, QSK, SIK, MARK 1-4, and MELK) that share a conserved N-terminal kinase domain activated by the upstream liver kinase B1 (LKB1). NUAK1 and NUAK2 proteins are broadly expressed, but enriched in cardiac and skeletal muscle. Muscle contraction and LKB1 phosphorylation can activate both NUAK proteins. NUAK2 activity is additionally stimulated by oxidative stress, AMP, and glucose deprivation in various cell types. Interestingly, NUAK2 expression increases during muscle differentiation and in response to stress or in aging muscle tissue, whereas dominant-negative (DN)-NUAK2 induces atrophy. Homozygous NUAK1 KO mice are embryonic lethal and <10% of NUAK2 homozygotes survive, precluding analysis of post-embryonic contributions. Because of this embryonic lethality, conditional NUAK1 KO mice were generated to examine muscle function . However, no change was observed in muscle mass or fiber size between control or muscle-specific NUAK1 KO mice, likely due to functional redundancy (Brooks, 2020).

The presence of single NUAK orthologs in worms (Unc-82) or flies (NUAK/CG43143) allows for the study of NUAK protein function without compensation from additional family members that may mask cellular roles. unc-82 associates with Paramyosin and likely Myosin B to promote proper myofilament assembly in C. elegans. The kinase domain in Drosophila NUAK shares 61% identity and 80% similarity to human NUAK1 and NUAK2. In flies, RNAi knockdown of NUAK phenocopies weak Lkb1 defects in regulating cell polarity during ommatidial formation and actin cone formation in spermatogenesis. NUAK kinase targets or additional functions in other tissues have not been reported (Brooks, 2020).

This study identified Drosophila NUAK as a key regulator of autophagic protein clearance in muscle tissue. NUAK physically interacts with and phosphorylates Fil [encoded by Drosophila cheerio (cher)]. NUAK also genetically and biochemically interacts with the Stv-Hsc-70-4 complex and Stv overexpression is sufficient to rescue NUAK-mediated muscle deterioration. The identification of Fil as a cargo protein that abnormally accumulates in muscle tissue deficient for NUAK, Stv, Hsc70-4, and Atg8a links protein aggregation to defects in autophagic disposal (Brooks, 2020).

Prior to this study, few substrates of NUAK kinase activity had been uncovered. One of these is Myosin phosphatase targeting-1 (MYPT1), a regulatory subunit of myosin light-chain phosphatase. Two Drosophila regulatory subunits, MYPT75D and Myosin binding subunit (Mbs), were tested in Stv NUAK sensitized genetic assay and no protein aggregation and/or muscle degeneration was observed. While negative, this data nevertheless argues that this family of phosphatases likely does not function with NUAK in muscle tissue. Since the mammalian NUAK1-MYPT1 interaction was identified in vitro and further validated in HEK293 cells, NUAK likely has cell and tissue-specific targets that regulate diverse biological outputs (Brooks, 2020).

Based upon the discovery of Fil as a novel NUAK substrate, two scenarios are envisioned that are not mutually exclusive to explain the molecular function of NUAK in preventing protein aggregation. First, the increase in sarcomere number upon muscle-specific NUAK RNAi suggests that at least one role of NUAK may be to negatively regulate the addition of proteins (such as Fil) into sarcomeres. This data is consistent with studies that show C. elegans unc-82 regulates myofilament assembly. Notably, one key feature of the misincorporated proteins in unc-82 mutants is their inclusion into aggregate-like structures, similar to the accumulation of Fil and CryAB in NUAK-/- muscles. An additional, or alternative possibility, is that NUAK phosphorylates unfolded or 'damaged' Fil for removal from the sarcomere, thereby triggering the Stv-Hsc70-4 complex to promote autophagic turnover. Thus, proteins such as Fil that fail to get incorporated into sarcomeres and/or sustain damage due to repeated rounds of tension-induced muscle contraction, may destabilize myofilament architecture and trigger abnormal protein (Brooks, 2020).

In both contractile muscle tissue and in adherent cells subjected to mechanical force, BAG3 acts as a hub to coordinate Fil-induced tension-sensing and autophagosome formation. The MSR of Fil is comprised of Ig repeats whose conformational transitions between open and closed states dictate differential protein-protein interactions and biological outputs. While the chaperones Hsc70/HSPA8 and HSPB8 weakly bind to the MSR of Fil, this biochemical interaction is greatly enhanced in the presence of BAG3. Interestingly, BAG3 interacts with Ig repeats 19-21 in the MSR, while the selected interaction domain of NUAK with Fil comprises Ig repeats 15-18. These data suggest that NUAK and Stv each bind to a separate region of the MSR in Fil (Brooks, 2020).

It remains to be determined if NUAK-mediated phosphorylation is a prerequisite for the removal of damaged Fil protein by BAG3. The rescue results suggest that this phosphorylation event is not required as Stv overexpression alleviates protein aggregation and muscle degeneration upon a loss of NUAK. An alternative possibility is that this excess Stv protein is present in sufficient amounts to interact with Fil and overcome the necessity for phosphorylation by NUAK. The inability of NUAK overexpression to restore muscle defects due to knockdown of Stv, Hsc70-4, or Atg8a suggests that NUAK functions upstream or parallel to this pathway. It seems likely that NUAK has additional target substrates for kinase activity that may regulate autophagic protein clearance in muscle tissue (Brooks, 2020).

Recent studies demonstrate that increased autophagic degradation of Fil by BAG3 also induces fil transcription as a compensatory mechanism to ensure steady-state Fil levels. Thus, whether loss of NUAK or Stv alters gene expression upon a block in protein clearance was investigated. While the mRNA levels of cher, CryAB, Hsc70-4, or Atg8a were not altered in NUAK or stv mutants, there was a large increase in p62 transcripts. Thus, this increase in p62 mRNA synthesis may contribute to the elevated p62 protein levels observed upon loss of NUAK or Stv as multiple stress conditions increase p62 transcription, including proteasome inhibition, starvation and atrophic muscle conditions. Data that support a role for an autophagic block include the localization of p62 and Atg8a to regions of protein aggregation (Brooks, 2020).

A model for NUAK is proposed that incorporates these new findings with existing roles for BAG3. Fil and CryAB are physically associated at the Z-disc in Drosophila larval muscle. The phosphorylation of Fil by NUAK may control the incorporation of Fil into the Z-disc during myofibril assembly and/or may be required for the disposal of damaged Fil protein. BAG3 and chaperones such as Hsc70/HSPA8 are thought to monitor the MSR of Fil to detect force-induced damage and to promote the addition of K63-linked polyUb chains. Recruitment of the ubiquitin autophagic adapter p62/SQSTM1 induces autophagosome initiation through the accumulation of Atg8a. Eventual fusion of these autophagosomes with lysosomes promotes protein client complex destruction (Brooks, 2020).

Upon loss of NUAK, excess Fil protein that fails to be incorporated into the Z-disc and/or is damaged due to tension-induced muscle contraction begins to accumulate near the Z-disc. The presence of CryAB in Fil-like aggregates may be due to the normal association of CryAB with Fil at the Z-disc, either to monitor Fil protein damage, or to prevent protein aggregation. It is interesting that while both Fil and CryAB contain actin-binding domains, these associations are lost in NUAK-/- muscle tissue as F-actin is displaced from regions of Fil-CryAB accumulation. At this point it cannot be determined if NUAK preferentially binds to the short (~90kD) and/or long (~240 kD) Fil isoforms since the mapped interaction domains (Ig domains 15-18) are present in both isoforms (Brooks, 2020).

In the initial stages of aggregate formation, nearly all Fil puncta are decorated with Ub. It is hypothesized that the observed decrease in Ub-Fil colocalization in large regions of aggregate formation may be due to intrinsic properties of aggregation-prone proteins whereby protein misfolding triggers aggregation of Fil with itself and other proteins. The accumulation of p62 and circular structures that stain positive for Atg8a in regions of Fil accumulation demonstrate that the autophagosome machinery is recruited to BAG3-client complexes. The absence of lysosomes in these aggregate regions suggest that either fusion and/or transport to sites of degradation are compromised (Brooks, 2020).

CASA-mediated autophagy via the BAG3-client complex includes Hsc70-4/HSPA8, HSPB8, and the E3 ligase CHIP/STUB1, the latter of which ubiquitinates Fil for the subsequent recruitment of p62 to initiate autophagosome formation. However, fibroblasts deficient for CHIP are not defective in autophagy and mice or flies lacking CHIP/STUB1 are viable. A failure to enhance protein aggregation defects upon CHIP RNAi knockdown in the sensitized NUAK+/- or stv+/- backgrounds suggests that additional Ub ligases cooperate with the Stv/BAG3 complex to remove damaged proteins. Future studies will also determine which Drosophila protein is the equivalent of HSPB8 since no genetic interactions were observed with putative CG14207 or Hsp67Bc RNAi lines. This negative data does not rule out the possibility that protein levels are not reduced enough to see phenotypes upon RNAi induction or possible functional redundancy exists between CG14207 and Hsp67Bc (Brooks, 2020).

An interesting hallmark of protein aggregate diseases is the accumulation of specific proteins in affected cells or tissues. Thus, proteins susceptible to aggregation in vivo may possess specific structural characteristics or shared biological functions. This latter feature is evident in a group of protein aggregate diseases termed myofibrillar myopathies (MFM). Laser microdissection of aggregates from normal or affected muscles reveal specificity in the types of proteins that accumulate in patients afflicted with MFMs. Common proteins present in these aggregates include Filamin C (FILC), αB-crystallin (CRYAB), BAG3, and Desmin (DES), among others. The inability of MFM patients to clear these aggregates results in myofibrillar degeneration and a decline in muscle function. Interestingly, mutations in Drosophila NUAK phenocopy both structural and functional deficits observed in MFM patients, including Fil and CryAB accumulation, muscle degeneration, and locomotor defects. The discovery of cellular degeneration and protein aggregation in muscle tissue upon loss of the single fly NUAK ortholog highlights the power of Drosophila as a model. Future studies will focus on identifying kinase targets of NUAK and defining additional proteins that function in NUAK and stv-mediated autophagy for the eventual development of therapeutic targets to treat MFMs and other protein aggregate diseases (Brooks, 2020).

Drosophila small heat shock protein CryAB ensures structural integrity of developing muscles, and proper muscle and heart performance

Molecular chaperones, such as the small heat shock proteins (sHsps), maintain normal cellular function by controlling protein homeostasis in stress conditions. However, sHsps are not only activated in response to environmental insults, but also exert developmental and tissue-specific functions that are much less known. This study shows that during normal development the Drosophila sHsp CryAB [L(2)efl] is specifically expressed in larval body wall muscles and accumulates at the level of Z-bands and around myonuclei. CryAB features a conserved actin-binding domain and, when attenuated, leads to clustering of myonuclei and an altered pattern of sarcomeric actin and the Z-band-associated actin crosslinker Cheerio (filamin). The data suggest that CryAB and Cheerio form a complex essential for muscle integrity: CryAB colocalizes with Cheerio and, as revealed by mass spectrometry and co-immunoprecipitation experiments, binds to Cheerio, and the muscle-specific attenuation of cheerio leads to CryAB-like sarcomeric phenotypes. Furthermore, muscle-targeted expression of CryAB(R120G), which carries a mutation associated with desmin-related myopathy (DRM), results in an altered sarcomeric actin pattern, in affected myofibrillar integrity and in Z-band breaks, leading to reduced muscle performance and to marked cardiac arrhythmia. Taken together, this study demonstrates that CryAB ensures myofibrillar integrity in Drosophila muscles during development, and it does so by interacting with the actin crosslinker Cheerio. The evidence that a DRM-causing mutation affects CryAB muscle function and leads to DRM-like phenotypes in the fly reveals a conserved stress-independent role of CryAB in maintaining muscle cell cytoarchitecture (Wojtowicz, 2015).

Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells

Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. This study provides evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. The data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. A revised model is provided for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery (Huelsmann, 2013).

The actin cross-linker Filamin/Cheerio mediates tumor malignancy downstream of JNK signaling

Cell shape dynamics, motility, and cell proliferation all depend on the actin cytoskeleton. Malignant cancer cells hijack the actin network to grow and migrate to secondary sites. Understanding the function of actin regulators is therefore of major interest. This study has identified the actin cross-linking protein Filamin/Cheerio (Cher) as a mediator of malignancy in genetically defined Drosophila tumors. In invasive tumors, resulting from cooperation of activated Ras with disrupted epithelial cell polarity, Cher is upregulated in a Jun N-terminal kinase (JNK)-dependent manner. Although dispensable in normal epithelium, Cher becomes required in the tumor cells for their growth and invasiveness. When deprived of Cher, these tumor clones lose their full potential to proliferate and breach tissue boundaries. Instead, the Cher-deficient clones remain confined within the limits of their source epithelium, permitting survival of the host animal. Through interaction with the myosin II heavy chain subunit, Cher is likely to strengthen the cortical actomyosin network and reinforce mechanical tension within the invasive tumors. Accordingly, Cher is required for aberrant expression of genes downstream of the Hippo/Yorkie signaling in the tumor tissue. This study identifies Cher as a new target of JNK signaling that links cytoskeleton dynamics to tumor progression (Kulshammer, 2012).

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. This study identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. It was also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression (Zheng, 2011).

Ten-m was thought to be the first non-transcription factor pair-rule gene. Three P-element insertion alleles were reported to lie in the same complementation group and be embryonic lethal. One allele, 5309, showed a severe pair-rule phenotype, one allele showed a moderate version of the same phenotype, whereas other lethal alleles did not have a significant cuticle phenotype. This study showed that the cuticle phenotype associated with the original 5309 stock segregates with the balancer chromosome. Indeed, the Df(3L)Ten-m^AL1 and Df(3L)Ten-m^AL29 deletion alleles exhibit fusions similar to the 5309 allele when balanced with the same balancer chromosome as the one used in 5309. The data show that the 'Ten-m' cuticle phenotype analyzed previously is an odd-paired mutation on the balancer chromosome and is not due to the Ten-m mutation itself. It should be noted that the embryonic CNS longitudinal connective discontinuity phenotype documented for Ten-m⁵³⁰⁹ is also due to a mutation on that original balancer (Zheng, 2011).

When Ten-m is not expressed, or is ectopically expressed in the ectoderm, aberrant motor axon growth cone guidance occurs. Growth cones use various kinds of substrates and guidance cues to navigate through a specific path to find their targets. In Drosophila and C. elegans, genetic screens have identified many secreted or transmembrane guidance cues including Netrins, Semaphorins, Slits, Nephrins, and classic morphogens that also act as guidance molecules. Most of these cues are expressed either by axonal tracts themselves or along the axonal trajectory by peripheral tissues, such as muscles. A transmembrane protein affecting migration, Ten-m, is expressed in epidermal stripes and in neurons. In both vertebrate and invertebrate embryos, axons must first exit the CNS, and then migrate along stereotyped pathways to reach their specific targets in the periphery. Since motor axons in both Ten-m loss of function mutants, and Ten-m ectopic expression embryos, exit the CNS normally but do not migrate along their specific paths, Ten-m appears to be dispensable for axonal extension but necessary for pathfinding decisions in the periphery (Zheng, 2011).

Ten-m is often required for correct choice point determination. During embryonic development, axons preferably extend along the surface of other axons to form axon bundles or fascicles (selective fasciculation), and exit those fascicles to navigate into their targets (selective defasciculation). These processes are regulated by both attractive and repulsive cues. These attractive and repulsive molecules can originate from the axonal tracts themselves or from the surrounding peripheral tissues. A guidance cue disruption of a repulsive molecule should cause abnormal defasciculation. In the case of Ten-m loss of function mutations, or gain of function, the ISN branches fail to maintain their segmental boundaries and invade the adjoining segments, occasionally to fuse with the adjacent segmental nerve. In mutants, loss of Ten-m activity in the motor axons could lead to failing to respond properly to cues, among other possible failings of the neurons. However, epidermal ectopic, or Paired-driven Ten-m expression leading to the same phenotypes suggest that Ten-m impacts peripheral cues, assuming that the overexpression effects were specific (Zheng, 2011).

Axon guidance disruptions observed when Ten-m is ectopically expressed suggests that Ten-m either: maintains peripheral cells to allow them to reach a stage to express a cue for motor axons; or more directly regulates the expression of cues to which motor axons respond. It is speculated that Ten-m might itself act as a peripheral cue for migration. Epidermal Ten-m, and perhaps specifically its expression spatially restricted to stripes, could help position a repulsive guidance cue for the ISN axons and prevent them from crossing into the adjacent segments. Given the collection of defects observed in motor neuron axons, Ten-m might induce a gene product that is, or might itself be, both a repulsive and an attractive guidance molecule, a situation that is not uncommon. For example, Netrins were first found as a chemoattractant for vertebrate commissural axons and circumferential axons in C. elegans. However, Netrins can also repel some axons, as demonstrated in unc-6/netrin mutant C. elegans and Drosophila. DCC/frazzled (deleted in colorectal carcinoma), a netrin receptor, mediates both attraction and repulsion while UNC-5, another netrin receptor, functions exclusively in repulsion (Zheng, 2011).

Filamins are very large proteins with an actin binding domain and more than 20 Ig-like repeats, that self associate as dimers. They act as Actin crosslinking proteins that are also scaffolds for a very large number and variety of binding partners. As such, they are involved in many functions, but especially relevant are cell adhesion and migration. This includes interactions with different cytoskeletal complexes. In flies, Filamin affects peripheral motor axon navigation in a manner similar to that of Ten-m. This function echoes vertebrate filamin activities. In contrast to Drosophila, mammals have three filamin proteins, A, B and C. Loss of function mutations in Filamin A are found in the human disease periventricular heterotopia, which is a defect in axonal migration that has been associated with the dynamic regulation of actin (Eksioglu, 1996; Kakita, 2002; Fox, 1998; Moro, 2002). However, detailed studies of patients carrying mutations shows evidence for a more complex regulation of axonal navigation than can be explained by simply an effect on growth cone motility. The current studies in flies suggest that in addition to growth cone motility, the context and restricted expression pattern of Filamin might influence axon guidance. In Drosophila, Filamin associates with the seg1 domain of Ten-m, a highly conserved domain within the Ten-m/Odz family that this study has named the FID. These two proteins are expressed in the epidermis, including co-expression in a series of 'belts' of epidermal cells, strongest laterally (Zheng, 2011).

How might Ten-m, together with Filamin, regulate axonal guidance? It is hypothesized that anchored epidermal Ten-m and Filamin might influence lateral motor axon navigation. These two proteins might set the stage for proper development leading to the expression of spatially restricted epidermal axon guidance cues, or directly impact the regulation of such cues, as ISN motor axon projections start to migrate out of the CNS and begin to reach their lateral and dorsal muscle targets. One speculation is that Ten-m linked to Filamin could itself be a candidate cue for motor axons (Zheng, 2011).

Fragile X mental retardation 1 and filamin a interact genetically in Drosophila long-term memory

The last decade has witnessed the identification of single-gene defects associated with an impressive number of mental retardation syndromes. Fragile X syndrome, the most common cause of mental retardation for instance, results from disruption of the FMR1 gene. Similarly, Periventricular Nodular Heterotopia, which includes cerebral malformation, epilepsy and cognitive disabilities, derives from disruption of the Filamin A gene. While it remains unclear whether defects in common molecular pathways may underlie the cognitive dysfunction of these various syndromes, defects in cytoskeletal structure nonetheless appear to be common to several mental retardation syndromes. FMR1 is known to interact with Rac, profilin, PAK and Ras, which are associated with dendritic spine defects. In Drosophila, disruptions of the dFmr1 gene impair long-term memory (LTM), and the Filamin A homolog (Cheerio) was identified in a behavioral screen for LTM mutants. Thus, this study investigated the possible interaction between cheerio and dFmr1 during LTM formation in Drosophila. LTM specifically was shown to be defective in dFmr1/cheerio double heterozygotes, while it is normal in single heterozygotes for either dFmr1 or cheerio. In dFmr1 mutants, Filamin (Cheerio) levels are lower than normal after spaced training. These observations support the notion that decreased actin cross-linking may underlie the persistence of long and thin dendritic spines in Fragile X patients and animal models. More generally, the results represent the first demonstration of a genetic interaction between mental retardation genes in an in vivo model system of memory formation (Bolduc, 2010).

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration, and mutations in human alpha-filamin (FLN1) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. This study reports the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development (Li, 1999).

Drosophila filamin encoded by the cheerio locus is a component of ovarian ring canals

The ring canals in the ovary of Drosophila provide a versatile system in which to study the assembly and regulation of membrane-associated actin structures. Derived from arrested cleavage furrows, ring canals allow direct communication between cells. The robust inner rim of filamentous actin that attaches to the ring-canal plasma membrane contains cytoskeletal proteins encoded by the hu-li-tao shao (hts) and kelch genes, and is regulated by the Src64 and Tec29 tyrosine kinases. Female sterile cheerio mutants fail to recruit actin to ring canals, disrupting the flow of cytoplasm to oocytes. cheerio was cloned and found to encodes a member of the Filamin/ABP-280 family of actin-binding proteins, known to bind transmembrane proteins and crosslink actin filaments into parallel or orthogonal arrays. Antibodies to Drosophila Filamin revealed that Filamin is an abundant ring-canal protein and the first known component of both the outer and inner rims of the ring canal. The cheerio gene also encodes a new Filamin isoform that lacks the actin-binding domain. It is concluded that localization of Filamin to nascent ring canals is necessary for the recruitment of actin filaments. It is proposed that Filamin links filamentous actin to the plasma membrane of the ring canal. Although loss of Filamin in human cells supports a role for Filamin in organizing orthogonal actin arrays at the cell cortex, the cheerio mutant provides the first evidence that Filamin is required in membrane-associated parallel actin bundles, such as those found in ring canals, contractile rings and stress fibers (Sokol, 1999).

Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency

Nurse cells are cleared from the Drosophila egg chamber by apoptosis. DNA fragmentation begins in nurse cells at stage 12, following the completion of cytoplasm transfer from the nurse cells to the oocyte. During stage 13, nurse cells increasingly contain highly fragmented DNA and disappear from the egg chamber concomitantly with the formation of apoptotic vesicles containing highly fragmented nuclear material. In mutant egg chambers that fail to complete cytoplasm transport from the nurse cells (dumpless chambers), DNA fragmentation is markedly delayed and begins during stage 13, when the majority of cytoplasm is lost from the nurse cells. These data suggest the presence of cytoplasmic factors in nurse cells that inhibit the initiation of DNA fragmentation. The dumpless mutants studied include cheerio and kelch, which both have aberrant ring canal morphology that does not permit cytoplasm to pass easily from the nurse cells to the oocytes. The chickadee, singed and quail gene products are necessary for the proper formation of cytoplasmic actin filament bundles that form in nurse cells at stage 10B, just prior to the onset of cytoplasmic transport. reeper and hid are expressed in nurse cells beginning at stage 9 and continuing throughout stage 13. The grim transcript is not expressed as strongly as rpr or hid. The negative regulators DIAP1 and DIAP2 are also transcribed during oogenesis. However, germline clones homozygous for the deficiency Df(3)H99, which deletes rpr, hid and grim, undergo oogenesis in a manner morphologically indistinguishable from wild type, indicating that genes within this region are not necessary for apoptosis in nurse cells (Foley, 1998).

Formation of the Drosophila ovarian ring canal inner rim depends on cheerio

In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. A gene is described, cheerio, that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since Hts and Kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization (Robinson, 1997; full text of article).

Formation of actin filament bundles in the ring canals of developing Drosophila follicles

Subsequent to the addition of F-actin, HTS-RC and phosphotyrosine protein(s), the Kelch protein (Kelch) also becomes localized to the inner rim of the ring canal. The function of Kelch is to maintain the compaction of the ring canal rim. In late stage kelch mutant egg chambers, F-actin diffuses into the inner lumen of the ring canals and partially blocks the transfer of nurse cell cytoplasm to the oocyte. In addition to these known components of the ring canal, the product of the cheerio gene is also required for proper ring canal formation. cheerio ring canals are small and lack F-actin, HTS-RC and Kelch. Furthermore, fusions between the nurse cell and the oocyte are frequently observed in cheerio egg chambers indicating that the integrity of the plasma membrane has been compromised. To date, the cheerio gene has not been cloned, so it is not known whether its product is a ring canal component. Once the ring canals are established, they do not remain static. The rims of newly formed ring canals have diameters of 0.5-1 mm. By stage 11, at the onset of rapid cytoplasmic transfer from the nurse cells to the oocyte, the ring canals have attained their maximum size, with a diameter of roughly 10 mm. EM studies have shown that the early phase of growth (prior to stage 5) is accompanied by the addition of new actin filaments to the ring canal. After stage 5, there is an increase in total F-actin at the ring canal, but it is unclear as to whether this increase results from the addition of new filaments or the lengthening of existing filaments. However, during this developmental period, the filaments become organized into large bundles (Tilney, 1996).

Filamin A mediates isotropic distribution of applied force across the actin network

Cell sensing of externally applied mechanical strain through integrin-mediated adhesions is critical in development and physiology of muscle, lung, tendon, and arteries, among others. This study examined the effects of strain on force transmission through the essential cytoskeletal linker talin (Rhea in Drosophila). Using a fluorescence-based talin tension sensor (TS), it was found that uniaxial stretch of cells on elastic substrates increased tension on talin, which was unexpectedly independent of the orientation of the focal adhesions relative to the direction of strain. High-resolution electron microscopy of the actin cytoskeleton revealed that stress fibers (SFs) are integrated into an isotropic network of cortical actin filaments in which filamin A (FlnA; Cheerio in Drosophila) localizes preferentially to points of intersection between SFs and cortical actin. Knockdown (KD) of FlnA resulted in more isolated, less integrated SFs. After FlnA KD, tension on talin was polarized in the direction of stretch, while FlnA reexpression restored tensional symmetry. These data demonstrate that a FlnA-dependent cortical actin network distributes applied forces over the entire cytoskeleton-matrix interface (Kumar, 2019).

Arterial myogenic activation through Smooth Muscle Filamin A

Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. This study used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states (Retailleau, 2016).

FilaminA and Formin2 dependent endocytosis regulates proliferation via the canonical Wnt pathway.

Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. This study reports that the actin-binding protein FilaminA (see Drosophila Cheerio physically interacts with the actin-nucleating protein Formin2 (see Drosophila Daam). Loss of FilaminA and Formin2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FilaminA interacts with the Wnt co-receptor Lrp6. Loss of FilaminA and Formin2 impairs Lrp6 endocytosis, downstream Gsk3β (see Drosophila Shaggy) activity, and β-catenin (see Drosophila Armadillo) accumulation in the nucleus. Finally, the proliferative defect in null FilaminA and Formin2 neural progenitors is rescued by inhibiting Gsk3β activity. These findings provide an unrealized mechanism whereby actin-associated endocytosis regulates proliferation by mediating molecules in the canonical Wnt pathway. Moreover, the Formin2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology Daam protein. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation (Lian, 2016).

The oxygen sensor PHD2 controls dendritic spines and synapses via modification of Filamin A

Neuronal function is highly sensitive to changes in oxygen levels, but how hypoxia affects dendritic spine formation and synaptogenesis is unknown. This study reports that hypoxia, chemical inhibition of the oxygen-sensing prolyl hydroxylase domain proteins (PHDs), and silencing of Phd2 induce immature filopodium-like dendritic protrusions, promote spine regression, reduce synaptic density, and decrease the frequency of spontaneous action potentials independently of HIF signaling. The actin cross-linker filamin A (FLNA) was identified as a target of PHD2 mediating these effects. In normoxia, PHD2 hydroxylates the proline residues P2309 and P2316 in FLNA, leading to von Hippel-Lindau (VHL)-mediated ubiquitination and proteasomal degradation. In hypoxia, PHD2 inactivation rapidly upregulates FLNA protein levels because of blockage of its proteasomal degradation. FLNA upregulation induces more immature spines, whereas Flna silencing rescues the immature spine phenotype induced by PHD2 inhibition (Segura, 2016).

Filamin a regulates neural progenitor proliferation and cortical size through Wee1-dependent Cdk1 phosphorylation

Cytoskeleton-associated proteins play key roles not only in regulating cell morphology and migration but also in proliferation. Mutations in the cytoskeleton-associated gene filamin A (FlnA) cause the human disorder periventricular heterotopia (PH). PH is a disorder of neural stem cell development that is characterized by disruption of progenitors along the ventricular epithelium and subsequent formation of ectopic neuronal nodules. FlnA-dependent regulation of cytoskeletal dynamics is thought to direct neural progenitor migration and proliferation. This study has shown that embryonic FlnA-null mice exhibited a reduction in brain size and decline in neural progenitor numbers over time. The drop in the progenitor population was not attributable to cell death or changes in premature differentiation, but to prolonged cell cycle duration. Suppression of FlnA led to prolongation of the entire cell cycle length, principally in M phase. FlnA loss impaired degradation of cyclin B1-related proteins, thereby delaying the onset and progression through mitosis. The cdk1 kinase Wee1 bound FlnA, demonstrated increased expression levels after loss of FlnA function, and was associated with increased phosphorylation of cdk1. Phosphorylation of cdk1 inhibited activation of the anaphase promoting complex degradation system, which was responsible for cyclin B1 degradation and progression through mitosis. Collectively, these results demonstrate a molecular mechanism whereby FlnA loss impaired G2 to M phase entry, leading to cell cycle prolongation, compromised neural progenitor proliferation, and reduced brain size (Lian, 2012).

MEK-ERK1/2-dependent FLNA overexpression promotes abnormal dendritic patterning in tuberous sclerosis independent of mTOR

Abnormal dendritic complexity is a shared feature of many neurodevelopmental disorders associated with neurological defects. This study found that the actin-crosslinking protein filamin A (FLNA) is overexpressed in tuberous sclerosis complex (TSC) mice, a PI3K-mTOR model of neurodevelopmental disease that is associated with abnormal dendritic complexity. Both under- and overexpression of FLNA in wild-type neurons led to more complex dendritic arbors in vivo, suggesting that an optimal level of FLNA expression is required for normal dendritogenesis. In Tsc1^null neurons, knocking down FLNA in vivo prevented dendritic abnormalities. Surprisingly, FLNA overexpression in Tsc1^null neurons was dependent on MEK1/2 but not mTOR activity, despite both pathways being hyperactive. In addition, increasing MEK-ERK1/2 activity led to dendritic abnormalities via FLNA, and decreasing MEK-ERK1/2 signaling in Tsc1^null neurons rescued dendritic defects. These data demonstrate that altered FLNA expression increases dendritic complexity and contributes to pathologic dendritic patterning in TSC in an mTOR-independent, ERK1/2-dependent manner (Zhang, 2014).

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. This study describes the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and it is suggested to be a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin binding, but not talin binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions (Kiema, 2006).

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REFERENCES

Search PubMed for articles about Drosophila Cheerio

Albin, S. D. and Davis, G. W. (2004). Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology. J Neurosci 24(31): 6871-6879. PubMed ID: 15295021

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Bolduc, F. V., Bell, K., Rosenfelt, C., Cox, H. and Tully, T. (2010). Fragile x mental retardation 1 and filamin a interact genetically in Drosophila long-term memory. Front Neural Circuits 3: 22. PubMed ID: 20190856

Brooks, D., Naeem, F., Stetsiv, M., Goetting, S. C., Bawa, S., Green, N., Clark, C., Bashirullah, A. and Geisbrecht, E. R. (2020). Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover. PLoS Genet 16(4): e1008700. PubMed ID: 32320396

DiAntonio, A., Petersen, S. A., Heckmann, M. and Goodman, C. S. (1999). Glutamate receptor expression regulates quantal size and quantal content at the Drosophila neuromuscular junction. J Neurosci 19(8): 3023-3032. PubMed ID: 10191319

DiAntonio, A. (2006). Glutamate receptors at the Drosophila neuromuscular junction. Int Rev Neurobiol 75: 165-179. PubMed ID: 17137928

Eksioglu. Y. Z., (1996). Periventricular heterotopia: an X-linked dominant epilepsy locus causing aberrant cerebral cortical development. Neuron 16: 77-87. PubMed Citation: 8562093

Foley, K. and Cooley, L. (1998). Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 125(6): 1075-1082. PubMed ID: 9463354

Fox, J. W., (1998). Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia. Neuron. 21: 1315-1325. PubMed Citation: 9883725

Huelsmann, S., Ylanne, J. and Brown, N. H. (2013). Filopodia-like actin cables position nuclei in association with perinuclear actin in Drosophila nurse cells. Dev Cell 26(6): 604-615. PubMed ID: 24091012

Huelsmann, S., Rintanen, N., Sethi, R., Brown, N. H. and Ylanne, J. (2016). Evidence for the mechanosensor function of filamin in tissue development. Sci Rep 6: 32798. PubMed ID: 27597179

Kakita, A., (2002). Bilateral periventricular nodular heterotopia due to filamin 1 gene mutation: widespread glomeruloid microvascular anomaly and dysplastic cytoarchitecture in the cerebral cortex. Acta Neuropathol. 104(6): 649-657. PubMed Citation: 12410386

Kiema, T., et al. (2006). The molecular basis of filamin binding to integrins and competition with talin. Mol. Cell 21(3): 337-47. 16455489

Kulshammer, E. and Uhlirova, M. (2012). The actin cross-linker Filamin/Cheerio mediates tumor malignancy downstream of JNK signaling. J Cell Sci. PubMed ID: 23239028

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Biological Overview

date revised: 12 December 2022

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